Molecular Biology of Human Cancers [2 ed.] 3031162854, 9783031162855

Table of contents :
Preface
How to Read This Book
Contents
Abbreviations
List of Boxes
Part I: Molecules, Genes, Cells, and Mechanisms
1: An Introduction to Human Cancers
1.1 An Overview of the Cancer Problem
1.2 Causes of Cancer
1.3 Characteristic Properties of Cancers and Cancer Cells
1.4 Metabolic Changes in Cancer
1.5 Characterization and Classification of Cancers in The Clinic
1.6 Cancer Treatment
Further Reading
2: Cancer Genetics
2.1 Cancer as a Genetic Disease
2.2 Genetic Alterations in Cancers
2.3 Inherited Predisposition to Cancer
2.4 Cancer Genes
2.5 Accumulation of Genetic and Epigenetic Changes in Human Cancers
Further Reading
3: DNA Damage and DNA Repair
3.1 DNA Damage and Repair: An Overview
3.2 DNA Mismatch Repair
3.3 Nucleotide Excision Repair
3.4 DNA Strand Break Repair
3.5 DNA Inter-Strand Crosslink Repair
3.6 Deficiencies in DNA Repair and Cancer Susceptibility
3.7 Cell Protection Mechanisms in Cancer
Further Reading
4: Oncogenes
4.1 Retroviral Oncogenes
4.2 Slow-Acting Transforming Retroviruses
4.3 Identification of Human Oncogenes
4.4 Functions of Human Oncogenes: Receptor Tyrosine Kinases and the MAPK Pathway
4.5 Functions of Human Oncogenes: MYC Oncogenes
Further Reading
5: Tumor Suppressor Genes
5.1 Tumor Suppressor Genes in Hereditary Cancers
5.2 RB1 and the Cell Cycle
5.3 TP53 and the Control of Genomic Integrity
5.4 Classification of Tumor Suppressor Genes
Further Reading
6: Cancer Pathways
6.1 Cancer Pathways
6.2 MAPK Signal Transduction Pathways
6.3 The PI3K Pathway
6.4 Interaction of MAPK and PI3K Signaling in Cell Cycle Regulation
6.5 Modulators of the MAPK and PI3K Pathways
6.6 Signaling by TGFΒ Factors
6.7 Signaling Through NFkB
6.8 Signaling Through STAT Factors
6.9 Developmental Regulatory Systems as Cancer Pathways
6.10 Signaling Through Nuclear Receptors
Further Reading
7: Cell Death and Replicative Senescence in Cancer
7.1 Limits to Cell Proliferation
7.2 Replicative Senescence
7.3 Mechanisms to Escape Replicative Senescence in Human Cancers
7.4 Mechanisms of Apoptosis
7.5 Mechanisms Diminishing Apoptosis in Cancer
Further Reading
8: Cancer Epigenetics
8.1 Mechanisms of Epigenetic Inheritance
8.2 DNA Methylation
8.3 Histone Modifications and Regulation of Chromatin Structure
8.4 Genomic Imprinting and X-Chromosome Inactivation
8.5 Epigenetics of Cell Differentiation
Further Reading
9: Invasion and Metastasis
9.1 Invasion and Metastasis as Multistep Processes
9.2 Genes and Proteins Involved in Cell-Cell and Cell-Matrix Adhesion during Invasion and Metastasis
9.3 Genes and Proteins Involved in Extracellular Matrix Remodeling during Tumor Invasion and Metastasis
9.4 Tumor Neoangiogenesis
9.5 Interactions in the Tumor Microenvironment
9.6 Interactions of Invasive Tumors with the Immune System
Further Reading
Part II: Human Cancers
10: Leukemias and Lymphomas
10.1 Properties of Hematological Cancers
10.2 Genetic Aberrations in Leukemias and Lymphomas
10.3 Molecular Alterations in Acute Myeloid Leukemia
10.4 Molecular Biology of Burkitt Lymphoma
10.5 Molecular Biology of Chronic Myeloid Leukemia
10.6 Molecular Biology of Promyelocytic Leukemia
Further Reading
11: Pediatric Cancers
11.1 Specific Characteristics of Pediatric Tumors
11.2 Histology, Etiology, and Clinical Behavior of Wilms Tumors
11.3 Genetics of Wilms Tumors and the WT1 Gene
11.4 Epigenetics of Wilms Tumors and the Imprinted 11p15 Region
11.5 Testicular Germ Cell Tumors
Further Reading
12: Cancers of the Skin
12.1 Carcinogenesis in the Skin
12.2 Squamous Cell Carcinoma
12.3 Basal Cell Carcinoma
12.4 Melanoma
12.5 Tumor Antigens
Further Reading
13: Colorectal Cancer
13.1 Natural History of Colorectal Cancer
13.2 Familial Adenomatous Polyposis Coli and Other Cancer Syndromes Predisposing to Colorectal Cancer
13.3 Molecular Subtypes of Colorectal Carcinoma
13.4 The WNT Pathway in Colorectal Cancers
13.5 TGFΒ Signaling in Colorectal Cancers
13.6 Inflammation and Colorectal Cancer
Further Reading
14: Bladder Cancer
14.1 Histology and Clinical Course of Bladder Cancer
14.2 Etiology of Bladder Cancer
14.3 Molecular Alterations in Invasive Urothelial Carcinoma
14.4 Molecular Alterations in Urothelial Carcinoma Subtypes
Further Reading
15: Lung Cancer
15.1 Etiology of Lung Cancer
15.2 Carcinogenesis by Cigarette Smoke
15.3 Small Cell Lung Cancer
15.4 Non-Small Cell Lung Cancer
Further Reading
16: Renal Cell Carcinomas
16.1 The Diversity of Renal Cancers
16.2 Cytogenetics and Genomics of Renal Cell Carcinomas
16.3 Molecular Genetics of Inherited Kidney Cancers
16.4 Von-Hippel-Lindau Syndrome and Clear-Cell Renal Cell Carcinoma
16.5 Molecular Genetics of Clear-Cell Renal Cell Carcinoma
16.6 Molecular Biology of Papillary Renal Cell Carcinomas
16.7 Immunotherapy and Targeted Therapy of Renal Carcinomas
Further Reading
17: Liver Cancer
17.1 Etiology of Liver Cancer
17.2 Genetic Alterations in Hepatocellular Carcinoma
17.3 Growth Factors and their Receptors in Hepatocellular Carcinoma
17.4 Viruses in Hepatocellular Carcinoma
Further Reading
18: Stomach Cancer
18.1 Etiology of Stomach Cancer
18.2 Helicobacter pylori as a Biological Carcinogen in Stomach Cancer
18.3 Diffuse-Type Gastric Cancer
18.4 Metaplasia and Gastric Cancer
Further Reading
19: Breast Cancer
19.1 Etiology of Breast Cancer
19.2 Biology of Mammary Tissue
19.3 Classification of Breast Cancers
19.4 Estrogen Signaling in Breast Cancer
19.5 ERBB Proteins in Breast Cancer
19.6 The BRCA Genes and Hereditary Breast Cancer
Further Reading
20: Prostate Cancer
20.1 Epidemiology of Prostate Cancer
20.2 Genetics, Genomics, and Epigenetics of Prostate Cancer
20.3 Targeting the Androgen Response in Prostate Cancer
20.4 Tumor–Stroma Interactions in Prostate Cancer
Further Reading
Untitled
Part III: Prevention, Diagnostics, and Therapy
21: Cancer Prevention
21.1 The Importance of Cancer Prevention
21.2 Primary Prevention
21.3 Cancer Prevention and Diet
21.4 Prevention of Cancers in High-Risk Populations
21.5 Prevention of Prostate Cancer by Screening
Further Reading
22: Cancer Diagnostics
22.1 The Evolving Scope of Molecular Diagnostics
22.2 Molecular Diagnosis of Hematological Cancers
22.3 Molecular Detection of Carcinomas
22.4 Molecular Classification of Carcinomas
22.5 Prediction of Response to Therapy
22.6 Pharmacogenetics
Further Reading
23: Cancer Therapy
23.1 Limitations of Current Cancer Therapies
23.2 Molecular Mechanisms of Cytotoxic Cancer Chemotherapy
23.3 Principles of Targeted Anti-Cancer Drug Therapy
23.4 Targeted Drug Cancer Therapies: Pioneers
23.5 Newer Targeted Drug Cancer Therapies
23.6 Cancer Immunotherapies
23.7 Genetic Cancer Therapies: Gene Therapy, RNA-Based Drugs, and Oncolytic Viruses
23.8 Prospects for Cancer Therapy
Further Reading
Index

Citation preview

Wolfgang A. Schulz

Molecular Biology of Human Cancers

Second Edition

Molecular Biology of Human Cancers

Wolfgang A. Schulz

Molecular Biology of Human Cancers Second Edition

Wolfgang A. Schulz Department of Urology Heinrich Heine University Düsseldorf Düsseldorf, Germany

ISBN 978-3-031-16285-5    ISBN 978-3-031-16286-2 (eBook) https://doi.org/10.1007/978-3-031-16286-2 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2007, 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Meinen Eltern gewidmet

Preface

The first edition of this book, written almost two decades ago, developed from a lecture series that I had organized at the Heinrich Heine University Düsseldorf. The book was intended to address in particular advanced students, like PhD students with a background in molecular cell biology or medical doctors taking up molecular cancer research. I have meanwhile learned that the book has also been useful in master courses, especially courses in molecular biomedicine curricula. This second edition has benefitted from lectures in our regular master course on molecular oncology and in our graduate school, the Düsseldorf School of Oncology. I owe to all those that contributed to that course and the lectures. The second edition likewise is primarily directed at advanced students, but it should be accessible to (hopefully many) others as well, with the help of a good textbook on molecular cell biology or human molecular genetics. One criticism on the first edition was that the text appeared very “dense”; that may still be so because I have tried to keep the book (relatively) concise, but I have deconvoluted sentences and clarified explanations. Colored figures in the second edition should also help to make the book more readable. Please see also the following comments on “how to read this book.” I am very grateful to many colleagues that contributed to the second edition by reading drafts of chapters or (valiantly) the entire manuscript, providing comments, figures, or important references. These were especially, at my home institution, Michèle Hoffmann, Gerhard Fritz, Wolfgang Göring, Annemarie Greife, Helmut Hanenberg, Csaba Mahotka, Hans Neubauer, Dieter Niederacher, Günter Niegisch, Julia Reifenberger, Simon Santourlidis, Margaretha Skowron, Rüdiger Sorg, and Nikolas Stoecklein, as well as Nadine Gaisa at the RWTH Aachen, Roman Nawroth at the TU München, Akinori Sato at the National Defense Medical College, Tokorozawa, and Jiři Hatina and his colleagues Vladimir Korinek and Jiři Sramek at the Charles University Prague at Plzen. I would particularly like to acknowledge Rainer Engers, Neuss, for providing a huge selection of photographs for preparing the histology figures and for correcting the according legends. At Springer Nature, I am indebted to Ina Stoeck who kindly guided and advised me during the process of writing and producing this edition, as well as to Gopalakrishnan Rajesh and the production team. I remain grateful as well to

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everybody who contributed to the first edition. As always, I have to thank my wife and family for encouragement and support (as well as patience…). I hope you will find this book informative and useful. Please feel free to send any comments and suggestions for improvement to my e-mail address [email protected]. Duesseldorf, Germany June 2022

Wolfgang A. Schulz

How to Read This Book

I recommend of course to read this book from front to back—reading one chapter per day, this will take less than a month. If you wish to skip a chapter, I suggest taking a look at its introductory key points to make sure that you will not miss anything that might be important to you. The book is divided into three main parts. Following a general introduction to human cancers in Chap. 1, the first part introduces molecular, genetic, and cellular mechanisms that are relevant to cancer development. The second, central part deals with individual cancers. In this second edition, the most common cancers each have their own chapter. Many additional cancers are treated in specific sections or briefly in boxes. Please consider that while I hope to have included basic information on the etiology, molecular genetics, pathobiology, and the treatment for most cancer types, the aim of the chapters in Part II is not to provide a comprehensive description of each cancer. More importantly, individual cancers rather serve as examples for how some of the mechanisms outlined in the first part operate in real cancers. For instance, carcinogenesis by physical, chemical, and biological agents is described in more detail in the context of skin cancers, bladder and lung cancers, and liver and stomach cancers, respectively. Similarly, the function of specific signal transduction pathways (“cancer pathways”) and processes is treated in more detail in cancer types where they are most relevant. For instance, WNT and TGFβ signaling are taken up again in the colorectal cancer chapter and tumor hypoxia in the chapter on renal cancers; mechanisms of metastasis are described in more detail in the context of prostate cancer. Collectively, Part II should moreover serve to illustrate the diversity of pathogenic mechanisms among human cancers. Part III deals with the applications (envisioned and realized) that originate from the insights into the molecular mechanisms of cancer development and progression. In these chapters, again, no comprehensive description is intended (nor possible), instead, general principles are outlined and instructive examples are discussed in detail. Note that the three-part structure implicates that some topics appear in several places, usually in more depth in later chapters. For instance, some basics of tumor immunology are introduced in Chap. 9, tumor antigens in the context of melanoma in Chap. 12 and tumor immunotherapy in Chap. 23. Crossreferences throughout the book refer back to figures, sections or chapters where terms and concepts were introduced, or refer forward to places where they are going to be treated in more detail.

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The general aim of this textbook is to make readers familiar with the most relevant genes and proteins as well as molecular and cellular mechanisms implicated in human cancers. I also aim at introducing and discussing many of the current concepts underlying molecular cancer research and its clinical applications. These concepts are crucial for cancer research and its “translation” to clinical applications, but they need to be critically considered and continuously refined. Each chapter is followed by a list of articles for further reading; these are mostly recent review articles, but some original papers are included, for example, on the comprehensive characterization of genomic alterations in specific cancers. You will find that this textbook does not treat the many different ingenious techniques and the often elegant (and usually laborious...) experiments that underlie our knowledge on cancer molecular biology in any appropriate detail. Doing that would have required a different kind of approach (and resulted in a very different and much bigger book); moreover, in molecular biology research and its applications, techniques change very rapidly. The cited reviews in each chapter ought however provide more information on these aspects. One important disclaimer: This is not a textbook of medical oncology. Therefore, mention of diagnostic and therapeutic procedures does not implicate that they are recommended (or not) by the author or publisher. Finally, note that names of individual human proteins (but not protein classes) are capitalized throughout the text. Gene names are italicized. Gene and protein names (with aliases) can be found at www.genecards.org and detailed descriptions of hereditary syndromes at www.omim.org. An index of genes and protein is provided at the end of the book; for genes and proteins mentioned frequently, the most pertinent section is indicated. A list of other abbreviations follows below.

How to Read This Book

Contents

Part I Molecules, Genes, Cells, and Mechanisms 1 An  Introduction to Human Cancers����������������������������������������������   3 1.1 An Overview of the Cancer Problem����������������������������������������   4 1.2 Causes of Cancer����������������������������������������������������������������������   7 1.3 Characteristic Properties of Cancers and Cancer Cells������������  13 1.4 Metabolic Changes in Cancer ��������������������������������������������������  20 1.5 Characterization and Classification of Cancers in The Clinic�����������������������������������������������������������������������������  23 1.6 Cancer Treatment����������������������������������������������������������������������  26 Further Reading ��������������������������������������������������������������������������������  27 2 Cancer Genetics ������������������������������������������������������������������������������  29 2.1 Cancer as a Genetic Disease ����������������������������������������������������  30 2.2 Genetic Alterations in Cancers��������������������������������������������������  31 2.3 Inherited Predisposition to Cancer��������������������������������������������  39 2.4 Cancer Genes����������������������������������������������������������������������������  44 2.5 Accumulation of Genetic and Epigenetic Changes in Human Cancers��������������������������������������������������������������������  47 Further Reading ��������������������������������������������������������������������������������  49 3 DNA  Damage and DNA Repair������������������������������������������������������  51 3.1 DNA Damage and Repair: An Overview����������������������������������  52 3.2 DNA Mismatch Repair ������������������������������������������������������������  58 3.3 Nucleotide Excision Repair������������������������������������������������������  60 3.4 DNA Strand Break Repair��������������������������������������������������������  62 3.5 DNA Inter-Strand Crosslink Repair������������������������������������������  67 3.6 Deficiencies in DNA Repair and Cancer Susceptibility ����������  69 3.7 Cell Protection Mechanisms in Cancer������������������������������������  70 Further Reading ��������������������������������������������������������������������������������  73 4 Oncogenes ����������������������������������������������������������������������������������������  75 4.1 Retroviral Oncogenes����������������������������������������������������������������  76 4.2 Slow-Acting Transforming Retroviruses����������������������������������  79 4.3 Identification of Human Oncogenes ����������������������������������������  82 4.4 Functions of Human Oncogenes: Receptor Tyrosine Kinases and the MAPK Pathway����������������������������������������������  87

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4.5 Functions of Human Oncogenes: MYC Oncogenes ����������������  92 Further Reading ��������������������������������������������������������������������������������  95 5 Tumor Suppressor Genes����������������������������������������������������������������  97 5.1 Tumor Suppressor Genes in Hereditary Cancers����������������������  98 5.2 RB1 and the Cell Cycle������������������������������������������������������������ 102 5.3 TP53 and the Control of Genomic Integrity ���������������������������� 107 5.4 Classification of Tumor Suppressor Genes ������������������������������ 116 Further Reading �������������������������������������������������������������������������������� 119 6 Cancer Pathways������������������������������������������������������������������������������ 121 6.1 Cancer Pathways ���������������������������������������������������������������������� 122 6.2 MAPK Signal Transduction Pathways�������������������������������������� 123 6.3 The PI3K Pathway�������������������������������������������������������������������� 126 6.4 Interaction of MAPK and PI3K Signaling in Cell Cycle Regulation���������������������������������������������������������� 131 6.5 Modulators of the MAPK and PI3K Pathways������������������������ 134 6.6 Signaling by TGFΒ Factors������������������������������������������������������ 136 6.7 Signaling Through NFkB���������������������������������������������������������� 138 6.8 Signaling Through STAT Factors���������������������������������������������� 140 6.9 Developmental Regulatory Systems as Cancer Pathways�������� 142 6.10 Signaling Through Nuclear Receptors�������������������������������������� 149 Further Reading �������������������������������������������������������������������������������� 150 7 Cell  Death and Replicative Senescence in Cancer������������������������ 153 7.1 Limits to Cell Proliferation ������������������������������������������������������ 154 7.2 Replicative Senescence ������������������������������������������������������������ 160 7.3 Mechanisms to Escape Replicative Senescence in Human Cancers�������������������������������������������������������������������� 163 7.4 Mechanisms of Apoptosis �������������������������������������������������������� 165 7.5 Mechanisms Diminishing Apoptosis in Cancer������������������������ 170 Further Reading �������������������������������������������������������������������������������� 174 8 Cancer Epigenetics�������������������������������������������������������������������������� 177 8.1 Mechanisms of Epigenetic Inheritance������������������������������������ 179 8.2 DNA Methylation �������������������������������������������������������������������� 181 8.3 Histone Modifications and Regulation of Chromatin Structure ������������������������������������������������������������ 186 8.4 Genomic Imprinting and X-Chromosome Inactivation������������ 193 8.5 Epigenetics of Cell Differentiation ������������������������������������������ 197 Further Reading �������������������������������������������������������������������������������� 203 9 Invasion and Metastasis������������������������������������������������������������������ 205 9.1 Invasion and Metastasis as Multistep Processes ���������������������� 206 9.2 Genes and Proteins Involved in Cell-Cell and Cell-Matrix Adhesion during Invasion and Metastasis������������ 210 9.3 Genes and Proteins Involved in Extracellular Matrix Remodeling during Tumor Invasion and Metastasis���� 217 9.4 Tumor Neoangiogenesis����������������������������������������������������������� 220 9.5 Interactions in the Tumor Microenvironment �������������������������� 224

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9.6 Interactions of Invasive Tumors with the Immune System������ 229 Further Reading �������������������������������������������������������������������������������� 233 Part II Human Cancers 10 Leukemias and Lymphomas ���������������������������������������������������������� 237 10.1 Properties of Hematological Cancers�������������������������������������� 238 10.2 Genetic Aberrations in Leukemias and Lymphomas�������������� 241 10.3 Molecular Alterations in Acute Myeloid Leukemia���������������� 243 10.4 Molecular Biology of Burkitt Lymphoma������������������������������ 246 10.5 Molecular Biology of Chronic Myeloid Leukemia���������������� 250 10.6 Molecular Biology of Promyelocytic Leukemia�������������������� 254 Further Reading �������������������������������������������������������������������������������� 258 11 Pediatric Cancers ���������������������������������������������������������������������������� 261 11.1 Specific Characteristics of Pediatric Tumors�������������������������� 262 11.2 Histology, Etiology, and Clinical Behavior of Wilms Tumors�������������������������������������������������������������������� 265 11.3 Genetics of Wilms Tumors and the WT1 Gene���������������������� 266 11.4 Epigenetics of Wilms Tumors and the Imprinted 11p15 Region�������������������������������������������������������������������������� 269 11.5 Testicular Germ Cell Tumors�������������������������������������������������� 271 Further Reading �������������������������������������������������������������������������������� 274 12 Cancers  of the Skin�������������������������������������������������������������������������� 275 12.1 Carcinogenesis in the Skin������������������������������������������������������ 276 12.2 Squamous Cell Carcinoma������������������������������������������������������ 279 12.3 Basal Cell Carcinoma ������������������������������������������������������������ 283 12.4 Melanoma ������������������������������������������������������������������������������ 285 12.5 Tumor Antigens���������������������������������������������������������������������� 288 Further Reading �������������������������������������������������������������������������������� 289 13 Colorectal Cancer���������������������������������������������������������������������������� 291 13.1 Natural History of Colorectal Cancer ������������������������������������ 292 13.2 Familial Adenomatous Polyposis Coli and Other Cancer Syndromes Predisposing to Colorectal Cancer �������������������������������������������������������������� 293 13.3 Molecular Subtypes of Colorectal Carcinoma������������������������ 299 13.4 The WNT Pathway in Colorectal Cancers������������������������������ 301 13.5 TGFΒ Signaling in Colorectal Cancers���������������������������������� 304 13.6 Inflammation and Colorectal Cancer�������������������������������������� 305 Further Reading �������������������������������������������������������������������������������� 307 14 Bladder Cancer�������������������������������������������������������������������������������� 309 14.1 Histology and Clinical Course of Bladder Cancer������������������ 310 14.2 Etiology of Bladder Cancer���������������������������������������������������� 313 14.3 Molecular Alterations in Invasive Urothelial Carcinoma������� 316 14.4 Molecular Alterations in Urothelial Carcinoma Subtypes �������������������������������������������������������������� 322 Further Reading �������������������������������������������������������������������������������� 324

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15 Lung Cancer ������������������������������������������������������������������������������������ 327 15.1 Etiology of Lung Cancer�������������������������������������������������������� 328 15.2 Carcinogenesis by Cigarette Smoke �������������������������������������� 329 15.3 Small Cell Lung Cancer���������������������������������������������������������� 331 15.4 Non-Small Cell Lung Cancer�������������������������������������������������� 333 Further Reading �������������������������������������������������������������������������������� 335 16 Renal Cell Carcinomas�������������������������������������������������������������������� 337 16.1 The Diversity of Renal Cancers���������������������������������������������� 338 16.2 Cytogenetics and Genomics of Renal Cell Carcinomas �������� 340 16.3 Molecular Genetics of Inherited Kidney Cancers������������������ 341 16.4 Von-Hippel-Lindau Syndrome and Clear-Cell Renal Cell Carcinoma ������������������������������������������������������������������������������ 345 16.5 Molecular Genetics of Clear-­Cell Renal Cell Carcinoma������ 349 16.6 Molecular Biology of Papillary Renal Cell Carcinomas�������� 351 16.7 Immunotherapy and Targeted Therapy of Renal Carcinomas�������������������������������������������������������������� 352 Further Reading �������������������������������������������������������������������������������� 354 17 Liver Cancer������������������������������������������������������������������������������������ 357 17.1 Etiology of Liver Cancer�������������������������������������������������������� 358 17.2 Genetic Alterations in Hepatocellular Carcinoma������������������ 362 17.3 Growth Factors and their Receptors in Hepatocellular Carcinoma ������������������������������������������������������������������������������ 364 17.4 Viruses in Hepatocellular Carcinoma ������������������������������������ 366 Further Reading �������������������������������������������������������������������������������� 371 18 Stomach Cancer ������������������������������������������������������������������������������ 373 18.1 Etiology of Stomach Cancer �������������������������������������������������� 374 18.2 Helicobacter pylori as a Biological Carcinogen in Stomach Cancer������������������������������������������������������������������ 379 18.3 Diffuse-Type Gastric Cancer�������������������������������������������������� 382 18.4 Metaplasia and Gastric Cancer ���������������������������������������������� 384 Further Reading �������������������������������������������������������������������������������� 386 19 Breast Cancer ���������������������������������������������������������������������������������� 387 19.1 Etiology of Breast Cancer������������������������������������������������������ 388 19.2 Biology of Mammary Tissue�������������������������������������������������� 390 19.3 Classification of Breast Cancers �������������������������������������������� 392 19.4 Estrogen Signaling in Breast Cancer�������������������������������������� 395 19.5 ERBB Proteins in Breast Cancer�������������������������������������������� 401 19.6 The BRCA Genes and Hereditary Breast Cancer ������������������ 404 Further Reading �������������������������������������������������������������������������������� 413 20 Prostate Cancer�������������������������������������������������������������������������������� 415 20.1 Epidemiology of Prostate Cancer ������������������������������������������ 416 20.2 Genetics, Genomics, and Epigenetics of Prostate Cancer������ 420 20.3 Targeting the Androgen Response in Prostate Cancer������������ 426 20.4 Tumor–Stroma Interactions in Prostate Cancer���������������������� 430 Further Reading �������������������������������������������������������������������������������� 435

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Part III Prevention, Diagnostics, and Therapy 21 Cancer Prevention���������������������������������������������������������������������������� 439 21.1 The Importance of Cancer Prevention������������������������������������ 440 21.2 Primary Prevention ���������������������������������������������������������������� 441 21.3 Cancer Prevention and Diet���������������������������������������������������� 447 21.4 Prevention of Cancers in High-Risk Populations ������������������ 452 21.5 Prevention of Prostate Cancer by Screening�������������������������� 455 Further Reading �������������������������������������������������������������������������������� 458 22 Cancer Diagnostics�������������������������������������������������������������������������� 459 22.1 The Evolving Scope of Molecular Diagnostics���������������������� 460 22.2 Molecular Diagnosis of Hematological Cancers�������������������� 464 22.3 Molecular Detection of Carcinomas �������������������������������������� 467 22.4 Molecular Classification of Carcinomas�������������������������������� 472 22.5 Prediction of Response to Therapy ���������������������������������������� 473 22.6 Pharmacogenetics ������������������������������������������������������������������ 477 Further Reading �������������������������������������������������������������������������������� 480 23 Cancer Therapy�������������������������������������������������������������������������������� 483 23.1 Limitations of Current Cancer Therapies������������������������������� 484 23.2 Molecular Mechanisms of Cytotoxic Cancer Chemotherapy������������������������������������������������������������ 486 23.3 Principles of Targeted Anti-­Cancer Drug Therapy ���������������� 496 23.4 Targeted Drug Cancer Therapies: Pioneers���������������������������� 502 23.5 Newer Targeted Drug Cancer Therapies �������������������������������� 506 23.6 Cancer Immunotherapies�������������������������������������������������������� 517 23.7 Genetic Cancer Therapies: Gene Therapy, RNA-Based Drugs, and Oncolytic Viruses ���������������������������� 523 23.8 Prospects for Cancer Therapy ������������������������������������������������ 530 Further Reading �������������������������������������������������������������������������������� 532 Index���������������������������������������������������������������������������������������������������������� 535

Abbreviations1

aa ADCC ALT AML APC APL ASO BCC BCG BER BL BMDC CAF ccRCC cfDNA CIMP CIN CIS CML CNV COMPASS COSMIC CT CTC ctDNA CTL CUP DC DISC DTC EBV

Amino acids Antibody-dependent cytotoxicity Alternative lengthening of telomeres Acute myeloid leukemia Antigen-presenting cell (also gene name!) Acute promyelocytic leukemia Antisense oligonucleotide Basal cell carcinoma Bacillus Calmette-Guérin Base excision repair Burkitt lymphoma Bone marrow-derived cells Cancer-associated fibroblast Clear-cell renal cell carcinoma Circulating free DNA CpG-island methylated phenotype Chromosomal instability (subtype) Carcinoma in situ Chronic myeloid leukemia Copy number variant (variation) Complex (of) Proteins Associated with Set1 Catalog of Somatic Mutations in Cancer Computed tomography Circulating tumor cell Circulating tumor DNA Cytotoxic T-lymphocyte Carcinoma with unknown primary Dendritic cell Death-inducing signaling complex Disseminated tumor cell Epstein–Barr virus

Standard units, common biochemical terms, gene and protein names, and abbreviations of syndromes are not listed. Gene and protein names (with aliases) can be found, e.g., at www. genecards.org and detailed descriptions of hereditary syndromes at www.omim.org. Note that throughout the text names of individual human proteins are capitalized. Gene names are italicized. 1 

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EC Embryonal carcinoma ECM Extracellular matrix EMT Epithelial–mesenchymal transition ESC Embryonal stem cell EV Extracellular vesicle FISH Fluorescence in situ hybridization GAP GTPase-activating protein GEF GTP exchange factor GGR Global genome repair GWAS Genome-wide association study HAT Histone acetyltransferase HCC Hepatocellular carcinoma HDAC Histone deacetylase HHV Human herpes virus HNSCC Squamous carcinoma of the head and neck HPV Human papilloma virus HRR Homologous recombination repair HSC Hematopoietic stem cell HSR Homogeneously staining region IARC International Agency for Research on Cancer IC Imprinting center ICGC International Cancer Genome Consortium ICI Immune checkpoint inhibitor ICL Inter-strand crosslink (repair) IGN Imprinted gene network InDel Small insertion or deletion lncRNA Long non-coding RNA LOH Loss of heterozygosity LOI Loss of imprinting LUAD Lung adenocarcinoma LUSC Lung squamous cell carcinoma MAF Minor allele frequency MAPK Mitogen-activated protein kinase MDSC Myeloid-derived suppressor cell MET Mesenchymal-epithelial transition (also gene name!) miR microRNA MMR DNA mismatch repair MRI Magnetic resonance imaging MSC Mesenchymal stem cell MSI Microsatellite instability NER Nucleotide excision repair NGS Next-generation sequencing NHEJ Non-homologous end-joining (repair) NK Natural killer (cell) NMD Nonsense-mediated decay OMIM Online Mendelian Inheritance in Man (database) PET Positron-emission tomography PRC Polycomb repressor complex

Abbreviations

Abbreviations

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pRCC RCC RTK SCC SCLC SNP SWI/SNF TAA TAM TCGA TCR TGCT TMB TNBC TNM UC UTR VUS WGD

Papillary renal cell carcinoma Renal cell carcinoma Receptor tyrosine kinase Squamous cell carcinoma Small cell lung cancer Single nucleotide polymorphism Switch/sucrose non-fermentable (protein complex) Tumor-associated antigen Tumor-associated macrophage The cancer genome atlas Transcription-coupled repair (also protein name!) Testicular germ cell cancer Tumor mutational burden Triple-negative breast cancer Tumor node metastasis (staging system) Urothelial carcinoma Untranslated region Variant of uncertain significance Whole-genome doubling

List of Boxes

Box 1.1   Reactive Oxygen Species���������������������������������������������������������� 10 Box 1.2   Hallmarks of Cancer������������������������������������������������������������������ 14 Box 3.1   The NRF2 Transcription Factor and Its Regulation������������������ 72 Box 4.1   Carcinogenesis by HTLV-I�������������������������������������������������������� 77 Box 5.1   Retinoblastoma-Like Proteins and Cell Cycle Regulation������ 105 Box 5.2   Cervical Cancer and Human Papilloma Viruses �������������������� 115 Box 6.1   Regulation of PTEN by an RNA Network������������������������������ 130 Box 7.1   Human Aging and Cancer ������������������������������������������������������ 158 Box 7.2   Carcinogenesis by HHV8�������������������������������������������������������� 171 Box 8.1   A Broader Definition of Epigenetics �������������������������������������� 180 Box 8.2   EZH2 Functions beyond PRC2 ���������������������������������������������� 189 Box 8.3   The p57KIP2 Cell Cycle Inhibitor���������������������������������������������� 195 Box 9.1   Signaling through RHO and RAC in Cancer�������������������������� 213 Box 9.2   MicroRNAs as Regulators of EMT���������������������������������������� 215 Box 9.3   Tumor Hypoxia and its Consequences������������������������������������ 221 Box 9.4   Co-carcinogenesis by HIV1���������������������������������������������������� 230 Box 10.1  Clonal Hematopoiesis ������������������������������������������������������������ 245 Box 11.1  Medulloblastoma�������������������������������������������������������������������� 264 Box 12.1  Head and Neck Squamous Cell Carcinoma���������������������������� 282 Box 13.1  Positional Cloning of Tumor Suppressor Genes in Hereditary Cancers������������������������������������������������������������������ 295 Box 13.2  The Microbiome in CRC�������������������������������������������������������� 306 Box 14.1  Tumor Suppressor Candidates at 9q in Bladder Cancer���������� 320 Box 16.1  E3 Ubiquitin Ligases in Cancer���������������������������������������������� 346 Box 17.1  Experimental Hepatocarcinogenesis in Rats �������������������������� 361 Box 18.1  EBV-Associated Stomach Cancer ������������������������������������������ 378 Box 18.2  Barrett’s Esophagus and Esophageal Adenocarcinoma���������� 385 Box 19.1  Ovarian Cancers���������������������������������������������������������������������� 406 Box 22.1  Positron-Emission Tomography���������������������������������������������� 462 Box 22.2  Pancreatic Adenocarcinoma���������������������������������������������������� 470 Box 23.1  Development of Anti-Cancer Drugs���������������������������������������� 497 Box 23.2 Mechanisms of Resistance to CDK4/6 Inhibitors in Breast Cancer������������������������������������������������������ 512 Box 23.3  CAR-T Cells���������������������������������������������������������������������������� 520

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Part I Molecules, Genes, Cells, and Mechanisms

1

An Introduction to Human Cancers

Key Points • In 2020, around 20 million new cases of cancer were registered and nearly 10 million deaths were caused by cancers worldwide. • “Cancer” comprises several hundred different diseases. Cancers of the breast, lung, prostate, large intestine, liver, and stomach provide the greatest shares of cancer mortality worldwide. Skin cancers are more prevalent, but are in general less lethal. Since most cancers arise more frequently in older persons, their overall incidence keeps rising in aging populations, although improvements in prevention, diagnostics, and therapy have reversed the trend towards increasing mortality. • Most cancers are carcinomas, arising from epithelial cells of various tissues. Cancers can also develop from other cell types. Leukemias and lymphomas originate from cells of the hematopoietic system (hence hematological cancers) and sarcomas from diverse mesenchymal cells. • Cancers are caused by the interplay of genetic predisposition, endogenous processes, and environmental factors. Genetic predisposition is highlighted by hereditary cancer syndromes but also modulates the risk from exogenous carcinogens. Exogenous carcinogens comprise physical, chemical, and various biological (bacteria, viruses, and parasites) agents. Physical and chemical carcinogens typically

act as mutagens, but may also promote carcinogenesis by causing additional damage to tissues and cells. Endogenous processes relevant for carcinogenesis include DNA replication errors, the production of endogenous carcinogens by metabolic processes, as well as chronic inflammation. • Cancers are characterized by specific properties. To varying extents, these include enhanced, often autonomous cell proliferation, insufficient apoptosis, altered cell and tissue differentiation, altered intermediary metabolism, genomic instability, and evasion of cellular senescence. Some of these properties are shared with other diseases and are also found in benign tumors, but invasion into different tissue layers and other tissues as well as metastasis to local lymph nodes and distant tissues defines malignant tumors, i.e., cancer. • In order to predict prognosis and select optimal treatments, cancers are categorized by their physical extension (stage), deviation from normal cellular and tissue morphology (grade), and their histology. Increasingly, molecular characteristics are employed for classification and for the prediction of responses to therapy. • Cancers are treated by surgery, radiation, immunotherapy, or chemotherapy (cytotoxic or molecularly targeted drugs and antibodies), or a variety of combinations of these measures.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 W. A. Schulz, Molecular Biology of Human Cancers, https://doi.org/10.1007/978-3-031-16286-2_1

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1  An Introduction to Human Cancers

4

1.1 An Overview of the Cancer Problem

(≈2.2), lung (≈2.2 million cases), and prostate cancers (≈1.4 million) had the highest incidence worldwide. Lung cancers were the most lethal What is commonly called “human cancer” com- causing ≈1.8 million deaths. Notably, cancers of prises in fact several hundred different diseases. the stomach and liver are much more frequent in Together, they account for approximately one-­ East Asian countries with more than 70% of all fifth of all deaths in highly developed countries. cases occurring in that region. Worldwide, these One person out of three will be treated for a cancers rank third and fourth in mortality, each severe cancer in their lifetime. Worldwide, almost causing ≈800,000 deaths in 2020. Apart from the major four, the most prevalent 20 million new cases and nearly 10 million deaths from cancer were registered in 2020. 1 In a typical cancers are basal cell carcinoma and squamous Western industrialized country like Germany carcinoma of the skin (not included in Fig.  1.1), with its 83 million inhabitants, more than 400,000 which are rarely lethal (→12). The second group persons are newly diagnosed with cancer each of cancers, not as prevalent as the “big four,” year (not counting skin cancers except for account each for a few percent of the total cancer ­melanoma), and more than 200,000 succumb to incidence in Western countries. In addition to canthe disease. Since the incidence of most cancers cers of the liver (→17) and stomach (→18), they increases with age, their overall incidence rises in comprise carcinomas of the urinary bladder (→14), kidney (→16), pancreas and esophagus, aging populations. If one considers incidence and mortality by and skin melanoma (→12.4) in both sexes, and organ site, disregarding further biological and carcinomas of the cervix (Box 5.2) and ovaries clinical differences, cancers fall into three large (Box 19.1) in females. Some of these cancers are groups (Fig. 1.1). In general, cancers arising from highly lethal, in particular pancreatic carcinoma, epithelia, designated “carcinomas,” are most which is increasing in incidence, but also liver canprevalent. In Western industrialized countries, cer, and stomach cancer, whose incidence is four carcinomas contribute the greatest share to decreasing (→17, →18). The most frequent non-­ incidence as well as to mortality. These are can- carcinoma cancers, leukemias and lymphomas cers of the lung (→15) and the large intestine (→10), fall into the second group with respect to (colon and rectum, →13) in both sexes, breast incidence and mortality, too. They are collectively cancer (→19) in females and prostate cancer known as hematological cancers since they origi(→20) in males. In 2020, female breast cancer nate from cells of the hematopoietic system. Females

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1.1  An Overview of the Cancer Problem

5

The third group with lower incidences includes other cancers of soft tissues (including various kinds of sarcoma forming in bone, fat, cartilage, and connective tissues), brain (including glioblastoma), testes, bone, and further organs. While comparatively rare, many of these cancers constitute a significant health problem in specific age groups or geographic regions. For instance, testicular cancer is generally the most frequent neoplasia affecting young adult males, with an incidence of over 1% in this group in some countries. Mortality from testicular cancer is now relatively low, but only because efficient treatments have been developed over the last half century (→11.5). The health situation in low-income countries differs substantially from that in high-income countries because of the continuing, recurring, or newly emerged threat of infectious diseases such as malaria, tuberculosis, and AIDS. Nevertheless, cancer is an important health issue in these countries as well, but with different patterns of incidence and often higher mortalities for the same entities. Cancers of the stomach (→18), liver (→17), urinary bladder (→14), esophagus, and cervix are each endemic in certain parts of the world (Fig. 1.2). Often, they manifest in younger patients than in Western industrialized countries. Of the major four cancers in industrialized countries, only lung cancer has a similar impact in low-income countries, where its incidence is in fact increasing.

This snapshot view of present cancer incidence conceals changes over time (Fig. 1.3). For instance, on the one hand, industrialization and the spread of cigarette smoking have been generally associated with an increased incidence of lung, kidney, and bladder cancer. On the other hand, improvements in general hygiene and food quality may have contributed to the spectacular decline in stomach cancer incidence that is continuing in many countries (→18.1). Prostate and testicular cancer appear to have increased over the last decades. In prostate cancer, a slight potential increase in the age-adjusted incidence is exacerbated by the overall aging of the population (→20.1). In some regions of the world, the incidence of melanoma has escalated in an alarming fashion. This increase is primarily related to lifestyle factors rather than the aging of the population (→12.1). One important aim of cancer research is understanding the causes that underlie the geographical differences in cancer incidence and its changes over time. This understanding is a prerequisite for optimal strategies to prevent cancer (→21). In principle, the prospects for prevention are best for those cancers with large geographical differences in their incidences or big changes over time. To give just one example: The incidence of prostate cancer in immigrants from East Asia growing up in the USA has been postulated to be more than 10-fold higher than that of their Lung 1 435 943 (15.4%)

Breast 2 261 419 (25.8%) Other cancers 3 215 738 (34.4%)

Other cancers 3 013 893 (34.4%)

Prostate 1 414 259 (15.1%)

Colorectum 865 630 (9.9%)

Stomach 369 580 (4.2%) Corpus uteri 417 367 (4.8%)

Lung 770 828 (8.8%) Thyroid 448 915 (5.1%)

Cervix uteri 604 127 (6.9%)

Total : 8 751 759

Fig. 1.2  Incidence of cancers by organ site worldwide Left: cancer incidence in females; right: cancer incidence in males. Nonmelanoma skin cancers are not

Oesophagus 418 350 (4.5%) Bladder 440 864 (4.7%)

Colorectum 1 065 960 (11.4%)

Liver 632 320 (6.8%)

Stomach 719 523 (7.7%)

Total : 9 342 957

depicted. Data source: GLOBOCAN, IARC, see Sung et al. (2021) l.c

1  An Introduction to Human Cancers

6

Fig. 1.3  Trends in the mortality of selected cancers in the USA since 1930 Death per year, age-standardized, for the USA. From top to bottom (in 2011): lung cancer male (yellow line), lung cancer female (yellow), breast cancer (red), prostate cancer (dark red), colorectal cancer male (purple), colorectal cancer female (purple), pancreatic cancer male (blue), liver cancer male (green), pancreatic cancer female (blue),

leukemia male (grey), uterine cancer (blue), liver cancer female (green), stomach cancer male (light blue), and stomach cancer female (light blue). Modified from Max Roser & Hannah Ritchie (2015) “Cancer”. Published online at OurWorldinData.org Retrieved from https://ourworldindata.org/cancer [Online Ressource]  Based on original data from the American Cancer Society

relatives residing in East Asia (→20.1). It is easy to imagine the potential for prevention if the causes for this difference were understood. Unfortunately, neither the incidence nor the mortality of most cancers have been greatly curbed by conscious human interventions over the last decades. The mainstay for the treatment of most cancers remains a combination of surgery, radiotherapy, and chemotherapy. Cytotoxic chemotherapy has been supplemented by biologics (natural compounds or their analogs) and targeted drugs (including small-molecule chemicals as well as antibodies) based on insights into the molecular biology of human cancers. Surgery and radiotherapy often provide successful cures for organ-confined tumors. Cytotoxic chemotherapy can cure certain hematological cancers and most testicular cancers but is at best moderately efficacious for most advanced stage carcinomas and other cancers. Similarly, modern targeted therapies developed from the insights of molecular cancer research (→23.3–23.5) can cure or contain specific cancer types and mitigate the progression of many others.

The same conclusion applies to recently introduced novel immunotherapies (→23.6). In general, thus, only modest improvements have been made in the cure and survival rates for advanced-stage cancers. Metastatic carcinomas in particular remain as a rule uncurable. Developing means for early detection of cancers at curable stages (→22.3) and developing therapies for metastatic cancers therefore remain important goals of current molecular cancer research. Notably, quality of life is now widely accepted as a criterion for successful therapy. Recognizing that not every malignant tumor can be cured by the presently available therapies, treatments are chosen to maximize the chance for a cure and provide palliation, while minimizing adverse effects to retain a maximum of life quality2. Providing a better basis for such choices in the form of “individualized” or “personalized” therapy constitutes another application of new insights into the molecular biology of cancers (→22.5). Given the high costs of many novel cancer therapies, minimizing expenses has become another consideration. 2 

1.2  Causes of Cancer

1.2 Causes of Cancer Cancers are caused by an interplay of genetic predisposition, environmental factors, and endogenous processes. The first factor, genetic predisposition, is dramatically illustrated by familial cancer syndromes that are caused by mutations in single genes. Penetrance in these syndromes varies, but may approach 100%, i.e., every person with a mutant gene will develop cancer in their lifetime (→2.3). The influence of environmental factors is evident from the changing incidences of cancers over time and their geographical variations, given that the genetic constitution of humanity does not perceptively change over a century and human populations in different parts of the world do not substantially differ in their genetic constitution (see however 2.3 and 2.4). Cancers can be caused or be promoted by exogenous chemical, physical, or biological carcinogens, or by endogenous processes in the human body that act on their own or together with exogenous agents. Differences in the genetic constitution among individuals and populations modulate susceptibility, i.e., the extent to which environmental carcinogens cause cancers (→2.3, →3.6). Moreover, social and economic conditions influence not only the access to prevention, diagnostic procedures, and therapy but also have an impact on carcinogenesis. Understanding the causes of cancers is a prerequisite for their prevention, by eliminating or avoiding exogenous carcinogens, ameliorating the effects of endogenous carcinogenic processes, detecting and intercepting cancers as precursor lesions or at early stages when they can still be cured, and identifying individuals and groups at increased risk (→21). The mechanisms of carcinogenesis in humans are often multifactorial and complex (→12.1, 14.2, 15.2, 17.1, 18.1). Different factors may act through different mechanisms and at different stages of tumor development. In experimental animals, carcinogens can be applied in a controlled fashion so that the individual steps and interactions between different factors can be precisely analyzed. In such mod-

7

els, initiating and promoting as well as complete carcinogens can be distinguished. Initiating carcinogens are typically mutagens, while promoting agents act by creating conditions that facilitate the expansion of cells with mutated genomes. Moreover, the genetic constitution of many experimental animals, especially mice, can now be modulated at will to investigate the influence of specific genes and genetic variants on carcinogenesis in the entire animal or in specific organs. Disentangling carcinogenic factors in real humans is much more difficult. Even identifying the steps at which carcinogens act may pose problems. For instance, tobacco smoke is doubtless a human carcinogen (→15.2). In fact, it contains a variety of different chemical carcinogens, some of which may act as initiators, some as promoters, and some as both. Nicotine itself is almost certainly not a direct carcinogen, but a potent alkaloid which acts not only on the central nervous system but may also influence cell signaling and cell interactions in the airways and in the lung. In particular, addiction to nicotine enhances exposure to actual carcinogens. Similarly, complex interactions take place during skin carcinogenesis caused by UV radiation (→12.1). Genetic variations in the human population influence, among others, the metabolism of tobacco smoke carcinogens and the penetrance of UV into the skin as well as the efficiency of repair of DNA damage induced by tobacco smoke carcinogens and UV radiation. As a consequence, it is often difficult to elucidate exactly by which mechanism a potential carcinogen acts in humans, even though it is clearly linked to a specific cancer by epidemiological data. Attempts at cancer prevention therefore often have to be begun before the relationship between a carcinogen and cancer development is fully understood (→21). Nevertheless, precise elucidation of the carcinogenic mechanisms is important and insights from molecular biology contribute more and more to improved cancer prevention (→21).

1  An Introduction to Human Cancers

8

Many carcinogens have been established as relevant to human cancers. For some carcinogens, the evidence is very strong, while for others the categorization as a possible carcinogen is intended to foster preemptive measures. The classification issued by the World Health Organization accordingly groups human carcinogens (single agents or mixtures) by the level of available evidence, as detailed in Table  1.1. 3 Exogenous carcinogens can be classified into chemical, physical, and biological agents. Table  1.2 provides an overview of established carcinogens, with prominent examples for each type. Chemical carcinogens: Chemical carcinogens comprise compounds from many different classes (Fig.  1.4) that originate from various sources. Inorganic compounds like cadmium, nickel, or arsenic are encountered in the workplace or contaminate water sources. Carcinogenic organic compounds can be aliphatic, like nitrosamines, which originate especially from smoked and pickled foods, or trichloro-ethylene, a compound used a. o. in industrial cleaning. Nitrosamines are thought Table 1.1  Classification of human carcinogens according to the WHO/IARC Group Group 1 Group 2A Group 2B Group 3 Group 4

Definition The agent is carcinogenic in humans. The exposure circumstance entails exposures that are carcinogenic to humans The agent is probably carcinogenic to humans. The exposure circumstance entails exposures that are probably carcinogenic to humans The agent is possibly carcinogenic to humans. The exposure circumstance entails exposures that are possibly carcinogenic to humans The agent (or exposure circumstance) is not classifiable as to carcinogenicity in humans The agent (or exposure circumstance) is probably not carcinogenic to humans

The IARC regularly publishes monographs on individual (or classes of) carcinogens that can be freely downloaded. 3 

Table 1.2  Types and examples of human carcinogens Type of carcinogen Chemical carcinogens

Physical carcinogens Biological carcinogens

Endogenous processes

Examples Cadmium, nickel, arsenic, ethanol, nitrosamines, trichloro-ethylene, arylamines, benzopyrene, aflatoxins, aristolochic acid, reactive oxygen species UV irradiation (specifically UVB), ionizing radiation Human papilloma virus (e.g., strain 16), Epstein-Barr-Virus, Hepatitis virus B, Helicobacter pylori, Schistosoma mansoni DNA replication, metabolic reactions generating reactive oxygen species, chronic inflammation

to contribute to stomach cancer in particular (→18.1) Aromatic compounds like benzopyrenes and arylamines are generated from natural sources by burning, and are among the many carcinogens in tobacco smoke. Arylamines are thought to cause bladder cancer in particular (→14.1). Polyaromatic compounds like benzopyrene are also released into the environment by burning of coal and fuels. They present a hazard in the workplace, e.g., during coal processing, dye production and use, but also from cooking fires and stoves in poorly ventilated rooms, where they may cause lung adenocarcinomas in nonsmokers (→15.4). A variety of synthetic polychlorinated (or brominated) chemicals are suspected or established carcinogens. Several natural compounds from plants and molds are also known to be highly carcinogenic. For instance, aflatoxin B1 is implicated as a carcinogen in liver cancer (→17.1). Birthwort species used in traditional herbal medicines contain carcinogenic aristolochic acids. Certain commonly used medical drugs can be carcinogenic, notably cytotoxic compounds used in tumor therapy such as cyclophosphamide, nitrogen mustards, and platinum complexes that react with DNA.  Various hormones and hormone-like chemical compounds from natural, industrial,

1.2  Causes of Cancer

9

Fig. 1.4  Structures of some chemical carcinogens NNK: 4-methyl-­nitrosamino-1(3-­pyridyl)-1-butanone

O N As2O3

N

CH3

Benzopyrene

CH3

Dimethyl-nitrosamine NH2 NH2

2HN

β-Napthylamine

Benzidine

O H

O H3C

O

O H

O

O

OH

HO

CH3

CH3

Aflatoxin B1

Diethyl-stilbestrole H N

CI CI

O 2-Acetylaminofluorene

C

C

H CI

Trichloro-ethylene O

O C

N CH2

N

and pharmaceutic sources influence cancer development in specific tissues, such as the breast (→19.1) and the prostate (→20.1). Oxygen may be regarded as the most abundant carcinogen. While it is relatively inert in its most common state, dioxygen, and is evidently safe when fully reduced towards H2O, partially reduced oxygen species or dioxygen activated to the singlet state (summarized as reactive oxygen species) are highly reactive compounds that can

CH2

NNK

CH2

N

CH3

Benzene

damage cells or act as mutagens (Box 1.1). Reactive oxygen species are formed at low levels during normal metabolism and are produced at increased rates during certain physiological processes like inflammation, but also during the development of many cancers. Their concentrations can become particularly critical during the metabolism of certain exogenous compounds, e.g., quinones, and by pathophysiological states such as iron overload.

1  An Introduction to Human Cancers

10

Box 1.1 Reactive Oxygen Species

Oxygen is present throughout the human body as the relatively unreactive dioxygen molecule in its triplet state (3O2). Singlet oxygen (1O2) is much more reactive. It can be generated by energy transfer from photoactivated compounds, e.g., porphyrins in the skin, and can be deactivated by transferring its energy to water molecules within a few μs. It also can add to double bonds in biomolecules, like those in unsaturated fatty acids of membrane lipids, yielding unstable endoperoxides that initiate chain reactions generating toxic and mutagenic compounds. Extensive oxidation of membrane lipids can initiate a type of cell death known as ferroptosis (→7.1). By taking up 4 electrons and 4 protons O2 yields H2O, e.g., in the Cytochrome oxidase reaction in the mitochondria. Water is of course innocuous, but all intermediate stages of O2 reduction are not. Superoxide (O2-) is a byproduct of several enzymatic reactions: it is especially produced by NADPH oxidases. In cells the main source is normally leakage of electrons from electron transport chains in the mitochondria and the ER.  Superoxide is highly reactive and initiates chain reactions with coenzymes, nucleotides and thiol groups of enzymes. Like H2O2, it reacts with metal ions. H2O2 is a general oxidant as well, although not quite as reactive as superoxide. However, via Fenton type reactions (see Fig. 17.3), it generates the extremely reactive hydroxyl radical (.OH) that oxidizes essentially any biomolecule it encounters during its nanosecond half-life. Acting upon DNA, it is highly mutagenic. Nitrous oxide (NO ) is another radical molecule produced in cells. It has a short half-life, and can react with multiple biomolecules, in particular with the amino groups of proteins and DNA bases. Moreover, it can combine with superoxide to form peroxynitrite (ONOO-), which has

a longer half-life. Therefore, this reaction decreases the toxicity of the parent compounds, but is a mixed blessing, because peroxynitrite still reacts with amino groups in proteins and DNA and may in fact be more mutagenic. The reactive molecules arising from oxygen are generally referred to as ‘reactive oxygen species’ (ROS). When using this summary term, it should be considered that the individual species differ vastly in reactivity and half-life and consequently in their effects. In fact, a certain level of reactive oxygen species and other radical molecules in the cell is physiological. Cells employ these reactive molecules to modulate the oxidation state of signaling molecules and metabolic enzymes. Nitrous oxide, e.g., elicits vasodilation in addition to other effects. Specialized cells like macrophages moreover generate superoxide, singlet oxygen and NO to kill infectious agents. The amount of reactive oxygen species in a cell is normally constrained by a variety of protective molecules, comprising low molecular weight compounds and enzymes (→3.7), which remove reactive oxygen and nitrous species and prevent or interrupt the radical chain reactions that they initiate. Specific repair mechanisms, like base repair by OGG (Oxoguanine glycosylase), deal with DNA damage caused by these molecules. If pro-oxidant molecules cannot control the level of reactive oxygen species, sustained ‘oxidative stress’ may damage cell membranes, organelles and DNA and cause cell death by ferroptosis, necrosis or apoptosis. Cheung EC & Vousden KH (2022) The role of ROS in tumour development and progression. Nat Rev Cancer 22:280–297 Sies H & Jones DP (2020) Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat Rev Mol Cell Biol 21:363–383

1.2  Causes of Cancer

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Physical carcinogens: In principle, any energyrich radiation can act as a carcinogen, depending on its dose and absorption. Visible light is not usually carcinogenic unless absorbed by “photosensitizers” which usually generate reactive oxygen species. UVB radiation is an important carcinogen in the skin (→12.1), and its effect is augmented by UVA.  In contrast, UVC can neither penetrate the atmosphere nor the upper (noncellular) protective layers of the skin. Therefore, it cannot act as a carcinogen under normal circumstances. γ-Radiation from natural, industrial, and iatrogenic sources (like in X-ray diagnostics) can penetrate into (and through) the body. It is carcinogenic to the extent to which it is absorbed, by damaging DNA and cells following direct absorption but also indirectly by generating ions and radicals including reactive oxygen species. Damage and carcinogenicity by γ-radiation therefore depend on the oxygen concentration in a tissue, but also on the capacities for cellular protection and repair (→3.7). Radioactive β-radiation and specifically α-radiation are most dangerous when nuclides are incorporated, e.g., of radon, cesium, uranium, or plutonium. The effect of radioactive isotopes depends moreover on their distribution in the body. For instance, radioactive iodine accumulates in the thyroid gland and therefore causes specifically thyroid cancers, whereas radioactive cesium isotopes are excreted with the urine and tend to become enriched in the urinary bladder. The noble gas radon is inhaled and causes predominantly lung cancer. 4 The potential carcino-

genicity of microwave and radio wavelength electromagnetic radiation is the subject of public debates, but at everyday exposure levels the risks are probably low. Biological carcinogens: Viruses can cause or facilitate carcinogenesis in various ways. They can express gene products inhibiting tumor suppressors or apoptosis or transduce oncogenes; many DNA viruses moreover activate the cell cycle to facilitate their replication. Integration of viral sequences into the host genome can disturb gene regulation at the integration site. The integration of viral genomes can elicit structural chromosomal changes and viral gene products may disturb the control of genomic stability. Moreover, viral infections alter the interaction of their target cells with other cells, altering immune responses and eliciting inflammation. Cells killed by the viruses or by immune cells that eliminate the infected cells must be replaced by tissue regeneration, increasing the risk for cancer development. Seven viruses are (at this time) recognized biological carcinogens in man (Table  1.3). Specific strains of human papilloma viruses (especially HPV16 and HPV18) are established as causative factors in cervical and penile cancers (Box 5.2) and a subset of oropharyngeal squamous carcinomas (Box 12.1). Specific herpes viruses can act as carcinogens or co-carcinogens, e.g., human herpes virus 8 (HHV8) in Kaposi sarcoma (Box 7.2) or Epstein-Barr virus (EBV) in certain lymphomas

Table 1.3  Human cancer viruses Virus Epstein-Barr virus

Acronym EBV

Genome DNA

Hepatitis virus B

HBV

Hepatitis virus C Human immunodeficiency virus-1

HCV HIV

Human papilloma virus Human T cell lymphotropic virus

HPV HTLV

Kaposi sarcoma herpesvirus

KSV/HHV8

DNA (RNA intermediate) RNA RNA (DNA intermediate) DNA RNA (DNA intermediate) DNA

‘While enrichment of “osteotropic” radioactive isotopes from elements with properties similar to calcium (like strontium) in bone tissue may cause damage it can be exploited for treatment of bone metastases. 4 

Cancers caused (examples) Burkitt lymphoma Gastric cancer Hepatocellular carcinoma Hepatocellular carcinoma Lymphoma Kaposi sarcoma Cervical cancer Adult T cell lymphoma/leukemia Kaposi sarcoma

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(→10.4) and carcinomas (Box 18.1). The hepatitis B virus (HBV) with its DNA genome and the RNA virus hepatitis C virus (HCV) cause a substantial proportion of liver cancers (→17.4). The retrovirus HTLV1 (human T cell leukemia virus) causes the rare leukemia ATLL by inappropriate growth stimulation of T cells (Box 4.1), whereas HIV facilitates the development of several cancers by indirect mechanisms  (Box 9.4). It is likely that additional viruses may act as carcinogens or facilitate tumor development indirectly. For instance, the β-polyomaviruses SV40 (simian virus 40), JCV, and BKV cause cancers in animals and partially transform human cells in culture (→5.3), but whether they actually cause human cancers is uncertain. Merkel cell polyomaviruses however are clearly involved in a rare skin carcinoma of older people. While a carcinogenic role is assumed for several bacterial species, definitive evidence exists for Helicobacter pylori as a carcinogen in stomach cancer (→17.3). Other potentially carcinogenic bacterial species encode toxins that may deregulate cellular signaling, cause cell death, or DNA damage (Box 13.2). More generally, bacterial infections contribute to chronic inflammation that can promote cancer development. The parasite Schistosoma trematodes definitely causes cancer in humans, especially in the urinary bladder. Two further parasite liver flukes, Opisthorchis viverrine and Clonorchis sinensis, are recognized biological carcinogens, too. Endogenous carcinogenic processes: How effective exogenous carcinogens elicit cancer in a specific person depends strongly on an individual’s exposure (dose and duration), specific responses, and general health. In that sense, endogenous processes are generally involved in cancer development by modulating the response to exogenous carcinogens. However, cancers may also be caused by a number of genuinely endogenous processes that include the following five. (1) Metabolic products: Normal intermediary metabolism generates carcinogenic compounds including nitrosamines, aromatic amines, quinones, reactive aldehydes (like acetaldehyde from ethanol and formaldehyde from demethylation reactions), in addi-

1  An Introduction to Human Cancers

tion to reactive oxygen and nitrous species. The concentration of these potential carcinogens varies, depending on factors like diet or physical activity, but a minimum level is associated with any level of metabolic activity and any type of diet. While potent protective and detoxification mechanisms exist for these compounds, they can be defective or become overwhelmed. (2) Spontaneous damage: In the same vein, damage to cells and specifically DNA occurs at a minimum rate spontaneously and particularly during cell proliferation, e.g., by errors during DNA replication or by spontaneous chemical reactions altering DNA bases (→3.1). The potentially huge number of such errors is kept at bay by highly efficient DNA repair mechanisms specifically directed at and eliminating erronous  products. In addition, overly damaged cells are removed by apoptosis or other modes of programmed cell death. These protective mechanisms however cannot be perfect either. Notably, mutations that impede the fidelity of replication or inactivate specific types of DNA repair (→3.6) are a frequent cause of cancer. (3) Aging: Damaged DNA and senescent cells (→7.2) accumulate with age and protective mechanisms may become less efficient at higher ages. Both factors may contribute to the increase of cancer incidence with age (Box 7.1). For instance, mutations converting C to T at CpG dinucleotides are observed in many human cancers; their frequency increases with the patient’s age even in normal tissues. (4) Cell proliferation: The risk of incurring and even more of “fixing” and propagating genetic changes is higher in proliferating cells. The cancer risk for specific tissues may therefore relate to their rate of proliferation, especially to the rate of proliferation of their tissue stem cells. Proliferation activity and accordingly the risk of carcinogenesis is higher during specific processes and periods, e.g., when tissues need to regenerate and cells proliferate after incurring damage. While tissue regeneration in general is associated with an increased cancer risk because of increased cell prolifera-

1.3  Characteristic Properties of Cancers and Cancer Cells

tion, it is particularly perilous if it includes prominent tissue remodeling as in liver cirrhosis (→17.1). One period in human life with particularly high proliferative activity is of course fetal development. Genetic and even epigenetic errors occurring during this period may especially lead to cancers in children (→11), but may also initiate cancers that manifest much later in life. (5) Chronic inflammation: It is associated with increased cancer risk in many organs, e.g., in the gut (→13.6) or stomach (→18.1). Overall, about 25% of all cancers are estimated to be associated with chronic inflammation, including those in which chronic inflammation is elicited by viral, bacterial, or parasitic infections. Several mechanisms contribute to cancer promotion by chronic inflammation. They include the increased production of mutagenic reactive oxygen species by inflammatory cells as well as the secretion of proteases, cytokines, and growth factors by various cell types in the inflamed tissue and an altered immune environment that together create favorable conditions for the expansion of genetically altered cell clones and for the spread of tumors (→9). In fact, some carcinogens have been proposed to act primarily by stimulating chronic inflammation, tissue growth, and remodeling without being actually mutagenic. In animal experiments, these can be identified as tumor promoters. In summary, both endogenous and exogenous factors can be responsible for human cancers. Typically, both contribute. In many  cases, the involvement of specific exogenous carcinogens or endogenous processes can be unequivocally identified by characteristic mutational signatures in the cancer cell genome (→3.1). However, not all carcinogens are mutagens and not only bacteria and parasites promote cancer via processes like chronic inflammation. Strong (complete) carcinogens like tobacco smoke act both as mutagens as well as by inducing tissue damage and chronic inflammation. Moreover, the carcinogenicity of some carcinogens like nickel and arsenic is mediated to a substantial extent by

13

disturbances of epigenetic regulation. For these reasons, the contribution of exogenous carcinogens to the overall cancer burden may be underestimated. Nevertheless, in some cancer types, the absence of mutational fingerprints, lack of epidemiological correlations, and of plausible evidence for the involvement of exogenous carcinogens point to a predominance of endogenous processes. However, in most cancers, it is difficult to estimate quantitatively how much genetic predisposition, occupational carcinogens, viruses, stem cell duplication rates, etc., each contribute to carcinogenesis. Nevertheless, such estimates may help to outline the potential for prevention (→21.1).

1.3 Characteristic Properties of Cancers and Cancer Cells In spite of their diversity, human cancers share several fundamental properties (Table  1.4, see also Box 1.2). Different cancers display each of these properties to different extents. They may moreover be acquired in a stepwise manner and become evident at various stages during cancer progression. One or the other characteristic property of cancers can also be found in other diseases and some are even associated with physiological adaptive responses or repair of tissue injury (although usually in a temporary and reversible manner). However, the combination of uncontrolled cell proliferation, altered differentiation and metabolism, genomic instability, and invasiveness with eventual metastasis is unique to and unequivocally defines cancer.

Table 1.4  Characteristic properties of human cancers Increased cell proliferation (often autonomous) Insufficient apoptosis Altered cell and tissue differentiation (with disturbed tissue architecture) Genomic instability Immortalization (growth beyond replicative senescence) Altered metabolism Invasion into different tissue layers and other tissues Metastasis to local lymph nodes and distant tissues

1  An Introduction to Human Cancers

14

Box 1.2 Hallmarks of Cancer

In a millennium issue article that appeared on January 7th, 2000, Hanahan and Weinberg proposed a set of characteristic properties of cancers. These traits considered as ‘hallmarks of cancer’ comprise • • • • • •

self-sufficiency in growth signals insensitivity to anti-growth signals evasion of apoptosis limitless replicative potential sustained angiogenesis tissue invasion and metastasis.

This concept has been highly influential and the criteria have been widely used in experimental cancer research and in preclinical development of anti-cancer therapies. A comparison shows that they are not too different from those listed in Table 1.3. They are not identical, though, because the two lists reflect somewhat different perspectives. The “hallmarks of cancer”’ are based to a greater degree on experience in experimental cellular and animal models: each trait can be observed and analyzed in cancer cell lines and in animal models. In appropriate models, these properties can be generated or suppressed by genetic manipulation. It is, however, not always straightforward to ascertain the hallmarks in actual human cancers, and, notably, the individual traits apply to different human cancers to different degrees. In comparison, the properties discussed in Sect. 1.3 are to a greater degree oriented at what can be observed in human cancers. They are consequently more descriptive and their molecular basis is not always completely understood. Unfortunately, at present, not all human cancers can be studied in adequate models. This is one of the more severe impediments to progress in cancer research. Optimal transfer (‘translation’) of experimental insights to the clinic requires appropriate and accessible experi-

mental models and an understanding of processes in real human cancers alike (cf. Part III). Indeed, in the best current cancer research, experimental data obtained using cell and animal models are combined with analyses of human cancer tissues. In a follow-up article that appeared 11 years later, Hanahan and Weinberg summarized conceptual progress on the six hallmarks of cancer and added two further ones as ‘emerging’, namely ‘deregulated cellular energetics’ and ‘avoiding immune destruction’. They additionally highlighted two processes important for cancer development and progression, ‘genomic instability mutation’ and ‘tumor-promoting inflammation’ as ‘enabling’ characteristics. Recent developments on the ‘hallmarks of cancer’ concept are discussed in the 2022 update by Hanahan l.c. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70 Hanahan D, Weinberg RA (2011) The hallmarks of cancer: the next generation. Cell 144:656–674 Hanahan D (2022) Hallmarks of cancer: new dimensions. Cancer Discov. 12:31–46

Increased and autonomous cell proliferation: The most obvious property of tumors is growth beyond normal boundaries. In fact, the term “tumor” denotes in a broader sense every abnormally large structure in the human body, including swellings or fluid-filled cysts. More precisely, then, cancers are tumors caused primarily by increased cell proliferation, i.e., a continuous and persistent increase in cell numbers. Increased cell proliferation per se is also observed during tissue regeneration, wound healing, adaptive tissue growth, and in some non-cancerous diseases, e.g., atherosclerosis. In general, an increased number of cells in a tissue is designated “hyperplasia.” Enlargement of tissues by extensive hyperplasia is considered a “benign” tumor (see Table 1.6 for a list of relevant terms). Hyperplasia

1.3  Characteristic Properties of Cancers and Cancer Cells

15

may be accompanied by changes in the morphology of the cells or the tissue, which is regarded as “dysplasia.” Dysplastic lesions in epithelia, designated carcinoma in situ, are one precursor of carcinomas that are, in such cases, considered “preneoplastic.” Substantial alterations in the tissue structure and in particular the invasion of tumor cells into different layers of the tissue define a malignant tumor or “cancer.” The difference between hyperplasia, benign tumors, and cancer can be discerned by microscopic inspection (see Fig. 1.5), or may even be macroscopically evident. Nevertheless, in some cases, additional criteria or biomarkers have to be employed to make the distinction. For instance, proliferating cells are generally marked by strong expression of the nuclear protein Ki67 (gene: MKI67; see Fig. 1.6). Cancerous glandular struc-

tures in the prostate (adenocarcinomas) may morphologically resemble normal glands but lack the cytokeratins of basal prostatic cells. Hyperproliferation in cancers is brought about by altered responses to exogenous growth regulatory signals (→6). On the one hand, cancers may be hypersensitive to growth-stimulatory signals or become largely independent of external signals as a consequence of activating mutations in signal transduction pathways or altered cell cycle regulation. On the other hand, sensitivity to growth-inhibitory signals by paracrine factors or cell–cell interactions is often diminished or abolished. Together, these altered responses result in the characteristic growth autonomy of cancers, which typically aggravates during progression. Insufficient apoptosis: Increasing cell numbers in cancers are caused by (variable) combinations

Fig. 1.5  Invasion and metastasis Top left: Invasion front of a squamous cell skin carcinoma (marked by arrows); Top right: stromal activation (desmoplastic reaction) in a ductal pancreatic adenocarcinoma;

Bottom left: liver metastasis (marked by the star symbol) of breast cancer. Bottom right: bone metastasis of an adenocarcinoma. Courtesy: R. Engers

16

1  An Introduction to Human Cancers

Fig. 1.6  Immunohistochemical staining of breast cancer tissues Immunohistochemical staining of breast cancers. Left: a lobular mammary carcinoma stained with a pan-­

cytokeratin antibody to better visualize the growth pattern of the cancer cells; right: an invasive ductal mammary carcinoma (actually: invasive mammary carcinoma of nonspecial type) stained for Ki67. Courtesy R. Engers

of three—by no means mutually exclusive—factors: (1) The rate of cell proliferation is enhanced by an increase in the proportion of cells actively progressing through the cell cycle, i.e., a higher “proliferative fraction,” and in some tumors by a more rapid transit through the cell cycle, which together result in increased DNA synthesis and a higher fraction of mitotic cells. (2) The rate of cell death is often decreased, especially by insufficient apoptosis (→7.5). In some cancers, blocked apoptosis is the primary driving force of cancer growth. In many other cancers, the rate of apoptosis is enhanced compared to the normal tissue, but not sufficiently high to compensate for the increase in proliferative activity. (3) In normal tissues, successive stages of differentiation are typically associated with progressively decreased p­roliferative capacity; in some tissues, fully differentiated cells are obliterated or lost at a steady rate (→7.1). Thus, a block to differentiation is sufficient to confer an increased proliferation rate in some tumors; more commonly, it augments tumor growth. Altered cell and tissue differentiation: Many cancers consist of cells that resemble precursor or stem cells of their tissue of origin and have not embarked on the normal course of differentiation, whereas others show properties of cells at intermediate stages of differentiation. Yet other cancers consist of cells with a near-complete set of differentiation markers, with the crucial differ-

ence that they continue to proliferate. In all these cancers, diagnosis of their tissue of origin can be made in a relatively straightforward manner. Many cancers however express markers that do not occur in their tissue of origin (→12.5). For instance, cancer cells may express proteins that otherwise are only found in fetal cells. Such “oncofetal” proteins include Carcinoembryonic antigen in colon carcinoma or α-Fetoprotein in liver cancer. These proteins can be useful as biomarkers for cancer detection and diagnosis. Other proteins expressed in cancers are never produced in the original cell type, e.g., “cancer testis antigens” in melanoma. This phenomenon is called “ectopic” expression. Some cancers change their phenotype to resemble cells from a different tissue in a process called “metaplasia.” Metaplasia is not per se a malignant state but may precede cancer development, e.g., during the development of a specific type of stomach cancer, gastric adenocarcinoma (→18.4). Other changes of cell differentiation obliterate the original cellular phenotype beyond recognition and generate “generic” cell types like a small epithelial-like cell with a large nucleus-to-cytoplasm ratio or a spindle-shaped cell resembling a mesenchymal fibroblast. These cell types are end-points of cancer progression in some carcinomas and are typically found more frequently in aggressive cases. If they constitute the main cell type in a metasta-

1.3  Characteristic Properties of Cancers and Cancer Cells

sis, it can be difficult to determine from which primary site it originates, especially if no primary tumor can be detected. Such cancers are termed carcinomas with unknown primary (CUP). Their treatment is accordingly difficult and prognosis is poor. Altered differentiation contributes to cancer growth and expansion in several respects. Prominently, in normal tissues, homeostasis is achieved by a finely tuned equilibrium between proliferation and differentiation. 5 Tissue stem cells have the ability to differentiate into each cell type of tissue. They often proliferate rarely, but usually by asymmetric divisions that generate one stem cell and a more differentiated tissue precursor cell. These precursor cells are responsible for the bulk of proliferative activity in the tissue, but undertake a limited number of (symmetrical) divisions with increasing commitment to terminal differentiation (hence also “transit amplifying fraction”). Ultimately, their progeny differentiates terminally, with irreversible loss of replicative potential or even of viability (→7.1). For instance, differentiated cells in keratinizing epithelia become crosslinked to each other, dissolve their nuclei, and become filled with structural proteins. Hyperproliferation in cancer may accordingly result from increased numbers of stem cells, increased proliferative activity of stem or precursor cells, or in particular, impediments to differentiation of stem or precursor cells to terminally differentiated cells. Moreover, the intimate link between full differentiation and loss of proliferative potential in terminally differentiated cells may be disrupted. In addition, cancer cells are typically characterized by increased “plasticity” compared to their normal counterparts. This manifests as an increased ability to revert to less differentiated states resembling precursor cells or even fetal cells or to switch (transdifferentiate) to entirely different phenotypes. In particular, plasticity and

What follows is a highly simplified crude model of tissue homeostasis. It neglects among others that stem cells may also undergo symmetric divisions to maintain their overall number and that early precursor cells may reverse to stem cells following damage. 5 

17

the loss of differentiation control allow epithelial cells to undergo a partial or complete transition to a mesenchymal phenotype (epithelial-­ mesenchymal transition, EMT) with enhanced abilities to migrate and survive independently of adhesion signals (→9.1). Such changes facilitate invasion and metastasis, which are further favored by the disruption of the normal spatial organization of the tissue as a consequence of disorganized proliferative activity and impeded differentiation. Genomic instability: A clear distinction between cancerous and non-cancerous cell proliferation can be made by the criterion of genomic instability. As a rule, cancer cells contain multiple genetic and epigenetic alterations (→2). Polyploidy, an increase in the number of genomes per cell, is found in many cancer cells, but also occurs as a physiological process in several normal tissues, usually in terminally differentiated cells. It can be ascertained by measuring cellular DNA content. Aneuploidy, i.e., aberrant numbers of several individual chromosomes, often with structural changes as well, is a more cancer-­ specific alteration. It can be detected by cytogenetic methods or by nextgeneration sequencing (NGS). In NGS studies, abnormal (gene) copy numbers are usually listed as CNVs (copy number variants). Polyploidy and aneuploidy are sometimes already discernible by microscopic observations of tumor tissues through the increased size and aberrant shape of the cancer cell nuclei (nuclear atypia) and by aberrant mitotic figures. While some cancers display a large number of chromosomal aberrations, others remain (nearly) diploid, but instead contain numerous point mutations. Genomic instability may therefore manifest as chromosomal instability or increased point mutation rate, or both. Some cancers moreover display grossly aberrant DNA methylation patterns (→8.2). As cancers progress, the number of alterations in their genome tends to increase. Even if outwardly homogeneous, cancers often consist of cell clones that differ in their genetic constitution. The variant clones are continuously selected for those proliferating most strongly, tolerating

1  An Introduction to Human Cancers

18

Hypoxia Immune response

Adaptation at metastatic site

Fig. 1.7  Clonal selection model of cancer growth Genomic instability in cancer continuously creates novel clones from the initial tumor (left). These clones are selected according to their abilities to proliferate in the face of hypoxia, immune responses, and many other challenges and to adapt at metastatic sites. One (as assumed here) or several clones may succeed

adverse conditions best, evading immune responses, etc., with the best-adapted cell clone dominating growth (Fig. 1.7). Notably, while one clone may dominate growth at the primary site, others may be better fitted to withstand the challenges presented during invasion and metastasis. Variations in the genetic constitution are moreover important during tumor treatment, e.g., by chemotherapy. Treatments exert a strong selection pressure by favoring cell clones with alterations that allow them to survive and continue to expand in spite of therapy. 6 Cancers may continuously acquire and gradually accumulate mutations during their development and progression. Alternatively, “punctuated evolution” with sequences of “catastrophic events” like chromoplexy (a chain of structural chromosomal alterations), chromothripsis (fragmentation and reassortment of a single chromosome), or clustered point mutations resulting from “kataegis” can be detected in some cancers. A degree of genomic instability with an increased rate of chromosome alterations, point mutations, or epigenetic defects is probably required for the Epigenetic changes may similarly serve as a means for adaptation during tumor expansion as well as under therapy. 6 

progression of all cancers (→2.5). In some cancers, genomic instability may precede and underlie tumor development from the very start. In others, genomic instability develops only during progression. For instance, chronic myeloid leukemia is initiated by a single chromosomal translocation, whereas genomic instability generates the ultimate lethal tumor stage and allows resistance to therapy (→10.5). As in this example, cancers with higher genomic instability generally display greater variability and are more prone to developing therapy resistance. Genomic instability in cancer cells can be caused by different mechanisms, including defective DNA repair or failure of mechanisms ensuring genomic integrity. The highest number of point mutations is found in cancers with defects in DNA mismatch repair or in cancers containing mutations that diminish the fidelity of DNA replication polymerases. Likewise, cancers with deficient DNA homologous recombination repair accumulate many chromosomal alterations, but also a high number of diverse types of mutations in the DNA sequence. Immortalization: Many cancer cells are “immortalized,” which means that they are theoretically capable of an infinite number of cell divisions. Most normal human cells can undergo only a finite number of divisions, at most 60–80, before they undergo “replicative senescence” and irreversibly lose their ability to proliferate (→7.2). Germline cells are obviously exempt from this restriction and so are tissue stem cells, as a rule. For instance, hematopoietic stem cells in the bone marrow can be successively transplanted across several recipients and still remain capable of reconstituting the entire hematopoietic system with its many types of blood and immune cells. Immortality in stem cells is maintained by specific mechanisms like the expression of Telomerase (→7.2), which is also found in many cancers. Many human cancer cells can indeed be maintained in tissue culture or as transplants in animals (designated “xenografts”) over many generations (passages). Replicative senescence can not only arise gradually following long-term proliferation, but can also be induced in a more rapid fashion following various cell stresses,

1.3  Characteristic Properties of Cancers and Cancer Cells

especially DNA damage and inappropriate hyperproliferation, that activate cellular checkpoints. Evasion of senescence induced by checkpoint signaling is a prerequisite for the growth of many cancers. On a note of caution, it is not proven that all human cancers consist of immortalized cells, since many cannot be grown in tissue culture or as xenografts. Telomerase expression likewise is not universal. A cancer may become life-­ threatening even without acquiring infinite growth potential. A single tumor cell could (in theory) yield up to 249 cells by undergoing 50 duplications, before reaching replicative senescence, as compared to 1013–1014 normal cells in an adult human. This calculation, though, ignores cell death and other constraints. Immortalization therefore does not have to be an early event in cancer development, but it clearly is in some cancer types. Altered metabolism: Increased cell proliferation requires cell growth and therefore increased biosynthesis of many metabolites, proteins, nucleic acids, and cell organelles as well as a sufficient supply of energy (in the form of ATP or GTP). Moreover, tumor cells have to cope with various metabolic (and other types of) stresses, such as hypoxia (→9.4) and other conditions that limit the supply of energy sources and biosynthetic precursors, and may require higher levels of protective compounds (Table 3.3). Therefore, many metabolic processes are changed in cancer cells. The following section (Sect. 1.4) gives a summary description of common metabolic alterations in cancer cells. Notably, the type and extent of these changes vary between different cancer types and often in the same cancer at different stages of its progression. Importantly, as cancers progress, they impose more and more metabolic demands on the entire organism and can interfere with normal metabolic regulation, e.g., by secreting hormones and cytokines. Both kinds of effects can substantially contribute to morbidity. Invasion and metastasis: A directly visible property of many human cancers is their ability for invasion and metastasis. Invasion and metastasis (→9) are definitive criteria that distinguish malignant from benign tumors (the expression

19

“malignant tumor” is synonymous with cancer, Table  1.6). Moreover, invasion and metastasis, with tumor cachexia and immune suppression, account for most of the lethality of human cancers. During invasion, cancers spread from their site of origin into different layers and parts of the same tissue, eventually growing beyond it and into neighboring structures. Invasion involves multiple steps and often substantial remodeling of the tissue by the cancer cells. The process is supported by other resident cells in the tissue (“stromal cells”) that interchange signals with the cancer cells, including inflammatory and immune cells in the stroma. Typically, in carcinomas, the basement membrane separating epithelium and mesenchyme is destroyed and tumors grow into the underlying connective tissue layers (Fig. 1.5). In some cancers, cells separate and migrate through the neighboring tissues, as single cells, in a single file pattern or as small, adherent cell clusters. Invasion is often accompanied by inflammation, so lymphocytes, granulocytes, and macrophages are present in the invaded tissue and in the tumor mass (→9.5). In the process, resident cells in the connective tissue change their phenotype through interactions with the carcinoma cells and actually support tumor growth by providing growth factors and aiding in remodeling of the extracellular matrix (a process histologically visible as “desmoplastic reaction,” Fig. 1.5). For instance, resident fibroblasts change their morphology and gene expression patterns towards a new phenotype, cancer-associated fibroblasts (CAFs). Immune cells that could eliminate cancer may be excluded or driven to an inactive, indolent state. The cellular composition of the tumor stroma may thus differ considerably from that in the connective tissue associated with a normal epithelium. Altered interactions of the cancer cells with other cell types in the local stroma, during the invasion of other structures and tissues and at metastatic sites are essential for successful tumor spread. An important component of malignant growth is neoangiogenesis (→9.4). The nutrient and oxygen supply provided by preexisting blood vessels does not usually suffice to support the extension

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of tumors beyond a few mm. Therefore, malignant but also some benign tumors induce neoangiogenesis, which comprises the growth and sprouting of new capillaries and the remodeling of existing blood vessels (→9.4). Lymph vessels too can be remodeled or newly formed. During metastasis, cancer cells separate from the primary tumor and migrate via blood or lymph vessels to distant organs where they form new tumors (Fig. 1.5). Depending on the route, “hematogenic” metastasis, which usually leads to metastases at distant organ sites, is distinguished from “lymphogenic” metastasis, which leads initially to the formation of metastases in the lymph nodes that drain the region from which cancer emerges. Metastases may also form by the spread across the lumina of tissues (like in the urinary bladder) or across the abdomen (trans-coelomic metastasis). Cancers differ in the extent and the sites to which they metastasize. Generally, preferred organs for metastasis are those with extended microcapillary systems such as liver, lung, and bone. Like invasion, metastasis is a multistep process and presents many challenges to cancer cells. Thus, many more cancer cells enter the blood or lymph than actually succeed in forming metastases. Important barriers to metastasis are posed by the necessity to survive in the bloodstream and in capillaries which carcinoma cells cannot pass, by the requirement for “extravasation” (i.e., traversing the capillary lining into the surrounding tissue) and by the need to survive and resume proliferation in the (different) microenvironment of another tissue. In fact, individual cancer cells or small groups may end up in a different tissue only to survive over long periods without net growth. These “micrometastases” are not detectable by current imaging techniques, although they may be biochemically detectable by proteins or nucleic acids from the cancer cells. Over time, such “dormant” micrometastases may adapt to their new environment and expand to larger metastases that threaten the patient’s life. This “reawakening” may occur many years after the primary tumor has been removed.

1  An Introduction to Human Cancers

1.4 Metabolic Changes in Cancer Cell proliferation, whether normal or abnormal, necessitates adaptations of the cellular metabolism (Table 1.5). Most obviously, DNA synthesis requires deoxynucleotides. Therefore, nucleotide biosynthesis enzymes, especially for deoxynucleotide biosynthesis, are induced and activated in proliferating cells. Biosynthesis of RNA, prominently for ribosomes to meet the demand for increased protein biosynthesis, must also be enhanced. Recognition of these requirements inspired the development of several first-­ generation anticancer drugs (“anti-metabolites”) that interfere with nucleotide biosynthesis; more sophisticated drugs of this kind are still in use (→23.2). Ribose sugars for nucleotide biosynthesis, needed in addition to pyrimidine and purine bases, are provided by the pentose phosphate pathway. This pathway also generates the necessary NADPH for increased lipid biosynthesis and redox regulation through glutathione. Like the genome and ribosomes, during cell proliferation all other cell components must be duplicated including structural proteins, the various organelles, and membranes. For this reason, lipid biosynthesis is enhanced in many cancer cells that cannot obtain enough fatty acids, phospholipids, and cholesterol from lipoproteins supplied by the gut and liver or from cells in the tumor microenvironment. As a consequence, expression and activity of key enzymes like ATP-­ citrate lyase, Malic enzyme, Acetyl-CoA carboxylase, Fatty Table 1.5  Metabolic changes in cancer Increased nucleotide biosynthesis Enhanced pentose phosphate pathway flux Enhanced glycolysis (with decreased gluconeogenesis) Altered usage (or direction) of the citric acid cycle Increased fatty acid and phospholipid biosynthesis Enhanced use of glutamine and asparagine Downregulation of urea biosynthesis Increased cholesterol (and isoprenoid) biosynthesis Altered methyl group (one-carbon) metabolism Enhanced serin biosynthesis Increased heme biosynthesis Increased glutathione biosynthesis and recycling Upregulation of polyamine biosynthesis Production of oncometabolites like α-hydroxyglutarate

1.4  Metabolic Changes in Cancer

acid synthase or Hydroxymethylglutaryl-coenzyme A reductase are often increased in cancer cells. Acetyl-CoA precursors for fatty acid and cholesterol biosynthesis can be derived from glucose through glycolysis or via the citric acid cycle. Cholesterol biosynthesis may be particularly important during cancer metastasis. Of note, conversely, fatty acid metabolism rather than synthesis may crucially contribute to maintaining the energy supply of cancer cells in certain conditions, especially during metastasis. In many cancer cells, the function of the citric acid cycle to provide intermediates for lipid biosynthesis is therefore more critical than in normal cells. Citric acid cycle metabolites are however often derived from glutamine or asparagine rather than from pyruvate (and glycolysis). Nevertheless, at least under aerobic conditions, the citric acid cycle remains an important source of ATP in most cancer cells. Exceptions to this rule occur under hypoxic conditions, where energy production relies increasingly on glycolysis, as well as in some cancers with genetic changes that inactivate citric acid cycle enzymes (→16.5). In the latter cancers, the direction of flux through the citric acid cycle is actually reversed towards citrate. Notably, genetic inactivation of Fumarate dehydrogenase and Succinate dehydrogenase is the primary cause of some cancers. A long-known characteristic of cancers is an increased reliance on glycolysis for energy (ATP) production. Increased glucose utilization by glycolysis is a normal adaptation to hypoxia, but in some cancers, it is also observed under aerobic (normoxic) conditions (known as the Warburg effect). Many tumor cells therefore express glycolytic isoenzymes that favor flux in the direction of pyruvate and lactate. Glucose uptake and phosphorylation are likewise enhanced by increased synthesis and membrane transport of glucose transporters (like GLUT1) and expression of Hexokinase 2. Notably, its product, glucose-­6-phosphate, can also be utilized in the pentose phosphate pathway. While glycolysis is increased, pyruvate entry into the citric acid cycle may be blocked because Pyruvate dehydrogenase is inactivated through phosphorylation by Pyruvate dehydrogenase kinase. As a conse-

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quence, lactate is generated and transported out of the cell, with both LDH and lactate exporters (monocarboxylate transporters, e.g., MCT1) being upregulated. Enhanced flux through glycolysis can be important for cancer cells beyond its function of providing energy. Pyruvate can be diverted towards lipid biosynthesis by Pyruvate decarboxylase. The glycolytic intermediate 3-­ phosphoglycerate is converted by Phosphoglycerate dehydrogenase to 3-­phosphohydroxypyruvate, a precursor for serine biosynthesis. While serine is a nonessential amino acid, increased serine biosynthesis is essential for the growth of some cancers, not only because serine is required itself, but also as a precursor of glycine for purine and glutathione biosynthesis and for the repletion of one-carbon units. In addition to serine, other nonessential amino acids may become limiting for tumor growth. Amino acid uptake is accordingly generally upregulated in proliferating cells to provide essential as well nonessential amino acids. One of the most critical amino acids is methionine as a carrier of methyl groups, beyond its function as a constituent of proteins. Choline biosynthesis (from phosphoryl-ethanolamine) requires three methyl groups from S-adenosylmethionine (SAM) and is also activated in many cancers. The backbone of SAM is furthermore used in the biosynthesis of the polyamine compounds spermidine and spermine, which are required at enhanced levels in proliferating cells. Methyl groups carried by folate coenzymes are furthermore needed for thymidine biosynthesis. Insufficient methionine levels moreover impede epigenetic regulation by methylation of histones and DNA (→8), where SAM is again the essential methyl group donor. Porphyrin biosynthesis and heme uptake are also increased in many cancers and may become limiting for cancer growth. In fact, intermediates of porphyrin biosynthesis may accumulate in tumor cells to higher levels than in normal cells. Of note, this accumulation can be used for the detection of cancers and in photodynamic therapy. Several more metabolites are found at supraphysiological levels in certain cancers, including

22

fumarate, succinate, sarcosine, glycine, kynurenine, and methylglyoxal. These are collectively denoted as “oncometabolites,” even though they are found (at lower concentrations) in normal cells too. An extreme case is α-hydroxyglutarate. Its concentration is several magnitudes higher in cancers with specific mutations in an Isocitrate dehydrogenase enzyme (either IDH1 or IDH2). These enzymes normally generate α-ketoglutarate but the mutations turn a rare side reaction into their main catalytic activity. The ensuing oncometabolite interferes with epigenetic regulation, adaptation to hypoxia, and many other processes that employ α-ketoglutarate as a co-substrate. Of note, in addition to insufficient concentrations of SAM and excessive α-hydroxyglutarate levels further metabolic changes in cancer cells may affect epigenetic regulation (→8). For instance, the activity of many histone demethylases is regulated by hypoxia and depends on the ratio of α-ketoglutarate to succinate. The prominent process of histone acetylation requires acetyl-­ CoA, which can be limiting in cancer cells. A large number of additional histone modifications have been discovered over the last years, which depend directly on the levels of specific metabolites and in turn regulate their metabolism. 7 These are likely to be affected in cancer cells as well. A key requirement for cell growth is increased protein synthesis, which is apparent  by several phenomena in cancer cells, such as the enhanced size and number of nucleoli, increased expression of translation initiation factors, and enhanced phosphorylation of ribosomal proteins. A particularly strong boost in protein synthesis may be required during invasion and metastasis (→9). Important stimulators of overall protein synthesis in cancer cells are the PI3K pathway through mTOR (→6.3) and oncogenic MYC transcription factors (→4.5). MYC transcription factors also stimulate mitochondrial biosynthesis to ensure that essential mitochondrial respiration and biosynthetic functions are available in proliferating (cancer) cells. These include lactylation of histones, e.g., which could be highly relevant in hypoxic cancers. 7 

1  An Introduction to Human Cancers

The PI3K/mTOR pathway and MYC transcription factors may be the most crucial regulators of the switch to a pattern of energy metabolism and biosynthesis enabling cell growth and proliferation. Both are activated by various genetic alterations in a broad range of cancers. In particular, the mTOR protein kinase in the mTORC1 complex regulates the shift towards an anabolic state, with increased protein  biosynthesis and decreased autophagy. In some cancers, specifically, the regulation of mTOR by AMPK is disturbed. As its name indicates, AMP-dependent kinase senses the energy level of the cell and inhibits mTOR if it is insufficient. Like TP53, AMPK also prevents the nuclear receptor SREBP from inducing fatty acid biosynthetic enzymes. Amino acid availability is also communicated to regulate mTOR activity. In the course of the cell cycle, E2F transcription factors induce nucleotide biosynthetic enzymes and other proteins required for DNA replication. Overactivity of E2F1 in cancer is often caused by inactivation of the tumor suppressor RB1 (→5.2). Similarly, loss of the tumor suppressor TP53 (→5.3) facilitates the establishment of many metabolic changes in cancers. TP53 is activated in response to different types of cellular stresses, including metabolic disturbances and especially insufficient GTP levels. Among others, active TP53 increases pentose phosphate pathway activity to provide metabolites for DNA repair and cell protection (→5.3). Specific changes, like increased glycolysis and decreased entry of pyruvate into the citric acid cycle take place especially when tumor cells adapt to hypoxia during tumor expansion (→9.4). This adaptation is directed by hypoxia-induced transcription factors like HIF1 which are constitutively activated in some tumors, especially the clear-cell subtype of renal carcinoma (→16.4). Notably, cancer growth imposes an overall enhanced energy demand on the body of the patient, which increases with the tumor load. Cancers moreover release waste products of their metabolism, such as lactate, with which the body has to cope. In addition, cancers often secrete enzymes and hormones that act on the host, some of which influence metabolic regulation in dangerous ways. In particular, secretion of cytokines

1.5  Characterization and Classification of Cancers in The Clinic

like tumor necrosis factor α (TNFα), interleukins 1 and 6 (IL1, IL6) can generate a systemic break-­ down of metabolic functions with evident wasting, termed “cachexia,” in addition to suppression of the immune system that facilitates opportunistic infections. Other tumor products, such as the CD95L protein (→7.4), can damage sensitive organs, such as the liver, and ectopically produced hormones can interfere with homeostasis. For instance, calcitonin production by small cell lung cancers may cause life-threatening deviations of serum calcium levels. Likewise, tumor secretion of parathyroid hormone-related peptide (PTHrP) and adrenocorticotropic hormone (ACTH) can critically disturb the systemic regulation of calcium and potassium levels, respectively. Such indirect disturbances of body homeostasis by cancers, designated as “paraneoplastic” symptoms, can impede the well-being of

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patients and endanger their survival in addition to the destruction brought about by the malignant growth per se.

1.5 Characterization and Classification of Cancers in The Clinic Many properties of cancers described in the previous sections are reflected in the terms and methodology used in the clinic and in diagnostic pathology to describe and classify cancers. A glossary of these terms is provided in Table 1.6. Classification of cancers is a prerequisite for appropriate treatment and for prognosis of their likely course. For these purposes, it is mandatory to obtain as precise as possible descriptions of the extension of a tumor, its degree of malignancy,

Table 1.6  Basic terms in oncology Term Meaning Adenocarcinoma A malignant tumor with resemblance to glandular structures Adenoma A benign tumor displaying a glandular structure Benign tumor A tumor not growing beyond a circumscribed region within a tissue Cancer A malignant tumor A solid malignant tumor formed from cells of epithelial origin Dysplasia Aberrant structure of cells or tissue organization Hyperplasia Increased number of cells in a tissue Leukemia A malignant tumor formed by cells of the hematopoietic cells and found in the blood Lymphoma A malignant tumor formed by cells of the lymphocyte cell lineage Malignant tumor A tumor characterized by permanently increased cell proliferation, progressive growth, and invasion or metastasis Metaplasia Change of a differentiated tissue into another

Remarks Often originated from gland tissue Often originated from gland tissue

Preferentially used for (suspected or verified) systemic disease

Carcinoma

Neoplasia Sarcoma Tumor Tumor grade Tumor stage

A (malignant) tumor A solid malignant tumor formed from connective tissue (mesenchymal) cells Any abnormal increase in the size of a tissue A measure of the cellular and/or tissue atypia of a tumor A measure of the physical extension of a (malignant) tumor

Can be restricted to specific lymphoid organs Corresponding to “cancer” in everyday language The related term transdifferentiation refers to according changes in cells Not always strictly used

Also used for swellings, unusual for benign hypertrophy or hyperplasia Different systems are in use for different (and even the same) cancer types Different systems are in use for different (and even the same) cancer types

1  An Introduction to Human Cancers

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Fig. 1.8  Tumor staging in bladder cancer Carcinoma in situ (CIS) is a severe dysplasia restricted to the epithelial (urothelial) layer. pTa tumors are papillary structures formed by hyperproliferative urothelium growing into the lumen of the urinary tract, but not into the underlying tissue layers. pT1 tumors extend into the

connective tissue layer, pT2 tumors into the muscular layers (with T2a and T2b distinguished by the depth of invasion), and T3 tumors into the bladder fat layer. T4 cancers have grown into neighboring organs, such as the prostate or the uterus. From Hurst C, Knowles MA (2015) l.c

and its histological subtype. These parameters are also relevant for cancer research that uses human cancer specimens. Staging: The extension of a tumor is described by “staging.” Prior to surgery or if none is ­performed, a “clinical stage” is defined by visual inspection, palpation, and various imaging techniques. Imaging techniques may include ultrasound, X-rays, computed tomography, magnetic resonance tomography, as well as functional imaging techniques like scintigraphy and positron emission tomography (Box 22.1). Some imaging procedures detect changes in tissue shape and density in cancers, whereas others register changes in metabolism and blood flow. If surgery is performed, a more precise delineation of the extension of the tumor can be made by inspection of the tumor site and by histopathological investigation of the specimen. The stage defined in this fashion is called pathological stage. It is denoted by a “p” prefix to distinguish it from clinical stage, which carries a “c” prefix. Several staging systems are employed. The most widely used systematic staging system is the TNM (tumor node metastasis) classification; others are in use for specific cancers. In the TNM system, the extent of the primary tumor is typically classified as T1–T4, where increasing numbers describe larger and/or more invasive tumors. Additional stages may be defined for some cancers, such as the pTa stage in bladder cancer

(Fig. 1.8). The presence of cancer cells in lymph nodes is denoted by N0, N1, and in some cancers also N2, with N0 meaning none detected. The presence of distant metastases is indicated by M0, meaning none detected, M1, or in some cancers also M2. Following surgery, it is important to know whether all of the local tumor has been removed. This is designated by the R value, where R stands for “residual” (tumor). R0 means that the tumor appears to have been entirely contained within the removed specimen. In all categories, the affix “x” is used for “not determined/ unknown.” Other staging systems, applied in specific cancer types, may combine T and N stages 8 or consider the size or invasion depth of the tumor. Grading: The degree of malignancy of a tumor is further estimated by grading. Several systems are in use for different tumors. To different extents, they score the degree of cellular and nuclear atypia and the degree of tissue disorganization in tumor sections, biopsies, or single tumor cells. The most prevalent system is G grading, which usually ranks from G0 to G4; it may be simplified to a high-grade/low-grade distinction (Fig. 1.9). The designation G0 typically denotes normal tissue structure and differentiation and no cellular atypia, as might be found in a benign e.g., as stage I–IV, where stage I corresponds to T1N0M0 and stage IV to T3/4N1M1. 8 

1.5  Characterization and Classification of Cancers in The Clinic

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Fig. 1.9  Tumor grading in bladder cancer Low-grade (left) and high-grade (right) intra-urothelial neoplasia of the urinary bladder urothelium. While both lesions are (still) noninvasive, the high-grade dysplastic

lesion on the right has a much higher propensity for progression. The center figure shows a transition region, at which low grade progresses to high-grade intraepithelial dysplasia. Courtesy: R. Engers

tumor. At the other end of the scale, G4 would be assigned to cancers with a cellular morphology completely different from the normal tissue and pronounced atypia of cancer cells and nuclei. The grades G1–G3 are considered well-differentiated, moderately, and poorly differentiated. Note that the term “differentiation” in cancer pathology is used in a different way than in cell and developmental biology. This can be confusing, the more so as in modern histopathology tumor grading based on morphology is often supported by staining for specific markers of cell differentiation, in order to determine the tumor subtype or cell of origin. Likewise, quantification of cell proliferation may supplement grading and support subtyping. Traditionally, mitotic figures were counted, but in current practice, immunohistochemical staining against PCNA (proliferating cell nuclear antigen), a subunit of the DNA replisome mainly expressed in S-phase cells, or Ki67, a nuclear protein expressed only in actively cycling cells, can be used to estimate the proliferative fraction in a tumor. Nucleolar activity, evident from the increased size or number of nucleoli, is another optional criterion. Histological classification: The location of a tumor is the first clue to its classification, but is, of course, not sufficient, since (1) a tumor mass may represent a metastasis or the extension of cancer from a neighboring organ and (2) different types of cancer may develop in the same tissue, possessing very different properties, taking different clinical courses and requiring different treatments (see Sect. 16.1 for an example).

Therefore, a tumor must be histologically classified from samples acquired by biopsy or from surgical specimens. Several of the designations used in this context have already been introduced in this chapter and are summarized in Table 1.6. Histological typing of tumors is performed by evaluating their morphology. Routine procedures use a variety of specific staining protocols developed over centuries in anatomy and pathology to highlight particular cell types as well as extracellular structures like basement membranes, fibers or mucus. Tumor classification by histopathological investigation is increasingly supported by molecular markers (→22). Immunohistochemical staining with antibodies directed against specific antigens of the presumed tissue of origin, such as cytokeratins, or tumor-specific antigens, e.g., Carcinoembryonic antigen, is often helpful. For leukemias, in particular, classification can be achieved by antibody staining of surface proteins that is then quantified by flow cytometry. Analyses of nucleic acids for specific genetic alterations or patterns of gene expression are increasingly used for the classification of cancers and the selection of therapies; for molecularly targeted therapies molecular diagnostics is often a prerequisite (→22.5). Cytogenetic techniques are mainly used for the classification of hematological cancers, but can be useful in other tumor types too (→22.2). Insights into the molecular biology of human cancers have begun to improve staging, grading, and histological classification. These improvements have an immediate impact on cancer ther-

1  An Introduction to Human Cancers

26

apy because in most cancers the choice of therapy is contingent on these parameters (→22.5). For instance, a tumor in the kidney will be treated differently, if it is a malignant renal cell carcinoma at an early or at an advanced stage, a benign tumor of mesenchymal origin, a metastasis of a melanoma, or a lymphoma. Molecular markers can provide further information. They may reveal differences between cancers with similar morphology that actually represent different diseases, like certain leukemias (→21.2). They may reveal morphologically undistinguishable subtypes within one disease, as in breast cancer (→19.3) or may predict different clinical courses for morphologically similar tumors at the same stage (→22.4). Moreover, molecular markers allow the selection of individual cancers for treatment with drugs tailored to specific targets and may allow to predict how well a patient tolerates cancer treatment by radiotherapy or cytostatic medication (→22.6).

1.6 Cancer Treatment In principle, a range of different therapies is available to treat human cancers. Surgery, irradiation, immunotherapy, or various kinds of chemotherapy can be employed, or any combination of these. The choice of therapy depends on the classification of cancer by the criteria described in the previous section and increasingly on molecular diagnostics (→22). Surgery or radiation is the prime treatment choices for localized cancers. In contrast, leukemias, lymphomas, and metastatic or locally advanced carcinomas and many soft tissue tumors require “systemic” chemotherapy, which may be supplemented by radiotherapy or surgery of primary cancers or (single) metastases. Medical treatment by chemotherapy or irradiation following surgery, to attack residual local tumors or micrometastases, is designated “adjuvant.” Medical treatment applied before surgery, in order to shrink the tumor mass to facilitate its complete resection, is termed “neo-adjuvant.” The standard medical treatment for cancer is usually designated as “first-line”; if it is unsuccessful

or if the tumor recurs, “second-line” therapy may be attempted. 9 The efficacies of chemotherapy and radiotherapy are extremely dependent on the tumor type. Some cancers, e.g., most testicular cancers and certain lymphomas, are highly sensitive, whereas others, e.g., renal cell carcinomas (→16.1) are overall less sensitive to traditional cytotoxic chemotherapy and radiotherapy than many normal tissues. In cancer chemotherapy, a wide range of different drugs are employed. Some drugs are aimed at the  cancer itself, whereas other medications are employed to stabilize specific body functions in the patient, for pain relief or to ameliorate other symptoms. The most important component in the pharmacological treatment of many cancers is still cytotoxic chemotherapy, the systemic administration of cytotoxic compounds (synthetic or from natural sources). In this kind of therapy, chemical compounds are employed that damage DNA, block DNA synthesis, transcription, or mitosis of cancer cells, in order to drive them into apoptosis or other forms of cell death (→23.2). A different type of anticancer drugs summarized as “biological agents” (or shorthand as “biologics”) are normal signaling molecules (or compounds modeled on them) that bind to receptor molecules in the cancer cells that are not directly involved in DNA replication or mitosis but regulate cell proliferation and survival. Examples for this type of drug are hormones and antihormones used in the treatment of breast cancer (→19.4) and prostate cancer (→20.3) and retinoids used to treat specific acute leukemia, promyelocytic leukemia (→10.6). Since the advent of recombinant DNA biotechnology, cytokines and growth factors can be produced at a reasonable cost and with sufficient purity to be used in cancer therapy. In some cases, they act directly on the cancer cells, in other cases, they stimulate the immune response against cancer, and in still another application, they stabilize the hematopoietic system of the patient against the effects of cancer and adverse effects of the treatment. Interferons and interleukins used in the therapy of leukemias (→10.4) and—until recently—renal With increasing numbers and varieties of therapeutic options, third line, etc., treatments have become possible. 9 

Further Reading

cell carcinoma (→16.7) are examples of the former two applications. Erythropoietin stimulating erythrocyte production and GM-CSF enhancing myeloid hematopoiesis are examples of agents used to ameliorate the effects of cytotoxic therapies. The increasing understanding of cancer molecular biology has allowed the development and application of many “molecularly targeted” therapies (→23.3). These chemical compounds or recombinant proteins supplement or replace traditional chemotherapy in many cancer types. Often, their application requires prior molecular diagnostics (→22.5). Several different types of radiation as well as particles and radioisotopes can be used in cancer radiotherapy. Most widely employed is ionizing radiation in the form of high energy γ-radiation. It damages cancer cells by direct effects on cellular macromolecules or by generating reactive oxygen species, as during carcinogenesis by ionizing radiation (→1.2). Typically, radicals induced by the radiation initiate chain reactions that lead to DNA double-strand breaks which prevent further cell proliferation or induce apoptosis. The radiation dose that can be applied is limited by its effects on normal tissues. Modern techniques allow improved physical focusing of the irradiation or use radioactive isotopes implanted into or targeted to the tumor. These improvements are most useful for the treatment of cancers at specific sites and their local environment. In some cases, the tumor cells can be sensitized to the radiation. For instance, the enrichment of porphyrins and biosynthetic intermediates in some tumors is exploited in photodynamic therapy. In this technique, intense light with a wavelength near the absorption maximum of protoporphyrin is applied by a laser beam to generate toxic levels of reactive oxygen species. In other cases, the preferential uptake of radioactive isotopes by cancer cells can be brought to good use, e.g., in the therapy of some types of thyroid cancers by radioactive iodine. Immunotherapy is already being applied in some cancers, albeit with highly variable efficacy. Several novel approaches have been introduced into clinical practice or are  under

27

investigation, including antibodies blocking immune checkpoints, vaccines, and genetically engineered T cells. Since the mechanisms contributing to successes and failures of immunotherapies are gradually becoming clearer, further improved immunotherapies may become a mainstay of therapy in the future (→23.6). Even where the full range of modern cancer therapies is available, many cancers cannot be cured today. Especially, in many advanced-stage cancers, state-of-the-art therapy manages to delay progression and to alleviate symptoms caused by cancer, but survival is not substantially prolonged. In general, over the last decades, improvements in therapy have increased the life expectancy of patients affected by many cancer types, but the gains vary widely between cancer types and individual patients (see Fig. 23.1). Therefore, novel cancer therapies remain urgently required. Expectations are accordingly high for molecular cancer research to reveal further therapeutic options. Indeed, many novel drugs based on insights from this kind of research are now in routine use (→23.5). How much they have improved cancer treatment, in general, is a matter of debate, though. Molecular cancer research has also deepened the understanding of established therapies like cytotoxic chemotherapy and radiotherapy (→23.2), likewise with a significant impact on cancer therapy. Immunotherapies are rapidly developing (→23.6) and currently experimental “genetic” therapies may make a significant impact in the future (→23.7).

Further Reading Amelio I et  al (2014) Serine and glycine metabolism in cancer. Trends Biochem Sci 39:191–198 Bergers G, Fendt SM (2021) The metabolism of cancer cells during metastasis. Nat Rev Cancer 21:162–180 Boroughs LK, De Berardinis RJ (2015) Metabolic pathways promoting cancer cell survival and growth. Nat Cell Biol 17:351–359 Boyd N et al (2016) Rare cancers: a sea of opportunity. Lancet Oncol 17:e52–e61 Chabner BA, Roberts TG (2006) Chemotherapy and the war on cancer. Nat Rev Cancer 2005:65–72 Cullin N et  al (2021) Microbiome and cancer. Nat Rev Cancer 21:1317–1341

28 Ferlay J et al (2021) Cancer statistics for the year 2020: an overview. Int J Cancer. https://doi.org/10.1002/ ijc.33588 Hanahan D (2022) Hallmarks of Cancer: new dimensions. Cancer Discov 12:31–46 Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70 Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674 Hoxhaj G, Manning BD (2020) The PI3K-AKT network at the interface of oncogenic signalling and cancer metabolism. Nat Rev Cancer 20:74–88 Kaelin WG, McKnight SL (2013) Influence of metabolism on epigenetics and disease. Cell 153:56–69 Knippel RJ et  al (2021) The cancer microbiome: recent highlights and knowledge gaps. Nat Rev Cancer 21:2378–2295 Kroemer G, Pouyssegur J (2008) Tumor cell metabolism: cancer’s Achilles’ heel. Cancer Cell 13:472–482 Liberti MV, Locasale JW (2016) The Warburg effect: how does it benefit cancer cells? Trends Biochem Sci 41:211–218 Martinez-Reyes I, Chandel NS (2021) Cancer metabolism: looking forward. Nat Rev Cancer 21:669–680

1  An Introduction to Human Cancers Nishikawa H et al (2021) Cancer cachexia: its mechanism and clinical significance. Int J Mol Sci 22:8491 Pavlova NN, Thompson CB (2016) The emerging hallmarks of cancer metabolism. Cell Metabol 23:27–47 Pitot HC (2002) Fundamentals of oncology, 4th edn. Marcel Dekker Siegel RL et al (2021) Cancer statistics, 2021. CA Cancer J Clin 71:7–33 Strachan T, Read AP (2019) Human molecular genetics, 5th edn. CRC Press Sullivan LB et al (2016) Altered metabolite levels in cancer: implications for tumour biology and cancer therapy. Nat Rev Cancer 16:680–693 Sung H et  al (2021) Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 71:209–249 Vineis P, Wild CP (2014) Global cancer patterns: causes and prevention. Lancet 383:549–557 Vogelstein B, Kinzler KW (eds) (2002) The genetic basis of human cancer, 2nd edn. McGraw-Hill

2

Cancer Genetics

Key Points • Cancer cells typically contain multiple changes in the base sequence of their DNA and alterations in the structure and number of genes and chromosomes. • The majority of these alterations are acquired by mutations occurring in somatic cells. Germline mutations underlie familial cancer syndromes, which can be inherited in a recessive or dominant fashion. Cancers in children and young adults can be caused by genetic or epigenetic alterations acquired during germ cell or fetal development. • Many different genetic alterations are observed in cancer cells. Individual genes contain mutations such as base changes (point mutations), insertions and deletions, or can be rearranged by chromosomal translocations or inversions. These changes lead to the expression of altered gene products, to aberrantly low or high gene expression, or may create novel gene products such as fusion proteins. Moreover, cancer cells are often aneuploid with numerical and structural alterations of their chromosomes. Such alterations comprise loss or gain of entire chromosomes or chromosomal segments. Their consequences for individual genes, summarized as copy number variations, range from complete loss by homozygous deletion through decreased copy numbers on the one hand to increased gene dosage by gains and gene amplification on the other

hand. Furthermore, rearrangements like translocations and inversions may generate fusion genes, disrupt genes or cause deregulation of gene expression. At heterozygous polymorphic loci, loss of heterozygosity may occur as a consequence of deletions or recombinations. • Infections by DNA viruses and retroviruses alter the composition of the genome, adding new sequences and mutating host genes by insertion of viral sequences or by inducing deletions or gene rearrangements. • These diverse types of genetic alterations occur to different extents in different types of cancers and even in cancers of the same histological type. In some cancers point mutations prevail, while in others chromosomal aberrations predominate. The highest frequency of genetic alterations is found in cancers caused by extensive exposure to environmental mutagens, like cancers of the skin and the lung, and in cancers with deficient DNA repair or DNA polymerases generating a “mutator” phenotype. • The diverse types of genetic changes result in altered patterns of gene expression and in the expression of altered gene products in cancer cells. Changes in gene expression are reinforced or caused by epigenetic mechanisms, which allow the stable propagation of alterations in gene expression without changes in the DNA sequence across cell divisions, in accord with the general concept of epigenetics.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 W. A. Schulz, Molecular Biology of Human Cancers, https://doi.org/10.1007/978-3-031-16286-2_2

29

30

• The mutations causing inherited cancer syndromes increase the lifetime risk of developing cancer several-fold. Overall, such mutations are relatively rare. In contrast, genetic variants in a large number of genes influence cancer risk only slightly or moderately but can be highly prevalent (“polymorphisms”). Relevant polymorphisms affect the metabolism of carcinogens, DNA repair, protection against cell damage, the regulation of immunity and inflammation, and the metabolism of hormones and growth factors. • Two particular important classes of genes affected by genetic and epigenetic alterations in cancer cells are oncogenes and tumor suppressor genes. Oncogenes contribute to tumor development through elevated or misdirected activity. In the case of tumor suppressors, conversely, deficient or absent function supports tumor development. Typically, both activation of oncogenes and inactivation of tumor suppressor genes are observed in one human cancer. More than 700 genes are considered as potential oncogenes or tumor suppressors in at least one human cancer type. • Most oncogenes and tumor suppressor genes encode proteins. However, the phenotype of many cancers is significantly modulated by overexpression or downregulation of regulatory RNAs like microRNAs (miRs) or long noncoding RNAs (lncRNAs). • Many genetic and epigenetic changes accumulate during the development of malignant tumors. Some of these are necessary for cancer development and considered as “drivers,” whereas many others are rather “bystanders” with no decisive effect on the tumor phenotype. Large-scale genomic analyses suggest an average of about four driver alterations per cancer, albeit with a large range of variation.

2.1 Cancer as a Genetic Disease The characteristic properties of cancer cells (→1.3) are predominantly the consequences of genetic changes in the tumor cells. Almost every cancer cell contains structural or numerical alter-

2  Cancer Genetics

ations in its genome. Their number and kind vary with cancer type and often with the stage of progression. According to NGS studies, some cancers contain thousands of point mutations, up to 100 or even 1000 per Mbp. These huge frequencies are typically observed in cancer types caused by environmental mutagens such as tobacco smoke (lung cancers) or UV irradiation (skin cancers), or in cancers with deficient DNA repair (e.g., colorectal cancers with mismatch repair deficiencies) or with defective replication DNA polymerases (like Pol ε). On the other end of the spectrum, some cancers may contain fewer than one point mutation per 10 Mbp, i.e., no more than a handful of changes in their protein-coding sequences. Low mutation rates are typical of cancers in children and young adults. Similarly, the number of detectable chromosomal alterations varies widely. Some cancers contain no detectable change in the number or structure of their chromosomes, whereas others present with grossly aberrant aneuploid genomes with an essentially uncountable number of changes. Again, specific defects in DNA repair and DNA damage signaling may underlie such cases. Yet other cancer types display one or a few distinctive chromosomal changes, which can be pathognomonic (i.e., they indicate the specific disease). It is therefore certainly appropriate to regard cancer as a “genetic” disease, even more so as some cancers can be transmitted in families as monogenic diseases, most often with an autosomal-­dominant mode of inheritance. Still, a few points must be kept in mind: (1) Only a minority of cancers are caused by inherited mutations in the germline. The vast majority of genetic alterations in cancers arise instead in somatic cells during the life of the affected person. Thus, most cancers are caused by “somatic” mutations. Even in cancers that are inherited over several generations in a family, the initial inherited mutation is almost always complemented by additional somatic mutations in the actual tumor. Cancers arising in young children or adolescents may be caused by mutations originating de novo in their parents’ germ

2.2  Genetic Alterations in Cancers

cells or during intra-uterine development. A typical cancer of this kind is Wilms tumor (→11), but similar mechanisms apply to testicular germ cell cancers and certain childhood leukemias. (2) The relationship between the mutant genotype and the disease phenotype is not at all straightforward in cancer. This is not per se unusual for genetic diseases, but in cancer the relationship can be extremely complex. Cancer cells as a rule contain many different mutations that may each contribute to various extents to the properties of the tumor. (3) Not all mutations in a cancer are causative or even relevant for the disease, as might be obvious in cancers with thousands of mutations in their genomes. Some genetic changes will be necessary for the disease and are considered “drivers,” irrelevant ones are ­ regarded as “bystanders” (or “passengers”). The distinction is not always easily made and some changes may actually lie in between the extremes, by affecting the phenotype of cancer, but not in an essential way. Other changes may be redundant and may only manifest contingent on others. Even mutations in well-defined “cancer genes” may be not essential for all cancers in which they are found. (4) Not all properties of cancer cells may result from genetic alterations. Many stable changes in cancer cells may be set up by stable regulatory loops without alterations in the DNA sequence or the copy numbers of genes. Such changes are considered as “epigenetic” (→8). Furthermore, the phenotype of a cancer cell is often strongly influenced by interactions with other cell types in the tumor microenvironment (→9).

2.2 Genetic Alterations in Cancers Many different types of alterations can be observed in the genome of human tumor cells by molecular or cytogenetic methods (Table 2.1). Some types of alterations affect single genes

31

only: the DNA sequence of an individual gene can be altered by point mutations, by smaller or larger deletions or insertions, or by rearrangements, with a wide array of potential consequences (see below). Larger deletions can affect several genes at once. Rearrangements can lead to the creation of novel genes from (two) others. Point mutations: Point mutations are due to base exchanges in DNA (hence also: base mutations). Base exchanges are classified as transitions (pyrimidine → pyrimidine, or purine → purine) or transversions (pyrimidine  →  purine or purine → pyrimidine). Base changes can have very different effects on the gene product, even those occurring within the coding region of a gene (Fig. 2.1). Silent (synonymous) mutations generate a different codon encoding the same amino acid. Silent mutations do therefore not change the coding potential of a mRNA, but they may affect its processing and stability. RNA stability as well as translational efficiency can also be influenced by mutations in the 3′-UTR or 5′-UTR (UTR: untranslated region). Missense (non-synonymous) base mutations change the amino acid sequence of the encoded protein, which may in consequence display increased, decreased, or unchanged activity. The functional effect of a specific missense mutation may to some extent be predicted by bioinformatical algorithms, but its evaluation ultimately requires functional biochemical and cellular assays. Nonsense mutations lead to a truncated protein product, which is often unstable. If not, the shorter protein may interfere with the function of the normal protein or its interaction partners in a “dominant-negative” manner. In particular, nonsense mutations occurring between the ATG start codon and the following splice site (i.e., in the first or second exon of a gene) destabilize the respective mRNA by making it prone to “nonsense-mediated decay (NMD),” a cellular quality control mechanism. Collectively, nonsense mutations and other types of mutations (such as frameshift mutations resulting from small insertions and deletions) that generate shortened protein products are designated “truncating mutations.” Importantly, point mutations can in principle be activating or inactivating. Evidently, the spec-

2  Cancer Genetics

32 Table 2.1  Types of genetic alterations in human cancers Genetic alteration Changes in the Base exchange DNA sequence

Typical consequences None, altered coding, altered splicing, protein truncation, read-through, altered mRNA stability, altered regulation Small insertion or deletion Frameshift mutations with protein (InDel) truncation, altered splicing, altered gene regulation Larger deletion Gene loss, protein truncation, or shortening by exon loss

Larger insertion

Structural chromosomal changes

Very large deletions Chromosomal translocation Chromosomal inversion Gene amplification

Numerical chromosomal changes

Aneuploidy Chromosome gain Chromosome loss

Viral infection

Introduction of viral genomes and insertion of viral sequences into the genome

Fig. 2.1  Effects of point mutations in the coding sequence of a gene. See main text for further explanations

Examples Activation of RAS proto-­ oncogenes, inactivation of TP53 tumor suppressor gene

Expansion or contraction of oligo-A stretch in TGFBR2 gene Deletion of CDKN2A, loss of pocket domain in RB1 tumor suppressor gene Inactivation of APC tumor Gene disruption with loss of suppressor gene by protein, altered splicing, altered retrotransposon insertion regulation Loss of several genes Deletions of chromosome 9q in bladder cancer Altered gene regulation, formation Activation of MYC oncogene in lymphomas, BCR-ABL of fusion genes encoding fusion fusion gene in CML proteins Altered gene regulation, formation Activation of RET oncogene in of fusion genes thyroid cancers Increased gene dosage, usually with Amplification of MYC or increased expression EGFR oncogenes Altered gene dosage Altered karyotype in many cancers Altered gene dosage Gain of chromosome 7 in papillary renal cell carcinoma Altered gene dosage Loss of chromosome 3p in clear-cell RCC Inactivation of TP53 and RB1 Introduction of novel regulatory proteins, altered gene regulation by by HPV E6 and E7 proteins, oncogene activation by viral insertion, chromosomal retroviral LTR instability at viral integration sites

mRNA

NH2

Protein

AAAAAAAA

ATG

STOP

COOH ATAAAA

E1 Start codon mutation

E2

Missense mutation

E3

Nonsense mutation

NH2

NH2

Gene

E4

Stop codon mutation

NH2

NH2

COOH COOH partial often unstable

COOH altered in some cases unstable

truncated often unstable

HOOC overlong often unstable

2.2  Genetic Alterations in Cancers

33

trum of mutations that increase the activity of a gene product is much more restricted than the spectrum of mutations decreasing or destroying its activity. For instance, mutations activating RAS oncogenes are almost entirely restricted to three particular codons (→4.3), while mutations inactivating the tumor suppressor TP53 are distributed across its gene (→5.3). The distribution and the type of mutations found in a gene in several cases of one cancer type may therefore help to identify its function as an oncogene or a tumor suppressor. Splice mutations: Splicing requires specific consensus sequences at the exon-intron junctions and additionally within the intron. The efficiency of splicing is additionally influenced by sequences within the exon. Mutations of sequences required for splicing may alter the protein product or the expression level of a gene more or less subtly (Fig.  2.2). Mutations disrupting a 5′-splice site usually result in the skipping of an exon, whereas mutations affecting a 3′-splice site may lead to the elongation of an exon until the next recognizable splice site is encountered or likewise to exon skipping. Exon skipping or elongation generates altered proteins that lack amino acids or contain additional amino acids. The consequences depend critically on whether the reading frame in the sequence following the alteration is changed. Fig. 2.2 Potential effects of splice site mutations. E: exon; I: intron. Note that the effects of splice site mutations depend on the number of nucleotides in the exon, i.e., whether it is divisible by 3. See the main text for further explanations

If the reading frame is changed, the ensuing protein product will usually be truncated and may be unstable; if not, the protein will contain additional amino acids that may alter its function. With analogous consequences, point mutations in introns or exons may create novel splice sites from “cryptic splice sites,” i.e., sequences resembling proper splice consensus signals. Altered splicing: The issue of altered splicing in human tumors is rendered difficult by the complexity of splicing in normal mammalian cells. The average human gene is estimated to generate at least five different mRNA variants by differential splicing, alternative promoter usage, and alternative termination signals. Altered splicing with an according change in the relative proportion of splice variants is common in human cancers. Several important genes display variations in splicing in human cancers that are probably functional-relevant. These include the tumor suppressor TP53 (→5.3) and its paralogs TP63 and TP73 (Fig. 2.3). They each encode several different proteins which—substantially or subtly—differ in function. Aberrant splicing in cancer is not necessarily caused by mutations in the affected gene. In some cases, it may be a consequence of alterations in splice regulators. For instance, mutations in the SF3B1 protein, a component of the ribonucleo-

mRNA E1

E2

E3

E4

AAAAAAAA

ATG

STOP AATAAA 3n bp

3n+1 bp

E2

E3

E1 5` splice mutation

3` splice mutation

E4

3` splice mutation

Gene E3 5` splice or E4 3` splice mutation

mRNA E1 I1 E2 E3 E4 AAAA

E1 E3 E4

E1 E2 E4 AAAA

E1 E2 E3 I3 E4 AAAA

AAAA

Protein truncated or frameshifted usually unstable

shortened lacking aa from exon 2

frameshifted often truncated often unstable

overlong often unstable

2  Cancer Genetics

34 ∆Np53

TP53

1

2

3

4

I4

5

6

7

8

9

9′

10

11

∆Np73

Tp73

TP53

12 ε

1

2

3

3′

4

5

6

7

8

9

10

11 γ

12 ξ

13 δ

14

TP73

β

∆Np63

TAp63

β 1

2

3

3′

4

5

6

7

8

9

10

11

12

13

14

15

TP63

γ

Fig. 2.3  Splice variants of TP53, TP63, and TP73. T P73 (p73) and TP63 (p63) are homologs of the important tumor suppressor protein TP53 (p53), with partly overlapping functions. In each gene, multiple protein forms result from the

use of different transcriptional start sites and, at least two alternative splices (Greek letters) in each gene. Untranslated regions are drawn in dark or light grey. In cancer cells, the relative frequencies of the different messages are often altered

protein complex recognizing the intron branchpoint during splicing, are quite common in certain hematological malignancies. Mutations in regulatory sequences: Mutations in noncoding sequences may alter the organization of the genome and affect transcriptional regulation. The regulatory sequences for a gene often extend over a large region, comprising the proximal promoter sequence as well as enhancers and silencers, which can be located upstream and downstream of the gene or in introns. Boundary elements organize chromatin domains into loops and restrict the possible interactions between enhancers and promoters. Mutations in the proximal promoter sequences contribute to the activation of certain oncogenes, prominently the TERT gene encoding the Telomerase protein subunit (see Fig. 7.7). Point mutations and deletions in enhancers and boundary elements have been detected, but make overall a relatively minor contribution to tumor development. Much more common is altered enhancer usage. This can be

caused by chromosomal rearrangements, like those causing Burkitt lymphoma (→10.4), or by epigenetic alterations. Prominently, aberrant formation of super-enhancers from several individual smaller regulatory regions may lead to the deregulation of oncogenes. Another mechanism for deregulation involves reactivation of cryptic (meaning: normally silent) promoters within endogenous retroelements leading to the expression of aberrant transcripts or to the overexpression of a gene. Deletions: In addition to base changes, smaller or larger deletions (or insertions) in the genomes of tumor cells disrupt the coding regions of genes or alter their regulatory elements. Small deletions can be detected by PCR-based analyses, while sufficiently large deletions can be revealed by cytogenetic techniques. Intermediate-sized deletions are often difficult to detect and require specific investigations. Small deletions and insertions of a size detectable by sequencing of shorter DNA fragments or short-range PCR are collec-

2.2  Genetic Alterations in Cancers

tively designated “InDels.” The effect of small deletions within a coding sequence depends on their size, in particular, on whether they affect the reading frame (like for mutations that affect splicing). Larger deletions can inactivate a single gene or several contiguous genes or can delete regulatory elements. In some cases, deletions inactivate both copies of a gene, e.g., by an internal deletion in one chromosome and loss of the second homologous chromosome. This defect is called “homozygous deletion.” Homozygous deletions may extend across several Mbp and even lead to the loss of several contiguous genes in tumor cells, like the adjacent CDKN2A and CDKN2B genes (see Fig. 5.9). Insertions: Insertion in the genomes of tumor cells can be short, comprising one to several bps. In the coding sequence of a gene, they often result in frame shifts, depending on the number of bps inserted and their position within the gene. They may also change the stability of its mRNA.  Insertions can arise by several mechanisms. Some are in fact duplications, e.g., in short repetitive sequences such as genomic polyA-­ sequences or minisatellites caused by the unrepaired slipping of DNA polymerases (→3.2). Others may arise during the repair of DNA strand breaks (→3.4). Larger insertions, but also deletions, can be caused by viruses or by endogenous retroelements, among others. Transpositions of endogenous retroelements take place in many human cancers, most by active retrotransposons of the LINE-1 family. While LINE-1 elements retrotranspose predominantly into gene-poor regions of the genome, insertions have been documented to disrupt or deregulate genes in a significant number of cases. Viral genomes: Viruses add extra genetic material to cells and often affect the genome of the host cell in additional ways. Retroviruses may transduce oncogenes (→4.1) and provirus  insertions deregulate the expression of genes near the insertion site (→4.2). Retroviral insertions are overall uncommon in human cancers, but insertions of DNA hepatitis B virus (HBV) sequences activate oncogenes in some cases of hepatocellular carcinoma (→16.4). DNA viruses are relevant in a

35

larger spectrum of human cancers. They contribute to the development of several human cancers by the expression of viral oncogene proteins. While human DNA viruses normally replicate as episomes, they may integrate into the genome of host cells. These insertions typically disrupt genes. Often, parts of the viral genomes are lost and the remaining expressed viral genes may act as oncogenes, as in the case of oncogenic HPV viruses (→5.3). Since the integration of viral sequences into the host genome involves the generation of DNA strand breaks the process may induce chromosome losses and rearrangements. Chromosomal translocations: Alterations in the structure or expression of individual genes can also be caused by chromosomal translocations (Fig. 2.4). These were first discovered in hematological and soft tissue cancers, but have now been characterized in many carcinomas as well. Obviously, a translocation may simply destroy and inactivate genes at the two sites involved in the translocation and sequences may get lost in the translocation process. However, translocations may also inappropriately activate genes, basically by two mechanisms. (1) A translocation may separate inhibitory regulatory elements from the coding region of a gene or place it under the influence of activating regulatory elements from another locus. In either case, overexpression or deregulation ensue. This mechanism accounts, e.g., for the deregulation of the MYC gene in Burkitt lymphoma (→10.4) or the activation of the ERG transcription factor gene in prostate carcinoma (→20.2). Intriguingly, the most common ERG fusion partner gene, TMPRSS2, resides several Mbp upstream of the ERG gene likewise on chromosome 21. Fusion genes can therefore be formed either by translocations between two different chromosomes 21 or by deletions of the intervening DNA on chromosome 21. (2) Translocations may position fragments of two genes next to each other, in the same orientation. Typically, the product of the novel gene is a fusion protein containing N-­terminal sequences from the upstream fusion partner and C-terminal sequences from the down-

2  Cancer Genetics

36 Chr 10 No gene

inv(10) (q11.2;q21)

Gene

Disrupted gene

RET

PTC-RET

PTC

Regulatory region

Gene A

Gene B

Deregulated gene B

Gene A

Gene B

Fig. 2.5  Activation of the RET gene by inversion in thyroid cancers. Inversion of a segment of chromosome 10 in some cases of thyroid cancers (induced, e.g., by ionizing radiation) yields a PTC/RET fusion gene activating the RET receptor tyrosine kinase. In other tumors, the RET protein is activated to an oncogene by point mutations

(Fig. 2.5). In this case, inversions within the long arm of chromosome 10q activate the RET oncogene by fusing it with other genes, most often the Fusion gene A-B PTC gene. 1 This particular inversion is a typical consequence of exposure to radioactive iodine. Fig. 2.4  Effects of chromosomal translocations in human The genetic alterations discussed above precancers. dominantly affect one or a few genes. Other Top: a gene is inactivated by disruption. Center: regulatory regions of gene 1 become positioned next to gene 2, genetic alterations in cancers impact the genome deregulating its expression. Bottom: gene 1 and gene 2 are more globally. fused resulting in the expression of a fusion protein conPolyploidy and aneuploidy: Most advanced trolled by regulatory regions from gene 1 cancers are aneuploid, exhibiting numerous structural and numerical chromosomal aberrastream partner. Transcription of the fusion tions. According to measurements of DNA congene is controlled by the promoter of the tent, many carcinomas appear to start out from a upstream fusion partner. Balanced transloca- near-diploid stage, duplicate their genome to a tions may form two fusion genes and pro- near-tetraploid stage, from which chromosomes teins. The fusion proteins can possess are lost and gained (with losses prevailing) until a properties that differ significantly from those metastable state is reached. Whole-genome of the original proteins. Two well-­ duplication (WGD) to a (more or less) tetraploid characterized proteins of this type are BCR-­ genome may in fact allow cancer cells to tolerate ABL (→10.5) and PML-RARα (→10.6) a wider range of chromosomal losses and gains causing chronic myeloid leukemia and acute than in a diploid state. Indeed, many established promyelocytic leukemia, respectively. carcinoma cell lines display a “pseudo-triploid” karyotype with a modal chromosome number Chromosomal inversions can have basically the same consequences as translocations since 1  they represent in essence translocations within The N-terminal PTC domain mediates constitutive homodimerization of the fusion protein leading to ligandone chromosome. A prominent example concerns independent activation of the RET tyrosine kinase, see the RET gene in papillary thyroid carcinomas Sect. 4.4.

2.2  Genetic Alterations in Cancers 1

2

3

6

7

8

13 T

19

14 20

T

37 4

9

15 21

10

16 22

5

11

17

12

18 X

Y

Fig. 2.6  Karyotype of a pseudo-triploid human bladder cancer cell line. Karyotyping of the cell line was performed by multicolor fluorescence in situ hybridization (chromosome painting), whereby each chromosome is depicted in a different color. The karyotype probably evolved by genome duplication (note the 2 X and 2 Y chromosomes each) followed by loss or gains of specific chromosomes. One copy each of chromosomes 9 and 11 was presumably already lost prior to genome duplication. Two chromosomes with translocations are indicated by “T”. Courtesy H. Rieder

≈70 (Fig. 2.6), where the “pseudo” indicates that the close-to-69 number of chromosomes obscures a multitude of changes in the number and structure of individual chromosomes. Some leukemias remain near-diploid through most of their course, displaying however distinctive chromosomal changes like specific translocations or losses of particular chromosomal fragments, but may then develop multiple and diverse chromosomal changes during progression (like CML, →10.5). Some carcinomas as well contain few chromosomal aberrations, but instead a high number of point mutations. In general, numerical and structural chromosomal aberrations are distinguished. Numerical chromosomal aberrations: In aneuploid cells, the numbers of chromosomes or chromosome arms deviate from the total number of complements present, e.g., three chromosomes of one kind may be found in a diploid cell. These numerical aberrations imply an altered gene dosage for the genes located on the respective chromosomes. The copy number of one gene in a tumor cell may therefore range from zero (i.e., homozygous deletion) to very high. However, even a tumor cell cannot manage to carry more than 5 or 6 copies of the same chromosome

because of imbalances in gene expression and increasing difficulties to conduct mitosis. Gene amplification: Gene copy numbers higher than five or six are reached by amplification of smaller chromosomal segments. These are called amplicons and may range from several hundred kbps to several Mbp in size, in quite regular to highly irregular arrangements. Their large sizes implicate that the amplified region may contain several genes, of which one or more may be overexpressed and be relevant for the tumor phenotype. Sufficiently large amplified regions located within a chromosome can become evident in cytogenetic analyses as “homogeneously staining regions” (HSR). Amplicons in tumor cells can also be episomal, forming circular DNA structures that present as small speckles in the nucleus and are designated “double minutes.” 2 Double minutes replicate autonomously, but do not possess kinetochores and are accordingly randomly distributed to daughter cells. Their numbers are therefore highly variable. Moreover, they may be in a dynamic exchange with corresponding HSRs on one or several chromosomes. Copy numbers of genes on double minutes can run up to thousands per cell, allowing massive overexpression. Extrachromosomal DNA on double minutes is moreover particularly susceptible to base and InDel mutations. Structural chromosomal aberrations: In tumor cells, structural chromosomal changes include the translocations and inversions discussed above as well as internal deletions of various sizes. Classical cytogenetic methods have identified “marker” chromosomes in many cancer cells that are composed of fragments from several different chromosomes (cf. Fig. 2.6). Molecular analyses reveal that in addition many chromosomes that appear grossly normal in cancer cells harbor deletions, inversions, or contain segments of other chromosomes. Numerical chromosomal changes can be most straightforwardly brought about by mitotic nondisjunction (Fig. 2.7). If a chromosome does not Extrachromosomal circular DNA can be generated by several processes, including recombination, breakagefusion-bridge cycles and chromothripsis. 2 

38

Fig. 2.7 Three mechanisms leading to chromosomal aberrations. Top: Mitotic nondisjunction typically yields trisomy and monosomy, respectively, of a chromosome in the daughter cells. Center: Double-strand breaks unrepaired until cell division can result in loss of genetic material. Bottom:

2  Cancer Genetics

Telomere fusions (or alternatively, double-strand breaks) may initiate breakage-fusion-bridge cycles. Note that the products resulting from disruption of the dicentric chromosome in the bottom figure are capable of starting further rounds of the cycle

2.3  Inherited Predisposition to Cancer

attach properly to the mitotic spindle, one daughter cell may end up with an additional chromosome and the other with one less. The mechanisms causing structural chromosomal changes are more varied and more complicated. Deletions or recombination are often initiated by double-­strand breaks in the involved chromosomes. In particular, double-strand breaks as well as telomere fusions may initiate breakage-fusion-bridge cycles (Fig. 2.7). If joined to the wrong site in the genome, one end of a double-strand break may initiate a series of rearrangements, where one end of the target sequence is joined to the starter sequence and the other end is joined to yet another site in the genome, until the repair process finally manages to link an open end to the second end of the initial double-strand break. This process, termed “chromoplexy,” can cause a substantial number of translocations in one instance. Chromoplexy was first discovered in prostate cancers, where it may be initiated at transcriptional hubs, in which several genes are brought closely together by androgen receptor-dependent transcription. Whereas chromoplexy can involve several different chromosomes, “chromotripsis” involves the shattering and reassembly of a single chromosome. Chromothripsis likewise results in a large number of rearrangements, but all within the same chromosome. Most likely, it occurs by accident, when a chromosome is separated in a micronucleus following mitotic missegregation, begins to be degraded, but is then reassembled. Upon closer analysis, chromothripsis can be detected at some frequency in various cancer types, with estimates ranging up to 10% overall. The multitude of structural changes in chromosomes reveals a high degree of illegitimate recombination in tumor cells. Illegitimate recombination may  moreover result in the exchange of genetic material rather than net loss or gain or structural changes like translocations. Indeed, molecular analyses of polymorphic DNA sequences reveal that tumor cells often contain only identical copies of DNA sequences that are heterozygous in normal cells of the patient. This alteration is called “loss of heterozygosity.” It can be caused by the deletion of one allele, but also by recombination without loss of genetic material (Fig. 2.8).

39

A

a

B

b

C

c

(1)

(4) (2) (3)

A

A

B C

a

A

a

A

a

B

B

c

B

B

C

C

C

c

Fig. 2.8  Mechanisms leading to loss of heterozygosity in cancer cells. Two homologous chromosomes are shown with polymorphic markers Aa, Bb, Cc (microsatellites or single nucleotide polymorphisms). (1) Loss of one chromosome leading to monosomy; (2) Loss of part of a chromosome arm leading to partial monosomy; (3) Interstitial deletion with loss of marker b; (4) Recombination leading to substitution of allele b by allele B

The various types of genetic alterations discussed in this section do not occur each to the same extent in every human cancer. Rather, in different tumors particular types of mutations tend to predominate. In some cancers point mutations are most prevalent, whereas chromosomal aberrations seem to be responsible for the majority of the genetic changes in others. In some cancers, distinct subtypes can be distinguished by this difference, e.g., in colorectal carcinomas (→13.3). Other differences may be more subtle. For instance, some cancers tend to lose or gain whole chromosomes, whereas others tend to delete, gain, or rearrange chromosomal fragments. The reasons underlying such differences are the subject of current research.

2.3 Inherited Predisposition to Cancer Although most genetic alterations in tumor cells develop during the lifetime of the patients, the predisposition to cancers can be inherited. Three different modes of inheritance can be distinguished.

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40

(1) In some families, cancers are very frequent is not an obligatory criterion. The increased and occur basically in each generation. This risk in cancer families with an autosomalpattern is a general hallmark of autosomal-­ dominant mode of inheritance is caused by dominant inherited diseases with high penan inherited mutation in a single gene. The etrance. The families may be afflicted by affected genes are usually tumor suppressor specific cancers, rarer types such as retinogenes and in exceptional cases oncogenes. blastoma or common types such as breast The inherited mutations are in general not cancer, or by several different cancers, like sufficient to cause cancer, but rather provide in Li-Fraumeni-syndrome (Table  2.2). the first of several required genetic alteraTypically, cancers manifest in these famitions (see Sect. 2.5). lies at a lower-than-average age of onset (2) In some families, predisposition to cancer is and also quite regularly at multiple sites or inherited in a recessive mode. More often bilaterally in paired organs, such as the than in the dominantly inherited cases, caneyes, kidneys, and breasts. Moreover, rare cer predisposition is found in the context of cancers like male breast cancer may cluster rare inherited syndromes with additional in such families. All of these criteria point manifestations. The affected patients may to inherited cancers. In some syndromes, initially be afflicted by other symptoms and cancer predisposition is associated with cancers appear later, albeit still at a relatively developmental defects, e.g., in the Gorlin early age. Syndromes in this category include and Cowden syndromes. If observed, assoxeroderma pigmentosum, ataxia telangiectaciation with developmental defects is sia, Fanconi anemia, Nijmegen breakage another indicator of inherited cancers, but it syndrome as well as the Bloom and Werner Table 2.2  Tumor syndromes inherited in an autosomal-dominant modea Syndrome Neurofibromatosis 1

Gene NF1

Location 17q11.2

Familial atypical multiple mole melanoma syndrome (FAMMM) Retinoblastoma Cowden syndrome Tuberous sclerosis

CDKN2A

9p21

RB1 PTEN TSC1, TSC2

13q14 10q23.3 9q34, 16p13

Basal cell nevus syndrome (Gorlin) Familial adenomatous polyposis coli Von Hippel-Lindau

PTCH APC VHL

9p22 5q21 3p25

Hereditary leiomyoma renal cell carcinoma Burt-Hogg-Dubè syndrome Hereditary gastric cancer Multiple endocrine neoplasia type 1 Neurofibromatosis 2 Li-Fraumeni syndrome Lynch syndrome (HNPCC)

FH

1q24.1

BHD CDH1 MEN1 NF2 TP53 MLH1, MSH2, others

17q11 16q22 11q13 22q 17p13.1 3p21, 2p15-­16

Hereditary breast and ovarian cancer

BRCA1, BRCA2

Multiple endocrine neoplasia type 2

RET

Hereditary papillary renal cancer

MET

Chapter 4.4

Eye, bone Many organs Soft tissues in several organs Skin, brain Colon, rectum, others Kidney, adrenal glands, others Uterus, kidney

5.2 6.3 6.3

Kidney, others stomach Endocrine glands CNS, peripheral nerves Many organs Colon, endometrium, stomach, others 17q21, 13q12 Breast, ovary (pancreas, prostate) 10q11.2 Thyroid and other endocrine glands 7q31 Kidney

See the OMIM online database for detailed information on these syndromes

a

Cancer site Peripheral nerves, eye, skin Skin, pancreas, others

5.2

12.3 13.2 16.4 16.3 16.3 18.3 – 6.9 5.3 13.2 19.6 5.1 16.2

41

2.3  Inherited Predisposition to Cancer Table 2.3  Tumor syndromes inherited in a recessive mode

Nijmegen breakage syndrome Bloom syndrome Werner syndrome

Function affected Nucleotide excision repair

Gene(s) involved Organ site XP (ERCC) genes, others Skin

Chapter 3.3

Strand break repair signaling ATM Crosslink repair FANC genes, BRCA2 (homozygous) Double-strand break repair NBS

multiple 3.6 Hematopoietic system, 3.5 others Hematopoietic system 3.4

Recombination Recombination repair, telomere function

multiple multiple

BLM WRN

syndromes (Table 2.3). These syndromes differ in the magnitude of the conferred cancer risk and in the predominant cancer types, but at least one type of cancer is substantially more prevalent in the affected persons than in the general population. In recessively inherited syndromes, predisposition to cancer is caused by germline mutations inactivating both copies of the same gene. The mutated genes are usually involved in DNA repair or cell protection (→3.6). In general, inherited defects favor the acquisition of genetic alterations in somatic cells that eventually lead to cancer. Inheritance of mutated genes in autosomal-­ dominant or recessive inherited cancer syndromes carries a greatly enhanced risk of developing cancer during an individual’s lifetime which may approach 100%, whereas the lifetime risk of “sporadic” cases in the general population may be minimal. Even in those cancers, like breast cancer, where the lifetime risk in the general population is high (up to 10%), the risk of developing cancer up to a certain age is strongly increased in persons with an inherited predisposition (Fig.  2.9). Fortunately, cancer predisposition of this kind is infrequent. Taken together, all high-risk mutations in dominantly and ­recessively inherited cancer syndromes may account for fewer than 10% of all human cancers. (3) Nevertheless, any individual’s cancer risk is contingent on their genotype. About one in a thousand base pairs differs between individual humans. More frequent variants (more

3.4 7.2

1.0

Cumulative incidence

Syndrome Xeroderma Pigmentosum Ataxia Telangiectasia Fanconi Anemia

0.8 0.6 0.4

BRCA1 c.4034delA c.5266dupC no mutations

0.2 0.0 28

48 68 Age at diagnosis

88

Fig. 2.9  Age dependency of cancers in hereditary and sporadic cases. Age at diagnosis in breast cancer patients with the indicated mutations in the BRCA1 tumor suppressor gene vs. patients without these mutations. The study was performed in Latvia, where the general prevalence of the indicated mutations is estimated as ≈0.5%, whereas around 5% of the unselected breast cancer patients in the study had these mutations, suggesting an overall ≈10-fold increased risk. From Plakins G et al. BMC Med. Genet. 2011

than 1% of all alleles in representative populations) are commonly called “polymorphisms.” Sequence variants are found in the coding regions of genes as well as in their regulatory sequences, but also in noncoding sequences throughout the genome. The differences comprise single nucleotide polymorphisms (SNPs), differences in the size of micro- and minisatellite repeats, but also insertions or deletions of various sizes. In addition, the copy numbers of some DNA sequences may vary between individuals; this phenomenon is designated as copy number variation (CNV). Sequence variants are

2  Cancer Genetics

comprehensively listed in dedicated databases. They list the allele frequencies in various populations, with MAF (minor allele frequency) denoting the frequency of the less common allele. Variants can be innocuous (neutral), elevate, or diminish the risk for a disease. Estimation of the conferred risk often requires a combination of experimental and clinical investigations and can therefore be difficult. Again, specific databases compile the knowledge on these relationships. For instance, up to 50% of individuals in some European populations lack the GSTM1 gene. It encodes a glutathione transferase enzyme metabolizing many chemical carcinogens, other xenobiotics, and some endogenous metabolites. This deletion is an example of a “null-allele” because no protein and thus no enzyme activity are present. Null-alleles can also result from nonsense mutations or missense mutations destabilizing a protein (e.g., in NQO1). In GSTM1−/− individuals, in particular, detoxification of carcinogenic metabolites of benzopyrene is less efficient. Since benzopyrene is an important carcinogen in tobacco smoke, the risk of GSTM1−/− smokers to develop cancer of the lung and other organs is increased, by a factor 84

Age [years]

Fig. 2.11  Age dependency of human cancers. Incidence of urological cancers in males in relation to age in Germany 2003 (according to data from the Robert

Koch Institute). Note the early peak in testicular cancers and the plateau in the kidney cancer curve

2.4 Cancer Genes

Tumor suppressor genes: The genes mutated in dominantly inherited cancers are obviously central to their development. Most of them belong to the category of tumor suppressor genes (→5). Characteristically, the function of tumor suppressor genes is strongly diminished or obliterated in cancers by genetic alterations or by epigenetic silencing. Not unexpectedly, those genes are often found mutated in sporadic cases of the same and additional cancer types (Table 2.5). Other tumor suppressors are inactivated only in sporadic cancers, where their inactivation can be shown to be essential for tumor development. As a rule, both alleles of a tumor suppressor gene are inactivated in cancer, but in specific cases, “­ haploinsufficiency” (where two intact copies of one gene are required for its full function) or dominant-­negative effects (where the altered gene product of one mutated gene impairs the function of the intact protein) may generate exceptions (see Sect. 5.4 for a more detailed discussion).

In many cancers, hundreds of genes are affected by point mutations, copy number alterations, or structural rearrangements. Moreover, the expression of many genes is not changed directly by genetic mechanisms such as altered gene dosage or mutation, but secondary to mutations in regulatory genes or by epigenetic mechanisms. It is therefore not immediately obvious which genetic changes are essential for tumor development, which changes contribute to the phenotype of cancer without being essential, and which changes are coincidental and irrelevant. Overall, according to the COSMIC database, ≈730 genes are considered as “cancer genes” in at least one type of human cancer at the time of writing. According to the database, ≈90% of these are affected by somatic genetic alterations, 20% by germline mutations, and 10% by both. Several kinds of cancer genes may be distinguished.

2.4  Cancer Genes

45

Oncogenes: The genes in oncogenic retroviruses known to cause cancers in animals were the first identified oncogenes (→4). Retroviral oncogenes usually differ in their sequence from the orthologous host genes and encode overactive, deregulated proteins. A few responsible genes in dominantly inherited cancers, such as RET in hereditary endocrine cancers and MET in hereditary renal papillary carcinoma (→16.3) are likewise activated to oncogenes by mutations. Germline mutations in oncogenes are however the exception to the rule. A much larger number of oncogenes in the human genome are activated recurrently in sporadic cancers by specific point mutations, by overexpression as a consequence of gene amplification, or by other mechanisms in somatic cells (Table  2.5). As a rule, only one copy of an oncogene is activated. DNA repair and DNA damage checkpoint genes: Most oncogenes and many tumor suppressor genes directly control cell proliferation, differentiation, or survival. Many genes inactivated by mutations in recessive cancer syndromes influence cancer development in a different manner (→3.6). Defects in these genes increase the rate of mutations, of which some alter the function of genes directly involved in cancer development. This type of tumor suppressor genes can be Table 2.5  Mechanisms inactivating tumor suppressor genes and mechanisms activating oncogenes Mechanism Missense mutations Truncating mutations Gene deletion

Tumor suppressor Frequent, distributed through the sequence Frequent, different sites Frequent

Gene amplification Translocation

Rare

Viral insertion

Infrequent (disruption) Never

Viral transduction Epigenetic deregulation

Rare (disruption)

Frequent (downregulation)

Oncogene Frequent, specific sites Uncommon Rare (only wild-type allele) Frequent Frequent, very selective Infrequent Rare (in humans) Common (overexpression)

regarded as “caretakers” (→5.4). In fact, mutations in genes of this type also underlie certain cancer predispositions inherited in a dominant fashion. Mutations in various caretaker genes also contribute to the development of sporadic human cancers. Risk-modulating genes: A related category of relevant genes is even less directly involved in cancer development. The products of genes in this group, exemplified by the GSTs, modulate the development of cancer, e.g., by influencing the level of active carcinogens, the reaction of the immune system to cancer cells, or the level of hormones and growth factors that stimulate tumor cell proliferation. Very often, their importance depends on environmental factors such as exposure to carcinogens. Once a cancer is established, their influence is often no longer relevant. Executor genes 4: In contrast, another category of genes becomes important only after cancer is established. These genes may be activated by mutations, but are more often induced as a consequence of the activation of oncogenes or the inactivation of tumor suppressor genes. They may not be absolutely necessary for the survival of the cancer cells, but support their sustained growth and specifically invasion and metastasis (→9). These complex processes appear to require well-­ coordinated programs of gene expression which can be initiated by mutations in a limited number of genes but require the activity of a much larger number of gene products for their execution. It is therefore not trivial to define a “cancer gene.” Some cases are clear-cut. For instance, almost every retinoblastoma displays inactivation of the gene RB1 (→5.1), and every Burkitt lymphoma shows activation of the gene MYC (→10.4), indicating that these are a crucial tumor suppressor and oncogene, respectively, in these tumors. Such bona fide oncogenes and tumor suppressor genes represent the extreme end of a continuum of more or less relevant cancer genes. Finally, while most well-characterized cancer genes encode proteins, accumulating evidence indicates important functions of diverse regulaThis term is not used consistently in the literature; one alternative designation is “enablers.” 4 

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46

tory RNAs in many cancers. Regulatory RNAs are arbitrarily classified by a size limit of 200 nt. One important class of small RNAs involved in cancer are microRNAs, which are about 20 nt long. In their canonical mode of action, they modulate gene expression by binding to specific mRNAs, blocking translation and enhancing their degradation. Each miRNA can bind to several mRNAs, with variable efficiency. In this fashion, miRNAs provide another level of gene regulation that may be particularly relevant in the adaptation to stress conditions. While point mutations in miRNA genes are rare in cancers, many miRNAs are deregulated by gene amplification, gene loss, or other mechanisms. In particular, downregulation of the Dicer protein, which is required for the maturation of miRNAs from their precursor RNAs, is common and leads to overall lower levels of miRNAs in many cancers. In fact, germline mutations in the rare DICER1 syndrome are associated with an array of pediatric and adult-onset cancers. However, while the expression of miRNAs at large is diminished in many cancers, individual miRNAs are overexpressed and contribute to the development and progression of cancers as “onco-miRs.” Other miRNAs act in a tumor-suppressive manner. It is in fact not unusual that the same miRNA promotes one type of cancer, but impedes the devel-

opment of another. A small selection of the many miRNAs implicated in cancer are listed in Table 2.6; most of them are discussed later in this textbook. RNAs that do not encode proteins and encompass more than 200 nt are summarized as long noncoding RNAs (lncRNAs). They form an extremely heterogeneous group, but most are transcribed by PolII, capped, spliced (albeit rarely as extensively as protein-coding genes) and poly-adenylated. Some originate as byproducts from the pervasive transcription of the genome and very likely do not serve a specific function. The importance of many lncRNAs may reside in the very act of their transcription, which helps to maintain open chromatin. Many “divergent” lncRNAs are transcribed from the same promoters as adjacent protein genes but in the opposite direction. Others overlap a protein-­ coding gene but are transcribed from their own promoters in the opposite direction as antisense transcripts. In both cases, transcription per se or the ensuing noncoding RNA may help to regulate the expression of the protein-coding gene in cis. Other lncRNAs are expressed from separate transcription units that are very similarly organized as those of protein-coding genes. A number of lncRNAs act in trans. They may regulate the expression of distant genes at vari-

Table 2.6  A selection of miRNAs implicated in cancer miRNA Let-7 miR-15 miR-16 miR-17 miR-19 miR-22-3p miR-29 miR-30 miR-34 miR-122 miR-145 miR-200 miR-222-3p miR-371 miR-375 miR-675

Chromosomal localization 9q22.2 13q14 13q14 13q31.3 13q31.3 17p13.3 7q32 6q13 1p36.1 18q21.3 5q32 1p36.1 Xp11.3 19q13.4 2q35 11p15.5

Function (selected) MYC regulation BCL2 regulation BCL2 regulation Mediates MYC effects PTEN downregulation Seminoma development Repressed by MYC Repressed by MYC Mediates TP53 effects Regulation of hepatic differentiation MYC regulation EMT regulation Regulation of KIT TGCT development Teratoma development EMT regulation

Comments Both ways

Affected by hepatitis viruses

Biomarker Biomarker Regulated with lncRNA H19

2.5  Accumulation of Genetic and Epigenetic Changes in Human Cancers Table 2.7  A selection of lncRNAs implicated in cancer Chromosomal Function lncRNA localization (selected) ANRIL 9p21 Represses CDKN2A CCAT1 8q24 Regulates MYC expression H19 11p15 miRNA regulation HOTAIR 12q13 Chromatin regulation MALAT1 11q13 Nuclear organization MEG3 14q32 Chromatin regulation NEAT1 11q13 Nuclear organization NKILA 20q13 NFκB regulation PCA3 9q21.1 May regulate RNA editing PCAT1 8q24 Regulates MYC expression PVT1 8q24 Regulates MYC expression Schlap1 2q31 Chromatin regulation TERRA 20q13 Telomere regulation XIST Xq13 X-chromosome inactivation

Comments

Imprinted

Imprinted

Prostate-­ specific

Prostate-­ specific

ous levels or exert structural functions in the nucleus or other cellular compartments, similar to the many noncoding RNAs involved in RNA processing and protein biosynthesis. A number of functionally important lncRNAs have been shown to be regularly upregulated or downregulated in specific cancer types and a few appear to contribute to the development and progression of one or several cancer types. Point mutations are not likely to affect the function of lncRNAs to a major extent, but a few have been detected nevertheless (e.g., in MALAT1 and NEAT1). However, deletions and amplifications of lncRNA genes ought to affect their expression levels, and lncRNA genes are involved in some gene fusions. A small selection of the many lncRNAs implicated in cancer are listed in Table  2.7; most of them are discussed later in this textbook.

47

2.5 Accumulation of Genetic and Epigenetic Changes in Human Cancers Advanced human cancers usually contain a multitude of diverse genetic alterations and additionally many epigenetic changes. In a manifest human cancer, it is difficult to determine at which stage of tumor progression they were acquired. Most data, e.g., from analyses of precursor stages and the comparison of mutational frequencies in mutated genes,5 suggest that they gradually accumulate during tumor progression. A gradual accumulation of multiple genetic changes over time would moreover explain why most cancers appear at older ages (Fig.  2.11) and would fit with mathematical models suggesting that 4–5 “hits” are necessary for cancer development. These mutations could arise in a gradual fashion over time, by spontaneous errors in endogenous processes or following exposure to exogenous carcinogens. In cancers arising in persons with inherited high-risk mutations, one essential mutation is already present in all cells, accounting for their earlier onset and the higher likelihood of the disease, including emergence of multiple tumors. However, not all data support the gradual accumulation model. In some cancers, a punctuated equilibrium model (named in analogy to concepts of phylogenetic evolution) fits better with the observations. In such cancers, catastrophic events like chromoplexy, kataegis, or the aberrant mitosis of a polyploid precursor cell may have constituted essential steps in their development. The acquisition of genetic alterations can be accelerated, if the cancer precursor cells acquire a “mutator phenotype” by defects in DNA repair, the fidelity of DNA polymerases, chromosome segregation, or cell cycle checkpoint signaling. Again, such deficiencies might be inherited in familial cases, increasing the likelihood of developing cancer and lowering the age of onset. Depending on the type of defect, a mutator phenotype can manifest as an increase in point mutaWhere the earliest “truncal” mutations should be present in 100% of the cancer cells. 5 

2  Cancer Genetics

48

tions or as one of various kinds of chromosomal instability. The most important driver genes affected by genomic alterations are oncogenes and tumor suppressor genes. Nevertheless, because of the multitude of genetic changes in advanced cancers, especially those with a mutator phenotype, identification of the drivers is not trivial. One criterion is that genetic changes relevant to one cancer type should be recurrent. However, only a few cancer types contain mutations in one particular gene or one particular chromosomal alteration in every tumor case. Mutations in certain oncogenes, like RAS family genes, or tumor suppressors, like TP53, are observed across a large spectrum of cancers and are good candidates for drivers whenever they are detected. Moreover, many cancers are characterized by activation or inactivation of particular signaling pathways (see Chap. 6), which can be caused by mutations in various of their components. In such instances of genetic heterogeneity, collectively, one change activating (or inactivating) a pathway may occur in every tumor, but not in the same gene in every case. Typically, the frequency of mutated genes in one cancer type exhibits a long-tailed distribution (Fig.  2.12). Thus, a few genes are mutated in a relatively large fraction of the tumors, but many others are only mutated in a few cases, where they may still be crucial for cancer development.

This observation implies that many human cancer types are highly genetically heterogeneous, in that each individual cancer has essentially its own set of genetic alterations, even with respect to drivers. One metaphor illustrates this distribution as a genomic landscape of “mountains and hills,” where the relatively few genes mutated in many cases represent the mountains in a landscape with many smaller hills of varying height formed by other relevant, but less commonly mutated genes. Actually, this metaphor applies to the distribution of driver genes across all cancer types too, with, e.g., RAS genes or TP53 representing mountains. In fact, the interpretation of genetic changes is even more difficult than discussed above because they differ not only between cancers in individual patients (inter-tumor heterogeneity), but also quite commonly between different parts of ­primary cancer and its metastases (intra-tumor heterogeneity). Finally, identifying driver genes in cancer is nontrivial because it is difficult to ascertain the significance of copy number alterations and of epigenetic alterations, even if they may have been comprehensively charted. Mathematical modeling and the results of experiments where genetic changes were deliberately induced in normal cells predict that in most cell types four to five genetic events are required to acquire full malignancy. This number fits well with the number of

20

Mutation frequency (%)

Non-recurrent missense Indel 15

10

Truncating Recurrent missense (hotspot, OncoKB, COSMIC)

5

TP53 SPOP KMT2C KMT2D FOXA1 AR ZFHX3 CDK12 PTEN ATM APC KDM6A SPEN PIK3CA CTNNB1 BRCA2 COL5A1 TAF1L CHD6 NCOR1 MED12 MGA STAB2 DHX30 ERF KMT2A CDC27 PTPRC JAK1 CHD3 ARIDIA USP28 ZNF292 SETD2 IGF2R ZMYM3 CDKN1B RB1 ANF43 BAPFI COL5A3 TRPM4 CHD1 CHD7 SAMD9 BRAF PPL RAG1 SLCAA2 USP7 ARID2 CNOT3 COL15AI CUL3 EHHADH ITSN1 RPRD2 ARID4A IL6ST MET NDST2 TBCID2 MATN4 SMARCA1 MYBSPIA PAX6 PYHIN1 SETDB1 SF3B1 SPATA18 TMPRSS2 AAMTS6D JADE2 ETV3 MBD1 NOX3 PALB2 PIK3R1 PIK3R2 RNF31 UNC13D PIK3C6 CDH1 ITSN2 KEAP1 PDS5A IDH1 PMS1 ASF3W KRAS HRAS TAP1 XPO1 AKT1 MRE11A U2AF1 SMARCAD1

0

Fig. 2.12  The long-tailed distribution of mutated genes in prostate cancers. Results of a comprehensive study of prostate cancer, indicating the frequency of cancers with mutations in each gene. From: Armenia J et al. (2018) Nat. Genet. 50,

645–651. Note that while prostate cancer is distinguished by a paucity of major driver mutations, similar distributions of the frequency of point mutations (including InDels) in different genes are observed in other cancer types

Further Reading

recognized drivers that comprehensive genetic analyses have yielded in many common cancers. However, in a substantial fraction of individual cancers, the number of unambiguously identified drivers is much lower. Thus, the identification of driver genes may remain wanting for technical reasons or for lack of knowledge on the function of poorly studied and uncommonly mutated genes. Another explanation for this discrepancy is that epigenetic changes make a greater contribution to cancer development and progression than is currently appreciated.

Further Reading Adams BD et al (2014) Aberrant regulation and function of microRNAs in cancer. Curr. Biol. 24:R762–R776 Bakhoun SF, Cantley LC (2018) The multifaceted role of chromosomal instability in cancer and its microenvironment. Cell 174:1347–1360 Brady SW et  al (2022) Therapeutic and prognostic insights from the analysis of cancer mutational signatures. Trends Genet. 38:194–208 Buisson R et  al (2019) Passenger hotspot mutations in cancer driven by APOBEC3A and mesoscale genomic features. Science 364:1251–1258 Burns KH (2017) Transposable elements in cancer. Nat Rev Cancer 17:415–424 Cervantes-Garcia K et  al (2021) APOBECs orchestrate genomic and epigenomic editing across health and disease. Trends Genet. 37:1028–1043 Chiu HS et al (2018) Pan-cancer analysis of lncRNA regulation supports their targeting of cancer genes in each tumor context. Cell Rep 23:297–312 Cosmic database (n.d.) https://cancer.sanger.ac.uk/census Elliott K, Larsson E (2021) Non-coding driver mutations in human cancer. Nat Rev Cancer 21:500–509 Foulkes WD (2008) Inherited susceptibility to common cancers. NEJM 359:2143–2153 Futreal PA (2004) A census of human cancer genes. Nat Rev Cancer 4:177–183 Garraway LA, Landes ES (2013) Lessons from the cancer genome. Cell 153:17–37 Gordon DJ et al (2012) Causes and consequences of aneuploidy in cancer. Nat Rev Genet 13:189–203 Hahn WC et  al (2021) An expanded universe of cancer targets. Cell 184:1142–1155 Hollstein M et  al (2017) Base changes in tumour DNA have the power to reveal the causes and evolution of cancer. Oncogene 36:158–167

49 Jang HS et al (2019) Transposable elements drive widespread expression of oncogenes in human cancers. Nat Genet 51:611–617 Koh G et  al (2021) Mutational signatures: emerging concepts, caveats and clinical applications. Nat Rev Cancer 21:619–639 Kong J, Lasko P (2012) Translational control in cellular and developmental processes. Nat Rev Genet 13:383–394 Lee E et al (2012) Landscape of somatic retrotransposition in human cancers. Science 337:967–971 Li Y et al (2020) Patterns of somatic structural variations in human cancer genomes. Nature 578:112–121 Lin S, Gregory RI (2015) MicroRNA biogenesis pathways in cancer. Nat. Rev. Cancer 15:321–333 Liu SJ et  al (2021) Long noncoding RNAs in cancer metastasis. Nat. Rev. Cancer 21:446–460 Mani RS, Chinnaiyan AM (2010) Triggers for genomic rearrangements: insights into genomic, cellular and environmental influences. Nat. Rev. Genet. 11:819–829 Martincorena I & Campbell PJ (2015) Somatic mutations in cancer and normal cells. Mitelman F, Johansson B, Mertens F (Eds.) (n.d.) Mitelman database of chromosome aberrations and gene fusions in cancer. https://mitelmandatabase.isb-­ cgc.org Noer JB et  al (2022) Extrachromosomal circular DNA in cancer: history, current knowledge, and methods. Trends Genet. 38:766–781 Obeng EA et al (2019) Altered RNA processing in cancer pathogenesis and therapy. Cancer Disc. 9:1493–1510 Online Mendelian Inheritance in Man (n.d.) (OMIM database), at the ncbi.nih.gov website Roberts SA, Gordenin DA (2014) Hypermutation in human cancer genomes: footprints and mechanisms. Nat. Rev. Cancer 14:786–800 Rodriguez-Martin B et  al (2020) Pan-cancer analysis of whole genomes identifies driver rearrangements promoted by LINE-1 retrotransposition. Nat. Genet. 52:306–319 The ICGC/TCGA Pan-Cancer of Whole Genomes Consortium (2020) Pan-cancer analysis of whole genomes. Nature 578:82–93 Tubio JMC (2015) Somatic structural variation and cancer. Brief Funct Genomics 14:339–351 Vogelstein B, Kinzler KW (2004) Cancer genes and the pathways they control. Nat Med. 10:789–799 Vogelstein B et  al (2013) Cancer genome landscapes. Science 339:1546–1558 Watson IR et  al (2013) Emerging patterns of somatic mutations in cancer. Nat Rev Genet 14:703–718 Yang L et  al (2013) Diverse mechanisms of somatic structural variations in human cancer genomes. Cell 153:919–929

3

DNA Damage and DNA Repair

Key Points Carcinogens that induce strand breaks may act • Genomic DNA is continuously subject to as “clastogens,” i.e., induce multiple structural damage. Damage may comprise chemical chromosomal aberrations. The involvement of modifications or loss of DNA bases, single-­ specifically acting carcinogens can be detected strand or double-strand breaks as well as intrathrough the type and distribution of mutations and inter-strand crosslinks. The vast majority found in cancer (mutational signature), such of the damage is appropriately repaired by as the spectrum of base changes in DNA repair mechanisms tailored to the varitrinucleotides. ous kinds so that few changes become perma- • The different DNA repair systems in human nent. Insufficient or inappropriate repair can cells are tailored towards the different kinds of however lead to mutations. DNA damage. They share components such as • DNA replication and physiological recombiDNA polymerases and DNA ligases, but each nation are particularly critical processes. employ additional specific proteins. During DNA replication misincorporation of • Specialized glycosylases remove damaged nucleotides and slipping of DNA polymerases bases. Alkylation at guanine O6 or N7 can be may result in point mutations, whereas stalldirectly reversed by enzymes like MGMT ing of replication forks may ultimately result (Methyl-guanine methyltransferase). AP (apuin DNA double-strand breaks and expose rinic/apyrimidinic) endonucleases prepare single-­stranded DNA to antiviral APOBEC3 sites lacking bases for short-patch or long-­ cytidine deaminases turning on the host patch base excision repair (BER). Mismatched genome. Physiological recombination is assobase pairs in DNA caused by mutagens or ciated with double-strand breaks and may DNA replication errors are the target of two incidentally cause translocations, e.g., during interlinked mismatch repair (MMR) systems. lymphocyte maturation. Alterations such as bulky base adducts and • In addition to endogenous processes such as pyrimidine dimers caused by UV light are oxidative stress and spontaneous reactions of removed by nucleotide excision repair (NER) DNA such as cytosine deamination, diverse systems. Double-strand breaks pose a major exogenous physical and chemical carcinogens challenge to genomic integrity and cell surchemically modify bases, form base adducts vival. They are recognized and handled by or cause DNA strand breaks. Some carcinoseveral alternative repair systems, homologens cause specific base mutations while othgous recombination repair (HRR), or nonhoers induce DNA strand breaks or crosslinks. mologous recombination repair (NHEJ), to © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 W. A. Schulz, Molecular Biology of Human Cancers, https://doi.org/10.1007/978-3-031-16286-2_3

51

3  DNA Damage and DNA Repair

52

•

•

•

•

prevent or minimize permanent damage. The inter-strand crosslink repair system (ICL) employs the FANC proteins to prepare crosslinked DNA for eventual repair by homologous recombination. DNA damage triggers cellular checkpoints that arrest cell cycle progression, stop DNA synthesis and block entry into mitosis. Alternatively, checkpoint activation may elicit apoptotic cell death. Double-strand breaks are particularly strong triggers of checkpoint activation. Viral infections activate cellular checkpoints and cellular stress responses as well. Inborn errors in DNA repair systems underlie syndromes associated with developmental defects, neurological disease, and cancer risk. For instance, nucleotide excision repair is defective in xeroderma pigmentosum, mismatch repair in Lynch syndrome (also HNPCC, hereditary nonpolyposis carcinoma coli), homologous recombination repair in hereditary breast and ovarian cancer syndromes, crosslink repair in Fanconi anemia and DNA damage signaling in ataxia telangiectasia, respectively. While each of these syndromes is individually rare, polymorphisms in DNA repair genes modulate the cancer risk in the general population. Acquired deficiencies in DNA repair systems likewise contribute to the development of many cancers. Deficiencies in MMR cause a distinctive subtype of colorectal cancer but are also observed in a fraction of cancers in other tissues. Deficient HRR is found particularly in a subset of breast, ovarian, and prostate cancers. Another layer of protective mechanisms helps to prevent DNA damage. Reactive mutagens are intercepted by low molecular weight protective compounds such as glutathione or by proteins such as metallothioneins or glutathione transferases. Specific mechanisms protect against reactive oxygen species and limit the effects of ionizing radiation. Genetic variation in individual humans influences the efficiency of these mechanisms, which are also affected by nutritional and additional environmental factors.

3.1 DNA Damage and Repair: An Overview The mutations and chromosomal alterations found in cancer cells (→2) represent only a fraction of those that arise during a human lifetime in normal tissues. In many tissues of older persons, cell clones can be detected that contain point mutations in numerous genes, even though chromosomal alterations are rare. Mutations are more frequent in tissues of individuals that are exposed to exogeneous mutagenic carcinogens. A yet much greater number of potential mutations are prevented by DNA repair mechanisms (Table 3.1). These diverse mechanisms are excellently tuned to the various types of DNA damage that could result in mutations. In addition, cells with substantially damaged DNA or aneuploid genomes are normally eliminated by cell death or prevented from proliferation by undergoing senescence or terminal differentiation. Therefore, in order to survive and proliferate, cancer cells need to circumvent or inactivate those cellular systems that signal damage, survey genomic integrity, and induce cell death and senescence in response to genome damage. Accordingly, defects in DNA repair systems and cellular surveillance mechanisms are an important, and often decisive factor in the development of human cancers. Such defects may be inherited or acquired. Damage to DNA can result from endogenous as well as exogenous sources. DNA replication is a particularly critical process and carries a substantial potential for spontaneous mutations. Replicating genomes are also more sensitive to damage induced by exogenous factors. This is one reason why proliferating cells are more susceptible to neoplastic transformation. Problems that may arise during DNA replication comprise deficiencies or imbalances in nucleotide precursors, misincorporation of bases, slippage of the replisome in tandem repeat sequences, single-­ strand breaks being converted into double-strand breaks by the replication process, as well as stalling of the replisome at “difficult” sequences, at bases modified by chemical reactions with exogenous mutagens, at crosslinked endogenous proteins, or by collisions with RNA polymerases. Collectively, these problems are summarized as “replication stress.” Replication stress is

53

3.1  DNA Damage and Repair: An Overview Table 3.1  An overview of DNA repair mechanisms in human cells Type of DNA damage

Repair Mechanisms

Base misincorporation

Mismatch Repair

Chemical modification of bases

Base excision repair Direct reversal Base excision repair

Base loss

Base excision repair

Intra-strand base dimers, crosslinks, bulky adducts

Nucleotide excision repair, 

Inter-strand crosslinks Double-strand breaks

Bypass repair Crosslink repair Homologous recombination repair Nonhomologous DNA end joining

most pronounced in cells exposed to genotoxic agents and is constitutive in many tumor cells, especially those with aneuploid genomes or with oncogenes that induce hyperproliferation. Replication of nuclear DNA is extremely precise with a nucleotide misincorporation rate of 10-7–10-6, since eukaryotic replication DNA polymerases discriminate well between the normal nucleotide triphosphates and the main replicases possess a 3′–5′ exonuclease proof-reading function. Repair polymerases do not achieve this level of precision and are usually more error-prone. In spite of the excellent fidelity of the replication proteins, in a genome of >3 × 109 bp, several hundred mistakes are expected during each replication. Most misincorporations are corrected by DNA base mismatch repair (MMR, see Sect. 3.2). Specific frequent errors are handled by specific enzymes. For instance, the relatively frequent misincorporation of uridine instead of thymidine is addressed by a dedicated base repair mechanism. As a consequence, the frequency of base changes per division is estimated as 1 × 10−10 in cells with proficient DNA repair systems. Mismatch repair (MMR) systems moreover take care of single-strand loops in replicated DNA that result from slippage of DNA polymerases at repeat sequences. Slippage tends to occur

Selected proteins involved MLH1, MSH2, MSH6, PMS1,  UGH, TDG MGMT, ALKB MBD4, MYH UGH, OGG, XRCC1 AP endonucleases, DNA polymerase, DNA ligase 3 XP (ERCC) proteins, CSA, CSB, XPG, DNA polymerases, DNA ligase 1 DNA polymerase η or ι FANC proteins + HRR components RAD52, NBS1, MRE11, RAD50, RAD51, BRCA2, BLM KU70, KU80, NBS1, MRE11, RAD50, FEN1, WRN, DNA ligase IV

in particular at microsatellites, which consist of 1–4 bp long tandem repeats. Defects in MMR therefore result not only in an increased frequency of base misincorporations but also in expansions or contractions of microsatellites. Even with fully functional MMR, microsatellites are subject to a higher-than-average mutation rate. As a consequence, they are often polymorphic. Mesomeric isoforms of DNA bases prone to mispairing with wrong partner bases can also lead to misincorporation of bases. The mesomeric isoforms of the standard four bases are short-lived but may become stabilized by chemical modifications. For instance, oxidation of guanine at position 8 yields 8-oxo-guanine (8-OG, also known as 8-hydroxy-guanine) which forms a relatively stable G:A (in fact, 8-OG:A) mismatch. If fixed by replication this mismatch can result in a G→T transversion. 8-Oxo-guanine is removed by base excision repair (BER) through OGG (Oxo-guanine glycosylase). Since OGG1 is polymorphic in man, individuals may differ in their capacity for removing this type of damage. The precision of DNA replication also depends on the nucleotide precursor pools. Disparities in the relative levels of the deoxynucleotide triphosphates

3  DNA Damage and DNA Repair

54

decrease the fidelity of base incorporation. As biochemistry textbooks discuss in detail, nucleotide biosynthetic pathways contain several cross-regulatory and feedback mechanisms to minimize such disparities. In addition, deregulation of precursor pools, in particular of guanine nucleotides, activates cellular checkpoints through the TP53 protein to arrest replication (→5.3). Next to dGTP, dTTP may be most critical, because DNA polymerases also accept dUTP. In normal cells, dUTP levels are kept low by enzymatic hydrolysis of the nucleotide. In cells with suboptimal thymidine biosynthesis, due, e.g., to low folate levels or to chemotherapy with methotrexate (see Fig. 23.2), significant levels of uracil bases are incorporated. These are removed by the specialized enzyme Uracil-N-­ glycohydrolase (UNG). Removal of the uracil base creates abasic sites and single-strand breaks in the DNA that undergo further repair. Stalling of DNA replication can cause severe problems, including point mutations, d­ ouble-­strand breaks resulting in chromosomal losses and rearrangements, and may eventually lead to cell death. Its resolution requires several specialized signaling and helper proteins to allow backtracking of the replisome or to initiate a restart from another origin of replication. Double-strand DNA breaks resulting from replication stalling require homologous recombination repair (HRR). HRR components also help in the backtracking and protect singlestranded DNA at the stalled replication forks. This is important as exposure of longer stretches of unprotected single-stranded DNA during DNA replication provides a substrate for APOBEC3 (A3) endogenous cytidine deaminases. Normally, these enzymes protect against retroviruses and endogenous retroelements by converting cytosine into uracil to mutagenize the viral genome. The nuclear A3B and A3A enzymes can however accidentally turn on the host cell genome, creating typical C→T mutations that cluster along a DNA segment. The resulting mutational pattern is known as “kataegis.” Kataegis is often found in the vicinity of doublestrand breaks, where unprotected stretches of single-stranded DNA may become exposed to the A3 enzymes. Many DNA bases undergo spontaneous chemical reactions at significant frequencies, during replication or other phases of the cell cycle. These are addressed by according, steadily proceeding base

Cytosine

Uracil

NH2

O NH

N NH

NH

O

O

Methyl cytosine

Thymine

NH2

O

H3C

H3C

N NH

O

NH NH

O

Fig. 3.1  Consequences of the deamination of cytosine and methylcytosine. See main text for details

repair mechanisms. Hydrolytic cytosine deamination (Fig. 3.1) is another frequent source of uridine in DNA, in addition to dUTP misincorporation. It is recognized and removed by UNG (Uracil-DNA glycosylase). The capacity of this enzyme is more than sufficient to remove the estimated ≈1000 uracil bases that are spontaneously generated in each cell per day. The rate of cytosine deamination can be increased by exogenous and endogenous compounds. For instance, nitrosation at its amino group promotes cytosine deamination. Another specialized glycosylase, DNA-deoxyinosine deoxyribohydrolase, eliminates hypoxanthine arising from the deamination of purine bases in a similarly efficient manner. Hydrolytic deamination of methylcytosine, which constitutes 3–5% of the cytosines in human cells (→8.2), is more of a problem since it yields the normal DNA base thymine. A specialized enzyme, TDG (G/T mismatch-­ specific thymine DNA glycosylase), removes specifically the thymine base from G/T mismatches. Nevertheless, the mutation rate at methylated cytosines is higher than elsewhere in the genome. Since methylcytosine is almost exclusively found at CpG dinucleotides1 in human cells, the resulting mutation is typically CG→TG.  In normal tissues and in cancers, these mutations accumulate over time and accordingly increase CpG is used to clarify that this refers to the dinucleotide C followed by G rather than the C:G basepair. 1 

3.1  DNA Damage and Repair: An Overview

with age. In some cancers, CG→TG mutations make a major contribution to the mutational spectrum. In the germline, during human evolution, this mutation has led to an overall depletion of CpG dinucleotides to approximately one-quarter of its expected frequency (see Sect. 8.2). Cytosine deamination resulting in C→T transition mutations can also be caused by specific enzymes. Whereas mutations caused by APOBEC3 enzymes in the human genome are erroneous, the activation-induced cytosine deaminase AID elicits C→T mutations in a controlled manner during the maturation of B cells in order to enhance the diversity of antibodies. These mutations are normally restricted to rearranged immunoglobulin genes, but may falsely target other genes and thereby contribute to cancer development. Next to the amino group of cytosine, functional groups of guanines constitute the most sensitive target for chemical reactions with DNA bases (see Fig.  3.4). In addition to oxidation at position 8, many electrophiles react rather spontaneously with its N7 amino or O6 hydroxyl functional groups. An important endogenous electrophile is S-adenosylmethionine, the standard methyl group carrier for biological methylation reactions. Methylation of guanine O6 can lead to base mispairing and methyl groups are therefore removed by a specialized enzyme, Methyl-guanine methyltransferase (MGMT). It transfers the methyl group to one of its own cysteines, inactivating itself in the course of the reaction. This enzyme also removes other alkyl groups from the O6 position.2 Alkyl adducts at N7 are removed by α-ketoglutarate-dependent dioxygenases (AlkB enzymes). Guanine is moreover a major target for alkylation by exogenous compounds, including several cytostatic drugs and major carcinogens. Again, MGMT acts to protect DNA from their impact. Downregulation of MGMT by epigenetic mechanisms in some cancers, including glioblastomas, increases their sensitivity to chemotherapy with alkylating agents (→22.5). It would also be expected to increase the overall rate of mutations at guanine bases in these cancers in general. It ought therefore properly be named AGAT.

2 

55

Many types of modified bases are removed by BER, which is initiated by one of the more than 10 base glycosylases with different specificities, such as UNG and OGG. As their name indicates, base glycosylases hydrolyze the N-glycosidic bond between modified bases and deoxyribose, leaving behind abasic sites (also known as AP, apurinic/apyrimidinic, sites) in DNA.  AP sites also arise regularly from spontaneous or induced hydrolysis of the N-glycosidic bonds of normal bases or of chemically modified bases. Purine bases are about 20-fold more susceptible to spontaneous loss from DNA than pyrimidines and more than 20,000 purine bases are estimated to be lost and (correctly) replaced in each human cell every day. AP sites, having arisen spontaneously, been induced, or generated by glycosylases, are filled in by short-patch repair (Fig.  3.2). Short-patch repair is initiated by the action of an AP-endonuclease like APE1 and APEXL2. They cleave the DNA strand with the missing base to provide a free 3′-hydroxyl group for DNA polymerase β. This enzyme removes the deoxyribose and inserts the correct nucleotide. DNA ligase 1 (LIG1) or DNA ligase 3 (LIG3) finally closes the strand break. The actions of the polymerase and the ligase are coordinated by XRCC1. While only one base is replaced during short-patch repair, an alternative mode, “long-patch repair,” replaces several nucleotides (Fig. 3.2). This repair system applies the FEN endonuclease, DNA polymerase δ/ε, PCNA, and DNA ligase 1. It is one of several backup systems for short-patch repair. Base excision repair thus encompasses a broad array of mechanisms through the combined action of glycosylases, AP-endonucleases, DNA polymerases, and DNA ligases. Processes like the hydrolytic deamination of cytosine or the oxidation of guanine lead to altered bases with an increased potential for mispairing. However, neither change interferes principally with DNA replication or transcription. This is not so for some other types of damage inflicted on DNA. Ultraviolet radiation (UV) causes various chemical reactions in DNA.  UV radiation with wavelengths in the absorption maximum of DNA (≈260 nm) cannot penetrate

3  DNA Damage and DNA Repair

56 Fig. 3.2  Short-patch vs. long-patch base excision repair. Damaged DNA bases (symbolized by the G with the red mark) can be replaced by short-patch (left) or long-patch (right) repair employing the indicated enzymes (in the order indicated from top to bottom). Long-patch repair is preferred or necessary if deoxyribose is as well damaged and/ or a phosphate group is missing

Short patch repair

Long patch repair

CTAGGCTA GATCCGAT

CTAGGCTA GATCCGAT Glycosylase APE1

Glycosylase APE1

CTA GCTA GATCCGAT

CTA GCTA GATCCGAT DNA pol β

DNA pol δ/ε

CTAG GCTA GATCCGAT

G

C

T

CTAGGCT A GATCCGAT FEN nuclease DNA ligase 1/3 DNA ligase 1

CTAGGCTA GATCCGAT

into the body (→12.1), but UVB (280–315 nm) can be absorbed to some degree. UVB radiation induces mainly reactions between adjacent pyrimidine bases such as thymine-thymine cyclobutane dimers and thymine-cytosine (or cytosine-cytosine) 6-4 photoproducts (Fig.  3.3). These intra-strand dimers present obstacles to transcription and the replication of DNA.  They are efficiently corrected by nucleotide excision repair (NER, see Sect. 3.3). If this repair system is not available, translesion DNA synthesis (see below) allows transcription and replication to continue, but at an increased risk for mutations, especially at CpC dinucleotides. Likewise, chemical reactions of endogenous compounds and activated chemical carcinogens can lead to modified bases that are too bulky to fit into the double helix. These adducts may block polymerases or cause base misincorporation and mutations. Adducts of the fungal toxin aflatoxin or the environmental contaminant benzopyrene at guanines are examples (Fig.  3.4). In contrast, aristolochic acid, a constituent of herbal medicines used in Eastern Asia and a sometimes contaminant of flour in Southeast Europa, reacts with adenine. In each case, insufficient or inappropriate repair leads to transversion mutations, G→T for aflatoxin and benzopyrene, and A→T for aristolochic acid.

CTAGGCTA GATCCGAT

Several commonly used cancer therapeutics react with DNA bases. Alkylating agents target reactive amino or hydroxyl groups at DNA bases. Cisplatin and mitomycin C (→23.2) crosslink adjacent bases, blocking transcription and replication. Again, NER is required to amend the damage. Removal of inter-strand crosslinks between opposite DNA strands by these compounds requires a specialized DNA repair system (inter-strand crosslink repair, ICL) plus HRR.  Nevertheless, such compounds are not only toxic but also mutagenic (to normal cells as well). Alkylation and crosslinking can also be performed by various aldehydes, from endogenous sources, or from the metabolism of dietary components. For instance, the metabolism of ethanol produces acetaldehyde. Even proteins can become covalently linked to DNA bases, spontaneously or by endogenous or exogenous chemical compounds. For instance, some reactions in the nucleus, like demethylation of modified histones, generate the crosslinking compound formaldehyde. The DNA methylation inhibitor 5-deoxy-azacytidine links DNA methyltransferases covalently to DNA to damage DNA and block DNA methylation by one stroke (Fig. 23.3). Because many mutagenic compounds and processes act on specific bases in more or less

3.1  DNA Damage and Repair: An Overview Fig. 3.3 Pyrimidine base reactions induced by UV irradiation. See main text for explanation

57 O

O CH3

O

CH3

CH3 NH NH

O N

O

NH

N

N O

O

N

O

NH

N O O

O P

P

Thymine-thymine cyclobutane dimer

Aflatoxin Aminofluorene Reactive oxygen species

Alkylating agents O

N NH O

Hydrolysis

NH N

NH2

Bifunctional agents Benzopyrene

Alkylating agents

Fig. 3.4 Reactions of chemical carcinogens with guanosine. See main text for explanation

specific sequence contexts, their respective footprints, mutational signatures, can be identified by large-scale sequence analyses. Mutational signatures are often based on the analysis of base changes in (the 64) trinucleotides. In many cases, the evaluation of mutational signatures in cancer genomes allows the identification of the respon-

Thymine-cytosine 6→4 product

sible mutagen, respectively carcinogen. Among others, specific mutational signatures highlight the CpG→TpG signature associated with aging, the G→T transversions for aflatoxin and benzopyrene, and A→T transversion for aristolochic acid, each in different sequence contexts, as well as the C→T transition signatures caused by APOBEC3 enzymes or AID.  The UV radiation mutational signature is distinguished by mutations in pyrimidine-pyrimidine dinucleotides. Mutagenic drugs used in chemotherapy like cisplatin also leave distinctive mutational signatures (Fig. 3.5). Notably, signatures of mutagenic processes are not restricted to base changes, but may also manifest in specific patterns of InDels or chromosomal alterations. Defects in HRR, e.g., result in an increased frequency of all three types of genomic alterations, each with a distinctive signature.

58

3  DNA Damage and DNA Repair

Fig. 3.5  The mutational signature of cisplatin in a bladder cancer cell line. See main text for explanation; from Skowron M et al. (2018) Sci Rep 9:14476. See Fig. 12.4 for additional mutational signatures

3.2 DNA Mismatch Repair The DNA mismatch repair system (MMR) takes care of mismatched bases and polymerase slipping during DNA replication. It comprises steps of damage recognition, incision, removal of a stretch of nucleotides in one DNA strand, resynthesis, and ligation (Fig. 3.6). Damage ­recognition is achieved by different protein dimers depending on the type of damage. Mismatched bases are recognized by the MSH2 and MSH6 protein pair, whereas—especially larger—insertion or deletion loops resulting from slippage are recognized by MSH2 pairing with MSH3. Following recognition of the mismatch in this manner, MLH1 and PMS2 (or alternatively MLH3) are recruited. Stimulated by PCNA, otherwise a subunit of the replisome, PMS2 introduces single-strand breaks (“nicks”) into DNA which serve as starting sites for the EXO1 exonuclease that removes the damaged strand, followed by restoration of the correct sequence by DNA polymerase δ and religation by LIG1. An evident dilemma during mismatch repair is how to decide which of the two mismatched bases is to be excised or, analogously, whether a single-strand loop constitutes a deletion or an

insertion. In some prokaryotes, this decision is facilitated by methylation of bases in the parental strand, whereas the daughter strand is methylated only later after replication has been finished. For instance, E. coli uses adenine (dam) methylation at GATC sequences for this distinction. In humans, DNA is methylated at cytosines immediately following replication (→8.2), but this modification does not serve the same purpose. More likely, newly synthesized strands are distinguished by the presence of single-strand breaks that serve as the starting points for nucleotide excision or by the occasional incorporation of RNA bases that are later removed. Inherited mutations that inactivate components of the mismatch repair system confer a strong predisposition to certain cancers. Since cancers in the colon and rectum are most conspicuous in the affected individuals, with a lifetime risk of up to 80% in some individuals, these syndromes were summarized under the heading of HNPCC, for “hereditary nonpolyposis colorectal cancer” (→13.2). The alternative name of Lynch syndrome is less misleading, since other tissues, like the endometrium, stomach, ovaries, hepatobiliary system, and upper urinary tract, are also at increased risk for cancers. The syndrome is genetically het-

3.2  DNA Mismatch Repair Fig. 3.6  Two important pathways in DNA mismatch repair. Left: repair of a T:G mismatch; Right: repair of an insertion/deletion at a TG microsatellite

59 Single base mismatch

Insertion (or deletion) loops GT GTGT GTGT CACACACA

CTAGGTTA GATCCGAT Recognition MSH6 MSH2

MSH6 MSH2

MSH3

Recruitment

PMS2 MLH1

Excision

MSH2

MSH3 PMS2 MLH1 MSH2 EXO1

EXO1

C TA GATCCGAT

G GTGT CACACACA Resynthesis

CTAGGCTA GATCCGAT

erogeneous because various components of the mismatch repair system can be defective, but it is almost always inherited in an autosomal dominant manner. Deleterious mutations in at least four different genes can underlie Lynch syndrome. Most cases are caused by germline mutations in one allele of MSH2 or MLH1, mutations in MSH6 and PMS1 contribute most of the remainder. Inactivation of MSH2 in particular is frequently due to larger deletions in the gene. A few cases of the syndrome are caused by “epimutations.” For instance, deletions at the end of the preceding TACSTD1 gene (encoding the epithelial adhesion protein EpCAM) elicit hypermethylation of the MSH2 promoter and repression of the gene. A phenotypically similar, but recessive syndrome causes colorectal cancers as a consequence of biallelic germline mutations inactivating the MYH gene, which encodes a protein recognizing adenine-oxo-guanine mismatches. MYH (or MUTYH) is a BER protein and its loss of function leads specifically to G→T transversions rather than the variety of missense mutations observed with defective mismatch repair. The dominant mode of inheritance in Lynch syndrome is not due to a dominant effect of the mutant gene product. Rather, retaining a single functional allele is generally sufficient for mismatch repair. Cancers arise instead when the sec-

GTGTGTGT CACACACA

ond, intact allele of the affected gene is accidentally mutated in somatic cells or replaced by recombination with the mutated allele (resulting in LOH). Cells with two inactive alleles then accumulate mutations during each round of DNA replication. Many of these mutations may be irrelevant, such as those in the length of intergenic microsatellite repeats which are not repaired after slippage. Other mutations however may activate oncogenes or inactivate tumor suppressor genes, because base exchanges arising from unrepaired mismatches or slippage in base repeats in their coding regions are not amended (→13.3). For instance, the MSH3 and MSH6 genes themselves contain cytidine and adenosine hexanucleotide stretches which tend to expand or contract in cells with defective mismatch repair. Microsatellite expansions and contractions occur regularly in cells with defective MMR, resulting in “microsatellite instability” (MSI). MSI can therefore be used to diagnose cancers arising from defective MMR. For this purpose, a standard set of five microsatellites that are most susceptible to changes has been defined. In many cases, inactivation can be detected by immunohistochemistry, since the MMR protein complex cannot be assembled on DNA if one of the components is missing. Cancers with MMR deficiencies and MSI are not restricted to Lynch syndrome families, but

3  DNA Damage and DNA Repair

60

also arise in sporadic (i.e., non-familial) tumors of various tissues at varying frequencies. The cause of sporadic MSI is mutation, deletion, or epigenetic silencing of mismatch repair genes. In sporadic colorectal cancers, the most frequent cause is silencing of MLH1 by promoter hypermethylation (→8.2). Overall, up to 15% of all colorectal cancers display MSI, but less than 5% of all colorectal cancers arise in Lynch syndrome families (→13.4). In addition to MSI, mismatch repair-deficient tumors in all tissues are characterized by exceptionally high rates of missense mutations. Whole genome or exome sequencing can therefore be used to identify such cancers, e.g., with the aim of prioritizing immunotherapy (→23.6). Finally, the mismatch repair system is prone to “futile cycles” which may actually mediate DNA damage and eventually cell death. Such cycles are started by mismatches resulting from modified bases, with the repair system repeatedly removing the opposite, normal base, resulting in a permanent gap and persisting strand breaks. This “weak point” of the mismatch repair system is exploited among others by cancer chemotherapeutic drugs, e.g., temozolomide, a prodrug that ultimately generates methyl-guanine and consequently DNA strand breaks (→22.5). MMR-­ deficient cancers are often also less sensitive to chemotherapy based on 5-fluoro-uracil, which acts partly by an analogous mechanism of causing lethal mismatches.

ERCC8 and ERCC6). Of note, some lesions impeding transcription by Pol II may also be removed by BER. In the actual TCR pathway, the protein complex assembled by CSA/CSB allows backtracking of the polymerase and subsequently the access of further factors that accomplish the repair. Global-genome repair is initiated by XPC when it recognizes distortions in the DNA helix, in transcribed and non-transcribed regions of the genome alike. Like recognition of lesions by CSA/CSB, recognition by XPC initiates binding of the TFIIH complex, which comprises around 10 proteins, including Cyclin H.  This cyclin regulates kinases that phosphorylate and activate Pol II during transcription, but also the DNA helicases ERCC2 and ERCC3 (also known as XPB and XPD) to start NER (Fig. 3.7).

Global genome

Transcription-coupled CSB

XPC

RNA pol II

CSA

Recognition

Recruitment

RPA

TFIIH

XPA

XPD XPB

Unwinding

3.3 Nucleotide Excision Repair Photoproducts and bulky adducts in DNA are removed by nucleotide excision repair (NER). Two interlinked systems are active in human cells, designated “global-genome” (GGR) and “transcription-coupled” repair (TCR). Transcription-coupled repair is more rapid, but is restricted to regions of the genome that are transcribed by RNA polymerase II (Pol II). When Pol II encounters an obstacle like a bulky base adduct or a cyclobutane photoproduct that impedes further progress of transcription, it activates repair via the CSA and CSB proteins (also known as

Excision XPF

Resynthesis

XPG

PCNA, RFC, POL δ/ε, LIG1/3

Fig. 3.7  Nucleotide excision repair. Nucleotide excision repair, e.g., of base dimers induced by UVB, can be performed by the converging global-­ genome (left) and transcription-­coupled (right) pathways. See main text for additional details.

3.3  Nucleotide Excision Repair

Following recognition of the lesion, the actual repair process then proceeds in the same manner in transcription-coupled and global-genome repair. Both repair systems use TFIIH components such as XPB and XPD to fully unwind the DNA helix in an ATP-dependent manner and the single-strand binding protein RPA to protect the ensuing single-strands. The XPA protein marks the actual lesion and stabilizes the repair assembly. The damaged segment of DNA is excised as a 22–30 nt single-strand fragment through 5′-incision by the ERCC1/XPF endonuclease and 3′-incision by the ERCC5 (XPG) endonuclease. The ensuing gap is filled by DNA polymerases δ, ε, or κ, supported by PCNA and RFC (replication factor C), and the restored doublestrand is sealed by DNA ligase 1 or DNA ligase 3 in concert with XRCC1. Notably, single-stranded DNA of sufficient length persisting for longer periods may activate the checkpoint kinase ATR (see Sect. 3.4). Delays in NER may therefore activate cell cycle checkpoints, even if not as regularly as double-strand breaks do. If not repaired by NER, photoproducts and other lesions encountered by the replisome during DNA replication can be bypassed through translesion DNA synthesis, which employs more tolerant DNA polymerases like POL η. These enzymes are capable of replicating DNA with different types of damage, but at the price of a higher error rate than regular replication by standard polymerases like Pol δ. For instance, Pol η tends to insert adenine opposite crosslinked pyrimidine bases. This is correct in the majority of cases where thymine dimers are encountered but causes base mutations if cytosines are involved. Mutations in genes encoding NER components underlie the diseases xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. These rare diseases are inherited in a recessive fashion. Patients with Cockayne syndrome and trichothiodystrophy suffer from growth defects and progressive mental retardation. Specific and less specific skin defects are apparent; in particular, scaly skin (ichthyosis) and brittle hair and nails are diagnostic for trichothiodystrophy.

61

Trichothiodystrophy patients are not particularly prone to cancers, but instead show some symptoms of premature aging and their life expectancy is diminished. The phenotypes in these patients are likely caused by accumulation of unrepaired DNA lesions in the affected tissues. In contrast, xeroderma pigmentosum patients, as a rule, do not display growth defects and mental retardation, except for those in a subgroup overlapping with Cockayne syndrome. The characteristic affliction in this syndrome is enhanced skin photosensitivity with abnormal pigmentation. UV-exposed parts of the eyes are also sensitive to damage. The main clinical problem is accordingly a huge increase in skin cancer risk, estimated as ≈2000-fold. Unprotected, almost all xeroderma pigmentosum patients develop multiple skin cancers in sun-exposed areas before the age of 30, often already during their first decade of life. All types of skin cancers are increased, basal cell carcinoma, squamous cell carcinoma and melanoma (→12). Cancers of internal organs also occur at increased frequencies. Typical Cockayne syndrome is caused by homozygous mutations in the CSA or CSB genes (hence their designation), whereas Xeroderma pigmentosum is caused by mutations in XP A–G genes (now officially renamed ERCC genes). Mutations in further genes may contribute, such as mutations in the gene encoding DNA polymerase η that are responsible for a disease variant, XP-V.  The very rare trichothiodystrophy syndrome is sometimes caused by mutations in a specific gene (TTD-A, encoding a TFIIH component), but more often by particular mutations in XPB or XPD. Specific XPD mutations account for most cases of combined xeroderma pigmentosum/Cockayne syndrome. The following hypothesis3 accounts for the different phenotypes resulting from mutations in CSA vs. XP genes. Mutations in Cockayne syndrome cause defects specifically in transcription-­ coupled repair to impair growth in general and the function of specific tissues such as the brain, since they diminish the efficiency of transcription in general. Apparently, alternative repair systems Somewhat simplified here.

3 

62

including GGR are not fast enough to compensate fully for the deficiency in TCR. Nevertheless, they eventually remove DNA damage and thereby prevent at least a major increase in cancer risk. In contrast, most defects in XP genes compromise transcription-coupled as well as global-genome repair, leading in particular to an increased sensitivity towards UV in exposed tissues with an associated high rate of mutations. Notably, in the case of XPC mutations, the defect is restricted to GGR, with TCR apparently intact.

3  DNA Damage and DNA Repair

DNA replication. Sometimes, two single-strand breaks may occur closely together by chance and lead to a factual double-strand break. Other double-­strand breaks are caused by exogenous agents. Some viral enzymes cut DNA in a similar fashion to restriction enzymes, e.g., retroviral integrases. Retrotransposition of endogenous retroelements (like LINE-1 or ALU SINEs, both mediated by the LINE-1 ORF2p) likewise involves double-strand breaks at the target site. Ionizing radiation can generate single-strand as well as double-strand DNA breaks. So do several chemical carcinogens and a number of drugs 3.4 DNA Strand Break Repair used in chemotherapy. Bleomycin, e.g., cuts DNA directly by producing hydroxyl radicals, Repair of damaged DNA bases by base excision and topoisomerase inhibitors generate strand repair (as described in Sect. 3.1) is a permanent breaks by inhibition of their target enzymes process in living cells. It is one of several pro- (→23.2).4 Also, double-strand breaks are a necescesses that generate single-strand breaks in sary intermediate in the repair of DNA inter-­ DNA.  Thus, up to 100,000 DNA single-breaks strand crosslinks. are formed in each cell every day. In the simplest Double-strand breaks are moreover created, in case, single-strand breaks are mended through a controlled fashion, during physiological recomreligation by DNA ligases. In other cases, com- bination processes. Important processes of this ponents of the short- and long-patch base exci- kind are meiotic recombination and the generasion repair or nucleotide excision repair systems tion of functional T cell receptor (TCR) and may be needed, depending on the type of damage immunoglobulin (IG) genes in lymphocytes, all to the base or the deoxyribose-phosphate of which occasionally result in errors. Unequal backbone. recombination in germ cells is an important cause Common accessory components in BER as of inherited diseases, including cancers. Aberrant well as in various forms of single-strand repair joining of gene sequences from T cell receptor or are the Poly(ADP-ribosyl) polymerases immunoglobulins genes to other genes, such as PARP1/2. In general, these enzymes attach chains MYC, is a frequent source of chromosomal transof ADP-ribose to many proteins including them- locations in lymphomas (→10.2). The selves. In the context of DNA repair, this modifi- lymphocyte-­ specific recombination system can cation helps to open up chromatin and locate create further translocations as well as deletions, repair components at the site of damage. if it acts accidentally at sites outside the TCR and Like mismatch repair during DNA replication IG gene clusters, and thereby contribute to the and base excision repair throughout the cell development of lymphomas. cycle, repair of DNA single-strand breaks goes Finally, as chromosomes in vertebrates consist on rather “quietly” under normal circumstances. of linear DNA molecules, their ends, telomeres, This changes dramatically when DNA double-­ can be regarded as permanent double-strand strand breaks appear. They can arise by physio- DNA breaks. To protect them from further damlogical or nonphysiological mechanisms, age and prevent the activity of DNA repair sysendogenous processes or through the impact of exogenous mutagens. In particular, a double-­ 4  Such inhibitors may moreover trap topoisomerases in a strand break can develop from a single-strand state covalently linked to DNA.  Reversal of these links break that is not repaired before it is encountered requires the specialized enzyme tyrosyl DNA phosphodiby the replisome or when replication stalls during esterase (TDP1) plus endonucleases.

3.4  DNA Strand Break Repair

63

tems at the chromosome ends, telomeres display Notably, NHEJ is also employed in the physiospecific base sequences that fold into a particular logical recombination processes in lymphocytes. structure that is covered (sheltered) by the The TP53BP1 protein is one of several factors Shelterin protein complex (→7.2). Damage to regulating the choice between these repair systelomeres can however expose DNA ends that tems. It inhibits the processing of double-strand become substrates for—inappropriate—DNA breaks for recombination and thereby prevents repair. repair by HRR. Independently of how they arise, double-­ In standard nonhomologous end joining strand breaks are dangerous as long as they exist, (NHEJ) (Fig. 3.8) double-strand breaks are proespecially to proliferating cells. They separate a tected by the KU70/KU80 protein heterodimer. fragment of DNA from the centromere, which These proteins prevent the DNA ends from illetherefore may become lost during mitosis  (see gitimate recombination and attempt to align Fig.  2.7). Moreover, the open DNA ends can them. Compatible ends may become directly recombine with other sequences in the genome, ligated, but in many cases the ends need to be starting chain reactions of recombinations and processed before they can be resealed. Processing chromosome alterations, such as breakage-­ is performed by the MRN complex consisting of fusion-­bridge cycles and chromoplexy, that may MRE11 (possessing nuclease activity), RAD50, lead to cell death or persistent genetic changes. and NBS1 (also known as Nibrin) and can involve Double-strand repair therefore encompasses filling in 5′-overhangs and degrading 3′-overblocking of the open DNA ends as a step as hangs. In addition, the FEN1 nuclease may be important as actually mending the break. In addi- involved as well as the WRN protein which may tion, active double-strand DNA break repair is supply additional helicase activity to KU70/ usually associated with the activation of cellular KU80 proteins. Apparently, processing, unwindcheckpoints that prevent the cell from entering or ing, and alignment of the strands proceed until further proceeding through replication and mito- short complementary base stretches are found sis. As a consequence of checkpoint activation, which can be used to hybridize the two ends. Any persisting unrepaired DNA double-strand breaks remaining overhangs are processed, gaps are may elicit apoptosis or induce senescence in nor- filled in and the sugar-phosphate backbone is mal cells. Checkpoint activation thus provides sealed by DNA ligase 4/XRCC4. The end prodanother important level of protection against car- uct of the repair process is a restored DNA doucinogenesis, in addition to DNA repair itself. ble helix with a deletion; its size is normally kept Several repair systems in human cells deal at a minimum. A distinctive characteristic of with double-strand breaks. They are classified sequences repaired by the major type of NHEJ into nonhomologous repair (NHEJ) and homolo- are microhomologies, i.e., short 1–12 bp stretches gous recombination repair (HRR) systems. Each that were identical in the original sequences at system involves recognition and protection of the both ends of the deletion. These homology double-strand break and processing of the ends. stretches are often much longer when deletions However, while NHEJ systems rejoin the ends of arise by illegitimate homologous recombination. the double-strand break directly, usually with In some cases, NHEJ repair involves the insertion loss5 of some DNA sequence at the ends, HRR of a few additional nucleotides, which may help uses homologous sequences to achieve (in most to anneal sequences. Specifically, during V(D)J cases) perfect restoration of the damaged joining in lymphocytes insertions of that sort proDNA. In general, nonhomologous repair systems vide additional variability in the TCR or IG genes. prevail in the G1 phase of the cell cycle, whereas When NHEJ is started, it elicits signals that HRR is preferred once homologous DNA activate cellular checkpoints. The KU protein sequences become available through replication. dimer constitutes a regulatory subunit of the DNA-dependent protein kinase (DNA-PK), 5  And in some cases, insertion of unrelated sequences. which is essential for proper DNA repair. Its cata-

3  DNA Damage and DNA Repair

64 Fig. 3.8  Repair of DNA double-strand breaks by nonhomologous end joining. See main text for details

Double-strand break DNA-PK (catalytic) End protection

End processing

Annealing Religation

lytic subunit phosphorylates not only itself and other proteins directly involved in the repair process but also activates the TP53 protein at the hub of cellular checkpoint regulation. Phosphorylation of TP53 by DNA-PK or other checkpoint kinases such as the ATM and ATR leads to cell cycle arrest or apoptosis (→5.3). In general, ATM is activated by double-strand breaks, whereas ATR rather responds to extended DNA single-strands. ATM and other proteins also regulate the NHEJ protein complex itself. Within the MRN complex, Nibrin appears to provide the major point of control. It is phosphorylated and activated by the ATM protein kinase. ATM and ATR furthermore phosphorylate and activate BRCA1 during HRR. While NHEJ generates deletions in most cases, HRR can in principle be performed in an error-free fashion and is the mechanism of choice in the G2 phase of the cell cycle when a second sequence homologous to the damaged one is available in the sister chromatid (Fig. 3.9). HRR also constitutes the preferred method for the repair of double-strand breaks that arise when breaks in one DNA strand are extended into double-­strand breaks during replication and when the replisome has stalled. The KU proteins are not involved in HRR, but the MRN complex is. It binds to the double-strand break

KU80/KU70 heterodimer

NBS MRE11

RAD50

LIG4 XRCC4

and the activated MRE11 nuclease processes the ends to yield a 3′-overhanging single-strand of several 100 bases. Activation of MRE11 is stimulated by CtIP, which is brought to the damage site by the BRCA1/PALB2 dimer. The interaction of BRCA1 with CtIP also ensures that the processing of the DNA ends does not become overly excessive. Initially, single-stranded DNA at the processed ends may be protected from degradation by (single-strand specific) nucleases by RPA.  To perform HRR however, the RAD51 protein is loaded on the single-strand DNA with the help of BRCA2. RAD51 too protects against nucleases, but moreover allows invasion of the intact homologous doublestranded (template) DNA by the single-strand DNA. This results in D-loop structures (“D” for “displacement”). This process again is stimulated by BRCA1. After homologous sequences in the invading and invaded DNA have paired, the 3′-hydroxyl ends of the single-strand are extended and a structure with two Holliday junctions forms, which is eventually resolved by endonuclease action followed by religation. The BLM helicase, deficient in the recessive Bloom syndrome, is involved in this process, as in other instances where complex DNA structures need to be processed. It is one of several “resolvases”

3.4  DNA Strand Break Repair Fig. 3.9  Mechanism of DNA double-strand break repair by homologous recombination. See main text for details

65

MRN

RAD52

RAD51

Resection

Strand invasion Homology search DNA synthesis

Resolution

that can contribute to the resolution of Holliday junctions. There are several possible outcomes of HRR, depending on how the Holliday junctions are resolved. Either both original sequences are restored, or a ­crossover takes place. Even crossover does not result in a change of sequence when the sister chromatid is used. However, if a different homologous sequence (e.g., from the homologous chromosome) was involved, gene conversion may ensue. Several components of the double-strand repair systems have been highlighted by their failure in inherited human diseases (see Table 3.2). Homozygous mutations in the NBS1 gene that compromise the function of Nibrin underlie the Nijmegen breakage syndrome. This very rare recessively inherited syndrome presents with mental retardation, immunodeficiency, and, tellingly, chromosomal instability and cancer susceptibility. Homozygous mutations in BLM are the cause of Bloom syndrome, which is characterized by genomic instability, premature aging, predisposition to cancer, and immunodeficiency. Homozygous mutations in the WRN gene encoding a related (RecQ family) helicase involved in double-strand break repair and telomere maintenance likewise increase the susceptibility to various types of cancer, particularly in soft tissues. However, the resulting Werner syn-

drome presents primarily as a premature aging disease (Box 7.1) manifesting typically around puberty. The most prevalent syndrome related to double-­ strand DNA repair is the recessively inherited ataxia telangiectasia (AT). AT is caused by homozygous mutations in the gene encoding the ATM protein kinase that regulates DNA double-­strand break repair and signals to checkpoints. Like NBS patients, AT patients are prone to infections and chromosomal aberrations. They have a ≈100-fold increased risk of cancers, mostly of leukemias and lymphomas. Both syndromes share, in particular, a hypersensitivity towards ionizing radiation. However, AT patients are usually not mentally retarded. Instead, they develop a gradual decline in the function of the cerebellum, which progressively impedes movements, speech, and sight. This distinctive ataxia led to the name of the syndrome along with the diagnostic telangiectasias, aggregates of small dilated blood vessels appearing in unusual places such as the conjunctiva of the eye. They are thought to be caused by inappropriate angiogenesis. The chain of events leading to these lesions may involve lack of ATM function leading to the incomplete function of TP53 alleviating suppression of hypoxia-induced angiogenesis (→9.4). Other aspects of the pleiotropic ATM phenotype

3  DNA Damage and DNA Repair

66 Table 3.2  Inherited deficiencies in DNA repair and predisposition to cancer Syndrome Xeroderma pigmentosum

Mode of inheritance Recessive

Cockayne

Recessive

Ataxia telangiectasia Fanconi anemia

Recessive Recessive

Nijmegen breakage

Recessive

Bloom

Recessive

Werner

Recessive

Hereditary breast and Dominant ovarian cancer Lynch syndrome (Hereditary Dominant nonpolyposis carcinoma coli)

Tissue with increased Gene(s) involved cancer risk XPA-G Skin (ERCC1-7), others Transcription-coupled CSA, CSB No significant nucleotide excision repair increase Checkpoint signaling ATM multiple Inter-strand crosslink FANC genes, Hematopoietic repair BRCA2 system, others Double-strand break NBS Hematopoietic system repair Double-strand break BLM multiple repair (HRR) Double-strand break WRN multiple repair Homologous BRCA1, BRCA2, Breast, ovary recombination repair others (prostate, pancreas) Mismatch repair MSH2, MLH1, Colon, endometrium, PMS2, others Stomach, others Repair system affected Nucleotide excision repair

are less well understood, including an elevation of the fetal Albumin homolog α-Fetoprotein. The chromosomal instability and hypersensitivity to ionizing radiation in the AT syndrome fit well with the known function of ATM as a crucial coordinator of double-strand break repair and as a checkpoint signaler. DNA double-strand breaks caused by physiological recombination, by viral or retrotransposon enzymes, by ionizing radiation or chemicals, or by oxidative stress all appear to activate ATM. Likely, this occurs by different routes. The MRN complex interacting through NBS1 is one established activator. A variant histone, H2AX, accumulating within 1  min at double-­strand breaks, is rapidly phosphorylated by ATM; this could well be another sensor protein. H2AX can alternatively be phosphorylated by DNA-PK. Of note, H2AX phosphorylated at S139, commonly known as γH2AX, is widely used in research to detect and quantify DNA double-­strand breaks. Following its activation, ATM goes on to phosphorylate further proteins involved in DNA repair such as FANCD2, BRCA1, and RPA.  Significantly, it also activates checkpoints that block further cell proliferation. Phosphorylation by ATM directly activates the TP53 protein, whereas it prevents the TP53

inhibitor protein MDM2 from binding to TP53 (→5.3). ATM also phosphorylates CHK2 (Checkpoint kinase 2), another activator of TP53. Together, these actions lead to cell cycle arrest at the G1/S checkpoint via induction of the p21CIP1 cell cycle inhibitor and at the G2/M checkpoint by other mediators (→5.3). DNA replication can be arrested via phosphorylation of CHK2 (checkpoint kinase 2) and Nibrin, while phosphorylation of TP53 and BRCA1 also activates the G2/M checkpoint. Some of the many functions of ATM may alternatively be provided by other protein kinases such as CHK2, ABL, and ATR. Severe damage by UV radiation and prolonged persistence of singlestranded DNA, e.g., are signaled by the ATR protein kinase in a quite similar fashion to ATM, including activating phosphorylations of TP53 and CHK1 (instead of CHK2). The partly overlapping functions of ATM and ATR are the likely explanation for why the cells of AT patients are hypersensitive to ionizing radiation, but not to UV light. Finally, another dominantly inherited cancer syndrome related to double-strand DNA repair is hereditary breast and ovarian cancer (HBOC, →19.6). It is most often caused by germline mutations in BRCA1 or BRCA2, or less frequently in other components of the HRR machinery.

3.5  DNA Inter-Strand Crosslink Repair

3.5 DNA Inter-Strand Crosslink Repair

67

to allow removal of one crosslinked strand, generating a double-strand break. The strand break is consequently recognized and processed—like in Crosslinks between DNA strands (inter-strand standard HRR—by MRN in conjunction with crosslinks) are a yet more severe impediment to CtIP. The opposite strand is treated like an unretranscription and DNA replication than the intra-­ paired adduct and can be replicated by translestrand crosslinks that can usually be removed by sion DNA synthesis. Various heterodimers of nucleotide excision repair. Repair of inter-strand FANC proteins with BRCA proteins and RAD51 crosslinks may often be only possible by sacrific- then initiate a regular round of HRR. Finally, the ing a fragment of DNA. FANC protein complexes are unloaded and the In non-replicating cells, several mechanisms remaining base adduct is repaired by NER. may in principle be used to deal with inter-strand Mutations in genes encoding FANC proteins crosslinks. They range from outright deletion of underlie the recessively inherited disease Fanconi the blocked double-stranded segment followed anemia (hence FANC proteins). Most common by NHEJ through error-prone excision/bypass-­ are mutations in FANCA, FANCC, and FANCG. repair by components from the NER arsenal to Patients with Fanconi anemia are small in stature essentially error-free repair by homologous and display an assortment of malformations in recombination with the homologous sister chro- different organ systems. Most typical are malformatid in G2 cells. mations of the lower arm (radius) and thumb. During DNA replication, a specialized repair Further parts of the skeleton may be affected as system with Fanconi anemia proteins (FANC) at well as the genitourinary system, the gastrointesits core combines with BRCA proteins, HRR, tinal tract, the heart, and the central nervous sysand NER as well as additional factors in a com- tem. “Café au lait” spots on the skin are an plex process to deal with inter-strand crosslinks additional diagnostic sign. (Fig.  3.10). More than 20 FANC proteins are The most problematic symptom of this pleioknown that are classified into three groups by tropic disease is a diminished function of the function. The proteins of group I constitute the hematopoietic system, often resulting in diminFANC core complex, which upon activation leads ished production of all blood cell types (pancytoto monoubiquitylation of the FANCD2-FANCI penia); this deficiency develops gradually during complex (constituting group II).6 The monoubiq- childhood. Malfunction of hematopoiesis leads to uitylated FANCD2-FANCI complex then enables bleeding, anemia, and increased susceptibility to DNA incision, translesion synthesis (TLS), infections. Conversely, the patients often suffer crosslink elimination, and replication rescue by from the preneoplastic myelodysplastic syndrome, group III FANC proteins in cooperation with the which is prone to progression towards outright HRR system. leukemias, typically AML (acute myeloid leukeWhen a replication fork encounters or two mia). The second-most prevalent cancer type is replication forks converge on a crosslink, it is head and neck squamous cell carcinoma (HNSCC). recognized and marked by FANCM together with Cells from Fanconi anemia patients are hypersenaccessory proteins. In the process, the ATR sitive to challenges with DNA-crosslinking comcheckpoint kinase is activated. Phosphorylation pounds like mitomycin C or diepoxy-butane and by ATR facilitates monoubiquitylation of typically arrest in G2. This assay provides a better FANCD2 and activates a cell cycle checkpoint. distinction from other diseases than the increased The ubiquitinated FANCD1/FANCD2 dimer in rate of spontaneous chromosomal breakage per se, turn activates endonucleases that make incisions which is more variable and can also be elevated in other diseases. Since DNA crosslink repair requires HRR fac6  FANCD2 monoubiquitylation can be used as a straightforward biochemical assay of the functionality of the tors in addition to the actual FANC proteins, it is upper part of the FANC system. also affected by defects in that repair system.

68

Fig. 3.10  DNA crosslink repair by FANC  and  BRCA proteins. An outline of DNA repair at a replication fork stalled at an inter-strand crosslink (bold step in the DNA ladder). BRCA proteins are thought to regulate the activity of FANC proteins

3  DNA Damage and DNA Repair

that coordinate excision and repair of the damaged DNA segment. The final stages of the process can be performed either by the HRR system (as in Fig. 3.9) to preserve the sequence or by NHEJ (as in Fig. 3.8) which regularly causes a deletion. From: Helbing-­Leclerc et al. (2021) l.c.

3.6  Deficiencies in DNA Repair and Cancer Susceptibility

Thus, while inherited mutations in one allele of BRCA1 and BRCA2 predispose to breast and ovarian cancers in a dominant manner (→18.3), homozygous mutations in BRCA2 (which is hence identical to FANCD1) lead to Fanconi anemia. The function of FANC proteins in signaling DNA damage and activation of HRR (and likely other systems) explains why cells from Fanconi anemia patients show a decreased ability to correctly repair double-strand DNA breaks in general, not only if they result from crosslinking. This regulatory function may also relate to some of the defects in hematopoiesis since maturation of B cells and T cells involves gene rearrangements that require joining of double-strand breaks introduced by the lymphocyte-specific recombinases. Indeed, these rearrangements have been found to be compromised and less precise in Fanconi anemia patients, contributing to the immunodeficiency in the syndrome. Finally, several commonly used cytostatic drugs are DNA crosslinkers. For instance, cisplatin is an essential component in many cancer chemotherapy regimens and has revolutionized especially the treatment of testicular cancer. Whether individual cancers respond to such drugs depends among others on their capacities for NER, ICL, and HRR.

3.6 Deficiencies in DNA Repair and Cancer Susceptibility It is clear from the previous sections that inherited deficiencies in DNA repair will confer susceptibility to cancer. A number of syndromes related to DNA repair indeed carry an increased risk of cancers, often in addition to other symptoms (Table 3.2). Homozygous mutations in the ATM, NBS1, WRN, FANC, and XP/ERCC genes underlie recessively inherited diseases, which confer an increased risk for cancers in the context of a syndrome with a wider range of afflictions. Heterozygous mutations in MMR and HRR genes and certain ATM mutations lead to cancer predisposition in a dominantly inherited fashion. As a rule, no other consistent symptoms are asso-

69

ciated with these mutations. With either type of predisposition, cancers develop at an increased rate as a consequence of an enhanced rate of mutations. These are predominantly point mutations in MMR deficiency and XP, and more frequently chromosomal aberrations in the other cancers. Typical of hereditary cancer syndromes, cancers appear at an unusually early age in all these diseases. An obvious question is to which extent defects in DNA repair are involved in sporadic cancers (i.e., the great majority of cancers), which arise in people without germline mutations in any of the above genes. Another question is, if defective DNA repair can be sufficient for cancer development, to which extent is it also necessary? In this regard, it needs to be considered that many genes involved in DNA repair are polymorphic. Several such genetic variants have been linked to an increased risk for at least one major cancer type. Typically, in each individual, the increases in cancer risk conferred by these variants are small compared to those resulting from mutations that completely inactivate a DNA repair system. However, frequent (polymorphic) variants of such genes could be important modulators of cancer frequency in the whole population (cf. 2.3). In addition to inherited mutations or variants, acquired mutations inactivate DNA repair genes in many cancers or these genes become silenced by epigenetic mechanisms. This is prominently observed in cancers with an MSI phenotype (→3.2) arising through somatic inactivation of MMR genes such as MLH1. Epigenetic inactivation of ATM, BRCA, ERCC2, FANC, NBS1, WRN, and other DNA repair genes may contribute to chromosomal instability in other sporadic cancers to various extents. Defects in cell cycle checkpoints that are activated by DNA damage are even more commonly detectable than the inactivation of DNA repair systems in many cancers. They are caused, e.g., by loss of checkpoint kinase or TP53 function. This may be so because defective checkpoint activation allows cell proliferation to continue in spite of DNA damage and replication stress, with the consequence that some defects persist and are

3  DNA Damage and DNA Repair

70

propagated through the following cell generations. In comparison, outright inactivation of DNA repair systems may compromise cell fitness. Thus, biallelic inactivation of BRCA1 or BRCA2 is lethal in many cells, even though it is (quite surprisingly) compatible with the aggressive growth of specific cancers. Finally, in the course of a human lifetime occasional DNA defects that have escaped repair accumulate, and DNA repair may become less efficient during aging. In addition, telomere dysfunction in aging cells (→7.2) may provide a new challenge to DNA repair systems which they cannot always master. These factors may contribute to the increased incidence of cancer with age.

3.7 Cell Protection Mechanisms in Cancer Exogenous carcinogens and mutagens arising from endogenous processes are often prevented from encountering DNA by cellular protection mechanisms that employ low molecular weight compounds as well as enzymes and binding proteins (Table  3.3). These highly diverse mechanisms serve as a further tier of protection against cancer in addition to DNA repair and the fail-safe mechanisms senescence and apoptosis. In many cases, they protect not only DNA from damage, but also preserve cell integrity more generally. Some of these mechanisms act against particular hazards, while others address a broader range. In the context of cancer, these protection mecha-

nisms are particularly important during two very different phases, viz., (1) during carcinogenesis and (2) during cancer therapy. One layer of cell protection is constituted by enzymes that metabolize potentially toxic compounds, such as chemical carcinogens and medical drugs. In general, these protective proteins are classified as phase I and phase II enzymes. As a rule, phase I enzymes like CYP family monooxygenases modify (usually hydroxylate) compounds to increase their solubility and allow conjugation with various hydrophilic moieties like glucuronic acid, sulfate, and glutathione in phase II reactions. The products of the combined phase I and II reactions can then be excreted by specialized transporters into the bile or the urine. Importantly, some products of phase I reactions (and more rarely products of phase II reactions) may be more reactive than the original compounds. Such toxic intermediates are produced among others from benzopyrene (→15.2) and aflatoxins (→16.1). Notably, many phase I and II enzymes are highly polymorphic in humans; these polymorphisms contribute to differences in the susceptibility to chemical carcinogens as well as to differences in the response to (anticancer and other) drugs between individuals (Table 2.4). A number of low molecular weight, chemically diverse compounds are employed in the cell to stabilize macromolecules and membranes, buffer against detrimental changes in osmolarity, stabilize the redox state, and quench radicals, especially reactive oxygen species. They comprise polyamines like spermidine and spermine,

Table 3.3  Some low molecular weight compounds and enzymes involved in cell protection Low molecular weight compound Glutathione Ascorbic acid Tocopherol

Function Protection against reactive oxygen species and electrophilic compounds Protection against hydrophilic radicals Protection against lipophilic radicals

Spermidine, spermine (polyamines) Taurine, betaine

Stabilization of ribonucleoprotein complexes Osmoprotection

Protein/enzyme Glutathione peroxidase Catalase Superoxide dismutase Glutathione transferases Thioredoxins Metallothioneins

Function Removal of H2O2 Removal of H2O2 Removal of superoxide Removal of electrophilic reactants Protection of protein thiols Binding of toxic metal ions

3.7  Cell Protection Mechanisms in Cancer

amino acids like taurine, the tripeptide glutathione, and the lipophilic and hydrophilic, respectively, vitamins E (α-tocopherol) and C (ascorbic acid). Glutathione, γ-glutamyl-cysteinyl-glycine (GSH), is crucial for maintaining a balanced cellular redox state; in addition, its thiol group reacts readily with dangerous electrophilic compounds. Oxidation of glutathione yields its disulfide GSSG, from which GSH can be regenerated by glutathione reductase, which uses NADPH as its co-substrate. For instance, GSH reacts spontaneously with hydrogen peroxide, but more readily upon catalysis by selenium-containing glutathione peroxidases (abbreviated GPx). Insufficient glutathione peroxidase function may be one reason why selenium deficiency may increase the risk of cancer. Similarly, glutathione spontaneously forms adducts with reactive electrophilic compounds, including activated carcinogens; again, these reactions are strongly accelerated by glutathione transferases (GSTs). The various isoenzymes in this family each catalyze the reaction of glutathione with several substrates, with each enzyme recognizing a different range of compounds. As a rule, the conjugation reaction inactivates the reactive carcinogen. The conjugates are further metabolized and eventually transported out of the cell and usually excreted as mercapturic acids with the bile. It is clear from this short description why genetic variants in GST enzymes modulate the risk of various cancers (→2.3). In addition, the level of glutathione itself and the ratio of GSSG:GSH determine the capacity for cellular protection. However, the same reactions protecting against carcinogens also influence the efficiency of many cancer therapies. Many important cytotoxic cancer drugs react with DNA and some act (at least partly) by inducing toxic levels of reactive oxygen species.7 Their conjugation to glutathione, catalyzed by GSTs, and the quench-

Cellular stress and increased production of reactive oxygen species can be secondary to damage to mitochondria, including damage to mitochondrial DNA, by carcinogens and other cytotoxic compounds. 7 

71

ing of reactive oxygen species by GSH and other radical quenchers protect normal cells, but also diminish the effects of therapy  on cancer cells. For instance, the GSTP1 isoenzyme is often overexpressed in cancers to confer resistance to cytotoxic chemotherapy, in some cases as a consequence of gene amplification. Conversely, this isoenzyme is downregulated in certain cancers like prostate carcinoma, possibly facilitating carcinogenesis (→20.2). Similar arguments apply to ionizing radiation. The effects of ionizing radiation on normal cells are mitigated by various cellular protection mechanisms, in addition to DNA repair. For instance, polyamines stabilize cellular macromolecules such as DNA and structural RNAs. Tocopherol, ascorbate, carotenoids, and glutathione can all act to quench the effect of hydroxyl radicals and other reactive (oxygen) species generated by high-energy radiation. Therefore, it can in some cases be helpful to deplete such compounds prior to therapy to enhance its effect. GSTs and glutathione peroxidase are examples of cell-protective enzymes that modulate the effects of many different exogenous and endogenous agents. Other enzymes are tailored towards specific compounds. Catalase and superoxide dismutase remove specific reactive oxygen species. Thioredoxins restore oxidized cysteine thiol groups, maintaining and regulating the function of many proteins. Metallothioneins are a group of small proteins protecting against toxic metal ions. They contain multiple thiol groups which react with and irreversibly bind potentially carcinogenic metal ions like cadmium and nickel. However, while they may prevent carcinogenesis by these and other substances, they may also contribute to resistance against chemotherapy employing metalorganic compounds, specifically against the widely used platinum complexes. Moreover, these proteins, too, react with free radicals induced by cancer treatments. Many protective enzymes, proteins, and transporters are induced by the NRF2 transcription factor, which is itself controlled by the cellular redox state and reacts to exposure to electrophilic compounds (Box 3.1).

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Box 3.1 The NRF2 Transcription Factor and its Regulation

Many proteins protecting against stress induced by exogenous and endogenous reactive chemical compounds are induced by the transcription factor NRF2. Together with its heterodimerization partner sMAF, it binds to promoter antioxidant-response elements to activate a large number of target genes. In this manner, NRF2 enhances glutathione biosynthesis, recycling and conjugation, detoxification and export of toxic compounds, lipid and heme metabolism, purine biosynthesis, and decreases apoptosis. Collectively, these activities mitigate cellular stress and prevent damage to cells and the genome. During exposure to chemical carcinogens and oxidative stress, NRF2 prevents carcinogenesis. Activation of NRF2 by compounds like the antioxidant sulforaphane (from certain cabbages) could therefore contribute to cancer prevention. However, persistent activation of NRF2 favors tumor development in some tissues and NRF2 protects cancer cells from many chemotherapeutic drugs and promotes their metabolism and excretion. NRF2 activity is normally tightly regulated by a variety of mechanisms and induced only in response to cellular stress. For instance, the NFE2L2 gene encoding NRF2 is induced by xenobiotics through the Aryl hydrocarbon receptor (AhR). Translation of NRF2 and the stability of its mRNA are positively and negatively regulated by several miRNAs. A major control mechanism acts via regulation of NRF2  protein turnover. Under basal conditions, cytoplasmic NRF2 is bound by the KEAP1 protein and presented to the Cullin-3 (gene CUL3) E3 ubiquitin ligase complex for polyubiquitination and subsequent degradation. The KEAP1 protein contains a comparatively large number of

cysteines with free thiol groups. A variety of chemical reactions at these thiols, especially at Cys151, inactivates KEAP and prevents degradation of NRF2. Newly synthesized NRF2 can consequently enter the nucleus and activate its target genes. In this manner, KEAP1 senses and relays the presence of reactive, potentially dangerous compounds in the cell. Additional mechanisms modulate NRF2 activity, including activating phosphorylation through MAPK and PI3K signaling and inhibition by certain nuclear receptors like RXRα. In the nucleus, phosphorylation by GSK3 marks NRF2 for degradation via the β-TRCP E3 ligase. Active NRF2 furthermore autoregulates its own activity by inducing KEAP1 and another feedback regulator, p62/SQSTM, which directs NRF2 towards autophagic degradation. NRF2 activation in cancers can be a consequence of activation of growth factor signaling or other changes in its regulators, but is—not infrequently—caused by mutations in components of the central regulator circuit, i.e., activating mutations in NFE2L2 that impair binding of NRF2 to KEAP1, or deleterious mutations or deletions of KEAP1 or CUL3. KEAP1 transcription can also be blocked by DNA-hypermethylation of its promoter. These mutations are observed in a variety of cancers but are especially frequent in squamous cell carcinomas of various tissues. Jaramillo MC, Zhang DD (2015) The emerging role of the Nrf2-Keap1 signaling pathway in cancer. Genes Dev 27:2179–2191 Menegon S et al. (2016) The dual roles of NRF2  in cancer. Trends Mol Med 22:578–593 Cloer EW et al. (2019) NRF2 activation in cancer: from DNA to protein. Cancer Res 79:889–898

Further Reading

It is important to realize that the efficiency of the cellular protection mechanisms discussed here (and others) is determined by interactions of genetic and environmental factors. On the genetic side, variants in a large number of genes involved in cell protection are expected to modulate the risk of cancers, but also the response to different therapies. For instance, common variants in many GST genes are associated with higher or lower risk for specific cancers. On the environmental side, the type, dose, and length of exposure are, of course, relevant, but also factors like diet and immune status which affect the levels of low molecular weight compounds and proteins involved in cell protection. Genetic and environmental factors may synergize with each other to promote or protect against cancer. Considering these factors is particularly important in the efforts to prevent human cancers (see Sect. 21.3).

Further Reading Alexandrov LB et al (2020) The repertoire of mutational signatures in human cancer. Nature 578:94–101 Brown JS et  al (2017) Targeting DNA repair in cancer: beyond PARP inhibitors. Cancer Disc 7:20–37 Burns MB et al (2013) Evidence for APOBEC3 mutagenesis in multiple human cancers. Nat Genet 45:1–8 Caestecker KW, Van de Walle GR (2013) The role of BRCA1 in DNA double-strand repair: past and present. Exp Cell Res 319:575–587 Caldecott KW (2014) DNA single-strand break repair. Exp Cell Res 329:2–8 Chatterjee N, Walker GC (2017) Mechanisms of DNA damage, repair and mutagenesis. Environ Mol Mutagen 58:235–263 D’Andrea AD (2010) Susceptibility pathways in Fanconi’s anemia and breast cancer. NEJM 362:1909–1919

73 Deans AJ, West SC (2011) DNA interstrand crosslink repair and cancer. Nat Rev Cancer 11:467–480 Die H et al (2011) Repair and biochemical effects of DNA repair crosslinks. Mut Res 711:113–122 Gaillard H et al (2015) DNA replication stress and cancer. Nat Rev Cancer 15:276–289 Garcia-de-Teresa B (2020) Chromosome instability in Fanconi Anemia: from breaks to phenotypic consequences. Genes 11:1528 Helbling-Leclerc A et  al (2021) Beyond DNA repair and chromosome instability—Fanconi anaemia as a ­cellular senescence-associated syndrome. Cell Death Differ. 28:1159–1173 Helleday T et  al (2014) Mechanisms underlying mutational signatures in human cancers. Nat Rev Cancer 15:585–598 Knijnenburg TA et  al (2018) Genomic and molecular landscape of DNA damage repair deficiency across The Cancer Genome Atlas. Cell Rep. 23:239–254 Krais JJ, Johnson N (2020) BRCA1 mutations in cancer: coordinating deficiencies in homologous recombination with tumorigenesis. Cancer Res. 80:4601–4609 Kucab JE et al (2019) A compendium of mutational signatures of environmental agents. Cell 177:821–836 Lynch HT et  al (2015) Milestones of Lynch syndrome 1895–2015. Nat Rev Cancer 14:181–194 Marteijn JA et al (2014) Understanding nucleotide excision repair and its roles in cancer and ageing. Nat Rev Mol Cell Biol 15:465–481 Martincorena I, Campbell PJ (2015) Somatic mutations in cancer and normal cells. Science 349:1483–1489 Pecori R et  al. (2022) Functions and consequences of AID/APOBEC-mediated DNA and RNA deamination. Nat Rev Genet 23:505–518 Roberts SA et al (2013) An APOBEC cytidine deaminase mutagenesis pattern is widespread in human cancers. Nat Genet 45:970–977 Roy R et al (2012) BRCA1 and BRCA2: different roles in a common pathway of genome protection. Nat Rev Cancer 12:68–78 Stok C et  al (2021) Shaping the BRCAness mutational landscape by alternative double-strand break repair, replication stress and mitotic aberrancies. Nucl Acids Res 49:4239–4257

4

Oncogenes

Key Points These alterations include chromosomal trans• The first oncogenes were discovered in the location, gene amplification, and specific genomes of acutely transforming retroviruses point mutations. As a consequence, the prothat cause hematological or soft tissue cancers tein products of the oncogenes become overin avian or mammalian hosts. These oncoexpressed, deregulated, overactive, or genes act in a dominant manner and alter the mislocated in the cell. A specific class of growth properties and morphology of specific oncogenes are generated by chromosomal target cells in mesenchymal tissues or the translocations or deletions that fuse two genes hematopoietic system. to create a new one with novel properties. • A second group of oncogenes comprises host • In their normal state, many cellular proto-­ proto-oncogenes that become activated by the oncogenes regulate cell proliferation, differinsertion of retroviruses which disrupts their entiation, and survival. Many act as regulation. extracellular growth factors, growth factor • The oncogenes of acutely transforming retroreceptors or juxta-membrane adaptors, and viruses originate from host genes and have transducers in signaling cascades emanating become deregulated and overactive by high from growth factor receptors. A large class expression directed by the retroviral long terof proto-oncogene products is constituted by minal repeat and by activating mutations. protein kinases, comprising in particular Several cellular genes such as NRAS, KRAS, growth factor receptors with a crucial tyroERBB1, and MYC orthologous to viral oncosine kinase activity (receptor tyrosine genes turned out to be overexpressed or kinases). Another large group of proto-­ mutated in human cancers. Many further celoncogenes consists of transcription factors lular orthologs of retroviral oncogenes are regulating cell growth, proliferation, and difalso involved in human cancers. ferentiation in the nucleus. Oncogenes can • Some paralogs of oncogenes may function as therefore be categorized according to their oncogenes, too. Thus, several RAS, ERBB, and cellular localization or by their biochemical MYC family members each are potent oncofunction. genes upon activation. • A large number of proto-oncogenes influence • Much more commonly than viral insertion, the mitogen-activated protein kinase (MAPK) other genetic changes activate cellular proto-­ cascade which links growth factor signaling to oncogenes to dominantly acting oncogenes. transcription in the nucleus and the regulation

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 W. A. Schulz, Molecular Biology of Human Cancers, https://doi.org/10.1007/978-3-031-16286-2_4

75

4 Oncogenes

76

•

•

•

•

of the cell cycle, while also influencing the cytoskeleton and protein synthesis. Growth factor signaling and MAPK pathway activity are tightly regulated in normal cells by feedback regulation and short half-lives of the activated states. Oncogenic mutations make oncogene proteins independent of input signals, prolong their active state, or disrupt feedback regulation. A single oncogene is rarely sufficient to transform a cell towards full malignancy. More commonly, a single oncogene confers some aspects of the malignant phenotype and cooperates with other oncogenes or with defects in tumor suppressors to completely transform cells. This relationship is illustrated in experimental systems, where two different ­ types of oncogenes are required for transformation. Human cancers accumulate many genetic and epigenetic alterations during their progression. In a typical cancer cell, many genes are overexpressed and their gene products are therefore overactive. While some of these genes may be required for tumor growth, the strict definition of an oncogene requires that its overexpression or overactivity are caused by genomic changes such as specific point mutations or gene amplification. Three members of the MYC family are potent oncogenes, MYC (also known as c-MYC), NMYC, and LMYC.  The MYC proteins are

transcription factors that promote cell proliferation and cell growth by activating or repressing a very large number of genes in the human genome. They are—alternatively— activated in many different human cancers by gene amplification, chromosomal translocation, and persistent deregulation. These alterations disrupt the tight control of MYC expression and activity in normal cells.

4.1 Retroviral Oncogenes Several different classes of viruses are involved in the development of cancers in humans and animals (Table 1.2). In humans, predominantly DNA viruses are implicated, e.g., several strains of human papilloma virus and herpes viruses like EBV.  In contrast, many retroviruses have been documented to cause cancers in animals. They act by two different mechanisms, which can be categorized as “acute” and “slow” or as “transducing” or “cis-acting” (cf. Sect. 4.2). In humans, HTLV1 (human T lymphotropic virus) is the only established directly acting oncogenic retrovirus (Box 4.1), whereas HIV1 contributes to carcinogenesis indirectly through more complicated mechanisms (Box 9.4). Of note, the DNA virus hepatitis virus B (HBV) replicates as well via an RNA intermediate and contributes to hepatocellular carcinoma through both genomic cis-acting and nongenomic mechanisms (→17.4).

4.1  Retroviral Oncogenes

77

proteins such as BCL-XL and XIAP, whereas proapoptotic proteins like BIM (→7.4) are downregulated. Deregulation of these factors is exacerbated by another viral protein, HBZ, which inhibits the FOXO3A transcription factor. In addition, Tax inhibits Caspase 8. Tax also influences cell cycle regulation directly by activating CDK4 and inhibiting INK4 CDK inhibitors. Interference with TP53 and ATM by Tax as well as deregulation of the expression of additional cell cycle regulators like Cyclin A contributes to the emergence of genomic instability that eventually leads to ATLL development. Even DNA mismatch repair is impeded by viral oncoproteins. Importantly, Tax expression in the infected cells is modulated by other viral proteins, especially HBZ and by interactions with other cells. This regulation maintains a critical level of Tax that allows persistence of the infected cells and evasion of immune responses. Moreover, infected cells secrete IL-10 to activate regulatory T cells (→9.6). Intriguingly, many of the effects elicited by Tax in infected T cells remain active in ATLL, although the viral protein is strongly downregulated in most leukemic cells. Mohanty S & Harhaj EW (2020) Mechanisms of oncogenesis by HTLV-1 Tax. Pathogens 9:543

Box 4.1 Carcinogenesis by HTLV-I

At least 10 million persons worldwide are infected by the retrovirus HTLV-I. HTLV-I infects primarily CD4+ T lymphocytes, but also many other lymphoid cell types. The virus persists in a latent state in many cells but may also spread through cell-cell contacts. Up to 5% of the chronically infected persons eventually develop a slowly but obstinately growing and usually fatal malignancy of clonal CD4+ CD25+ cells, termed adult T cell lymphoma/leukemia (ATLL). This cancer is clearly initiated by the retrovirus. As it progresses however it develops chromosomal aberrations and often mutations in TP53 and CDKN2A and may become less dependent on the viral oncoproteins. Throughout much of its development, ATLL appears to moreover require stimulation by the antigen recognized by the particular T cell clone. In addition to the standard Gag, Pol, and Env proteins of retroviruses (cf. Fig. 4.1), HTLV-I expresses several accessory proteins from the 3′-end of its ≈9 kb genome, which help to establish and maintain viral infection. They are thought to be involved in the initial immortalization and clonal expansion of infected T cells. The most important of these proteins is Tax, a transactivator protein resembling the HIV1 Tat protein in some respects. Tax acts in a pleiotropic fashion, influencing the expression of many proviral and cellular genes. It binds to cellular transcriptional activators like CREB and augments their interaction with transcriptional coactivators, in particular with CBP/p300, to activate the proviral LTR.  Tax contributes to constitutive activation of NFκB signaling (→6.7) in the infected cells by activation of IKK and interactions with inhibitory IκB proteins as well as NFκB transcription factors. This leads to the increased production of cytokines like IL2, of cytokine receptors like IL2R, and of antiapoptotic

Acute transforming retroviruses such as ALV (avian leukosis virus) or RSV1 (Rous sarcoma virus) elicit leukemias, lymphomas, or sarcomas rapidly after infection of their animal hosts. For instance, many avian acute transforming retroviruses were identified as the cause of epidemics ravaging fowl farms. They were shown to be capable of transforming their target cells in a dominant fashion, without any apparent requirement for a co-carcinogen. Soon after their discovNote that this chicken virus is unrelated to the human pathogen respiratory syncytial virus that is also abbreviated RSV. 1 

4 Oncogenes

78 LTR

LTR U3 R U5

ψ

gag

pol

env

ψ

gag

pol

env

U3 R U5

LTR U3 R U5

LTR src

U3 R U5

RSV

LTR

LTR U3 R U5

ALV

ψ

gag

onc

env

U3 R U5

Oncovirus

Fig. 4.1 Prototypic retrovirus and onco-retrovirus genomes. Genomes of ALV (avian leukosis virus), RSV (Rous sarcoma virus), and a typical oncogene-transducing avian

retrovirus (like avian myelocytomatosis virus) as proviruses

ery in the early twentieth century, these viruses were predicted to carry genes that cause cell transformation and that were accordingly termed oncogenes. The existence of these genes was formally and physically demonstrated in the second half of the twentieth century and the first oncogene protein, v-Src from RSV, was biochemically characterized in the late 1970s. The v-Src protein is a protein kinase located at the inner face of the plasma membrane. While most protein kinases phosphorylate serine or threonine residues in their substrates, v-Src phosphorylates tyrosine, which came as a surprise at the time of discovery. In the RSV genome, v-src is carried as an additional gene 3′ to the standard gag, pol, and env gene complement. This is unusual for acutely transforming retroviruses. In most of them, rather, one or two oncogenes replace parts of the standard genes (Fig.  4.1). Typically, oncogenes replace the pol gene and a part of the gag gene and the oncogenic protein is expressed as a gag-fusion protein at high levels, its transcription being driven by the viral long terminal repeat (LTR). This high expression level contributes to the dominant mode of action of ret-

roviral oncogenes. Of course, the replacement of pol or other viral genes by an oncogene sequence obliterates the ability of the virus to replicate autonomously, rendering it “defective.”’ For replication and propagation, defective retroviruses need intact replication-competent helper viruses that supply the proteins required for reverse transcription, integration, packaging, and maturation. This requirement may explain why acutely transforming retroviruses are (fortunately) rare. Conversely, the unique ability of RSV to replicate autonomously may have contributed to its early isolation a century ago. To date, dozens of oncogenes have been isolated from retroviruses and have been biochemically characterized. A selection is listed in Table  4.1, with their origin, cellular localization, and main biochemical function. Several points can be noted in this compilation. (1) Some oncogenes seem to be similar. In some cases, this is due only to the nomenclature: v-Myb and v-Myc are not overtly similar beyond being transcription factors but have both been discovered in retroviruses causing myeloid leukemias. In contrast, Ki-Ras and Ha-Ras are indeed highly similar proteins,

4.2  Slow-Acting Transforming Retroviruses

79

Table 4.1  Some important retroviral oncogenes Species Monkey Chicken Cat

Tumor type Sarcoma Leukemia Leukemia

Localization in the cell Extracellular Cell membrane Cell membrane

Main biochemical function Growth factor Tyrosine kinase Tyrosine kinase

Cat

Sarcoma

Cell membrane

Tyrosine kinase

Chicken Sarcoma Mouse Leukemia

Inner cell membrane Cytoplasm

Tyrosine kinase Tyrosine kinase

Mouse

Sarcoma

Ha-ras

Virus Simian sarcoma virus Avian erythroblastosis virus Feline sarcoma virus (SM strain) Feline sarcoma virus (HZ2 strain) Rous sarcoma virus Abelson murine leukemia virus Murine sarcoma virus (3611 strain) Harvey sarcoma virus

Rat

Sarcoma

Ki-ras

Kirsten sarcoma virus

Rat

Sarcoma

akt

AKT8 virus

Mouse

Thymoma

myc

Several avian myelocytomatosis viruses Avian myeloblastosis virus avian reticuloendotheliosis virus Murine osteosarcoma virus Avian sarcoma virus Avian erythroblastosis virus HTLV1

Chicken Leukemia

Inner cell membrane/ Tyrosine kinase cytoplasm Inner cell membrane GTP-binding protein Inner cell membrane GTP-binding protein Inner cell membrane/ Serine protein cytoplasm kinase Nucleus Transcription factor

Chicken Leukemia Turkey Leukemia

Nucleus Nucleus

Transcription factor Transcription factor

Mouse Chicken Chicken Human

Nucleus Nucleus Nucleus Nuclear

Transcription factor Transcription factor Transcription factor Transcriptional regulator

Oncogene sis erbB fms kit src abl raf

myb rel fos jun erbA tax

Osteosarcoma Sarcoma Leukemia Leukemia, lymphoma

hinting at the existence of oncogene families. (2) The products of retroviral oncogenes appear to cover a relatively limited range of biochemical functions, with protein kinases like v-ErbB, v-Src, v-Pi3k, v-Raf and transcriptional activators like v-Fos, v-Jun, v-Myb, and v-Myc comprising the majority. Some act as receptors at the cell membrane, like v-ErbB or v-Fms, or proteins transducing signals from receptors, like the Ras proteins. (3) Some oncogenic retroviruses carry two oncogenes that cooperate during transformation. For instance, the erythroblastosis virus contains two oncogenes, v-ErbA and v-ErbB. The first gene is a transcriptional repressor protein (related to the nuclear receptor family, →6.10) and the second one a constitutively active cell membrane receptor tyrosine-protein kinase. Their cooperation likely results from v-ErbA blocking the differentiation of erythrocyte precursors and v-ErbB stimulating the proliferation and supporting survival of these cells to cause erythroblastosis.

4.2 Slow-Acting Transforming Retroviruses Slow-acting transforming retroviruses are as a rule replication-competent and do not transduce oncogenes. Instead, they cause transformation by integrating into or in the vicinity of cellular genes and altering their expression. In this fashion, they convert cellular genes from proto-oncogenes into oncogenes in a cis-acting manner. Integration of a slow-acting retrovirus activates transcription of the targeted gene by the transcriptional regulatory sequences of the retroviral LTR and may additionally disrupt negative regulatory elements. While several mechanisms are conceivable by which the viral regulatory sequences could cause gene overexpression, the predominant mechanism seems to be activation of the cellular gene promoter by the enhancer function of the retroviral LTR (Fig. 4.2). Oncogene activation may result from the effect of the enhancer of the “idle”

4 Oncogenes

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provirus 3' LTR on the proto-oncogene promoter as shown in the figure. The retrovirus may alternatively integrate in reverse orientation. Subsequent recombination may remove viral coding sequences to leave an intact LTR that can still act as an enhancer. Often, provirus insertion additionally disrupts or separates negative regulatory elements of the host gene. For instance, the cellular avian Myc gene (initially called c-myc, where c stands for cellular) comprises three exons. Several negative regulatory elements are located upstream of its two transcriptional start sites and in the first intron. Transcription from a physiological start site proceeds into the first intron and pauses there until further signals arrive (see Sect. 4.5). A typical

retroviral insertion into the avian Myc gene disrupts these negative control mechanisms and activates uncontrolled transcription from an otherwise inactive (cryptic) promoter near the end of intron 1. In this fashion, Myc transcription becomes independent of extracellular signals. Specifically, the expression of the gene is not downregulated in response to signals for proliferation arrest and cell differentiation. The avian Myc gene activated by slowly transforming retroviruses is very similar to the oncogene carried by the avian myelocytomatosis virus (Fig. 4.3). Apparently, the cellular gene is the precursor of the viral gene and was picked up and transduced by a precursor of the myelocytomatosis virus. This may have occurred by recombination of

E1

E2

E3

Proto-oncogene Insertion U3 R U5

ψ

gag

pol

env

U3 R U5

Retroviral DNA

U3 R U5

ψ

gag

pol

env

E1

U3 R U5

Provirus

Fig. 4.2  Oncogene activation by retroviral insertion. A frequent mode of oncogene activation involves enhancer activity of the “idle” provirus 3'  LTR on the proto-­ oncogene promoter as shown in the figure. The retrovirus may also integrate in reverse orientation and activate the

E2

E3

Oncogene

host gene through the enhancer activity of its 5′ LTR. Recombination may remove viral coding sequences to leave an intact LTR that can act as an enhancer. Additionally, provirus insertion may disrupt or disconnect negative regulatory elements of the host gene v-Myc

Fusion with viral gag protein Transactivation Gag

Heterodimerization DNA binding basic HLH

Thr61 Linker amino acids

Fig. 4.3  The v-Myc protein. In avian and murine transforming retroviruses, the v-Myc protein retains all functional domains of its cellular ortholog. It is always overexpressed, usually as a fusion protein

LZ

Missense mutations in different viral strains

with viral gag sequences at the N-terminus plus linker amino acids. In individual viral strains, individual amino acids in the N-terminal part of the protein are mutated, especially Thr61, a site for regulatory phosphorylation

4.2  Slow-Acting Transforming Retroviruses

81

the avian Myc mRNA with the retroviral genomic mRNA. Alternatively, the recombination may have involved a retroviral genomic transcript and a transcript from a Myc locus into which a retrovirus had been inserted. In this fashion, the more abundant slow-acting transforming retroviruses may give rise to the rarer acutely transforming types. When a slowly transforming retrovirus integrates into the Myc gene, the target gene becomes deregulated and overexpressed (Fig.  4.2). The v-myc gene contained in the myelocytomatosis retrovirus is likewise strongly expressed. In addition, there are several changes in the amino acid sequence of v-Myc compared to the original avian Myc gene. These are not random. For instance, an amino acid exchange in most viral strains removes a threonine which upon phosphorylation promotes the degradation of the Myc protein, further enhancing its oncogenicity (cf. 4.5). In summary, the acutely transforming retrovirus overexpresses an altered cellular protein, whereas a slowly transforming retrovirus deregulates the endogenous protein, which may remain unchanged. As in the example of cellular Myc/v-myc, retroviral oncogenes are almost always altered compared to the cellular orthologs from which they originated. These alterations are often more severe than in the case of Myc. They may comprise truncation, mutation, or fusion to viral proteins which increase the activity, affect the regulation, and alter

the localization of the oncoproteins within the cell. For instance, the v-ErbB product is derived from the cellular Erb-b1 gene which encodes the epidermal growth factor receptor Egfr (Fig.  4.4). This receptor is basically composed of three domains, namely an extracellular domain binding the growth factor ligands, a transmembrane domain, and a cytoplasmic tyrosine kinase domain, which is controlled by autoinhibitory sequences. The viral product lacks most of the extracellular domain but contains a small segment of Gag that causes oligomerization of the protein, which would normally need to be induced by ligand (like EGF) binding. Furthermore, a point mutation and a C-terminal truncation in the cytoplasmic domain relieve autoand feedback inhibition. In summary, thus, the virus encodes and overexpresses a constitutively active protein. The different time-courses of transformation by acutely and slowly transforming viruses are partly accounted for by these additional alterations in the transduced oncogene. Further differences contribute. Acutely transforming retroviruses transduce the activated oncogene into each cell they infect and thereby create a large pool of potentially transformed cells. In contrast, slowly transforming retroviruses tend to integrate into different sites in each infected cell and only rarely “hit” a cellular proto-oncogene, yielding only a few potentially transformed cells.

EGF receptor

Extracellular

Transmembrane domain

Intracellular

tyrosine kinase

H2N

COOH

v-ErbB point mutation Gag

Env tyrosine kinase

Fig. 4.4 Activation of ErbB1 towards the v-erbB oncogene. In the oncogenic receptor, truncation of the extracellular domain abolishes ligand binding, while fusion to a Gag

peptide leads to its constitutive oligomerization independent of ligands. A truncation at the C-terminus and a point mutation in the autoinhibitory loop relieve autoinhibition and further enhance constitutive activity

82

One reason for the slower tumor development is thus the additional time required for the expansion of one or a few tumor cell clones. There are likely two more reasons. The first is that transduction of an activated oncogene into a large number of cells is bound to have a substantial effect on their interaction with each other and further host cells. For instance, the levels of autocrine or paracrine cytokines secreted by these cells could be significantly altered and antitumor immune response could be overwhelmed. The second reason is that a larger pool of cells containing an oncogene increases the probability of a second mutation that might be required for complete transformation and subsequent tumor progression. There is indeed very good evidence that transformation by slowly transforming retroviruses often requires a second genetic alteration. Occasionally, this is provided by the insertion of a second retrovirus elsewhere in the genome. Some acutely transforming retroviruses like the erythroblastosis virus carry two oncogenes thereby achieving “two hits” at one stroke. A comparison of genes that have been found to be activated by retroviral insertion with those listed in Table  4.1 reveals that several possess viral homologs, in keeping with the idea that acutely transforming viruses arose from slowly acting precursors. Notably, many genes activated by viral insertion have never been observed to be transduced. Some genes may simply be too large to be accommodated in a retrovirus, but functional limitations are also conceivable. Viral insertions moreover tend to activate cellular proto-oncogenes that are activated in cancers predominantly by gene amplification like MYC and CCND1. Additional oncogenes were discovered by analyses of integration sites in animal tumors induced by experimental infection with retroviruses but were never detected in natural infections. For instance, the deregulated gene at one integration site of the mouse mammary tumor viruses (named int-1) turned out to encode an important growth factor, later named WNT12 (→6.9). Importantly, there is a cellular homolog for every retroviral oncogene discovered to date. Therefore, all retroviral oncogenes are thought to A composite of Wingless (a Drosophila mutant) and int-1. 2 

4 Oncogenes

have evolved from cellular precursors. For instance, the homolog of the v-src gene of RSV is c-Src (the human ortholog is SRC), encoding a protein kinase located at focal adhesion points, where the actin cytoskeleton is attached to the cell membrane. The SRC kinase relays cell adhesion signals to the cytoskeleton as well as to other kinases that control cell proliferation (→6.5). These signals may be mimicked by the viral oncoprotein, which is altered in comparison to the cellular protein by several point mutations and the replacement of the C-terminal 17 amino acids by an unrelated peptide.

4.3 Identification of Human Oncogenes With the exception of the rare HTLV-1 virus (Box 4.1), acutely transforming retroviruses have not been observed in humans and even activation of cellular genes by insertion of viruses such as HBV (→17.4) is rare. An accidental case of iatrogenic oncogene activation took place when during gene therapy of children with severe hereditary immunodeficiency a recombinant retrovirus carrying the therapeutic gene caused leukemia by integrating into and activating the MLO2 proto-oncogene. Nevertheless, the elucidation of the mechanisms by which retroviruses cause cancers in animals has been highly instructive for the understanding of human cancers. Even if genes in the human genome are almost never activated by retroviruses to become oncogenes, many genes orthologous to the viral (and animal) oncogenes can be activated by different mechanisms in humans. Following rapidly on the discovery of cellular oncogenes, many human cancers were screened for oncogenic alterations in these genes. Indeed, many genes related to retroviral oncogenes are now established as oncogenes in human cancers. Table 4.2 lists a selection of those identified in the two decades after the molecular characterization of the first retroviral oncogenes. By now, the COSMIC database lists more than 500 genes as likely oncogenes in at least one cancer type. Many of the older oncogenes were identified through one of the following lines of research.

4.3  Identification of Human Oncogenes

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Table 4.2  Some important oncogenes in human cancers Oncogene Tumor type TGFA Many carcinomas FGF1 Many solid tumors WNT1 Selected carcinomas IGF2 Many cancers ERBB1 Many carcinomas ERBB2 Selected carcinomas KIT Testicular cancers, gastrointestinal stromal tumors RET Thyroid and other endocrine cancers MET Renal and other carcinomas IGFRI Liver and other carcinomas SMO Skin cancers HRAS Many cancers

Activation mechanism Overexpression Overexpression

Cellular localization Extracellular Extracellular

Main biochemical function Growth factor Growth factor

Overexpression

Extracellular

Growth factor

Overexpression Overexpression, mutation Overexpression

Extracellular Growth factor Cell membrane Tyrosine kinase Cell membrane Tyrosine kinase

Mutation

Cell membrane Tyrosine kinase

Mutation, chromosomal inversion

Cell membrane Tyrosine kinase

Mutation, overexpression

Cell membrane Tyrosine kinase

Overexpression

Cell membrane Tyrosine kinase

Mutation Mutation

Cell membrane Inner cell membrane NRAS Many cancers Mutation Inner cell membrane KRAS Many carcinomas Mutation Inner cell membrane BRAF Melanoma, colon Mutation Inner cell membrane, cytoplasm PIK3A Many cancers Overexpression Inner cell membrane, cytoplasm CTNNB1 Colon and liver Mutation Inner cell carcinomas, membrane, others cytoplasm, nucleus MYC Many cancers Translocation, overexpression, mutation Nucleus MYCN Selected cancers Overexpression Nucleus MYCL Lung cancers Overexpression Nucleus RELA Leukemias Translocation Nucleus MDM2 Sarcomas and Overexpression Nucleus, other solid cytoplasm tumors SKP2 Selected cancers Overexpression Nucleus, cytoplasm CCND1 Many cancers Overexpression Nucleus CCND2 Selected cancers Overexpression Nucleus CDK4 Selected cancers Overexpression, mutation Nucleus BCL2 Follicular Translocation, overexpression Mitochondria lymphoma, others

Receptor GTP-binding protein GTP-binding protein GTP-binding protein Tyrosine kinase

Phospholipid kinase

Cytoskeleton, transcriptional activation

Transcription factor Transcription factor Transcription factor Transcription factor Transcriptional regulator, ubiquitin ligase Ubiquitin ligase Cell cycle regulation Cell cycle regulation Cell cycle regulation Apoptosis regulation

4 Oncogenes

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Analysis of human orthologs of retroviral oncogenes: An obvious approach was to investigate the human orthologs of viral oncogenes for mutations and dysregulation in human cancers. Indeed, many orthologs of viral oncogenes are recurrently mutated or overexpressed in human cancers and are clearly involved in their development. For instance, the human ortholog of v-erbB, ERBB1 (EGFR), and the human v-myc orthologue MYC are overexpressed, mutated, and causally involved in various human cancer types. Of note, overexpression of ERBB1 or MYC is not in every case caused by primary changes in these genes. Rather, expression of their gene products, the EGFR receptor tyrosine kinase, and the transcription factor MYC, is regulated by several pathways influencing cell growth and proliferation. Other oncogenes are only directly involved in a restricted range of cancers, such as RELA (→6.7) in selected lymphomas. On the other hand, some potent viral oncogenes have not turned up as common dominantly acting oncogenes in humans, prominently v-src and v-fos. Instead, the products of the corresponding human genes are more subtly involved in shaping the phenotype of human cancers. In summary, human orthologs of almost all viral oncogenes contribute in some way to one or the other human cancer. 3T3 cell transformation assay: Cell culture assays such as the 3T3 fibroblast focus formation assay were originally developed for the identification of viral oncogenes and were consequently also used to discover human cellular oncogenes. In the original 3T3 assay, the immortalized mouse fibroblast cell line 3T3 was infected with an oncogene-carrying retrovirus and yielded foci of transformed cells with altered morphology that emerge from the monolayer at confluence (Fig. 4.5). Foci can also be obtained by transfection of DNA from human tumor cells and the gene responsible for the altered phenotype can be isolated. This assay led to the identification of several human oncogenes. The most dramatic insight was that three human orthologs of v-ras genes can act as oncogenes, namely orthologs of the Ki-ras (KRAS) and Ha-ras (HRAS) genes as well as a third relative, NRAS. All three possess similar structures and functions (see Sect. 4.4). When they were recovered from 3T3 focus formation assays, each car-

ried point mutations at specific sites, either codons 12, 13, or 61. Thus, some cellular genes like the RAS genes can be converted from proto-oncogenes to oncogenes by a single point mutation. The 3T3 assay has also yielded several additional candidate oncogenes, as well as a number of interesting artifacts from genes rearranged or truncated by the transfection procedure. Other transformation assays: The 3T3 assay is limited by a strong bias towards a type of oncogenes that are “Ras-like,” while others, like the potent MYC oncogene, do not score. This limitation arises because the assay selects for the ability of oncogenes to keep fibroblasts growing beyond confluence and to change their morphology. Mutant RAS genes confer these properties, but by far not all oncogenes do. Therefore, further cellular assays were developed to identify oncogenes acting on other cancer-relevant properties. For instance, in the REF assay, primary rat embryo fibroblasts are infected with retroviruses or transfected with purified oncogene or tumor cell DNA.  In this assay, two oncogenes are required for focus formation, one “Ras-type” and one “Myc-type” oncogene. This is another instance of oncogene cooperativity, as discovered initially in some animal retroviruses. In the REF assay, the “Myc-type” genes prevent rodent embryo fibroblasts from senescence and stimulate their proliferation, while “Ras-type” genes elicit overproliferation and altered morphology. Obviously, in 3T3 cells, the first step has already happened, as these cells are “immortalized” (Fig. 4.5).3 Gene amplification: Since overexpression often contributes to the function of oncogenes, genes that are recurrently strongly overexpressed in human cancers are obvious candidates for oncogenes. Many cancers contain recurrent amplifications of particular chromosomal regions, often detectable even at the resolution of cytogenetic techniques. For instance, the overexpression of MYC and EGFR in human cancers often results from an amplification of their genes located at 8q24 and 17p12. Investigation of other The relevant change is probably a mutation of mouse Tp53 (→5.3). Indeed, mutant Tp53 acts as a “myc-like” gene in the REF assay. 3 

4.3  Identification of Human Oncogenes Fig. 4.5  A comparison of the 3T3 and REF focus formation assays. See main text for explanation

85

Embryo fibroblast

Oncogene #1 myc-like

Oncogene #2 ras-like

Oncogenic Change myc-like

3T3 cell line

Oncogene ras-like

Transformed 3T3 cells

amplified regions has accordingly revealed further oncogenes. A segment of chromosome 2p24 recurrently amplified in neuroblastoma, but also in certain carcinomas, contained a gene related to MYC that was accordingly named MYCN (N for neuroblastoma). Another related gene, MYCL, was found amplified and overexpressed in lung cancers. A different region originating from chromosome 17q11-12 amplified in breast cancer and other carcinomas yielded an overexpressed gene homologous to ERBB1. This is now officially named ERBB2, but still doubles commonly as HER2. Findings like these confirm that consistently amplified regions in the genome of cancer cells often contain oncogenes and that genes related to known oncogenes can be oncogenes (see below). However, the identification of oncogenes from amplified regions is not always straightforward, since amplicons can encompass several Mbp. For instance, a region from 12q14 amplified in various human tumors contains the genes GLI1, CDK4, and MDM2.4 As detailed in later chapters, each of these genes possesses properties that make it a good candidate for an oncogene. Perhaps, in different tumors one or the other or more than one could be relevant. A different kind Both the acronyms HDM2 and MDM2 are in use for the latter gene, as MDM2 stands for mouse double minute. 4 

of complication is that amplifications may be associated with gene silencing rather than overexpression in a few cases. Chromosomal translocations: Another type of chromosomal aberration in human cancers are translocations. In hematological cancers, in particular, they activate genes at the translocation sites to become oncogenes (→2.2, →10.2). Therefore, systematic investigation of recurrent translocations in hematological cancers by cytogenetic and molecular cloning techniques, and more recently, by NGS analyses, has revealed many oncogenes. More recently, characterization of translocations in soft tissue tumors and carcinomas revealed oncogenes as well. One gene activated by several different translocations is MYC, emphasizing what a potent oncogene it is. A second oncogene identified at a recurrent site of translocations in 11q13  in different cancers was initially named PRAD1 or BCL1 but is now known as CCND1 since it encodes Cyclin D1, a crucial regulator of cell cycle progression through the G1 phase (→5.2). Interestingly, CCND1 is also overexpressed as a consequence of amplifications in some carcinomas. Not untypical, several neighboring genes are often co-amplified with CCND1, including GSTP1 (→3.7), and genes encoding fibroblast growth factors (FGF). The typical translocation in follicular lymphoma activates the BCL2 gene (for “breakpoint cluster

86

2”). This gene belongs to an entirely different functional class: the BCL2 protein is a direct regulator of apoptosis at the mitochondrion (→7.4) and was the first discovered member of a large family of apoptosis regulators. Oncogene families: Since oncogenes often seem to come in families (Table  4.2), it is ­tempting to speculate that genes homologous to oncogenes might also be oncogenes. This idea has proven correct in some instances, but perhaps less consistently than originally expected. In some cases, the reasons are obvious (with hindsight). For instance, the MYC family includes members like MXI1 and MAD that are actually antagonists of the proto-oncogene proteins MYC, NMYC, and LMYC, while MAX encodes a heterodimerization partner for all members of the family. It is less straightforward to understand, why ERBB1 and ERBB2 are more often activated to oncogenes than two further members of the family ERBB3 and ERBB4. Similarly, the closest human ortholog of the viral v-raf gene, CRAF, is much less frequently activated in human tumors than its homolog BRAF (→12.4). In general, homology to a proven oncogene hints at a relevant function in cancer but does not allow firm conclusions on the oncogenic potential of a gene. Cancer pathways: The dilemma of how to test the functional importance of a gene in human cancer has been partly alleviated by the recognition that many oncogenes and tumor suppressor genes interact in signal transduction pathways (→6) and networks that control cell proliferation, survival, and genomic stability. If the product of a gene can be demonstrated to influence the activity of a cancer pathway that has been proven to be important in a specific tumor type, its overexpression or mutation is more likely to represent oncogenic activation. This line of investigation has yielded sufficient data to regard genes like CDK4 (→5.2), MDM2 (→5.3), or CTNNB1 (→6.9) as proto-oncogenes. Lineage-specific oncogenes: Investigations of specific cancers by traditional techniques and more recently by NGS analyses have highlighted another class of oncogenes that acts in a tissue-­ specific or cell-type specific manner. The products of these genes, typically transcription factors, are essential for the proliferation, sur-

4 Oncogenes

vival, and function of specific tissues and cell types. In cancers originating from these cells, they remain essential for proliferation and survival, while their effects on differentiation and tissue-specific functions are diminished or abolished. Like for other oncogenes, activation of lineage-specific oncogenes may be brought about by overexpression via amplification, mutation, or deregulation. In some cases, activation of lineage-­ specific oncogenes does not occur by genetic alterations in the gene itself, but rather by mutations in cofactor genes. For instance, the Estrogen receptor α and the Androgen receptor are essential for the growth and survival of (many) breast cancers and (almost all) prostate cancers, respectively. They are very rarely affected by genetic alterations in primary tumors, but their activity is distorted. Their character as oncogenes is unmasked upon therapy with anti-hormonal drugs when the genes (ESR1 and AR, respectively) develop mutations that allow them to keep active despite therapy. Since human cancers accumulate many genetic and epigenetic alterations over long periods of progression, in a typical cancer many genes are overexpressed and many of their products are overactive, and many genes may contain mutations. Many cancers display chromosomal instability or an increased rate of point mutations. Comprehensive investigations by next-­generation sequencing (NGS) have accordingly identified hundreds of mutant genes in many tumors including good candidates for novel oncogenes, e.g., because they show a pertinent biochemical property such as a protein kinase activity or DNA-­ binding ability or interact with known regulators of cell proliferation and survival. Likewise, many genes are consistently overexpressed in human cancers but the functional relevance of overexpression can be unclear in individual cancers even for bona fide oncogenes like MYC and EGFR. Many of the genes highlighted by mutations or overexpression may thus well be functionally important and contribute to tumor growth. Proving that a gene of this sort is a true oncogene requires that its overexpression or altered activity is caused by substantial changes in the gene, such as a mutation or an amplification accompanied

4.4  Functions of Human Oncogenes: Receptor Tyrosine Kinases and the MAPK Pathway

by overexpression or deregulation. Furthermore, it needs to be shown that the altered or overexpressed gene product dominantly confers an essential property for the survival and sustained growth of cancer.5 This is not only an issue of scientific interest but highly relevant for the development of successful targeted therapies (→23.3). Today, fortunately, many experimental models are available to investigate the ability of oncogenes (singly or combined) to elicit or promote cancers. Prominently, expression of mutant oncogenes, at physiological or elevated levels, can be engineered in specific tissues or cell types of mice to investigate the consequences. Notably, such models often use inducible oncogenes in order to avoid developmental defects caused by their constitutive expression.

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(extracellular) peptide growth factors like TGFα (transforming growth factor alpha), FGF1 (fibroblast growth factor), or WNT1 (wingless/int-1) which are mitogens for epithelial or mesenchymal cells. These factors are overproduced by many carcinomas resulting in autocrine growth stimulation. Cancer cells may moreover, in a paracrine fashion, stimulate the production of growth factors from various stromal cells in tumor tissues that in turn act on the cancer cells. Peptide growth factors bind to and activate receptors at the cell membrane such as the EGFR (epidermal growth factor receptor, Fig. 4.4), the product of the ERBB1 gene, or one of several FGF receptors. The EGFR is overexpressed in different carcinomas, often as a consequence of gene amplification. In certain cancer types, such as lung adenocarcinoma (→15.4), point mutations or in-frame deletions, usually in its tyrosine 4.4 Functions of Human kinase domain, activate the receptor constituOncogenes: Receptor tively. In others, structural alterations in the Tyrosine Kinases extracellular domain relieve the dependency on and the MAPK Pathway growth factor binding or widen the range of EGF-­ like growth factors that elicit strong activation of The oncogenes of acutely transforming retrovi- the receptor. FGFRs are also altered in many ruses (Table  4.1) as well as human oncogenes human tumors. Whereas FGFR1 may be overex(Table 4.2) can be categorized by their biochemi- pressed as a consequence of gene amplification, cal function or by the localization of their prod- the FGFR3 is activated by specific point mutaucts. A sketch of these localizations (Fig.  4.6) tions in cancers of the urinary bladder (→14.3) suggests that the spatial distribution of these pro- and the cervix or may be activated through the teins in the cell may not be incidental. Indeed, formation of fusion proteins. Further growth facmany proven or suspected human oncogenes tor receptors such as ERBB2 (→19.5), MET belong to functional pathways (or networks, (→16.5), IGFR1 (→17.3), KIT (→11.5), and since they interact mutually) which transmit sig- RET (→2.2) are likewise crucial oncogenes in nals for proliferation and survival from the cell human cancers. These receptors share structural exterior to the nucleus. The first pathway of this domains and the principal mechanisms of activakind to be elucidated was the (canonical) tion and signaling and are summarized as recepmitogen-­ activated protein kinase (MAPK) tor tyrosine kinases (RTKs, also: TRKs). RTKs pathway. constitute one of the biggest classes of oncogenes Normal cells proliferate in response to extra- and are prominent targets for chemotherapy by cellular signals that are conferred by soluble novel drugs and antibodies (→23.5). Of note, growth factors and are modulated by signals con- some oncogene products are receptors belonging veyed by cell adhesion to the ECM and by cell-­ to different classes, e.g., cytokine receptors to-­cell interactions. The first group of presumed (→6.8). human oncogene products accordingly comprises Binding of a growth factor to the extracellular domain of a RTK leads to the formation of receptor homodimers (e.g., EGFR/EGFR) or heterodi5  The COSMIC database classifies cancer genes into two mers (e.g., ERBB2/ERBB3). It furthermore tiers according to the level of evidence for functional causes (stepwise) conformational changes by relevance.

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4 Oncogenes

Fig. 4.6  Cellular localization of viral and cellular oncogene proteins.

The presumed localizations of some early discovered oncogenes are shown. See main text for further discussion

which an inhibitory pseudo-substrate peptide loop (also called activation loop) moves out of the active center of the intracellular catalytic tyrosine kinase domain. Likewise, inhibitory sequences from the receptor C-terminus dissociate from the catalytic center allowing binding of ATP and other substrates. As a consequence, the tyrosine kinase becomes fully active and phosphorylates primarily itself (autophosphorylation) as well as other substrates. Typically, the receptor subunits phosphorylate each other in trans. Several receptor dimers may then be assembled to larger complexes in the cell membrane followed by internalization via endosomes. Often, signaling continues inside the cell until the RTK is poly-ubiquitinated and degraded, or is recycled to the cell membrane in an inactive state. Overexpression of RTKs in tumor cells favors dimer formation. Thereby, it makes cells responsive to lower concentrations of ligand growth factors or even leads to growth factor-independent activation. Oncogenic point mutations often occur in or around the inhibitory loop thereby

causing constitutive activation of the tyrosine kinase activity. Cross- and autophosphorylation of RTKs provide phosphotyrosines for recognition by adaptor proteins that dock onto the activated receptor via SH2 (src-homology 2) or PTB (phosphotyrosine binding) domains or—for some receptors—onto primary substrates of the receptor like the FRS (FGF receptor substrates) or IRS (insulin receptor substrate) proteins. The SH2 and PTB domains of various proteins all recognize and bind phosphotyrosines but in different peptide contexts. As a rule, multiple proteins bind to one receptor through different phosphotyrosines. In this fashion, one receptor can activate several signaling pathways, like the MAPK and PI3K (→6.3) pathways. For instance, the activated EGFR binds the adaptor proteins GRB2 and SHC (which again interacts with GRB2), PLCγ (Phospholipase Cγ), the regulatory subunit of PI3K (Phosphatidylinositol-3′-kinase), and GAP (GTPase activator protein). EGFR may moreover

4.4  Functions of Human Oncogenes: Receptor Tyrosine Kinases and the MAPK Pathway

lead to phosphorylation of the STAT1 and STAT3 transcription factors (→6.8) and modulate cell attachment via the FAK (Focal adhesion kinase) and SRC kinases. In a negative feedback loop phosphorylation of CBL, which is recruited to the EGFR by GRB2 and a phosphotyrosine, stimulates receptor polyubiquitination and degradation. A positive feedback loop activates ADAM proteases that process membrane-bound HB-EGF to an active EGFR ligand (Fig. 4.7). In comparison, FGFRs tend to activate more selectively the MAPK pathway. GRB2 is again the major adaptor protein, but in this case interacts with phosphorylated FRS2 rather than the RTK itself. In contrast, the RTK MET (→16.5) which is activated by its ligand hepatocyte growth factor (HGF) employs the adaptor proteins GAB1 and GRB2 to activate MAPK and PI3K signaling, but influences cell adhesion and migration more strongly via RAC1/CDC42 and FAK. In order to activate MAPK signaling, the adaptor protein GRB2 assembles in turn a further adaptor protein named SOS into the complex, which interacts with and activates RAS proteins (Fig.  4.8). All RAS proteins, HRAS, KRAS, or NRAS, are ≈21 kDa proteins that are linked to the inner face of the cell membrane through their C-terminus which is posttransla-

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tionally modified by myristylation, farnesylation, and methylation (see  Fig. 23.15). RAS proteins belong to a larger superfamily of small monomeric G proteins that can bind alternatively GTP or GDP. In the active state, GTP is bound. Hydrolysis of GTP to GDP by the combined action of RAS and a GTPase activator protein (GAP) restores the basal inactive state. In the normal process of signaling from RTKs, activation of RAS depends on the interaction with SOS which acts as a guanine nucleotide exchange factor (GEF), loading the RAS protein with GTP to replace GDP. The activated state of normal RAS proteins is short-lived since RTKs activate GAPs that stimulate GTP hydrolysis in parallel to GEFs like SOS. However, mutations in RAS amino acids 12, 13, or 61, which surround the GTP-binding site, hinder the access of the GAP protein and prolong the active state. RAS proteins are the next branching point in the signaling network since activated RAS acts on several pathways that affect cell proliferation, protein synthesis, the cytoskeleton and cell survival, prominently the PI3K pathway (→6.3). Of note, components of the PI3K pathway like the AKT kinase and the PI3K catalytic subunit were also initially discovered as viral oncogenes (Table 4.1).

Fig. 4.7  Signaling from the EGFR. See main text for further details

EGF P

Adap

tors

CBL P

P

P

P

GAP

Deg rad atio n Term

inat

MAPK

PLC/PKC PI3K

JAK/STAT

ion

4 Oncogenes

90 Fig. 4.8  The RAS activation cycle. See main text for further description

Receptor activation

GEF GDP

GTP

RAS-GDP

RAS-GTP

Activation of pathways

P GAP

The MAPK pathway (Fig. 4.9) is one major route by which RAS proteins relay proliferation signals. It proceeds via interaction of activated RAS with RAF proteins. The three RAF proteins (A, B, C) in human cells are Ser/Thr protein kinases. Like many protein kinases, they contain a regulatory domain and a C-terminal catalytic domain. In the inactive state, the protein resides in the cytosol and the kinase activity is blocked by the regulatory domain. Activated RAS directs RAF to the cell membrane and relieves inhibition by its regulatory domain. RAF activity is moreover modulated by phosphorylation of the CR2 segment in its regulatory domain. A tyrosine in this domain is phosphorylated by SRC family kinases; serine and threonine residues are phosphorylated by protein kinase C isozymes which can be activated by RTKs via PLCγ (→6.5). These interactions are further links in a wider signaling network. In some human cancers, BRAF is altered by specific point mutations such as V600E to become overactive (→12.4). In these cancers, BRAF mutations occur in a mutually exclusive manner to mutations in RAS genes. Since RAS proteins act on several pathways, this finding is an important argument for the conclusion that signaling via RAF is decisive for transformation in these cancers. Tellingly, the main modification in the

retroviral v-raf oncogene is the inactivation of the regulatory domain of the cellular protein. Activated RAF is the first one in a cascade of protein kinases that next comprises MEK and ERK proteins (Fig.  4.9). Alternative names for these are MAPK (mitogen-activated protein kinase) for ERK and MAPKK for MEK; RAF proteins can therefore be considered as one type of MAPKKK (or MEKK). There are several additional MEKKs in parallel pathways in human cells (→6.2). Most human cells express two MEK and two ERK protein kinases (ERK1/2 and MEK1/2). MEK are highly specific kinases and phosphorylate predominantly ERK proteins at both tyrosine and threonine in a TEY aa sequence. Activated ERKs phosphorylate a variety of substrates to activate protein synthesis, alter the structure of the cytoskeleton, and following relocation to the nucleus, to induce gene expression. Interaction of MEKs, ERKs, and MAPKKs is supported and its specificity is enhanced by scaffold proteins. Phosphorylation of MAPKKs, MEKs, and ERKs is removed by several specifically as well as broadly acting protein phosphatases in order to terminate the signal. Some of these phosphatases, like MKP1 (MAP kinase phosphatase 1), are themselves activated or induced by MAPK signaling, while others may be constitutively active. As a consequence of this and further feedback

4.4  Functions of Human Oncogenes: Receptor Tyrosine Kinases and the MAPK Pathway Fig. 4.9  The main MAPK pathway. See main text for further explanation

91

Receptor tyrosine kinase GF

SOS

RAS

P

RAF

Grb2 SHC

P

MEK1/2 P

P

ERK1/2

KSR/MP1

P

P P ERK1/2 P ELK-1 SRE

mechanisms, active MAPK signaling normally proceeds in bursts rather in the form of continuously high activity. Increased protein synthesis and cytoskeletal changes elicited by MAPK signaling in the cytoplasm are important for cell growth and facilitate cell migration. Stimulation of cell proliferation requires ultimately an altered pattern of gene expression and stimulation of cell cycle progression in the nucleus. The mitogenic signal from growth factors to redirect transcription towards cell proliferation is to a large extent relayed by the MAPK signaling cascade. Activated ERKs phosphorylate several transcription factors (Fig.  4.9) directly or stimulate other protein kinases like p90RSK1 (RSK)  to do so. In this fashion, they

P SRF

P FOS AP1

JUN

P MYC

MAX

E-Bòx

induce the transcription of a larger set of genes required for cell proliferation, which again are organized as a cascaded network in the nucleus. In previously resting (quiescent) cells, the initial set of genes induced as a consequence of MAPK pathway stimulation by growth factors are the “early-response” genes. They are, e.g., induced by treatment of quiescent cells in cell culture with growth factors or reprovision of serum. Several early-response genes are related to viral oncogenes, such as FOS, JUN, MYB, and MYC. Among these, MYC is certainly the most important oncogene in the context of human cancers, as it is frequently activated by genetic changes leading to its overexpression or deregulation so that its expression becomes independent

92

of growth factor signaling. FOS, JUN, and MYB are necessary for the growth of many human normal and cancer cells but do not commonly act as actual oncogenes. This may be so because the viral counterparts of these genes are severely altered and overexpressed. The cells of long-lived humans moreover may have better checks against tumor formation than rodent and avian cells. Indeed, in some human cells, overexpression of proteins like FOS or JUN induces apoptosis rather than stimulating cell proliferation. Heterodimers of FOS and JUN are one isoform of the transcription factor AP1, which may alternatively be composed of several other related proteins. Depending on its constitution, its interaction with other transcription factors and the cell context, AP1 can serve many different functions, which include driving proliferation but also inducing differentiation. The products of the “early-response” genes induce the expression of genes required for cell cycle progression, directly or indirectly. In many cells, one of the most important proteins linking mitogenic signals to cell cycle regulation is Cyclin D1, the product of the CCND1 gene. Overexpression of Cyclin D1 caused by gene amplification or chromosomal translocations deregulating CCDN1 is important in several different human cancers. Moreover, Cyclin D1 is often overexpressed without alterations in the gene itself as a consequence of mutation in MAPK pathway components or upstream regulators like EGFR. Cyclin D1 overexpression partly mediates the oncogenic effect of those alterations. The related gene CCDN2 is expressed in a more restricted range of tissues. Accordingly, overexpression and amplification of this gene occur in a limited range of human tumors, e.g., in testicular germ cell cancers. D-Cyclins activate the cyclin-dependent kinases 4 (CDK4) or 6 (CDK6) to promote progression through the G1 phase of the cell cycle (→5.2, →6.4). Amplification and overexpression of CDK4 as well as oncogenic point mutations are also observed in human cancers alternatively to genetic changes in CCDN1. Finally, physiological signaling for cell proliferation requires a parallel signal for cell survival,

4 Oncogenes

either by the same growth factor and receptor or by a complementing pathway; for MAPK signaling this is often the PI3K pathway (→6.4). For instance, insulin-like growth factors stimulate the MAPK cascade, too, to some extent, but confer a particularly strong survival signal through PI3K signaling (→6.3). Therefore, the overexpression of the insulin-like growth factors IGF1 and IGF2, their receptor IGFR1 as well as mutations directly activating PI3K (most often by oncogenic mutations in PIK3CA) are oncogenic events in many human cancers. Moreover, cell proliferation requires cell growth and metabolic adaptations, which are also achieved crucially through PI3K signaling and MYC-induced transcription (→1.4). Apoptotic cell death due to inappropriate stimulation of cell proliferation can be prevented by oncogenic overexpression of specific anti-­ antiapoptotic regulators like BCL2 (→7.5). Overexpression of BCL2 by a chromosomal translocation is the most common genetic cause of follicular lymphoma, but overexpression of BCL2 or other anti-apoptotic proteins like MCL-­ 1 and BCL-XL occurs also in many other hematological cancers and carcinomas. Synergisms between oncogenes that stimulate cell proliferation (like MYC) and oncogenes that prevent fail-­ safe apoptosis elicited by this stimulation (like BCL2) is evident in many human cancers and can be modeled in mouse lymphomas. Of note, this provides another example of oncogene cooperation.

4.5 Functions of Human Oncogenes: MYC Oncogenes Different genomic alterations activate one of the three MYC genes (genes: MYC, MYCL, and MYCN; proteins MYC, LMYC, NMYC; Fig.  4.10) to oncogenes in a broad range of human cancers. Activation of MYC is most common, in keeping with its widespread expression in many tissues and cell types. MYC may turn into an oncogene by amplification—in up to 20% of cancers overall—or by translocations destroying its normal regulation, e.g., in certain lymphomas and leukemias (→10.2). Even gains of parts

4.5  Functions of Human Oncogenes: MYC Oncogenes Fig. 4.10  The MYC family proteins. MB: Myc box; NLS: nuclear localization signal; BR: basic region; HLH: helix-­loop-­helix; LZ: leucine zipper. Slightly modified from Chen H et al. (2018) Signal Transduct Target Ther 3:5

MB1

MB2

93 MB3

NLS

BR HLH

LZ

439

MYC 462 NMYC 364 LMYC 180 MAX

or the entire long arm of chromosome 8 (8q) may cause relevant increases in MYC expression. Overexpression of MYC can moreover result from the activation of several cancer pathways, not only MAPK but also PI3K, WNT/β-Catenin, MAPK, and Notch signaling (see Chap. 6), as MYC regularly mediates some of their effects on (normal and cancer) cell growth. MYC amplification is consequently uncommon in cancer cases with mutations that constitutively activate PI3K (like PIK3CA and PTEN mutations) or WNT/β-­Catenin (through APC inactivation) signaling. Genomic alterations of MYCN and MYCL, especially amplifications, are each found in a more limited range of cancer types, presumably reflecting their more restricted pattern of expression in human tissues. MYCN amplification is in particular the decisive event distinguishing aggressive pediatric neuroblastomas but is also observed in several tumor types presenting or assuming a neuronal-like phenotype such as small cell lung carcinoma. MYCL, MYCN, and MYC amplifications are alternative genetic changes in that tumor type. This is one of several observations suggesting that the three family members have largely similar effects, differing in essence only in their expression pattern. MYC expression is normally tightly controlled at several steps. Transcription of the gene is promoted by distant upstream and downstream enhancers, which are activated in a cell type-­ specific fashion. Several smaller enhancers can coalesce to form a super-enhancer for maximal activation. Enhancer activity is regulated by transcription factors responding to various signaling pathways, as well as by noncoding RNAs. While several cancer pathways (see above) activate

MYC, normal TGFβ signaling represses its transcription. Aberrant enhancer activation with super-enhancer formation is one mechanism leading to MYC overexpression. This activation is often caused by epigenetic mechanisms, but in some cases it is due to duplication of enhancer sequences. Closer to the gene as well, positive and negative elements regulate transcription. Positive elements include a G-rich promoter region that can form a non-canonical quadruplex DNA structure. Negative elements include a region in the first intron, where Pol II pauses and requires additional signals to continue transcription. Intriguingly, the MYC locus is located in a region on 8q24 containing few protein-coding genes, but a large number of lncRNA genes. Many of these lncRNAs, like PVT1, CCAT1, and PCAT1, can regulate MYC expression—at various steps—or modulate MYC action. PVT1 and MYC, in particular, form a positive regulatory circuit. Note that gains and amplifications of the 8q24 region in cancer also increase the copy number of lncRNA genes along with MYC. Both MYC mRNA and proteins are short-­ lived, with protein half-lives normally below 20  min. Turnover and translation of the MYC mRNA may be regulated by several miRNAs, including let-7a, miR-34, and miR-145, which are considered tumor-suppressive. MYC protein turnover is regulated by phosphorylation. Phosphorylation of Ser62 by Cyclin-­ dependent kinase 1 (CDK1) or ERK stabilizes the MYC protein. However, it primes MYC for phosphorylation of Thr58 by GSK3β. Subsequent dephosphorylation of Ser62 by protein phosphatase PP2A allows polyubiquitination of MYC by the E3 ubiquitin ligase FBXW7 and consequen-

4 Oncogenes

94

tially leads to its  proteasomal degradation. Inhibition of GSK3 by the PI3K pathway (→6.3) and increased MAPK signaling therefore increase MYC expression at the protein level as well. Furthermore, FBXW7 is inactivated by mutations in a range of cancers. Therefore, mutations in the MBI region of MYC and MYCN observed in some tumors influence predominantly protein stability. Interestingly, the threonine corresponding to Thr58 is mutated in the v-myc gene of many myelocytomatosis virus strains. An ultimate fail-safe mechanism against MYC overactivity is apoptosis. MYC induces several pro-apoptotic proteins, partly through the transcription factor E2F1, and in particular, the p14ARF protein encoded at the CDKN2A locus. The p14ARF protein activates TP53 and can hereby elicit apoptosis of cells with inappropriately sustained MYC activation. Cancers with MYC overactivity are therefore under pressure to acquire genetic alterations that limit induction of apoptosis (see 10.4 for a prominent example). MYC proteins act as transcription factors and can regulate genes positively or negatively. Expression of at least one MYC family  protein may be required for the proliferation of almost all normal cells. In normal cells however MYC expression is dependent on growth factors and rapidly downregulated when the stimulus ceases. Active MYC is particularly important for the expansion of precursor cell populations, but persistent activation can lead to the depletion of stem cell populations, and may thus favor differentiation, including terminal differentiation (→7.1). Upon terminal differentiation however MYC expression is downregulated along with cell cycling.6 In many experimental models, MYC can stimulate cells to enter the cell cycle from a G0 state MYC is often mentioned as a stem cell factor. This concept is based on the observation of Yamanaka and coworkers that MYC together with three further transcription factors can turn many differentiated cell types into induced pluripotent cells. However, unlike the other three factors, MYC is primarily required to stimulate the proliferation required for epigenetic reprogramming. In a nutshell, MYC is required for stem cell proliferation rather than stem cell specification.

(quiescence), although in vivo additional signals may be required. These additional signals moreover counteract the induction of apoptosis and activation of cellular checkpoints that are elicited by excessive MYC activity. MYC stimulates cell cycle progression and even DNA replication licensing and activity. Several Cyclin and CDK genes (→6.4) are direct targets as well as CDC25C, which encodes a CDK-activating phosphatase, and SKP2, which encodes a ubiquitin ligase directing the proteolytic degradation of the CDK (and thus cell cycle) inhibitor p27KIP1 (and interacting both positively and negatively with MYC). Notwithstanding these important influences, the most singular effect of MYC may lie in reprogramming of transcriptional activity and cellular metabolism towards growth, in other words to the doubling of cell mass and cell components required to generate two daughter cells. Thousands of genes are influenced by MYC proteins, directly or indirectly (see a small selection of MYC target gene listed in Table  4.3).7 They encode ribosomal proteins, rRNAs (where MYC stimulates transcription by Pol I and Pol III), nucleolar proteins, translation initiation factors and other proteins required for RNA processing and protein biosynthesis, components of mitochondria, nucleotide, polyamine and lipid bioTable 4.3  A selection of MYC target genes Function Cell growth

Selected proteins regulated by MYCa ↑ RNA polymerase, nucleolar proteins, ribosomal proteins, splice factors, eIF proteins, CAD, polyamine biosynthesis (ODC, Spermidine synthase), chaperones Cell ↑ Cyclin D2, Cyclin B1, CDK4, proliferation CDC25A, CDC25C, E2F1, SKP2, CUL1, Telomerase, MCM proteins, ↓ p21CIP1, p27KIP1, p15INK4B, MYC Cell ↓ Cell type-specific bHLH differentiation transcriptional activators, ID proteins Metabolism ↑ LDH, PFK, Enolase Adhesion ↓ LFA1, PAI1, ITGB1 Apoptosis ↑ E2F1 (p14ARF), BAX

6 

↑ upregulation or activation ↓ downregulation or direct interference a

These are mainly targets identified in earlier studies.

7 

Further Reading

synthesis, glutamine and carbohydrate metabolism, among many others. The broad range of genes activated by MYC genes is partly explained by the wide distribution of E-boxes in gene promoters. E-boxes have the simple symmetrical consensus sequence CANNTG and can be bound by various transcription factors, which may compete with MYC.  MYC binds to E-boxes as a heterodimer with MAX, a protein containing a homologous helix-loop-helix binding domain, but lacking the transcriptional activation and protein-protein interaction domains of MYC proteins. Transcription factors binding to promoter E-boxes typically stimulate transcription initiation. MAX can however interact with other helix-­ loop-­helix proteins like MXI1 or various “MAD” proteins to repress genes. Notably, some of these antagonists, especially the MAD factors MGA and MNT1, are subject to downregulation in cancer cells, caused by gene deletions and other mechanisms. In addition to inducing genes as a transcriptional activator at E-boxes, MYC promotes transcription via stimulation of transcription elongation, usually by binding to sites in the first gene intron. In this case, MYC promotes the transition of Pol II from an initiated, but “paused” to an actively transcribing state, which is achieved mainly by phosphorylation of the polymerase by CDK7 and CDK9. Capping of mRNAs is likewise stimulated. These functions of MYC may require MAX, but not necessarily consensus E-boxes. To some extent moreover increased transcription by MYC could be an indirect effect, namely the consequence of the increased biosynthesis of many components required for RNA biosynthesis (proteins, RNA, and nucleotides) that forms part of its action. Gene repression by MYC can likewise be achieved by different mechanisms. One mechanism—estimated to apply to 25–40% of MYC-­ repressed genes—involves an interacting protein, MIZ-1. MIZ-1 requires NPM (Nucleophosmin) to act as a transcriptional activator. Increased levels of the ribosomal protein RPL23 (one of many ribosomal components induced by MYC) displace NPM and the co-activating histone acetyltransferase p300 and thereby turn MIZ-1 into a

95

repressor, e.g., at the CDKN2B gene that encodes the cell cycle inhibitor p15INK4B (→5.2). Interactions of MYC with other repressive transcription factors have also been described. MYC moreover induces miRNAs like those in the miR-­ 17 cluster that mediate posttranscriptional downregulation of genes involved in the regulation of cell cycle progression and cell death. Conversely, MYC represses some miRNA such as miR-29, miR-30, and let-7 that tend to impede tumor growth, and especially the miR-200s that regulate the epithelial-mesenchymal transition (see Box 9.2). Transcriptional activation and repression by MYC are associated with changes in chromatin structure. Again, both direct and indirect effects are involved. MYC proteins interact with co-­ activators that modify histones, especially histone acetyltransferases like TIP60 (gene: KAT5), GCN5, CBP, p300, and histone deacetylases like HDAC3 (→8.3). MYC proteins are not pioneer factors, though, meaning that they cannot bind to closed chromatin; rather, they prefer actively transcribed genes and genes with CpG-islands (→8.2). However, several MYC target genes encode chromatin regulators which can mediate long-term activation or repression of genes (and enhancers).

Further Reading Amjadi-Moheb F et  al (2021) Insights into the links between MYC and 3D chromatin structure and epigenetics regulation: implications for cancer therapy. Cancer Res 81:1925–1936 Blume-Jensen P, Hunter T (2001) Oncogenic kinase signalling. Nature 411:355–365 Bretones G et al (2015) Myc and cell cycle control. BBA 1849:506–516 Cascon A, Robledo M (2012) MAX and MYC: a heritable breakup. Canner Res 72:3119–3124 Comoglio PM (2018) Known and novel roles of the MET oncogene in cancer: a coherent approach to targeted therapy. Nat Rev Cancer 18:341–358 Dang CV (2012) MYC on the path to cancer. Cell 149:22–34 Kales SC et  al (2010) Cbl and human myeloid neoplasms: The Cbl oncogene comes of age. Cancer Res 70:4789–4794 Kalkat M et  al (2017) MYC deregulation in primary human cancers. Genes 8:151

96 Klein G (2002) Perspectives in studies of human tumor viruses. Front Biosci 7:d268–d274 Knights V, Cook SJ (2010) De-regulated FGF receptors as therapeutic targets in cancer. Pharmacol Ther 125:105–a17 Kress TR et  al (2015) MYC: connecting selective transcriptional control to global RNA production. Nat Rev Cancer 15:593–607 Larsson LG, Henriksson MA (2010) The Yin and Yang functions of the Myc oncoprotein in cancer development and as targets for therapy. Exp Cell Res 316:1429–1437 Lemmon MA, Schlessinger J (2010) Cell signaling by receptor tyrosine kinases. Cell 141:1117–1134 Mercer KE, Pritchard CA (2003) Raf proteins and cancer: B-Raf is identified as a mutational target. BBA 1653:25–40 Mikkers H, Berns A (2003) Retroviral insertional mutagenesis: tagging cancer pathways. Adv Cancer Res 88:53–99 Papke B, Der CJ (2017) Drugging RAS: know the enemy. Science 355:1158–1163 Pelengaris S, Khan M, Evan G (2002) c-MYC: more that just a matter of life and death. Nat Rev Cancer 2:764–776 Posternak V, Cole MD (2016) Strategically targeting MYC in cancer. F1000 Res 5:408

4 Oncogenes Prior IA et al (2021) The frequency of Ras mutations in cancer. Cancer Res 80:2969–2974 Rickman DS et  al (2018) The expanding world of N-MYC-driven cancers. Cancer Disc 8:150–163 Schaub FX et al (2018) Pan-cancer alterations of the MYC oncogene and its proximal network across the Cancer Genome Atlas. Cell Systems 6:282–300 Schlessinger J (2000) Cell signaling by receptor tyrosine kinases. Cell 103:211–225 Simanshu DK et al (2017) RAS proteins and their regulators in human disease. Cell 170:17–33 Truica MI et  al (2021) Turning up the heat on MYC: progress in small-molecule inhibitors. Cancer Res 81:248–253 Tu WB et  al (2015) Myc and its interactors take shape. BBA 1849:469–483 Wagener C, Stocking C, Müller O (2017) Cancer signaling: from molecular biology to targeted therapy. Wiley-VCH Wilson C, Kanhere A (2021) 8q24.21 Locus: a paradigm to link non-coding RNAs, genome polymorphisms and cancer. Int J Mol Sci 22:1094 Wu YM et al (2013) Identification of targetable FGFR gene fusions in diverse cancers. Cancer Disc 3:636–647 Yaeger R, Corcoran RB (2019) Targeting alterations in the RAF-MEK pathway. Cancer Discov 9:329–341

5

Tumor Suppressor Genes

Key Points • While oncogenes promote tumor development as a consequence of their increased activity or deregulation, tumor suppressor genes have to undergo loss of function to allow tumor development and progression. • Many hereditary cancers result from germline mutations in tumor suppressor genes that can be passed on in families. In most cancer syndromes with a dominant mode of inheritance, one mutant allele of a tumor suppressor gene is inherited. When the second allele as well becomes inactivated by mutation, deletion, recombination with the mutated allele, or by epigenetic repression, cancer development is initiated. In sporadic cancers, two mutations must occur in the two alleles of the tumor suppressor gene within one cell line. Therefore, inherited cancers typically occur at earlier ages and are more often multifocal than sporadic cancers of the same type. Of note, while inheritance takes place in a dominant mode, most tumor suppressors behave as recessive genes at the cellular level. • Loss of a tumor suppressor allele by deletion or recombination often becomes apparent as loss of heterozygosity (LOH) of polymorphic markers, like microsatellites or SNPs, in its vicinity. LOH analysis is a useful method to detect deletions or recombinations in tumors. Recurrent LOH in a region of the genome in a

tumor type may hint at the location of a tumor suppressor gene. • Retinoblastoma has provided the paradigm for tumor suppressor genes that also fits for many other “classical” tumor suppressors. The tumor suppressor gene inactivated in almost all retinoblastomas, RB1, encodes a central regulator of the cell cycle, prominently of the G1→S transition. The RB1 protein also aids in ensuring correct mitotic segregation, affects chromatin structure, and regulates apoptosis. It acts by binding and controlling other proteins, prominently as a corepressor of E2F transcription factors. These activate genes required for entry into the S phase and for DNA synthesis, but E2F1 may also induce apoptosis in certain circumstances. RB1 itself is regulated through phosphorylation by cyclin-dependent kinases (CDKs) which in turn are only active as heterodimers with their regulatory cyclin subunits. • Multiple mechanisms are employed in human cells to regulate the cell cycle and RB1 phosphorylation, including the consecutive induction and proteolysis of several cyclins, the phosphorylation and dephosphorylation of CDKs and the induction, stabilization, and proteolysis of several CDK protein inhibitors. CDK inhibitors comprise the INK4 family which specifically inhibits CDK4 and CDK6 and the CIP/KIP family which inhibits other

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 W. A. Schulz, Molecular Biology of Human Cancers, https://doi.org/10.1007/978-3-031-16286-2_5

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5  Tumor Suppressor Genes

98

•

•

•

•

CDKs. CDK inhibitors can act as tumor suppressors. The most important one in this regard is CDKN2A/p16INK4A, but p27KIP1, p21CIP1, p15INK4B, and p57KIP2 are also relevant. Alterations in cell cycle regulation in different tumors are brought about by either loss of tumor suppressor (like RB1 or CDKN2A) function or oncogene (like CCND1 or CDK4) activation. This example illustrates that many tumor suppressors act antagonistically to respective oncogenes. Another example is the tumor suppressor NF1, a GTPase activating protein (GAP) that limits the activity of RAS proteins. According “pairs” of tumor suppressors and oncogenes can be identified in other signal transduction pathways that regulate cell proliferation and differentiation. Loss of tumor suppressors and activation of the ­corresponding oncogenes have often similar, albeit rarely fully equivalent consequences. Whereas loss of RB1 function directly impinges on cell proliferation and differentiation, loss of tumor suppressors like TP53 promotes tumor formation in a different manner. Inactivation of TP53 compromises the ability of a cell to react appropriately to genomic damage, chromosomal instability, hyperproliferation induced by oncogenes, viral infections, and certain metabolic imbalances. Specifically, loss of TP53 function permits survival and proliferation of cells that accumulate genomic alterations and thereby the emergence of cancers. Several other tumor suppressors, like the BRCA1 and BRCA2 DNA HRR proteins that are defective in familial breast cancers, also act primarily by protecting against genomic instability. MMR components also behave as tumor suppressors. Such “caretakers” can be considered as a different class of tumor suppressors from direct negative regulators of the cell cycle and proliferation-stimulating signal transduction pathways. TP53 is the most frequently altered gene across all human cancers. Several upstream pathways responding to different kinds of genomic instability activate TP53, mainly

through a variety of posttranslational modifications that stabilize the protein. Following its activation, TP53 functions as a transcriptional activator to influence several downstream processes. Prominently, activated TP53 can arrest the cell cycle via induction of cell cycle inhibitors like p21CIP1 or can elicit apoptosis by induction of BAX and other pro-apoptotic proteins. TP53 exerts a range of additional functions that as a rule protect against tumor development. Its action is limited in particular by a feedback mechanism that involves the induction of MDM2 which inhibits TP53 and initiates its proteolytic degradation. • In different human cancers, TP53 is inactivated by different mechanisms. In many carcinomas, one gene copy is inactivated by point mutations in the central DNA-binding domain of the protein, whereas the other one is lost by deletion or recombination with the mutant allele. Nonsense mutations are also not infrequent. In some tumors, MDM2 is overexpressed. The CDKN2A gene encodes not only the CDK inhibitor p16INK4A, but also an activator of p53, p14ARF, in a different reading frame. Therefore, homozygous deletions and certain point mutations in CDKN2A impede the function of both TP53 and RB1. • TP53 and RB1 cooperate to induce cell cycle arrest or replicative senescence in response to checkpoint activation and hyperproliferation. • Many DNA tumor viruses like tumorigenic strains of human papilloma virus (HPV) express oncogenic proteins inactivating both TP53 and RB1, underlining the crucial role of these two proteins in the protection against human cancers.

5.1 Tumor Suppressor Genes in Hereditary Cancers As described in Chap. 3, several recessively or dominantly inherited syndromes caused by defects in DNA repair genes are associated with an increased cancer risk (Table 3.2), such as HBOC and Lynch syndrome. Other cancer syndromes inherited in a dominant mode are not due

5.1  Tumor Suppressor Genes in Hereditary Cancers

99

to defects in DNA repair. These syndromes may predispose to a restricted range of cancers or even to a single tumor type, but a few are rather unselective, e.g., the rare Li-Fraumeni and Cowden syndromes (Table 5.1). Typically, these syndromes are highly penetrant and the lifetime risk of cancer may approach 100%. As a further important characteristic, patients with familial cancers often develop cancers at a significantly lower age than in corresponding “sporadic” cases. For instance, whereas sporadic colon or breast cancers are typically present in patients in their 60s or 70s, familial cases can appear already in the third or fourth decade of life. In addition, patients with inherited cancers may develop multiple cancers of the same type or cancers of different types. Multifocality or bilaterality is obvious in cancers of paired organs such as the breasts, kidneys, or eyes. Patients with FAP (→13.2) or HPRCC (→16.3) can have literally hundreds of individual tumors in their bowel or kidneys, respectively. A theoretical explanation for these properties of hereditary cancers was developed for hereditary retinoblastoma and also applies to other heredi-

tary cancers that are caused by mutations in tumor suppressor genes. Retinoblastoma is a rare tumor occurring in young children. It is composed of incompletely differentiated retinal cell precursors (retinoblasts) that form an expanding cell mass in the back of the eye. The incidence, in general, is around 1 per 20,000 live births, but in some families approximately every second child is affected, indicating an autosomal dominantly inherited disease with high penetrance. In these families, bilateral tumors may develop, which are otherwise extremely rare. While all patients suffering from this disease are children, the cancers in familial cases manifest on average a few years earlier. Retinoblastoma was recognized as a specific disease and was treated by surgery already in the nineteenth century. Attentive surgeons of the time noted that patients cured of retinoblastoma tended to develop other cancers later in life, notably of the bone (osteosarcoma), and that their children were again frequently affected by the disease. Based on the assembled statistical data, in the early 1970s, a model was developed that accounted for these observations.

Table 5.1  A selection of dominantly inherited cancer syndromes in humans Syndrome Retinoblastoma

Gene RB1

Location 13q14

Li-Fraumeni

TP53

17p13.1

Hereditary melanoma and pancreatic cancer Familial adenomatous polyposis coli Cowden

CDKN2A

9p21

APC

5q21

PTEN

10q23.3

BCNS (Gorlin)

PTCH

9p22

Von Hippel-Lindau

VHL

3p25

Hereditary breast and ovarian cancer Lynch (HNPCC)

BRCA1, BRCA2

17q21, 13q12 3p212, p15-­16 10q11.2

MLH1, MSH2, others RET

Multiple endocrine neoplasia type 2 Hereditary renal papillary cancer MET

7q31

Cancer site Eye, bone

Function Tumor suppressor Many organs Tumor suppressor Skin, pancreas, others Tumor suppressor Colon, rectum, others Tumor suppressor Many organs Tumor suppressor Skin, brain Tumor suppressor Kidney, adrenal glands, Tumor others suppressor Breast, ovary (prostate, Tumor pancreas) suppressors Colon, endometrium, Tumor stomach, others suppressors Thyroid and other endocrine Oncogene glands Kidney Oncogene

100

5  Tumor Suppressor Genes

The two-hit (or Knudson) model (Fig.  5.1) assumes that the development of retinoblastoma requires two mutations (“hits”) within one cell line to generate the initial tumor cell clone. In hereditary cases, one hit is already present in the germline of the affected families. Retinoblastoma develops if a single additional mutation takes place in any retinoblast during the critical period of development when these cells proliferate. The probability of a mutation in a specific human gene is in the order of 10-6–10-7 per cell generation. It is thus not unlikely that one or several mutations occur that lead to one or two retinoblastomas. If no mutation is inherited, two (successive) mutations in two cells within the same line are required. The probability of this double accident is very low and it is even lower for two

events in different cell lines within the same person. Thus, in familial cases, tumors are much more likely to arise at all, tend to develop more rapidly (because their expansion begins already after a single hit), and they can be bilateral. In comparison, tumors form much less frequently, on average later, and almost never in both eyes in children without an inherited defective allele. The original mathematical model did not make an assumption on which genes are affected by hit 1 and hit 2. They could be one allele each of distinct genes or two alleles of the same gene. However, the model required that the genes behave recessively at the cellular level. Since oncogenes act in a dominant fashion, the model indicated a different sort of tumor gene characterized by inactivation in cancers. Accordingly,

Fig. 5.1  The two-hit model for retinoblastoma. Right: a single mutation (denoted by the exploding star) does not suffice to cause cancer. This requires the rare event of a second mutation in the same cell line (center).

Since in hereditary cases (left) the first mutation is already present in the germline (top tier), cancers are frequent and may even occur in both eyes

5.1  Tumor Suppressor Genes in Hereditary Cancers

some retinoblastomas occur in patients lacking segments of chromosome 13, with a common region of deletion within band 13q14.1. Deletions of this region were also seen in sporadic cases. Therefore, at least one of the hits leading to retinoblastomas involved the loss of a gene and its function. In fact, the second hit in retinoblastoma regularly inactivates the second allele of the same gene. As tumor formation requires inactivation of its function, this gene was termed a “tumor suppressor.”1 The gene that is defective in the overwhelming majority of retinoblastomas is accordingly known as RB1. It encompasses ≈180 kb of genomic sequence, in which 27 exons code for a 4.7 kb mRNA and a 107 kDa phosphoprotein (also known as pRB or pp110RB1, the apparent molecular size being influenced by phosphorylation). Mutations of this gene are observed in familial as well as in sporadic retinoblastomas and osteosarcomas, but also in sporadic cases of several other cancers including lung, glioma, bladder, breast, and prostate cancer. In familial cases of retinoblastoma, mutations in RB1 are passed on in the germline. Mutations running in different families comprise deletions of various sizes, ranging from cytogenetically detectable (i.e., comprising several Mbp) to single base deletions, small insertions, nonsense and splice mutations, and specific missense mutations that usually alter amino acids in the central portion of the protein, the “pocket” domain. The second RB1 allele can be inactivated in tumors in a separate event by deletions, insertion, or various kinds of point mutation (Fig. 5.2). In a few cases, an entire chromosome 13 is lost, e.g., by mitotic nondisjunction, more frequently, large deletions obliterate segments including 13q14.1. In some cases (only in childhood cancers), the otherwise intact second allele is not transcribed as a consequence of hypermethylation of the RB1 promoter (→8.3). Another important mechanism involves the replacement of the intact allele by a defective Historically, the designation tumor suppressor was also derived from other types of experiments, including cell fusion experiments.

101

copy. This can again occur by several means. In some tumors, the cells contain two identical chromosomes 13, either by duplication of the single chromosome remaining after loss of the other or by misdistribution of chromosomes during mitosis. In other cases, recombination between the two different chromosomes leads to replacement of larger or smaller regions in one chromosome by sequences from the other one and specifically to conversion of the intact RB1 allele to a defective version. Several of the above mechanisms, such as larger deletions, unequal recombinations, or recombination followed by chromosomal loss, leave the cell not only without a functional allele of the RB1 tumor suppressor gene but also abolish other differences between the two homologous chromosomes. As a consequence, base variants in single nucleotides (SNPs) or microsatellite sequences disappear, as heterozygous sequences become homozygous (Fig.  5.2). This phenomenon, known as LOH (see Fig. 2.8), therefore marks regions in a tumor genome where deletions or illegitimate recombination have taken place. Since such processes are often involved in the inactivation of tumor suppressor genes, the observation of recurrent LOH in one region in a particular tumor type hints at the location of a tumor suppressor gene. Many further “classical” tumor suppressors also fit the two-hit model well (Table 5.1), among them APC in colon adenocarcinoma (→13.2), VHL in clear-cell renal cell carcinoma (→16.4), and CDH1 in one type of gastric cancer (→18.3).2 Others follow the model at least partly. BRCA1 and BRCA2 behave according to the model in some cancer types like breast cancer (→19.6), but not generally. Conversely, several tumor suppressor genes are never mutated in the germline, but both copies may become inactivated in sporadic cancers. It is also conceivable that mutation of one copy of a tumor suppressor gene could be sufficient to initiate the development of cancer, albeit not as efficiently as the inactivation of both

1 

Such tumor suppressors are called “classical” in this text, note that this designation is not universally used. 2 

5  Tumor Suppressor Genes

102

a

A

RB1

RB1 B

b

(1)

(3)

A

A

a

RB1

RB1

b

B

B

(5) (4)

(2)

A RB1 B

a RB1 b

A RB1 B

a RB1 B

A RB1 B

a RB MRB1 b

Fig. 5.2  Mechanisms causing inactivation of the second RB1 allele. (1) Loss of chromosome 13 carrying the intact allele; (2) deletion; (3) independent mutation; (4) recombination; (5)

promoter hypermethylation. Mechanisms (1), (2), and (4) are associated with LOH, cf. Fig. 2.8. The mutated first allele is marked by an exploding star. A/a and B/b denote allelic markers flanking the RB1 gene

alleles. This situation is called “haploinsufficiency.” It is discussed for several tumor suppressors, including PTCH1 in basal cell carcinomas of the skin (→12.3) and PTEN in a variety of cancers (→6.3). In the case of inherited mutation in PTCH1 and PTEN, the case for haploinsufficiency is supported by the observation that the patients may have developmental abnormalities unrelated to cancer. Thus, the dependency on a tumor suppressor may be gradual; this is probably true for PTEN (see Box 6.1). An entirely different situation is provided by the (rare) germline mutations that activate oncogenes to cause dominantly inherited cancer syndromes. While this situation can be created experimentally in mice with high efficiency, e.g., by introduction of a mutant Ras gene, in humans, very few inherited cancers are caused by ­activated oncogenes. Multiple endocrine neoplasia type 2 is caused by mutations in the RET proto-­oncogene and predisposes to cancers of endocrine glands, notably the thyroid and adrenal glands. RET is a receptor tyrosine kinase and the inherited mutations lead to its constitutive activation. Comparable mutations in the MET gene, which

likewise encodes a receptor tyrosine kinase, cause papillary renal cell carcinoma, although tumor development requires duplication of the mutant oncogene (→16.3). The fact that tumor suppressor mutations rather than oncogene mutations predominate in (dominantly) inherited human cancer may reflect better protection of human cells against oncogenic transformation compared to rodent cells or a pronounced sensitivity of human development to disturbances by mutated oncogenes. Tellingly, some inherited developmental syndromes are caused by germline RAS mutations, but never in the hotspot codons 12, 13, and 61. Similarly, congenital oncogenic mutations in PIK3CA always occur in a mosaic fashion leading to overgrowth of specific body parts.

5.2 RB1 and the Cell Cycle The product of the RB1 gene, the RB1 phosphoprotein, is most of all a central regulator of the cell cycle (Fig. 5.3). It controls the G1→S phase transition by binding to E2F1, E2F2, or E2F3

5.2  RB1 and the Cell Cycle

103 P P P

Cyclin D RB1

P RB1

Cyclin E

Corepressor CDK4

E2F

Coactivator

CDK2 E2F

DP1

G0/G1

Fig. 5.3  Function of RB1  in the regulation of the cell cycle at the G1 → S transition. DP1 is a heterodimer partner of E2F proteins. Corepressors usually encompass or interact with histone deacetylases

transcription factors as a corepressor and thereby repressing the transcription of genes needed for entry into S phase. Binding to E2F factors is alleviated and repression is relieved when RB1 becomes hyperphosphorylated3 towards the end of G1. Two successive phosphorylation events are essential to inactivate RB1. The first phosphorylation is normally performed by a CDK4/Cyclin D holoenzyme and the second one subsequently by the CDK2/ Cyclin E holoenzyme. Hyperphosphorylated RB1 is inactive as far as G1/S cell cycle regulation is concerned, but likely exerts other functions in the S phase, where it may be involved in chromatin organization, and during mitosis, where it may help to organize chromosome segregation. Following mitosis, RB1 is partly dephosphorylated and the hypophosphorylated state is restored. In this fashion, RB1 switches

DP1

S

(HDACs) and coactivators with histone acetyltransferases (HATs), respectively. The functions of HDACs and HATs are further explained in Sect. 8.3

between hypophosphorylated and hyperphosphorylated states during the cell cycle. Loss of RB1 function therefore upsets cell cycle regulation and can lead to unrestrained cell proliferation. Specifically, in the absence of RB1 immature cells like retinoblasts may not spend sufficient time in G1 to enter a differentiated state or may not be able to establish a stable quiescent state (i.e., enter G0). Worse, since RB1 may also be required for proper chromatin structure and chromosome segregation, cells lacking functional RB1 tend to become genomically unstable and may acquire additional alterations that favor tumor progression. At least one mechanism may protect against loss of RB1 function: Overactivity of E2F factors, particularly E2F1, can induce apoptosis. The mechanism of cell cycle regulation sketched so far is in fact much more complex, even when only the G1→S transition is considered (see also Box 5.1) and comprises several 3  The RB1 protein is almost always phosphorylated at layers of control (Fig.  5.4). The CDK protein some sites; its phosphorylation therefore varies between hypo- and hyperphosphorylation rather than between non-­ kinases are only active when bound to their (positive) regulatory subunits; these are D-Cyclins (1, phosphorylated and phosphorylated.

5  Tumor Suppressor Genes

104 p15 p16 CDK4

Cyclin D destruction

Cyclin D

p27 CDK2

E2F

p21 Cyclin E

RB1

Cyclin B destruction

E2F

G1 M Cyclin B

P S

RB1

CDK2 Cyclin A

G2

p21

CDK1

Cyclin A destruction CDK1 Cyclin A+B

Cyclin A

CDK1

Fig. 5.4  Three layers of cell cycle regulation. The figure focuses on the G1→S transition, which is the main point of regulation in human cells (mechanisms regulating G2→S transition are used in specific cells). The inner layer consists of the RB1 phosphorylation cycle which determines E2F activity. It is dependent on the second layer formed by the CDK/cyclin cycle (CDK1 is also

known as CDC2). This second layer is controlled by a third one involving phosphorylation and dephosphorylation of the CDKs and CDK inhibitor proteins. This layer interacts mutually with the inner layers and is influenced by various mitogenic and antiproliferative signals

2, or 3) for CDK4 and CDK6 and Cyclin E for CDK2 (in G1). These cyclins fluctuate in a coordinated fashion over the course of the cell cycle. Cyclin D expression in particular is directly stimulated by growth factors and other exogenous signals; Cyclin E expression is as well influenced by signal transduction pathways (→6.4). Moreover, CDKs are only active, if they are phosphorylated at multiple sites in a specific pat-

tern. Phosphorylation at a specific threonine residue by the CDK activating kinase (CAK, identical to CDK7) in complex with its regulatory subunit Cyclin H is activating, while phosphorylation at two other threonine residues is inhibitory. For activation of the CDKs, the phosphate groups at these latter sites need to be removed by CDC25 phosphatases, which likewise respond to external signals (→6.4).

5.2  RB1 and the Cell Cycle

Box 5.1 Retinoblastoma-Like Proteins and Cell Cycle Regulation

Like RB1, the two related “pocket proteins” p107 and p130 (genes RBL1 and RBL2) are involved in the control of the cell cycle and of cell differentiation. However, they appear to lack the ability to establish repressive chromatin structures and the additional functions in the control of genomic stability ascribed to RB1. Cell differentiation depends on RB1 or its homologs to various extents. These differences may give a reason why RBL1 and RBL2 are not common tumor suppressors. Conversely, redundancies in the functions of the three pocket proteins may explain why RB1 loss contributes to the development of cancers in many tissues, but to different degrees. Like RB1, p107 and p130 interact with E2F transcription factors. However, whereas RB1 binds to and inhibits transcription activation by E2F1, E2F2, and E2F3, p107 and p130 interact with the transcriptional repressors E2F4 and E2F5. In this fashion, the three pocket proteins cooperate to repress genes required for the transition from G1 to S-phase of the cell cycle. In addition, p107 and p130 regulate the expression of G2/M genes. To this end, they cooperate with the “DREAM” complex formed from the proteins LIN52, LIN9, LIN54, and LIN37, which is anchored to chromatin by RBBP4. Via E2F4/DP p107 or p130 bind to CDE sites (with the consensus sequence GGGCGC), while the DREAM complex, linked via LIN52 to one of the pocket proteins, recognizes CHR sites (TTTGAA) in the vicinity of CDE sites. In the course of the G2 phase, p107 and p130 are successively replaced by B-MYB and FOXM1 and G2/M-specific genes become transcribed. B-MYB is activated through phosphorylation by CDK2. Additional transcription factors support the expression of G2/M-specific genes, including NF-Y (by binding to CCAAT boxes), NRF1, and CREB1. Notably, while rarely

105

acting as true oncogenes (in a strict definition), B-MYB and FOXM1 are overexpressed in many cancers, often correlating with progression and worse outcome. Their overexpression may of course simply signify a higher fraction of cells in G2/M and thus more rapid proliferation, but the two transcription factors may have additional functions. FOXM1 in particular appears to facilitate metastasis in some cancer types. Fischer M, Müller GA (2017) Cell cycle transcription control: DREAM/MuvB and RB-E2F complexes. Crit Rev Biochem Mol Biol 52:638–662

A further layer of control is provided by protein inhibitors of the CDKs (Table 5.2). There are two classes of CDK inhibitors, CIP/KIP and INK proteins. The first class comprises the p21CIP1, p27KIP1, and p57KIP2 proteins, the second class encompasses p15INK4B, p16INK4A, p18INK4C, and p19INK4D. The corresponding genes are systematically designated CDKN1A–CDKN1C and CDKN2A–CDKN2D. All proteins are named according to their molecular weights. The function of the INK4 (inhibitor of kinase 4) proteins is straightforward: they compete with d-Cyclins for binding to CDK4 and CDK6 and thereby block kinase activity. The function of the CIP/KIP proteins (CDK/kinase inhibitory proteins) is more complicated. At high concentrations, they inhibit the activity of CDKs in general, but whereas INK4s inhibit CDK monomers, CIP/ KIPs block the CDK-Cyclin holoenzymes and only at higher concentrations. At lower concentrations, they stimulate the assembly of CDK-­ Cyclin complexes. On the one hand, this mode of action helps to coordinate the cell cycle in proliferating cells, in particular through p27KIP1: Until late G1 phase, p27KIP1 remains bound to the CDK2/Cyclin E complex delaying its activity until CDK2 activity is required to inactivate RB1 fully. At this stage, the p27KIP1 inhibitor is phosphorylated and rapidly degraded (cf. 6.4). On the other hand, high levels of CIP/KIP inhibitor proteins arrest the cell cycle (at several points) in cells that are not supposed to proliferate.

5  Tumor Suppressor Genes

106 Table 5.2  Inhibitor proteins of cyclin-dependent kinases CDK Inhibitor p21CIP1 p27KIP1 p57KIP2 p16INK4A p15INK4B p18INK4C p19INK4D

Gene CDKN1A CDKN1B CDKN1C CDKN2A CDKN2B CDKN2C CDKN2D

Location 6p21.2 12p13.1-p12 11p15.5 9p21 9p21 1p32 19p13

The different CDK inhibitors respond to different signals that can induce cell cycle arrest, allowing fine-tuned cellular responses to different signals. For instance, in some cell types, p15INK4B is induced by inhibitory growth factors like TGFβ (→6.6). High levels of this inhibitor protein dissociate Cyclin D/CDK4 complexes, blocking CDK4. They also redirect any p21CIP1 or p27KIP1 in the complex towards CDK2/Cyclin E thereby blocking this kinase as well. By comparison, p16INK4A has a long half-life and accumulates gradually in continuously proliferating cells until its concentration becomes high enough to slow down the cell cycle or arrest it irreversibly (→7.2). The p21CIP1 inhibitor accumulates in a similar fashion during frequent replication, but also in response to various growth factors and especially to DNA damage (see next section). Together, p16INK4A and p21CIP1 establish the proliferation arrest upon induction of cellular senescence (→7.2). p57KIP2 is expressed in a restricted range of cell types, most prominently during embryonic development where the protein appears to help establish a terminally differentiated state in specific tissues (Box 8.3). The gene encoding p57KIP2, CDKN1C, is normally expressed only from the maternally inherited allele, i.e., it is imprinted (→8.4). In summary therefore RB1 forms a node in the cell cycle regulation network which ensures that cell proliferation occurs only in response to proper signals, e.g., following stimulation by growth factors via the MAPK and PI3K signaling pathways (→6.4). It is easy to conceive how this regulation network becomes disrupted by loss of function of the central RB1 protein. Alternately to loss of RB1 function itself, other components of the regulatory network may be affected in human cancers. Of note, not all these changes may be equivalent. Often, the loss of RB1 itself appears to carry the most severe consequences, as one might guess.

CDK inhibited Several CDK2 Several CDK4 CDK4 CDK4/6 CDK4/6

Inducers (selected) TP53, growth factors TGFβ Cell differentiation Overproliferation TGFβ Cell differentiation Overproliferation

Overexpression of D-Cyclins or CDK4 as a consequence of gene amplification or mutations of CDK4 that make it unresponsive to CDK inhibitors may substitute for RB1 inactivation in some cancers. Amplifications of the CCNE gene are found in specific cancer types. Alterations in CDK inhibitors can be detected in a broad variety of human cancers. Prominently, the CDKN2A gene is inactivated by point mutation, promoter hypermethylation, or homozygous deletion in a wide range of human cancers. CDKN2A is in fact another classical tumor suppressor gene, since as a rule both alleles are inactivated in such cancers, and germline mutations of the gene have been found in families prone to pancreatic cancers and melanoma in a syndrome known as familial atypical multiple mole melanoma syndrome (FAMMM). Mutations or deletions of the genes encoding CDK inhibitors other than p16INK4A are much less frequent in human cancers. The CDKN2B gene encoding p15INK4B is located within 40 kb of CDKN2A (cf. Fig.  5.9) and is often deleted together with its neighbor. Inactivation of this gene may be most crucial in certain leukemias. CDKN2C and CDKN2D are only mutated in selected cancers. Inactivation of CDKN1C, in accord with its more circumscribed expression, may be relevant in a smaller range of cancers. Mutations in the CDKN1A and CDKN1B genes encoding p21CIP1 and p27KIP1 are infrequent. Instead, downregulation of their expression is highly prevalent in many different cancers. Their downregulation often indicates tumor progression and that cancers may take a more aggressive clinical course. It is not fully understood, why these genes are rarely mutated in cancers. Presumably, the two inhibitors remain necessary for proper cell cycle progression and stress responses in many cancer cells. However, their

5.3  TP53 and the Control of Genomic Integrity

regulation may be substantially altered. For instance, p27KIP1 may be downregulated as a consequence of increased PI3K/AKT signaling or MYC activity, or due to overexpression of the ubiquitin ligase SKP2 that directs its degradation (→6.4). SKP2 overexpression can be caused by the gain or amplification of its gene at 5p13.2. RB1 is a tumor suppressor gene primarily because its inactivation removes an essential point of regulation of the cell cycle that is required for coordinated proliferation and differentiation during the development and maintenance of normal tissues. Its loss of function advances a tumor cell directly towards unrestricted proliferation and diminished dependence on extracellular signals. Similar effects are caused by the loss of other negative regulators of the cell cycle like CDKN2A/p16INK4A. Conversely, positive regulators of the cell cycle like Cyclin D1 and Cyclin E may act as oncogenes if they become deregulated as a consequence of amplification of their respective genes (CCND1 and CCNE). An interesting, but largely unanswered question is why germline RB1 mutations only lead to a rather narrow spectrum of (childhood) cancers, despite the largely ubiquitous expression of RB1 and its basic function in cell cycle regulation. In fact, somatic RB1 inactivation occurs in many cancer types but is likewise far from universal. CDKN2A inactivation is much more widespread in comparison (see below). Part of the answer may be that related proteins (Box 5.1) and other control mechanisms of the cell cycle may provide additional barriers to tumor development in many tissues.

5.3 TP53 and the Control of Genomic Integrity Mice lacking Rb1 are not viable because of defects in the development of several tissues. In contrast, mice lacking the tumor suppressor Tp53 are overall viable. However, these animals, and even heterozygous mice with only one functional allele, rapidly succumb to tumors within the first few months of life. In the rare human disease, Li-Fraumenisyndrome (Table 5.1), too, one mutant copy of the TP53 gene is inherited. The affected persons develop various types of tumors in different tissues,

107

including blood, lymphoid organs, soft tissues, the nervous system, and various epithelia, likewise often early in life.4 In accord with the two-hit model, all tumors contain point mutations inactivating the second TP53 allele, have lost the segment of chromosome 17p where the gene resides, or have undergone LOH in that region. The TP53 gene is also inactivated in many different sporadic cancers in man; it is overall the most frequently mutated gene across human cancers. Intriguingly, the main function of the TP53 protein appears to reside in the prevention of genomic instability and cancers, making it literally a tumor suppressor. In comparison, the tumor suppressor function of RB1 appears as more of a byproduct of its normal physiological function as a regulator of cell proliferation and differentiation (but see below). The approximately 20 kb TP53 gene on chromosome 17p13.1 comprises 11 exons transcribed predominantly into a 2.2 kb mRNA, from which a 39 kD protein is translated. On SDS-PAGE gels, it shows up at 53 kDa, which leads to its name. The TP53 protein (often simply: p53) has a structure typical of transcriptional activators (Fig. 5.5). It is composed of a central DNA-­binding domain, an N-terminal region comprising two transcription activation domains and a proline-rich region, and a C-terminal domain which contains the nuclear localization signal, mediates tetramerization, and regulates DNA-­binding. Of note, several splice variants encode variants of the protein that lack one or the other domain (see Fig. 2.3); these are functionally important in specific circumstances. TP53 functions as a transcriptional activator at several hundred genes. The range and selection of target genes depend on the expression level and the pattern of posttranslational modifications at the protein. At many genes, TP53 binds as a tetramer to specific symmetric binding sequences (half site consensus: RRRCWWGYYY) in the promoter or in the first intron. The strength of transcriptional activation by TP53 decreases at distances beyond 1 kb from the TSS, but TP53 may nevertheless activate some genes by binding to their enhancers. TP53 can also repress transcription, but mostly through indirect mechanisms. One mechanism The tumor spectrum differs however between mice and humans with Tp53/TP53 germline mutations. 4 

5  Tumor Suppressor Genes

108 DNA binding 1

2

3

4

5

6

TP53 7

8

9

10

11

20 kb Sequence features

TAD

PXXP

Basic domain

C-ter TP53

Mutation hotspots

R175

G245

R249

R248

R282

NLS/NES

R273

Transactivation Specific DNA binding Domains

Tetramerization Regulation Unspecific DNA binding NLS 1

100

200

300

393

aa

Fig. 5.5  Structure and functional domains of TP53. Top: Organization of the TP53 gene. The introns are not to scale. The central DNA-binding domain is encoded by exons 4–8. Bottom: Features of the TP53 protein. C-ter:

C-terminal interaction domain; NLS/NES: nuclear localization/export signals (aa 305–322 and 340–351, respectively); PXXP: proline-­rich domain

involves the p21CIP1 protein that is prominently and strongly induced by TP53. It inhibits various CDKs to arrest the cell cycle via RB1. CDK inhibition results moreover in hypophosphorylation of the RB1-related proteins p107 and p130 (RBL1 and RBL2). RBL1 and RBL2 associate with E2F4 and E2F5 (repressors from the E2F transcription factor family) to stabilize the repressive DREAM complex (see Box 5.1) at genes required for cell cycle progression into mitosis (like CCNB1, CCNB2, and CDC25A). In other cases, TP53induced miRNAs like miR-34 mediate posttranscriptional downregulation of gene expression. Through various target genes (see Table 5.3 for examples), TP53 coordinates the cellular response to many kinds of damage to the genome, including DNA double-strand breaks induced by chemical mutagens and ionizing radiation, as well as to a variety of cellular stresses, such as guanine nucleotide imbalance, hypoxia, viral infection, retrotransposon reactivation, and oncogene-induced

Table 5.3  A selection of TP53 target genes Process Cell cycle regulation DNA repair Apoptosis Growth factor signaling Metabolism Angiogenesis Feedback

Target genes CDKN1A, GADD45A XPC, DDB2, RRM2B, POLH BAX, PUMA, NOXA, TP53AIP1, APAF1 IGFBP3, PTEN TIGAR, SLCA2, SLCA4 TSP1 MDM2, PPM1D

hyperproliferation (Fig.  5.6). Upon activation, TP53 can induce cell cycle arrest or apoptotic cell death as well as other effects (see below). TP53 is moreover involved in the regulation of stem cell plasticity and replicative senescence (→7.2). TP53 activity is regulated mostly posttranslationally by phosphorylation and other modifications of the protein (Fig. 5.7) that alter its stability and activity. Increased transcription of the TP53

5.3  TP53 and the Control of Genomic Integrity Fig. 5.6  TP53 as a network node. Only a part of the huge network is shown. Inputs are framed in red, outputs are framed in green. See main text for details

109 Hyperproliferation

DNA damage Replication stress

Ribonucleotide depletion

Telomere shortening

Metabolic and oxidative stresses TP53 Metabolic adaptation

Feedback ragulation

Signaling to stroma

DNA repair Cell cycle arrest

Apoptosis

363

42

1

393 aa

N-terminal domain

6

15

CK1

DNAPK ATM ATR

18

20

CHK 1/2

DNA damage

C-terminal domain

23

37

CAK

46

81

p38MAPK

JNK

315 317 371 373 376 378 382 386 392

CDK2 PCAF

Cell cycle

GFs

Cell cycle

PKC

PKR

SUMO

CBP/p300

GFs

CK2

DNA damage

dsRNA

Cell cycle

Fig. 5.7  Posttranslational regulation of TP53. Note that only the N-terminal and C-terminal domains of the protein are shown and only a selection of modifications, mostly phosphorylations (in orange) except at

Lys320 and Lys373 (acetylation) and 386 (sumoylation). The modifying enzymes are boxed. Some of the signals eliciting the modifications are indicated

gene contributes to its activation under specific conditions, e.g., following exit from G0 or due to induction by interferons. Under normal circumstances, TP53 undergoes rapid turnover, with a half-life of ≈10–20 min. The MDM2 protein is a major factor responsible for this short half-life. It binds to the N-terminal domain of TP53 blocking its transcriptional activity and initiating its export

from the nucleus. Furthermore, MDM2 acts as a specific E3 ubiquitin ligase for TP53, which following poly-ubiquitination at its C-terminal domain, is rapidly degraded by the proteasome. A related protein, MDM4, stabilizes MDM2 and blocks transcriptional activation by TP53 by binding to its N-terminal domain. Ubiquitination of TP53 by MDM2/MDM4 is counteracted by

5  Tumor Suppressor Genes

110

specific deubiquitinases like HAUSP (gene USP7) which accordingly stabilize TP53. Different pathways signal the various types of DNA damage and cellular stress to TP53 through various posttranslational modifications (Fig. 5.7); more than 300 different modifications have been described overall. Double-strand breaks induced by ionizing radiation activate ATM or DNA-­dependent protein kinase, while extensive UV damage and persisting singlestranded DNA rather activate ATR (→3.3). These kinases phosphorylate TP53 at Ser15 and Ser37. Downstream of ATM and ATR, the checkpoint protein kinases CHK1 and CHK2, which respond to DNA damage and additionally to mitotic disturbances, phosphorylate TP53 Ser20. Phosphorylation at Ser15, Thr18, or Ser20, in particular, blocks the interaction of TP53 with MDM2 and increase the half-life of the protein. Phosphorylation at Ser46 by ATM or HIPK2 (Homeodomain interacting protein kinase 2) may shift the TP53 response towards apoptosis. Moreover, phosphorylation at most sites in the N-terminal transactivation domain enhances the strength of TP53 as a transcriptional activator and its interaction with co-­ activators like the p300 histone acetyltransferase. Phosphorylation at Ser392 by the double-strand RNA-dependent protein kinase PKR activates TP53  in response to viral infections. Conversely, phosphorylation at different sites, e.g., at Thr18 by CK2 (Casein kinase 2), at several more C-terminally located serine residues (371, 376, and 378) by PKC and at Ser315 by CDK2 rather restrain TP53 activity. Phosphorylation by p38MAPK and JNK (→6.2) at Ser46 and Thr81 appear to modulate the proapoptotic function of TP53. In the C-terminal regulatory domain, sumoylation at Lys386 helps to guide TP53 within the nucleus. Acetylation at Lys320, Lys373, and Lys382 modulates DNAbinding, transcriptional activation, and nuclear localization. Acetylation in the DNA-binding domain at Lys120 and Lys164 may modulate TP53 activity on specific genes, likely promoting apoptosis over cell cycle arrest. Furthermore, methylation of several lysine residues in the C-terminal domain modulates transcription activation.

CDKN2A

p16INK4A

p14ARF

CDK4

MDM2

RB1

TP53

E2F1

Fig. 5.8  Regulation of TP53 and RB1 by proteins from the CDKN2A locus. See main text for details

Inappropriate cell proliferation, induced, e.g., by oncogenic RAS proteins, also activates TP53, predominantly through an indirect mechanism (Fig.  5.8). Increased proliferation is associated with increased activity of E2F transcription factors such as E2F1. In addition to genes required for cell cycle progression, E2F1 activates the transcription of p16INK4A mRNA from the CDKN2A gene, but also a second promoter in the gene directing the expression of a 14 kDa protein in an alternative reading frame, which was accordingly named p14ARF (p19Arf in mice). The p14ARF protein inhibits MDM2. Thus, inappropriate proliferation signals induce p14ARF which blocks MDM2 leading to stabilization and activation of TP53. The CDKN2A gene is thus in fact a double locus with two common exons, of which exon 2 codes for parts of both p16INK4A and p14ARF, but in different reading frames (Fig. 5.9). Alterations in this locus may therefore compromise the functions of both TP53 and RB1 (Fig.  5.8). While many mutations in exon 2 inactivate both ­proteins, the most efficient mechanism for complete CDKN2A inactivation is by homozygous deletion. This relationship accounts for the observation that CDKN2A is the gene most frequently subject to homozygous deletions across all cancer types.

5.3  TP53 and the Control of Genomic Integrity Fig. 5.9 Organization of the CDKN2A locus at 9p21. See main text for details

111

CDKN2B p15INK4B

E1

E2

centromere

CDKN2A p14ARF1

E1 β

p16INK4A

E1α

E2

E3

telomere 40 kb

Following its activation by one of the above pathways, TP53 can in principle induce multiple and diverse cellular responses (Fig.  5.6), which include cell cycle arrest, DNA repair, apoptosis, ferroptosis, autophagy, changes in cell metabolism, altered secretion of growth factors (particularly of anti-angiogenic factors) and, last not least, its own inactivation by MDM2. Several factors influence which responses are induced and to which extent, such as the cell type, the type and degree of DNA damage or cellular stress, as well as competing signals. Following TP53 activation, cell cycle arrest and apoptosis occur alternatively to each other, and the fate of a cell may sometimes simply depend on which response is induced more rapidly. The duration of TP53 activation also matters. The ultimate outcome is mediated through the pattern of posttranslational modifications, including the basal and induced pattern of phosphorylation of TP53 by multiple kinases (Fig. 5.7). For instance, ATM/CHK2 tend to favor apoptosis, whereas ATR/CHK1 tend to favor cell cycle arrest, by phosphorylating and inactivating the CDK phosphatases CDC25B and CDC25C in addition to TP53. This differential response may relate to the activation of the kinases by double-strand breaks and singlestranded DNA, respectively. Overall, several hundred genes can be activated or repressed by TP53 in various circumstances. Therefore, many different proteins act downstream of TP53 as mediators of its action in human cells with different outcomes. Arrest of the cell cycle by TP53 is mediated through rapid and strong induction of cell cycle inhibitory proteins, prominently p21CIP1, as well as through repression of several proteins required

for the G2→M transition. This response appears to be common to all cell types and to follow the activation of TP53 by a variety of signals. Importantly, cell cycle arrest induced by TP53 may be irreversible and take the form of replicative senescence (→7.2), especially if RB1 is concomitantly activated. Another common response to TP53 activation is the induction of GADD45, a protein involved in DNA repair and in the maintenance of activated checkpoints. In addition, activated TP53 induces several factors actively contributing to DNA repair, such as the NER proteins XPC and DDB2 (also known as XPE), Ribonucleotide reductase subunits, and the repair DNA polymerase POLH. Induction of apoptosis by TP53 appears to proceed via different routes in different cells, most broadly via induction of BAX, which thereby overcomes inhibition by its homolog BCL2 to activate apoptosis at the mitochondrial membrane (→7.4). Conversely, BCL2 is repressed by TP53. Several further proteins of the “mitochondrial” or “intrinsic” apoptotic pathway (→7.4) can be induced by activated TP53, including NOXA, PUMA, p53AIP, and APAF1. In parallel, TP53 increases the sensitivity towards exogenous apoptotic signals, e.g., by increasing expression of the death receptor CD95 encoded by the TNFRSF6 gene (→7.4). This variety of redundant pro-apoptotic signals might have evolved as interlinked backup mechanisms to ensure that apoptosis cannot easily be circumvented if TP53 is fully activated. Alternatively, this variety may allow a better choice between apoptosis and survival (with cell cycle arrest) depending on further intracellular and exogenous signals and on the cell type.

112

Several signal transduction pathways (→6) modulate the cellular response to TP53. Whereas increased activity of ERK and JNK MAPK pathways tends to activate TP53 and sustained activity of the WNT pathway in cancers like colorectal cancer (→13.4) and liver cancer (→17.2) appears to be incompatible with functional TP53, activation of the PI3K and NFκB pathways in cancers tends to counteract the effects of TP53. In the case of the PI3K pathway, this may be prominently due to activation of MDM2 by the AKT kinase and to increased “survival signaling” at large, including induction of antiapoptotic proteins like BCL2 (→6.3). Conversely, TP53 strongly induces IGFBP3, a secreted protein that binds and sequesters IGFs (insulin-like growth factors), which activate PI3K signaling particularly strongly. In addition, TP53 induces PTEN, the major inhibitory protein in the PI3K pathway. Autonomous activation of the PI3K pathway, e.g., by loss of PTEN function, therefore obliterates this branch of TP53 action. In addition to impeding the pro-apoptotic effect of TP53, phosphorylation of p21CIP1 and p27KIP1 following PI3K activation impair growth arrest induced by TP53. Overall therefore the PI3K pathway limits the effects of TP53 activation on cell growth and survival. In comparison, NFκB signaling (→6.7) appears to more selectively impede activation of apoptosis by TP53 by inducing antiapoptotic proteins like BCL-XL and IAPs (→7.4) as key elements.5 Since the AKT kinase also phosphorylates and activates NFκB factors, PI3K and NFκB signaling may combine to antagonize the induction of cell death by TP53. Conversely, TP53, NFκB, and FOXO3 factors (which are negatively regulated by AKT phosphorylation) target some of the same genes to protect cells following stress. TP53 effects on metabolism can also be interpreted as cell-protective, namely aimed at allowing repair and recovery following cellular damage and cell cycle arrest. By direct and indirect mechNFκB activation, like TP53 activation, can be a response to cellular stress, but rather favors cell survival and additionally induces inflammatory factors (see Sect. 6.7). The two stress responses thus cooperate in some situations, but act antagonistically in others. 5 

5  Tumor Suppressor Genes

anisms, activated TP53 can influence glutamine and glucose metabolism and lipid biosynthesis (→1.4), enhance autophagy and strengthen protection against oxidative stress. One prominent direct target of TP53 is the ingenuously named TIGAR gene (for TP53-induced glycolysis regulating phosphatase). It encodes a fructose bisphosphatase capable of hydrolyzing both fructose-1,6-biphosphate as well as fructose-­2,6-­ biphosphate, thereby redirecting glucose flux from glycolysis towards the pentose phosphate pathway that generates NADPH for cell protection and ribose sugars for nucleotide biosynthesis. In addition, TP53 diminishes glucose uptake by indirect downregulation of the GLUT1 (gene: SLCA2) and GLUT4 (gene: SLCA4) transporters. A further group of genes induced or repressed by TP53 functions in the communication with neighboring tissue cells, such as endothelial cells. For instance, TP53 induces Thrombospondin (TSP1) which inhibits the proliferation of endothelial cells and thereby angiogenesis (→9.4). Finally, TP53 limits its own action through feedback mechanisms. The MDM2 gene is a direct transcriptional target of TP53 and becomes relatively rapidly induced following TP53 activation. Accumulating MDM2 blocks transcriptional activation by TP53 and causes its degradation. The efficacy of this mechanism is dramatically illustrated by an experiment in mice. Mice lacking Tp53 are in general viable, although they die of tumors at an early age. In contrast, mice lacking Mdm2 die in utero from widespread apoptosis, especially if exposed to genotoxic stress. Additional knockout of the Tp53 gene largely corrects this defect. In addition to MDM2, several further factors limit the duration and intensity of TP53 activation, including the MDM2 homolog MDM4 (also: MDMX) and various phosphatases. The protein phosphatase WIP1 (gene: PPM1D) reverses particularly the crucial phosphorylation in the amino-terminal TP53 domain and is linked to TP53 in a similar feedback loop as MDM2 (Table 5.3). In summary, TP53 can be regarded as the central node in an important network which regu-

5.3  TP53 and the Control of Genomic Integrity

lates the cellular response to many kinds of genomic damage and many types of cellular stress (Fig.  5.6). This explains of course why defects in TP53 are so widespread in human cancers. Cancers lacking functional TP53 will tolerate more DNA damage, including DNA strand breaks and aneuploidy, will condone inappropriate proliferation signals and nucleotide imbalances (that favor mutations), and will less easily enter replicative senescence or undergo cell death. Compared to cancers of the same type with wild-type TP53, cancers with mutant TP53 therefore tend to accumulate more genomic alterations and often run a higher risk of progression, spontaneously or under treatment with genotoxic cancer drugs. Of note, like RB1, TP53 is part of a (small) protein family (see Fig. 2.3). Its homologs TP63 and TP73 have mostly different functions from TP53, but TP73—like TP53—can elicit apoptosis, by inducing a similar range of proapoptotic genes. In specific circumstances, it may therefore serve as a backup to TP53. For this reason, presumably, TP73 is subject to inactivating mutations and gene deletions in specific cancer types. A prominent function of TP63 is the maintenance of the basal cells in squamous epithelia. Its function is therefore especially relevant in squamous cell carcinomas (→12.2). In different human cancers, the inactivation of TP53 function is brought about by several mechanisms, to different extents (Table 5.4). Missense mutations plus LOH: The most common mechanism of TP53 inactivation in many different human cancer types consists of missense mutations in one allele and loss of the second functional allele by deletion or recombination, resulting in LOH at 17p13. It is likely favorable for the two changes to occur in this order, since some missense mutation in the first allele may already impede the function of TP53, with LOH completing its inactivation. In experimental models, certain TP53 mutants compromise the function of unaltered (“wild-type”) protein. They act as dominant negatives, probably by sequestering wild-type protein monomers in inactive (tetra-

113 Table 5.4  Mechanisms of TP53 inactivation in human cancers Mechanism of inactivation Missense mutations Nonsense and splice mutations Deletion of one allele Overexpression of MDM2 by gene amplification or deregulation Loss of p14ARF function by gene deletion, mutation, or promoter hypermethylation Loss of function of upstream activators, e.g., ATM Loss of function of downstream effectors, e.g., BAX Inactivation by altered posttranslational modification Inactivation by viral oncoproteins, e.g., HPV E6

meric) complexes.6 Accordingly, mutations in the C-terminal TP53 domain mediating tetramerization are rare. Nevertheless, how important this effect actually is during the development of human cancers is debated. Conceivably, the initial missense mutation might increase the probability of the loss of the second allele through a dosage effect that diminishes the ability of the cell to react to genomic damage such as illegitimate recombination or deletion of chromosome 17p. Most missense mutations in TP53 occur in the central domain of the protein that is necessary for specific binding to DNA (Fig. 5.5). This domain comprises a larger structure made up of individual β-sheets stabilized by Zn2+ ions, which supports the α-helix that directly contacts DNA through arginine and further residues. The arginine residues in this α-helix are mutation hotspots, but other amino acids in the central domain can also be mutated. The exact site of the mutation also depends on which carcinogen was involved (→3.1). Mutations in the N-terminal and C-terminal domains are found at lower frequencies. They may compromise the activation and oligomerization of TP53, respectively. Nonsense and splice mutations: Nonsense and splice mutations inactivating TP53 are also found in many cancer types, albeit in general at a lower frequency. Apparently, the more frequent misGain of function like transcriptional activation of new target genes has also been described. 6 

114

sense mutations provide some sort of advantage, either by acting as dominant-negatives as described above or by retaining selectively specific functions of TP53 which are advantageous to tumor cells (like metabolic regulation and induction of DNA repair proteins). MDM2 overexpression: Some tumors harbor amplifications of MDM2 or the related MDM4 gene. Their overexpression likely diminishes the function of TP53. Amplification of MDM2 and mutation of TP53 occur in a mutually exclusive fashion in many cancer types. This is a strong argument that they are indeed complementary. Nevertheless, this mechanism of TP53 inactivation is frequent only in some cancer types, e.g., certain sarcomas. This specificity may be explained by functions of MDM2 beyond the regulation of TP53 that are important in specific cell types. Notably, in cancers with mutant TP53, MDM2 cannot be induced by TP53. If the mutation does not destabilize the TP53 protein in other ways (such as by truncation), this will lead to an accumulation of the mutated TP53 protein, since MDM2 is the ratelimiting enzyme for its degradation. This is one reason for the paradoxical observation that TP53 protein levels are higher in many cancers than in the corresponding normal tissues. Another reason may be the increased level of genomic instability and consequently increased stability of (mutant) TP53. Thus, in some cases, detection of increased TP53 protein levels (i.e., accumulated mutant protein) can be used for the detection of tumor cells and may serve as a simple (albeit imprecise) surrogate for TP53 mutation analysis. Upstream activator inactivation: The functions of TP53 are expected to be impeded by mutations in upstream pathways that provide signals for its activation (Fig.  5.6). This is likely the case in ataxia telangiectasia (→3.6). In this disease, activation of TP53 in response to ionizing radiation is diminished as a consequence of defects in the ATM kinase. ATM mutations are also found in some sporadic cancers, but the loss of p14ARF may be more frequently relevant. The CDKN2A gene is inactivated in many different types of human cancers by deletions or point mutations, in specific cancers also by promoter hypermethylation. The loss of p14ARF would be predicted to specifically disrupt the activation of TP53 in response to inappropriate cell prolif-

5  Tumor Suppressor Genes

eration, e.g., as induced by oncogene activation. Of note, loss of p14ARF by CDKN2A deletions also occurs in cancers with TP53 inactivation by other mechanisms, but this apparent paradox may be accounted for by p16INK4A presenting a mutation target in the same gene. Intriguingly, loss of p16INK4A and RB1 instead occur in a mutually exclusive fashion. Cell-type specific downregulation: There are a few cancers in which mutations of TP53 are extremely rare. In testicular germ cell tumors, especially seminomas, mutations may be very rare, because the gene is only weakly expressed at the stage of development from which the tumor cells arise. However, TP53 can be activated by strong signals and the presence of an intact gene is proposed as one factor responsible for the high efficacy of radiotherapy and chemotherapy in these cancers. Inactivation by viral proteins: Yet another mechanism is found in most cervical squamous carcinomas, in a subset of head and neck squamous cell carcinomas, and occasionally in other cancer types that are caused by specific oncogenic strains of human papilloma virus (HPV). These DNA viruses express an oncoprotein, E6, which binds to TP53 and promotes its degradation, quite similar to MDM2. This mechanism inhibits one of the cellular responses to the infection that would prevent the replication of the virus. HPV furthermore inhibits RB1 through a second oncoprotein, E7, thereby promoting cell cycle progression towards S phase with optimal conditions for viral replication. In addition to E6 and E7, additional viral proteins may contribute to deregulation of the cell cycle and escape from antiviral responses. HPVs have circular double-stranded DNA genomes of ≈7.9 kb that encode seven early and two late proteins, with expression and replication directed by one control region (CR). While the viruses replicate in a circular episomal form, in most cancers the viral genome is integrated into the cellular genome in a linear form. Typically, during integration, the circular genome is opened within the E2 gene and this gene is inactivated; additional viral genes may also be lost. E6 and E7 expression is then activated by cellular regulatory elements. Sustained expression of E6 and E7 represents the first step towards cancer development

5.3  TP53 and the Control of Genomic Integrity

(Box 5.2). In particular, inhibition of both TP53 and RB1 prevents induction of replicative senescence (→7.2). Moreover, it disrupts checkpoint activation in response to DNA damage and favors the accumulation of further genetic alterations that lead to cancer In a few cancers that express only HPV16 E6, CDKN2A is disrupted and p16INK4A is inactivated instead of RB1. In the others, p16INK4A is strongly overexpressed since it is under feedback control by RB1 and further induced by continuous cell proliferation.

Box 5.2 Cervical Cancer and Human Papilloma Viruses

Multiple strains of human papilloma viruses (HPV) are transmitted by sexual or other close contacts. Many strains cause transient benign lesions of the epidermis and other squamous epithelia, but oncogenic strains elicit eventually squamous cell carcinomas of the cervix, the penis, the anogenital region, and the oropharynx in a fraction of the infected individuals. The differences between the strains can be traced to differences in the potency of their encoded E6 and E7 proteins. The most prevalent oncogenic strains are HPV16 and HPV18. About 90% of all uterine cervix carcinomas (cervical cancers) are squamous carcinomas, with adenocarcinomas constituting most of the remainder. The vast majority of cervical squamous carcinomas are initiated by infections with high-risk strains of HPV like HPV16 and HPV18. More than 10 further HPV strains are also oncogenic. In a fraction of the infected women, they induce progressively dysplastic lesions known as cervical intraepithelial neoplasia (CIN) that eventually turn into carcinomas. In the process, production of infectious viruses becomes gradually repressed, but the viral genes integrated into the host genome remain expressed, especially E6 and E7. The HPV oncogenes inactivate TP53 and RB1 and further promote cancer development through effects on the tumor microen-

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vironment, suppressing immune responses and inducing neoangiogenesis. Cancer development is accompanied by additional genomic changes, such as mutations in ERBB family receptors and FGFR3, which in particular result in activation of the PI3K pathway. Localized cancers can often be successfully treated by surgery followed by adjuvant chemoradiotherapy. Metastatic cancers remain difficult to cure, even by chemotherapy and modern immunotherapies. Worldwide, cervical cancers are still frequent. More than 570,000 new cases were diagnosed and more than 300,000 women died from these cancers in 2018. Almost 90% of these cancers occurred in low- or middle-income countries. In contrast, incidences in higher income countries have strongly decreased as a consequence of screening programs that aim at the detection of dysplastic precursor lesions. These were initially based on cytological investigations of cervical mucosa samples (“Pap smears”). Over time, these rapid and cheap, but not always accurate tests have been supplemented or replaced by molecular assays. For instance, HPV genomes can be detected by in situ hybridization in infected cells. However, immunohistochemical staining for p16INK4A has become the most commonly used assay. This procedure is a straightforward and usually reliable surrogate as it reflects the inactivation of RB1 by the E7 protein of oncogenic HPVs. Another step forward to decrease morbidity and mortality caused by cervical carcinoma is the vaccination of adolescent girls against HPV, which has proven to be effective and safe. Given the options of vaccination for primary prevention and screening by a relatively simple procedure, squamous cervical carcinomas are now regarded as one of the most preventable cancer types. Cohen PA et al. (2019) Cervical cancer. Lancet 393:169–178.

5  Tumor Suppressor Genes

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Other DNA viruses have developed similar mechanisms to cope with TP53 during infection. Adenoviruses, which are tumorigenic in rodents, employ the E1B protein to sequester and inhibit TP53, but the protein is not degraded following adenovirus infection. Notably, TP53 was originally discovered as a protein regularly associated with the major transforming protein of the DNA tumor virus SV40, a β-polyomavirus infecting many primates including man. This virus encodes two regulatory proteins, named large-T and smallT (tumor) antigens. Large-T antigen is a multifunctional protein that regulates SV40 transcription and replication. It moreover binds several host proteins, including TP53. A second important protein sequestered and inactivated by SV40 large-T is RB1 (Fig. 5.10). Inhibition of RB1 by the large-T antigen is thought to allow progression into S phase for optimal viral replication. Other DNA tumor viruses too inactivate RB1, HPV through E7, and adenoviruses through their E1A proteins. Notably, while certain adenoviruses cause tumors in laboratory animals, they are probably not transforming in man. There is no conclusive evidence that SV40 and related viruses such as BKV and JCV may cause human cancers. The fact that very different DNA viruses specifically inactivate TP53, as well as RB1, suggests that these two proteins not only impede tumor development but also interfere with viral infections. In fact, even viruses that can replicate outside S phase may deploy factors to inhibit TP53 as well as RB1. Inactivation of the two tumor suppressors by viral products may thus aim at preventing antiviral responses in the infected cell, but may also contribute to redirection of cellular resources towards viral replication. In particular, as in carcinogenesis, inactivation of TP53 and RB1 will prevent the induction of replicative senescence as a protective response in virus-infected cells.

Adenovirus E1B

E1A

Large T

RB1

TP53

SV40 E7

E6

Papilloma viruses

Fig. 5.10  Inactivation of TP53 and RB1 by DNA tumor virus proteins. See main text for details

5.4 Classification of Tumor Suppressor Genes The fact that DNA tumor viruses so specifically target TP53 and RB1 is another indication that these proteins are of central importance in both normal and cancer cells. They reside at nodes of (linked) networks that control cell proliferation and genomic integrity. These networks must be subverted and their controls must be overcome for tumor growth. This can be achieved in a straightforward manner by inactivation of the tumor suppressors by deleterious mutations in their genes. Alternatively, negative regulators of TP53 or RB1 may be overexpressed or positive regulators of the cell cycle may acquire mutations that allow them to escape from regulation by the tumor suppressors. To this end, MDM2 or CDK4 among others can be activated to oncogenes. As discussed in the previous chapter (→4.4), many cellular oncogenes discovered as analogs of retroviral oncogenes act upon or within the MAPK signaling pathway. Conversely, the tumor suppressor NF1, which is mutated in familial neurofibromatosis type 1, is a tissue-specific

5.4  Classification of Tumor Suppressor Genes

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GAP that regulates RAS function and, in this Similarly, inactivation of tumor suppressors like fashion, inhibits MAPK signaling (→4.4). MLH1, MSH2, BRCA1, BRCA2, and ATM has Mutations in NF1 in the syndrome and in spo- little direct effect on cell proliferation and differradic cancers truncate the large protein or selec- entiation. Rather, inactivation of these factors tively impede the function of its RAS interacting predominantly enhances the rate of mutations or domain. allows to tolerate genetic instability. Genomic Similar examples of tumor suppressors and alterations favored in this manner eventually oncogenes acting as mutual antagonists can be affect genes like RB1 which are directly responfound in other signal transduction pathways that sible for unchecked growth at the cell and tissue regulate cell proliferation and differentiation. For level. It was therefore suggested to categorize instance, PTEN is a negative regulator of the tumor suppressors into two classes, gatekeepers, PI3K pathway and a classical tumor suppressor, and caretakers, respectively. According to that whereas PIK3CA mutations and amplification concept, inactivation of gatekeepers like RB1 turn the PI3K kinase into an oncogene (→6.3). directly leads to the deregulation of proliferation, PTCH1 is a negative regulator of SMO in the differentiation, and cell death, whereas caretakHedgehog pathway, which becomes overactive ers act indirectly by favoring the accumulation of by either loss of tumor-suppressive PTCH1 or genetic alterations. The designation “gatekeeper” oncogenic point mutations in SMO (→6.9), APC implies in addition that cancer of a certain type promotes the inactivation of β-Catenin (gene: may only arise if the function of this particular CTNNB1) in the canonical WNT pathway tumor suppressor is abolished. This may indeed (→6.9). APC inactivation or oncogenic activation be true in some cases, like APC in a subtype of of β-Catenin alternatively deregulates WNT sig- colorectal cancer, where the concept was develnaling. Based on the existence of such obvious oped (→13.3). However, many classical tumor antagonisms, it has been suggested to name suppressors do not so neatly fit into either catetumor suppressors “anti-oncogenes.” This desig- gory. Thus, TP53 can influence cell cycle regulanation is a bit misleading in so far as is no strict tion beyond its function in checkpoint execution pairwise complementarity between oncogenes and RB1 as well as APC appear to be relevant for and tumor suppressors. Not uncommonly, several the control of mitotic fidelity. In particular, many tumor suppressors and several oncogenes may gatekeeper tumor suppressors influence cell cycle each be involved at different steps of the same checkpoints, which link the control of genomic pathway. Moreover, activation of proto-­stability to cell cycle regulation in general. oncogenes and inactivation of tumor suppressors Additional classes of tumor suppressor genes from the same pathway typically have similar, may exist. For instance, some genes may be irrelbut not identical consequences. Nevertheless, the evant for the growth of a primary tumor, but their presence of alternatively occurring alterations in loss of function would be essential for metastasis. tumor suppressor genes and oncogenes suggests Such genes have been proposed to constitute the that it is primarily the altered activity of cancer category of “metastasis suppressor” genes. Genes pathways that is crucial for cancer development, of this kind have been found (→9.2), but the conrather than that of individual genes (→6.1). cept is not broadly used. In addition, other genes Usually, cancer pathway deregulation can be recurrently inactivated in some cancers do not fit achieved in various ways, of which tumor sup- straightforwardly into the “caretakers and gatepressor inactivation and proto-oncogene activa- keepers” categories, for instance, epigenetic regtion are only the most obvious ones. ulators mutated in various cancers (→8.3). Another problem with the concept of “anti-­ So, what is the precise definition of a tumor oncogenes” is that it does not cover tumor sup- suppressor gene? The strictest definition, accordpressors like TP53 well. Unlike inactivation of ing to the Knudson two-hit model, would encomRB1, loss of TP53 function does not usually pass all those genes that if affected by inactivating directly lead to altered cell proliferation. mutations lead to cancers inherited in an

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autosomal-­ dominant fashion, but behave in a recessive mode at the cellular level. Thus, one mutant, functionally inactive allele is inherited and the second one is inactivated in the ensuing tumors by point mutation, deletion, insertion, recombination, or promoter hypermethylation. Often, both alleles of these same genes are also functionally inactivated in sporadic cases of the same type. No conceptual problem arises from extending this definition to all genes with recurrent inactivation of both alleles in many sporadic cases of a specific tumor type if the functional relevance for tumor development or progression can be demonstrated. In this textbook, genes of this kind are regarded as “classical” tumor suppressors. The concept is more difficult to apply when inactivation of both alleles does not occur regularly or only one allele of a gene is consistently lost. The first case could be a random event and the second case could, e.g., be a consequence of proximity to a real tumor suppressor gene.7 Another possible explanation in such cases is haploinsufficiency, i.e., loss of one allele leading to expression at half the normal level permits tumor formation. Haploinsufficiency has been discussed for several established tumor suppressors, especially PTEN. In particular, inactivation of the first allele of tumor suppressors like TP53, RB1, or APC that impinge on the stability of the genome may not directly transform cells, but may increase the likelihood of inactivation of the respective second allele. Unfortunately, haploinThe MTAP gene encoding Methylthioadenosine phosphorylase is located next to CDKN2A and therefore often co-deleted by homozygous deletions. The loss is however rather a liability than an advantage to the tumor cells because methylthioadenosine (a product of cellular methylation reactions) accumulates. 7 

5  Tumor Suppressor Genes

sufficiency can be difficult to ascertain in the context of human cancers, as exemplified by losses of NKX3A at 8p21  in prostate cancer (→20.3) or by the elusive tumor suppressor gene at chromosome 9q in bladder cancer (Box 14.1). Moreover, recurrent chromosomal losses may diminish the dosage of several genes located in the same region with an additive or synergistic effect. A prime example is a recurrent loss of the short arm of chromosome 3  in clear-cell renal carcinoma which abolishes one allele of the crucial VHL tumor suppressor along with one or several additional tumor suppressors (→16.3). Notably, some genes appear to act as oncogenes in one type of cancer and as tumor suppressors in others. A notorious case concerns NOTCH genes. NOTCH1 is mutated in many different cancers, but the spectrum of mutations in the large gene is substantially different between squamous carcinomas and other cancer types (Fig.  5.11). In squamous cell carcinomas, the type and distribution of mutations correspond to the pattern expected for a tumor suppressor. In other cancers, e.g., in T-ALL, the predominance of point mutations at specific sites suggests oncogenic activation. A particularly difficult case is raised by genes whose expression is consistently downregulated in one type of cancer, but which do not undergo genetic (structural or copy number) alterations and do not even exhibit a clear-cut epigenetic mark of stable silencing such as promoter hypermethylation (→8.2). This is analogous to the issue of to what extent overexpressed genes can be considered oncogenes (→4.3). As in that case, defining the precise function of the gene and protein in question is the first essential requirement for approaching the issue.

Further Reading

Fig. 5.11  Distribution of mutations in NOTCH1 in different cancer types. Mutations in NOTCH1 are distributed across the entire gene in squamous cell carcinomas (350 cancers overall) and encompass many truncating mutations. In T-ALL (1349 cases overall), point mutations cluster in hotspots.

Further Reading Ak P, Levine AJ (2010) p53 and NFκB: different strategies for responding to stress lead to a functional antagonism. FASEB J. 24:3643–3652 Amelio I, Melino G (2015) The p53 family and the hypoxia-inducible factors (HIFs): determinants of cancer progression. Trends Biochem Sci 40:425–434 Baugh EH et al (2018) Why are there hotspot mutations in the TP53 gene in human cancers? Cell Death Differ 25:154–160 Berger AH et  al (2011) A continuum model for tumour suppression. Nature 476:163–169 Bieging KT et  al (2014) Unraveling mechanisms of p53-mediated tumour suppression. Nat Rev Cancer 14:359–370 Coschi CH, Dick FA (2012) Chromosome instability and deregulated proliferation: an unavoidable duo. Cell Mol Life Sci 69:2009–2024 de Oliveira GAP et al (2020) The status of p53 oligomeric and aggregation states in cancer. Biomolecules 10:548 Dick FA et al (2018) Non-canonical functions of the RB protein in cancer. Nat Rev Cancer 18:442–451 Dyson NJ (2016) RB1: a prototype tumor suppressor and an enigma. Genes Dev 30:1492–1502 Evan GI, Vousden KH (2001) Proliferation, cell cycle and apoptosis. Nature 411:342–348 Fiorentino FP et  al (2013) On the role of retinoblastoma family proteins in the establishment and maintenance of the epigenetic landscape. J Cell Physiol 228:276–284

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Truncating mutations remove primarily the C-terminal PEST domain which promotes degradation of the NOTCH1 (ICD) protein; they are therefore probably oncogenic. From: Shah PA et  al. (2020) Cells 9:2677. Originally published under the terms of CC BY. See Fig. 6.13 for more details on the NOTCH1 protein

Fischer M (2017) Census and evaluation of p53 target genes. Oncogene 36:3943–3956 Fridman JS, Lowe SW (2003) Control of apoptosis by p53. Oncogene 22:9030–9040 Gazdar AF, Butel JS, Carbone M (2002) SV40 and human tumours: myth, association or causality? Nat Rev Cancer 2:957–964 Greenman CD (2012) Haploinsufficient gene selection in cancer. Science 337:47–48 Hafner A et al (2019) The multiple mechanisms that regulate p53 activity and cell fate. Nat Rev Mol Cell Biol 20:199–210 Hermeking H (2012) MicroRNAs in the p53 network: micromanagement of tumour suppression. Nat Rev Cancer 12:613–626 Hofseth LJ, Hussain SP, Harris CC (2004) p53: 25 years after its discovery. Trends Pharmacol Sci 25: 177–181 Inoue K, Fry EA (2017) Haploinsufficient tumor suppressor genes. Adv Cancer Biol 118:83–122 Irwin MS, Kaelin WG (2001) p53 family update: p73 and p63 develop their own identities. Cell Growth Different 12:337–349 Kastenhuber ER, Lowe SW (2017) Putting p53 in context. Cell 170:1062–1078 Kinzler KW, Vogelstein B (1997) Gatekeepers and caretakers. Nature 386:761–762 Kruse JP, Gu W (2009) Modes of p53 regulation. Cell 137:609–622 Mandigo AC et al (2022) Relevance of pRB loss in human malignancies. Clin. Cancer Res 28:255–264

120 Muller PAJ, Vousden KH (2014) Mutant p53  in cancer: new functions and therapeutic opportunities. Cancer Cell 25:304–317 Munro S et al (2012) Diversity within the pRb pathway: is there a code of conduct? Oncogene 31:4343–4352 Satyanarayana A, Kaldis P (2009) Mammalian cell-cycle regulation: several Cdks, numerous cyclins and diverse compensatory mechanisms. Oncogene 28:2925–2939 Sherr CJ (2004) Principles of tumor suppression. Cell 116:235–246 Sherr CJ (2012) Ink4-Arf locus in cancer and aging. Wiley Interdiscip Rev Dev Biol 1:731–741 Sherr CJ, McCormick F (2002) The RB and p53 pathways in cancer. Cancer Cell 2:103–112

5  Tumor Suppressor Genes Tiwari B et al (2018) Transposons, p53 and genome security. Trends Genet 34:846–855 Vogelstein B, Lane D, Levine AJ (2000) Surfing the p53 network. Nature 408:307–310 Walter DM et al (2019) RB constrains lineage fidelity and multiple stages of tumour progression and metastasis. Nature 569:423–427 Xue W et  al (2012) A cluster of cooperating tumor-­ suppressor gene candidates in chromosomal deletions. PNAS 109:8212–8217 Zhu L et al. (2015) Antitumor mechanisms when pRb and p53 are genetically inactivated. Oncogene 34:4547–4557

6

Cancer Pathways

Key Points • In addition to canonical MAPK signaling, • Proliferation, differentiation, and survival of many growth factors activate PI3K signaling normal cells are regulated by a limited number through receptor tyrosine kinases and RAS of interlinked signal transduction pathways. proteins. Following the activation of the They transmit and integrate signals from phosphoinositide-­ 3-phosphate kinase PI3K growth factors, hormones, cell-cell and cell-­ and the PKD and AKT protein kinases the matrix interactions. As a consequence of pathway branches to protect against cell death, deregulation, these pathways can turn into increase cell growth and protein synthesis, “cancer pathways.” Inappropriate activation of and to support cell proliferation. The PTEN cancer pathways in many cases and inactivaphosphatase is a major antagonist of the tion in others are crucial for the development upstream part of the pathway. Cell growth and progression of human cancers. In particustimulation by the pathway is mediated espelar, many proto-oncogenes and tumor supprescially by the mTOR kinase, which is negasors act as regulators within or upon cancer tively regulated by the TSC tumor suppressors pathways. and the AMP-dependent kinase (AMPK). • MAP kinase pathways transduce signals from Like MAPK signaling, PI3K signaling is the cell membrane to the nucleus, as well as to enhanced in many cancers as a consequence the translational machinery and to the cytoof mutations in receptor tyrosine kinases or skeleton. The canonical MAPK pathway is diverse pathway components, most commonly activated by receptor tyrosine kinases and oncogenic mutations in PIK3CA (encoding a other proteins at the cell membrane and procatalytic subunit of PI3K) or genetic changes motes proliferation in response to growth facinactivating the tumor suppressor PTEN. tors in many cell types. The pathway proceeds • In the stimulation of cell proliferation, the through RAS proteins via a cascade of RAF, PI3K pathway acts synergistically with the MEK, ERK, and additional protein kinases. canonical MAPK pathway. The activity of the • Further, similarly composed MAPK pathways two pathways converges in particular on cell mediate among others stress responses and cycle regulation. may elicit apoptosis in some circumstances. • Another frequent target of membrane recepWhile the functions of the various MAPK tors is phospholipase C, which activates PKC pathways are overlapping, the ERK pathway isoenzymes that can crosstalk with MAPK is usually the most relevant one for the control and other signal transduction pathways. The of cell proliferation. diverse PKC enzymes are targets of other pro© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 W. A. Schulz, Molecular Biology of Human Cancers, https://doi.org/10.1007/978-3-031-16286-2_6

121

6  Cancer Pathways

122

•

•

•

•

teins and compounds, including the tumor-­ promoting phorbol esters. Signals from cell-to-cell and cell-matrix adhesion likewise modulate MAPK and PI3K activity. As a rule, TGFβ factors stimulate the proliferation of mesenchymal cells but inhibit the proliferation of epithelial and many immune cells. In response to TGFβ receptor activation, SMAD transcription factors are phosphorylated and translocated to the nucleus where they activate genes encoding a. o. CDK inhibitors or proteins involved in the formation and remodeling of the ECM. Other SMAD factors act as feedback inhibitors of the pathway. Since TGFβ signaling tends to limit the proliferation of epithelial cells, disruption or diversion of the pathway is particularly important for the progression of many carcinomas. Moreover, TGFβ synthesized by ­nonresponsive carcinoma cells activates tumor stroma and inhibits immune cells. Signaling through NFκB transcription factors regulates activation and proliferation of immune cells and inflammation. In epithelial cells, NFκB transcription factors modulate especially apoptosis during cellular stress and the interaction with other cell types during immune responses and inflammation. In carcinoma cells, NFκB remains important for prevention of apoptosis and regulation of interactions with stromal and immune cells. Stimulation of cell proliferation by cytokines often proceeds via the JAK/STAT pathway in hematopoietic cells and lymphocytes, but the pathway is also important in epithelial cells. In this straightforward pathway, JAK protein kinases recruited by membrane receptors for cytokines and other growth factors phosphorylate STAT transcription factors which translocate into the nucleus to activate transcription. The JAK/STAT pathway is feedback-regulated by SOCS proteins. Overactivity of the pathway is in particular responsible for increased proliferation in many hematological cancers. The patterning of tissues during fetal development involves several specialized regulatory

systems, in particular, the WNT, Hedgehog (SHH), and Notch pathways. These “morphogenic” pathways remain important in adult tissues, especially for the maintenance of stem cell compartments and the regulation of cell fates. Disturbances in these pathways are fundamental for the development of specific human cancers. Thus, colorectal carcinomas invariably display constitutive activation of the WNT pathway, basal cell carcinoma of the skin of the Hedgehog pathway, and certain T cell leukemias of the Notch pathway, respectively. Conversely, Notch signaling is frequently inactivated in squamous cell carcinomas. The responsible alterations are alternatively mutations in proto-oncogenes or tumor suppressor genes in the respective pathways. • Hippo signaling controls the size of tissues during normal development and regeneration. Inactivation of upstream regulators or enhanced nuclear localization of downstream effectors contributes especially to cancer progression. • Nuclear receptors are ligand-dependent transcription factors. The large family includes hormone receptors like the Estrogen receptors α (ERα) and β, the Progesterone receptor (PR), and the Androgen receptor (AR), whose altered activity are crucial to breast and prostate cancers, respectively. Other family members respond to specific paracrine signaling factors like retinoic acid receptors (RARs) or metabolites like the peroxisome-proliferation activating receptors (PPARs).

6.1 Cancer Pathways With more than 750 different genes shown to be causally involved in human cancers and many more implicated, one might suspect that a bewildering variety of mechanisms cause human cancers. Nevertheless, a great number of currently known oncoproteins can be assigned to a limited number of “cancer pathways.” These pathways

6.2 MAPK Signal Transduction Pathways

123

Table 6.1  An overview of important cancer pathways Oncogenes in the pathway (examples) RAS, BRAF

Tumor suppressors in the pathway (examples) NF1

PI3K, AKT, mTOR

PTEN, TSC1/2 TGFBR2, SMAD2, SMAD4

NFκB JAK/STAT

RELA JAK2, STAT3

CYLD SOCS1

WNT/β-­Catenin Hedgehog Notch Hippo

WNT1, β-Catenin

APC, AXIN, RNF43

SMO, GLI1 NOTCH1 YAP, TAZ

PTCH1, SUFU NOTCH1 Merlin, FAT1

Pathway MAPK (canonical) PI3K/AKT TGFβ

regulate the fate, survival, proliferation, differentiation, and function of normal cells. In cancer cells, their overactivity or inactivation causes their characteristic properties such as uncontrolled proliferation, insufficient apoptosis, altered differentiation and tissue structure (→1.3). Some cancer pathways are only involved in a few cancer types, whereas others contribute to a broad range of malignant tumors. A working definition of “cancer pathway” might read as follows: A cancer pathway is a cellular system regulating cell proliferation, survival, or differentiation whose activation or inactivation by one or several genetic or epigenetic alterations is essential for the development of at least one human cancer. Cancer pathways become most clearly apparent if alterations in different components of the same regulatory system are observed in individual cases of the same cancer type (or in different cancer types). By this criterion, several regulatory systems already treated in the previous two chapters are prototypic cancer pathways (Table  6.1). They comprise the MAPK signal transduction pathway (→4.4), the TP53 control network (→5.3), and the cell cycle regulatory network centered around RB1 (→5.2). These three systems actually interact with each other. Further pathways and proteins are connected to them, especially the PI3K pathway, but also signaling from PKC kinases and cell adhesion proteins, the TGFβ response

Cancers (examples) Many Many Carcinomas, selected soft tissue cancers, selected leukemias Selected leukemias, many carcinomas Selected carcinomas, many leukemias, and lymphomas Carcinomas of colon, liver, breast, stomach, and others Skin basal cell carcinoma T cell lymphomas, carcinomas Several

pathway, the NFκB pathways, and the JAK/STAT pathway. Another group of cancer pathways comprises the WNT, Hedgehog, Notch, and Hippo regulatory systems. These “morphogenic pathways” are essential for the development of tissues, the determination of cell fate, and cell differentiation during ontogeny. They remain important in adult humans by contributing to tissue homeostasis and especially to the specification and regulation of stem and precursor cells, particularly in tissues that undergo rapid turnover or regeneration. Morphogenic pathways tend to be particularly important in specific cancers. Likewise, altered signaling through nuclear receptors is especially important in specific cancer types.

6.2 MAPK Signal Transduction Pathways Signaling through the canonical MAPK pathway composed of RAF, MEK, and ERK proteins (→4.4) is required for the proliferation of many normal cells as well as of many cancer cells. In normal cells, it relays especially signals from receptor tyrosine kinases (RTKs) activated by growth factors to the nucleus to induce the transcription of genes directing cell proliferation. This particular MAPK pathway is known as the “classical mitogenic cascade” or the “canonical”

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MAPK pathway. It also responds to a range of further extra- and intracellular stimuli and it elicits effects besides altered gene expression, including changes in the cytoskeleton and protein biosynthesis. As discussed in Sect. 4.3, MAPK signaling normally proceeds in bursts due to several feedback loops in the pathway. In some cell types sustained MAPK signaling may rather induce differentiation. Likewise, persistently high levels of MAPK signaling may be lethal in some tumor cells because they disturb regular replication, i.e., induce replication stress. In many cancers, however MAPK signaling is enhanced, prominently as a consequence of oncogenic activation of various RTKs or one of the three RAS proteins HRAS, KRAS, or NRAS (→4.4). Downstream of RAS, overactivity of the pathway can be caused by activating mutations in RAF kinases, most commonly BRAF, or occasionally in MEK kinases. In contrast, activation of ERK kinases, ARAF, and CRAF by point mutations are rare, but their activity is of course required for MAPK signaling in many normal and cancerous cell types. Moreover, the catalytic domains of RAF kinases have been discovered as constituents of fusion oncogenes in a few cancers of various types. Activation of MAPK signaling can also be caused or be enhanced by the loss of feedback inhibitors acting upstream or downstream of the core pathway. The GAP NF1 is a classical tumor suppressor that is mutated in neurofibromatosis type I, but also in a variety of sporadic cancers. It acts upstream of the pathway by limiting the duration of the active state of RAS proteins. Sprouty proteins, which are induced as a consequence of MAPK activation, interrupt interactions between adaptor proteins and RAS, and additionally inhibit RAF activity. The DUSP5/6 protein phosphatases are likewise induced following MAPK activation and dephosphorylate ERK and MEK to limit signaling through the pathway. These inhibitors are lost in some cancers by mutations or by epigenetic repression. The canonical pathway is one of several similar modules. Each is composed of a MAP kinase (corresponding to ERK), a MAPK kinase (like MEK), and a MAP kinase kinase (like RAF)

6  Cancer Pathways

(Fig.  6.1). Other MAPK modules respond likewise to extracellular signals from growth factors or from hormones and cytokines relayed through G-proteins and to signals from cell adhesion molecules. They may also be activated by stress signals generated within the cell or at the cell membrane. Like the canonical pathway, alternative MAPK pathways can stimulate cell proliferation, but also apoptosis, cell differentiation, or specific cell functions. None of them however has been as straightforwardly implicated in the control of cell proliferation and in as many cancers as the canonical pathway. The best-studied of the parallel pathways involves MEKK1, MEK4, and JNK1 (JUN N-terminal kinase). It leads ultimately to the phosphorylation of the transcriptional activator JUN. JUN acts as a component of AP1 transcription factors, homo- or heterodimers composed of JUN homologs, and other proteins like FOS. This alternative pathway is also often activated by RAS proteins, in response to cellular stress or by certain cytokines. Alternatively, other GTPbinding proteins such as RAC or CDC42 activate the pathway through MEKK1, 2, or 3 in response to cell adhesion signals. The outcome of activation of this MAPK pathway may be variously stimulation or arrest of cell proliferation, or in some circumstances, apoptosis. JNK1 and the MAPK p38α in particular can be strong activators of apoptosis, but the outcome of their action is dependent on the cell type and on other signals. For instance, p38α (MAPK14) may induce apoptosis, but at the same time increase the production and secretion of growth factors and cytokines. In some cell types, p38α promotes differentiation. JNK signaling may contribute to the maintenance of tumor stem cells. In cancers overall therefore JNK1 and p38α not only tend to inhibit cancer development but also to promote cancer progression and contribute to drug resistance. Possibly because of this ambivalence, the components of the JNK pathway do not seem to constitute primary targets of mutations in human cancers. Crosstalk between different MAPK pathways is substantial, e.g., at the level of the MAPKKs. Moreover, JUN is phosphorylated as a consequence of activation of the JNK MAPK pathway,

6.2 MAPK Signal Transduction Pathways Fig. 6.1 MAPK signaling modules. The box on the left illustrated the general setup of MAPK modules. Three actual modules are shown on the right. These are in reality more diverse than shown and may crosstalk with each other and the canonical pathway on the left. Additional partial modules exist. Interactions within each module is facilitated by scaffolding proteins (not shown)

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Growth Factors

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while the protein is transcriptionally induced by the ERK MAPK pathway. Thus, these two MAPK pathways synergize during regular cell proliferation. Both the ERK and the JNK MAPK pathways can be activated through RAS proteins. In their active state as RAS-GTP they function at the level of the MAPK kinase kinases by recruiting RAF proteins or MEKK1 to the cell membrane and relieving their autoinhibition. RAS proteins also act in a more indirect manner on the G-proteins RAC and RHO (Box 9.1) and RAL, by activating their GTP-exchange factors (GEF) or GTPase activating proteins (GAP). Through these routes, RAS is thought to elicit the changes in the cytoskeleton and consequently cell shape, adhesion, and migration that are commonly seen in cells with oncogenic RAS. Notably, they form the basis of the classical 3T3 assay system that helped in the discovery of the RAS oncogenes (→4.3). Of course, these effects are also relevant to the properties of tumor cells in vivo (→9.2). The activation of the JNK MAPK pathway points to another facet in the pleiotropic function of RAS proteins. Overactivity of RAS proteins can induce growth arrest, apoptosis, or replicative senescence in normal cells. In some cell

SRE

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types, RAS activation may moreover induce differentiation. Therefore, in addition to their effect on cell proliferation, RAS proteins in their active state stimulate responses that eventually terminate their effects or evoke fail-safe reactions against hyperproliferation. Depending on the circumstances, the JNK MAPK pathway may act in similarly varied manners. Another important fail-­ safe mechanism is induction of CDKN2A with increased transcription of p14ARF that leads to stabilization of TP53 (→5.3). Cell transformation by oncogenic RAS therefore requires as a rule inactivation of this fail-safe mechanism, e.g., by homozygous deletion of CDKN2A. RAS proteins are almost invariably activated when signals emanate from active growth factor receptors. They therefore mediate many effects of the physiological or oncogenic activation of receptor tyrosine kinases. In addition to activation of the MAPK pathway, activation of the PI3K pathway (see next section) by RAS is important for normal cell proliferation. It remains particularly important in those cancers where that pathway is not independently activated, e.g., by mutations in PIK3CA. The high prevalence of RAS mutations in human cancers, overall ≈20%, is thus well explained by the function of these

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proteins as nodal regulators of several pathways fostering essential properties of cancer cells. Which of the pathways downstream of activated RAS is most relevant may vary between cancers. For instance, in melanoma mutations in RAS genes and BRAF are mutually exclusive (→12.4) suggesting that the canonical MAPK pathway is most crucial. Finally, RAS proteins mediate not only signaling from receptor tyrosine kinases but are also involved in cellular responses to cytokines and hormones that signal through G-protein coupled serpentine receptors. Conversely, receptor tyrosine kinases and others modulate the activities of the MAPK, PI3K, and additional cancer path-

ways not only through RAS but also via other routes (cf. sections 6.3 and 6.5).

6.3 The PI3K Pathway The PI3K pathway regulates cell proliferation and cell growth, in particular protein biosynthesis, prevents apoptosis and contributes to the control of cellular metabolism, particularly that of glucose transport and utilization (cf. 1.4). The pathway (Fig. 6.2) is stimulated by active receptor tyrosine kinases, by RAS proteins and some cytokine receptors. It is distinguished as a cancer pathway by a variety of genetic alterations in

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Fig. 6.2  An overview of the PI3K pathway. Main PI3K pathway components are ringed by bold lines; inhibitory proteins are marked in grey. Note that PKD and PI3K effects beyond AKT phosphorylation are not detailed. Some important proteins affected by AKT phos-

phorylation are indicated. In this graphic, the pathway is directly stimulated by interaction of a PI3K regulatory subunit with an activated RTK.  Alternatively, it may be activated through RAS or by interaction with receptor substrate proteins like IRS1/2

6.3 The PI3K Pathway R1

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Fig. 6.3 Phosphorylation and dephosphorylation of PI(3,4)P by PI3K and PTEN. PI3K introduces a phosphate group at the 5-position of the phospholipid (blue circle) that is removed by

PTEN. Inositol-4-phosphatases (INPP4) remove the phosphate group at position 4 (green circle) of the PI3K substrates

various cancers that enhance its activity. The phosphoinositide-3-kinase (PI3K), for which the pathway is named,1 is one of the most regularly mutated oncogenes across all human cancer types. A major inhibitor of the pathway, PTEN, is a classical tumor suppressor, inactivated not only in the hereditary Cowden syndrome but likewise in a broad range of sporadic cancers. Two further tumor suppressors, TSC1 and TSC2, modulate the branch of the pathway regulating cell growth and protein biosynthesis. Deleterious mutations in their genes cause the human tumor syndrome tuberous sclerosis. The inactive PI3K heterodimer comprises one of five inhibitory regulatory subunits (p85 as a rule, genes PIK3R1 and PIK3R2) and one of three catalytic subunits (p110). The catalytic subunit PI3Kα (gene: PIK3CA) is the most relevant one in the context of cancer. In the basal state, the activity of the p110 catalytic subunit is restricted by the p85 regulatory subunit. Following its release from PI3KR, the kinase subunit phosphorylates the membrane phosphatidylinositol (PI) phospholipids PI(4)P and PI(4,5)P to yield PI3P, PI(3,4)P and PI(3,4,5)P, respectively (Fig. 6.3).

Following activation and autophosphorylation of receptor tyrosine kinases, the regulatory subunit p85 of PI3K binds to the tyrosine phosphates via its SH2 domains, which releases the catalytic subunit. The catalytic subunit is also—independently or concomitantly—stimulated by direct interaction with RAS-GTP.  Lipid phosphorylation generating PI(3,4,5)P at the inner face of the plasma membrane creates binding sites for proteins containing a pleckstrin homology (PH) domain. These become relocated to the membrane. Among them are the protein kinases PDK1 and AKT that relay the actual PI3K pathway signal. Notably, several other proteins contain PH domains, among them GEFs for RAC and other RAS-like proteins involved in the organization of the cytoskeleton. In the main PI3K pathway (Fig.  6.2), at the inner face of the cell membrane, PDK1 phosphorylates and activates AKT2 (also known as PKB) the most important nodal point and effector in the pathway. Of note, PDK1 phosphorylates further protein kinases, including the PKC isoenzyme ξ (→6.5) and p70S6K, which is also a target of ERK MAP kinases. Full activation of AKT requires phosphorylation by the mTORC2 kinase There are actually three isoenzymes, AKT 1-3, with similar properties. 2 

PI3K/AKT pathway is also in common use

1 

128

complex at a different serine residue. This additional phosphorylation influences the substrate choice of AKT; mTORC2 is moreover a substrate for feedback inhibitors of the pathway. Depending on its phosphorylation by PDK1, the mTORC2 complex, and other kinases, AKT protein kinase can phosphorylate a variety of proteins to activate the various branches of the PI3K pathway. Phosphorylation of TSC2 inhibits the TSC1/TSC2 protein complex which—via the intermediate GTP-binding Rheb protein—controls the mTOR kinase. The acronym mTOR stands for “mammalian target of rapamycin,” since rapamycin inhibits the mTORC1 complex in which mTOR is the central subunit. mTOR is also a component of the mTORC2 complex which modulates the activity of PI3K signaling (see above). The active mTORC1 complex is the main mediator of cell growth stimulation by the PI3K pathway. In particular, its kinase activity influences protein biosynthesis by phosphorylation of EIF4BPs and the p70S6K kinase which then phosphorylates the S6 subunit of the ribosome. The overall result is an increase in cap-dependent protein biosynthesis for cell growth. This increase promotes the translation of several cancer-­ relevant factors like HIF1 (hypoxia-inducible factor) and VEGF (vascular endothelial growth factor). In addition to the PI3K pathway, the mTORC1 complex responds to the energy state and amino acid supply of the cell. Information on their states is relayed among others by mutual interaction with the protein kinase AMPK (AMP-dependent kinase). AMPK is crucially controlled through inhibitory phosphorylation by LKB1 (gene: STK11); the LKB1 kinase is inactivated in the hereditary Peutz-Jeghers cancer syndrome. Additional regulator proteins signal the supply of critical amino acids like leucin, arginine, and methionine (or SAM, respectively). If substrate and energy levels are sufficient for growth, active mTORC1 moreover inhibits autophagy through phosphorylation of its regulator ULK1. The

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effects of mTORC1 on protein synthesis and cellular metabolism are particularly important for the anabolic action of insulin and insulin-like growth factors (IGFs) in many tissues, but they are also a more general prerequisite for cell growth and therefore central to tumor growth. The mTOR inhibitors TSC1 and TSC2 are also known as Tuberin and Hamartin. Either protein is mutated in tuberous sclerosis (TSC), an autosomal dominant disorder affecting ≈1/6000 persons. The TSC genes, which are located at chromosomes 9q34 and 16p13, respectively, behave as classical tumor suppressors. Thus, in affected persons, one defective copy of a TSC gene is inherited. Mutation of the second allele leads to hamartomas, usually small benign tumors consisting of different connective tissue components. They develop in several organs including brain, heart, lung, and kidneys. Although carcinomas are not much more frequent in tuberous sclerosis, TSC1 and TSC2 mutations are found in several carcinomas, e.g., of the urinary bladder and the kidney. Inhibitors of mTOR are applied to treat tuberous sclerosis and renal carcinomas (→16.7). In addition to cell growth and protein synthesis (via mTORC1), the AKT protein kinase stimulates cell survival and cell proliferation by phosphorylation of several substrates. Phosphorylation of forkhead (FKH) transcription factors like FOXO1 prevents their transcriptional activation of proapoptotic and growth-inhibitory genes like CD95, BIM, and PUMA, and CDKN1A, CDKN1B, and GADD45A, respectively. Phosphorylation by AKT moreover directly inhibits the apoptosis activator BAD (→7.4). Many proteins phosphorylated by AKT are recognized by 14-3-3 proteins. These prevent phosphorylated FOXO factors from entering the nucleus and phosphorylated BAD from interacting with mitochondrial proteins. Through the same mechanism, phosphorylation by AKT limits not only the transcription of CDKN1B but also the activity of the encoded p27KIP1 cell cycle

6.3 The PI3K Pathway

inhibitor. TP53 activity may also be diminished, since phosphorylation by AKT activates MDM2 (→5.3). Further effects on cell proliferation and metabolism result from phosphorylation of glycogen synthase kinase 3 (GSK3) by AKT. Both isoforms of GSK3 (α and β) are inhibited. GSK3 is well known as an important regulator of glucose metabolism and as a major target of insulin action. Phosphorylation by GSK3β moreover activates TSC2. In addition, the GSK3s regulate cell proliferation and survival by phosphorylating MYC and the antiapoptotic protein MCL-1, among others. Another GSK3β function, likely not affected by AKT phosphorylation, is the regulation of β-Catenin levels within the canonical WNT pathway (Sect. 6.9). As in the regulation of MYC and β-Catenin, phosphorylation by GSK3 often “primes” substrates for further phosphorylation and subsequent polyubiquitination and proteasomal degradation. While the TSC proteins inhibit one branch of PI3K signaling, PTEN antagonizes the pathway

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at its root, by hydrolytically removing phosphates from the position 3 of phosphatidylinositol compounds (Fig. 6.3). Mutations in the PTEN gene, located at 10q23.3, cause the hereditary Cowden tumor syndrome. Like mutations in the TSC genes, PTEN mutations in the germline predispose to multiple hamartomas, but unlike tuberous sclerosis, the Cowden syndrome confers increased risks for major common cancers, such as colon and breast cancer. Deletions, mutations, and inactivation of the PTEN gene are also observed in many different sporadic cancers and are often associated with tumor progression and a more aggressive clinical course. PTEN expression is delicately regulated at many different levels (Box 6.1). Further antagonists at the early stages of the pathway are the INPP4 phosphatases which hydrolyze the phosphate at position 4 of PI(3,4)P, the most crucial substrate of PI3K (Fig. 6.3). Interestingly, INPP4B is an imprinted gene (→8.4) and is subject to epigenetic inactivation in cancer.

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Box 6.1 Regulation of PTEN by an RNA Network

Many functions of the tumor suppressor PTEN are dosage-dependent. Transcription of the PTEN gene is regulated by positive and negative inputs. Among others, Early growth response protein 1 (EGR1), Peroxisome proliferator-activated receptor γ (PPARγ) and TP53 bind to its promoter region to activate the gene. The AP-1 factor JUN is an example of a negative regulator; it represses PTEN upon MAPK pathway activation. Monoallelic or biallelic deletion of its gene or epigenetic repression by DNA hypermethylation or repressive histone modifications downregulate PTEN expression in cancers or obliterate its expression. Posttranscriptional regulation is likewise important. A number of miRNAs have been reported to prevent translation of PTEN mRNA or promote its degradation, including (but not limited to) miR-21, miR-­ 17, miR-214, miR-19, miR-20, miR-93, miR-106b, and miR-26. Most of these miRNAs bind to the 3’UTR of the PTEN mRNA via their seed regions in a canonical fashion. Some of these miRNAs are upregulated in specific cancers and may significantly contribute to the downregulation of PTEN. PTENP1 is a pseudogene highly homologous to PTEN that is transcribed, but not translated into a protein. The PTENP1 transcript contains one or several binding sites for the miRs that also recognize PTEN mRNA.  By binding these miRs (“sponging”), PTENP1 transcripts decrease the miRNA concentration available to inhibit translation of PTEN from its actual mRNA.  Since its discovery, the PTENP1 RNA has become a prototype of a competing endogenous RNA (ceRNA). Meanwhile, many other ceRNAs have been postulated to influence cancer development and progression. In particular, many

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lncRNAs have been suggested to function as ceRNAs, among them are several which bind miRNAs that regulate PTEN expression. These include CASC2 and GAS5, which are upregulated in various cancers, the MEG3 lncRNA from an imprinted locus at 14q32, which is downregulated in many cancer types, and the XIST RNA which is prominently involved in establishing X-chromosome inactivation for dosage compensation. To make matters even more complex, the PTENP1 gene can also be transcribed in an anti-sense direction. The ensuing antisense RNA pairs with and stabilizes the sense transcript. While it is generally accepted that a RNA network contributes to the regulation of PTEN in normal cells, cancer, and other diseases, there are many discrepant or contradicting observations in the literature on this topic in detail. Given the intricacy of the network, this may not be too surprising. Li W et al. (2018) Regulation of PTEN expression by noncoding RNAs. J  Exp. Clin Cancer Res 37:223 Sellars E et  al. (2020) The complex landscape of PTEN mRNA regulation. Cold Spring Harb Perspect Med 10:a036236 Ghafouri-Fard S et al. (2021) Regulatory role of microRNAs on PTEN signaling. Biomed. Pharmacother 133:110986

Of note, PTEN may have functions beyond hydrolyzing phosphatidylinositol in the PI3K pathway by acting as a protein phosphatase on the SRC and FAK kinases, which signal in response to cell adhesion (section 6.5), and by regulating the expression of the RAD51 DNA repair protein in the nucleus. Overactivity of the PI3K pathway is a consequence of mutations activating receptor tyrosine kinases or RAS proteins in many cancers. In many other cancers, its overactivity is caused by genomic alterations that directly affect compo-

6.4 Interaction OF MAPK and PI3K Signaling in Cell Cycle Regulation

nents of the pathway (Fig.  6.2). Mutations in PIK3CA are among the most frequent ones across all types of cancers. Although they are all oncogenic, they are found in different domains of the protein. Depending on their location, they make kinase activity independent of RAS interactions or diminish binding of the regulatory subunits. Inactivation of the regulatory subunit genes PIK3R1 and PIK3R2 genes is infrequent. Oncogenic mutations activating AKT and mTOR kinases are found in some cancers. The most commonly mutated inhibitory component of the pathway is PTEN, which is subject to frequent biallelic inactivation by point mutations, deletions at 10q, or by epigenetic repression. Of note, compared to other tumor suppressors, PTEN loss may occur in a more gradual manner and partial loss of function may already favor tumor development in some tissues. PTEN biosynthesis is moreover regulated by an RNA network (Box 6.1) which includes onco-miRs that may exert their effects largely via PTEN downregulation. Further downstream in the pathway, the inactivation of TSC1 or TSC2, of LKB1 and even AMPK in specific cancers may result in enhanced mTOR activity.

6.4 Interaction of MAPK and PI3K Signaling in Cell Cycle Regulation During normal cell proliferation, MAPK and PI3K pathways complement each other (Fig. 6.4). Together, these pathways elicit a panoply of changes that complement the actual stimulation of cell proliferation, such as increased metabolism, enhanced protein synthesis, reorganization of the cytoskeleton, and inhibition of proapoptotic signals. Generally speaking, the MAPK pathway predominantly stimulates cell proliferation (i.e., DNA synthesis and mitosis), but requires additional signals from the PI3K pathway for cell growth (including protein biosynthesis) and in order to compensate proapoptotic signals caused by isolated strong MAPK signaling. Physiological stimulation of cell proliferation by (one or several) growth factors therefore

131

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Fig. 6.4 Synergism between the PI3K and MAPK pathways. A schematic interpretation of the interaction of the two pathways activated by growth factors and cytokines. These may act through receptor tyrosine kinases, but also other types of receptors responding to secreted factors or cell adhesion. See the main text for details. MYC activity is notably stimulated by both PI3K and MAPK signaling (not shown here)

often requires the coordinated activation of both pathways (see below). The receptors of certain growth factors, prominently members of the insulin family like IGF1 and IGF2, preferentially stimulate the PI3K pathway, but also other receptor tyrosine kinases (e.g., ERBB2/ERBB3). In this fashion, they may complement growth factors and receptors that act predominantly through the MAPK pathway. Accordingly, in tumor cells, mutations leading to the constitutive activation of the MAPK pathway may need to be complemented by increased activity of growth factors like the IGFs or by mutations directly activating the PI3K pathway. Conversely, therapeutic inhibition of one pathway may—at least partly—be compensated by crosstalk activation of the other one. For instance, inhibition of AKT may lead to increased RAF activity and consequently ERK activation and the inhibition of TSC2 by this route. Stimulation of cell proliferation by MAPK signaling is supported by PI3K signaling in several ways (Fig.  6.5). Following their translocation into the nucleus as a consequence of MAPK activation, activated ERK protein kinases phosphorylate several transcriptional activators that induce genes required for cell cycle progression.

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catalyzes the hydrolysis of inhibitory threonine phosphates groups from CDK4 as well as CDK2. As the CDK4/Cyclin D holoenzyme accumuP27KIP1 GSK3 lates, it sequestrates more p27KIP1, diverting it from CDK2. Since Cyclin E begins to accumulate at this stage of the cell cycle, CDK2/Cyclin E CDK 2 MYC enzyme activity increases. The Cyclin E gene CDC25A (CCNE) promoter is activated by E2F1. ERK RB1 Therefore, as RB1 starts to become hyperphosCDK 4 TFs phorylated and less capable of inhibiting E2F1, Cyclin D more Cyclin E is produced further increasing the activity of CDK2. This autocatalytic loop eventuFig. 6.5  Cell cycle activation by coordinated action of ally leads to irreversible commitment to S phase; the MAPK and PI3K pathways. KIP1 Following activation by mitogenic signals (top left cor- it is held in check by p27 . For full CDK2 activation, the p27KIP1 inhibitor ner), the pathways interact to stimulate cell cycle progression towards S phase through several factors; a selection is must be removed. This occurs by successive shown. Negative regulators are shaded phosphorylation, polyubiquitination, and proteolytic degradation. In normal cells, the downreguIn addition, ERK kinases phosphorylate the lation of p27KIP1 is a gradual process which pp90RSK kinases which then likewise translocate accelerates when CDK2 becomes sufficiently to the nucleus to phosphorylate a further set of active to phosphorylate a fraction of the inhibitor transcriptional activators. Among the more protein thereby liberating further CDK2 proteins important targets are the SRF, ETS1, ELK1, and to phosphorylate more p27KIP1, in another autoMYC factors, which are all stimulated by this catalytic loop. A crucial component in this protype of phosphorylation, and the AP1 compo- cess is MYC. MYC is induced by many growth nents FOS and JUN, which are also induced at factors, but its half-life and activity are regulated the transcriptional level. by phosphorylation. Phosphorylation at Ser62 by In many cells, an important consequence of ERK leads to increased stability and activity, these events is the stimulation of Cyclin D tran- whereas GSK phosphorylation at Thr58 has the scription3. Accumulation of Cyclin D drives cell opposite effect. In the stimulation of MYC activcycle progression during most of the G1 period. ity, again, the inhibitory phosphorylation of GSK It is counteracted by the degradation of the pro- by AKT synergizes with MAPK signaling. tein, which is promoted through phosphorylation Activation of MYC contributes to the induction by GSK3. This inhibition is relieved by phos- of Cyclin D1, CDK4, and CDC25A, and contribphorylation of GSK3 by AKT.  The coordinated utes to repression of p27KIP1. Moreover, MYC activation of Cyclin D is thus an important point induces CUL1, a component of the protein comof synergy between the MAPK and PI3K path- plex that polyubiquitinates p27KIP1. A final obstaways (Fig. 6.5). cle to cell cycle progression is induction of CDK Further genes activated as a consequence of inhibitors by FOXO transcription factors. These MAPK pathway stimulation code for the are phosphorylated by AKT and consequently CDC25A phosphatase and the G1 cyclin-­ maintained in the cytoplasm. Regulation of MYC dependent kinase CDK4. CDC25A induction and CDK inhibitors like p27KIP1 are thus further synergizes with Cyclin D accumulation to points of synergy between the two pathways. increase CDK4 activity since the phosphatase The various synergisms between the MAPK and AKT pathways that drive cell cycle progression during normal cell proliferation are reflected 3  In most cell types, this would be Cyclin D1, Cyclin D2 and D3 are involved in a more restricted range of cell in the alterations observed in cancers (Fig. 6.6). types. Receptor tyrosine kinases may be such potent AKT

6.4 Interaction OF MAPK and PI3K Signaling in Cell Cycle Regulation

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Fig. 6.6  An overview of alterations in MAPK and PI3K pathways in human cancers. Alterations in the MAPK (right) and PI3K (left) pathways synergize to increase cell proliferation and growth and to decrease apoptosis. Pathway inhibitors inactivated by deletion, mutation, and/or promotor hypermethylation in

cancers are marked in green. Pathway activators activated by mutation or overexpression in cancer are marked in red, dark red indicates frequently observed alterations. In addition, oncogenic activation of RTKs is a common cause of the overactivity of the two pathways

oncogenes because they are capable of activating both pathways in parallel. Oncogenic RAS may likewise activate both pathways. Conversely, oncogenic MYC itself, while very efficient in promoting cell growth and stimulating cell cycle progression, tends to drive cells into apoptosis, which needs to be counteracted by factors that activate the PI3K pathway, like IGF growth factors or RAS mutations. This explains the cooperation of RAS-like and MYC-like oncogenes in experimental rodent cell transformation assays (→4.3), that is indeed at work in many human cancers. However, these combinations of alterations are probably still not sufficient to cause human cancers, since they are counteracted by further fail-safe mechanisms. The most important ones, arguably, are activation of TP53 by hyperprolif-

eration signals and accumulation of CDK inhibitors like p16INK4A and p21CIP1 during sustained rapid proliferation of human cells. These fail-safe mechanisms ultimately induce replicative senescence (→7.2). Thus, at some point in the ­development of human cancers, they need to be inactivated. The most fundamental way to achieve this is through the loss of both RB1 and TP53. Another frequent mechanism is deletion of the CDKN2A locus that encodes p16INK4A and p14ARF. Of note, while the prime significance of these genomic alterations may be the inactivation of fail-safe mechanisms against hyperproliferation, they concomitantly diminish the dependence of tumor cells on external growth factors and increase their tolerance of genomic instability. Alterations that lead to increased or constitutive activity of the MAPK and PI3K pathways

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may in some cases be self-limiting, inducing apoptosis or senescence, but they exert a strong selection pressure favoring further changes that lead to progression towards a more aggressive phenotype. This sort of progression is observed in many human cancers, in carcinomas as well as hematological cancers.

6.5 Modulators of the MAPK and PI3K Pathways The MAPK and PI3K pathways are not only activated by receptor tyrosine kinases, but also modulated by further signals. These emerge from other kinds of receptors, such as cytokine receptors and G-protein-coupled serpentine receptors (GPCR), as well as from various cell adhesion molecules. This part of the intracellular signaling network involves a number of additional protein kinases as well as other enzymes. Several of those were in fact discovered (in often substantially altered form) as retroviral oncogenes. Nevertheless, in human cancers, they rarely appear as oncogenes or tumor suppressors that would fit the stringent definitions discussed above (4.3 and 5.4). These proteins are instead important in human cancers by enabling the establishment of typical properties of tumor cells and as modulators of the MAPK and PI3K cancer pathways. Most receptor tyrosine kinases (RTK) and some GPCRs activate the MAPK cascade through RAS. Many GPCRs and some RTKs additionally employ phospholipases in this context, typically PLCβ in the case of GPCRs and PLCγ in the case of RTKs. These phospholipases cleave phosphatidylinositol diphosphate in the membrane to yield diacylglycerol and inositol triphosphate (IP3). IP3 is a second messenger molecule that regulates cytosolic Ca2+ levels which affect many cellular functions, including metabolism, transcription, the cytoskeleton, and motility. The diacylglycerol moiety liberated by the PLC reaction acts as another signal molecule by binding and activating PKC protein kinases. This protein kinase family comprises more than 10 members in humans, with different tissue distri-

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butions and specificities. The most widely distributed member is PKCα. This enzyme, like other “classical” PKCβ and PKCγ isoenzymes, is activated synergistically by diacylglycerol and Ca2+, whereas other (“novel”) isoenzymes such as the likewise widely distributed PKCδ are independent of Ca2+ levels and tend to be located at intracellular membranes. Other (“atypical”) family members, represented by PKCξ, are independent of both activators. The structure of PKCs is modular, like that of many other protein kinases. A distal catalytic domain containing the ATP and substrate binding site is auto-inhibited in the inactive state by a pseudo-substrate in the proximal regulatory domain. In the conventional PKC isoenzymes, the regulatory region contains binding sites for diacylglycerol and for calcium ions. Binding of both activators relieves their autoinhibition. Like the RAF kinases, PDK and AKT, many PKCs are activated at the inner face of the plasma membrane. The diacylglycerol binding site in PKCs strongly binds phorbol esters like tetradecanoyl phorbol acetate (TPA, also known as phorbol myristate acetate, PMA). This natural compound from croton oil is a strong irritant and stimulates the proliferation of some cells. In animal experiments, it promotes the growth of skin tumors, although it is not mutagenic. It is thus the prototype of a “tumor promoter.” However, promotion is dependent on the tissue and the species. Moreover, as repeated application of PMA results in permanent downregulation of PKCs, it is unclear whether promotion by PMA in vivo is mediated by short-term activation or long-term downregulation of PKCs. PMA activity may depend on which PKC isoenzymes are expressed and how crucial they are for the regulation of proliferation in a particular tissue. PKCs are serine/threonine protein kinases with a (broad) range of substrates that differ between the individual isoenzymes. Since PKCs such as PKCα and PKCε can phosphorylate RAF, they can augment MAPK signaling independent of or synergistically with RAS. By phosphorylating cytoskeletal proteins like MARCKS, they affect the structure of the actin cytoskeleton.

6.5 Modulators of the MAPK and PI3K Pathways

Through MARCKS and by direct phosphorylation, they influence cell motility and transport processes. PKCs also phosphorylate selected nuclear receptors like the Vitamin D receptor (VDR). Last, but not least, PKCs can phosphorylate and regulate receptor tyrosine kinases, including the EGFR. Phosphorylation at Thr654 by PKCs not only promotes internalization of the EGFR but also diminishes its kinase activity. Similarly, PKC phosphorylation decreases PI3K activity and promotes its internalization. Conversely, the maturation and stability of PKCs are regulated by the PI3K pathways through PDK1 and mTORC2. One could say that PKCs are in effect used to relay signals in the cell from one activated pathway to another, i.e., for crosstalk. Their function may thus be characterized as coordinating rather than determining. This could explain why they have not been observed to act as strong oncogenes in spite of their close connection to the control of cell proliferation and motility. The (overall infrequent) mutations in PKC genes found in cancer predominantly inactivate their function, but PKCs are neither classical tumor suppressors. Inhibition of PKCs does block the proliferation of some cancer cells and clearly, activation by phorbol esters can principally stimulate the proliferation of normal and particularly of transformed cells. In addition, PLCs and PKCs appear to be critical mediators of the effects of several oncogenes, including retroviral oncogenes like v-yes and cellular oncogenes like TRK that act in lymphoid cells. They may also be necessary in those (rarer) cases, where the proliferation of cancer cells is caused by activated G-coupled receptors. PKCs also participate in the crosstalk between cell adhesion molecules and growth-regulatory pathways. Signals emerge from cell-to-cell adhesion as well as from adhesion to the ECM. Cell-­ to-­ cell adhesion is prominently mediated by cadherins which are connected to the cytoskeleton and influence its activity (→9.2). Different cadherins activate or inhibit specific cancer pathways to different extents. E-Cadherin, the major cadherin in many epithelia, binds β-Catenin and may thereby limit WNT/β-Catenin pathway

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activity (→6.9) in some cell types. Moreover, E-Cadherin supports PI3K pathway activation to yield a robust survival signal. Both signals make sense in a cell that is firmly integrated into a relatively quiescent epithelium. In many invasive carcinomas, E-Cadherin is downregulated or becomes replaced by other cadherins that activate different pathways. For instance, N-Cadherin activates signaling through RHO GTPases and stimulates the dimerization and thereby the activation of FGF receptors. Integrins mediate cell interactions with the basement membrane and the ECM (→9.2). At the inner surface of the cell membrane, they provide a focus to organize the attachment of the actin cytoskeleton at focal adhesion contacts. These contacts are the spatial hub of a signaling network that impacts the cytoskeleton, cell proliferation, and survival (Fig. 6.7). Once integrins are connected to ECM proteins and the actin cytoskeleton, additional proteins like Talin are recruited to the focal adhesion protein complex on the inner face of the cell membrane and a number of signaling pathways can become activated. In particular, the tyrosine kinases FAK (Focal adhesion kinase), SRC (the human ortholog of the RSV v-src oncogene, →4.1), and the pseudo-kinase ILK (Integrin-linked kinase)4 relay signals from focal adhesion sites. Following autophosphorylation, FAK can be recognized by SH2 domains of the SRC protein kinase, which is linked to the inner face of the plasma membrane through a myristate anchor. Upon binding to FAK, SRC is activated and in turn further phosphorylates FAK. Activated SRC behaves similar to activated receptor tyrosine kinases, binding adaptor proteins like SHC/GRB2, phosphorylating and activating a number of proteins, including RAS as well as PLCs and consequentially PKCs, and in this fashion ultimately stimulates the MAPK and PI3K pathways. SRC furthermore interacts mutually with the EGFR. It binds to the EGFR when it is activated by EGF-like growth ILK contains a kinase-like domain and binds ATP, but does not phosphorylate any of its multiple binding partners. 4 

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136 Fig. 6.7  Signaling from integrins. See main text for explanation

Integrins

P

FAK

SRC

ILK

P PLC PKC

Paxillin complex

RAS RHOA

PI3K

RAF

factors, or following the activation of SRC by other receptors or adhesion signaling. SRC phosphorylates specific tyrosine residues in the EGFR intracellular domain, e.g., Y845, to amplify signaling through MAPK and other mediators, including PLC, PKC, and STAT5. Integrins are furthermore capable of activating PKCs directly and interact with small GTPases like RAC that regulate the structure of the cytoskeleton. SRC is often overexpressed and overactive in human cancers, but, unlike its viral ortholog, the human protein is rarely activated by mutations. Increased activity of SRC is however often associated with carcinoma progression, where cells become highly invasive and migratory. Increased activity of FAK and expression of ILK are likewise observed in such cases. On the one hand, these changes likely result from differences in the molecules mediating adhesion in invasive cancer cells. On the other hand, the increased activity of the protein kinases contributes to increased ­motility. In particular, their activation may simulate an attachment signal that prevents anoikis, a specific type of programmed cell death elicited by lack of adhesion. Anoikis presents another barrier to the systemic spread of cancer cells during metastasis, especially to that of single cells.

Actin cytoskeleton

6.6 Signaling by TGFΒ Factors The TGFβ superfamily of growth factors comprises ≈30 members with diverse functions. For instance, members of the family such as Mullerian inhibitory factor or the diverse bone morphogenetic proteins (BMPs) were discovered for their functions in shaping tissues during development. In the context of cancer, TGFβ1 has received the most attention, as it is a potent growth inhibitory factor for epithelial cells and because mutations in pathways that relay TGFβ signals in the cell are frequent during the progression of many carcinomas. TGFβ1 is also an inhibitory factor for many immune cells. In contrast, it stimulates the proliferation of many mesenchymal cell types. This last property had originally led to the designation “transforming growth factor” because TGFβ cooperates with the EGF-like TGFα to stimulate anchorage-independent growth of mesenchymal cells in culture, a characteristic of a transformed phenotype in these cells. In carcinoma tissues, not only the loss of response to TGFβ in the tumor cell, but also its effects on immune and mesenchymal cells are significant. TGFβ secreted by carcinoma cells or liberated from pre-proteins bound to the ECM inhibits anticancer immune responses while stim-

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Fig. 6.8 Intracellular signaling by TGFβ and BMPs. R-SMADs are labeled in green, Co-SMADs in grey, and I-SMADs in yellow, respectively

TGFβs

BMPs

P

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Cytosol

SMAD2/3

SMAD1/5/8

SMAD7

SMAD2/3

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SMAD6

SMAD4 P SMAD4

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Nucleus P SMAD2/3 SMAD4

ulating stromal cells to proliferate and produce ECM and other factors supporting carcinoma growth. In particular, the ability of carcinoma cells to secrete or activate TGFβ for action on other cells while being themselves unresponsive to its growth-inhibitory effect helps to establish an environment favorable for invasion and metastasis. In carcinoma cells with intact TGFβ signaling, TGFβ may facilitate invasion and metastasis by promoting epithelial-mesenchymal transition (EMT). These relationships may explain why changes in TGFβ signaling often portent the onset of invasion and metastasis in the course of carcinoma development (cf. Sect. 13.5). The main intracellular pathway for TGFβ signaling is quite straightforward (Fig.  6.8). The growth factor associates with a type II TGFβ receptor (TGFBR2) at the cell membrane to form a tetrameric complex with a type I receptor (TGFBR1). The receptor type I protein becomes phosphorylated which activates its serine/threonine kinase. It then phosphorylates SMAD2 or

SMAD1/5/8

P

SMAD4

SMAD3 proteins which are presented by the SARA protein at the inner face of the cell membrane. The phosphorylated SMADs are released and can heterodimerize with SMAD4 to become transported into the nucleus where they activate or repress various genes. SMAD2 and SMAD3 are categorized as R-SMADs (“R” denoting receptor-regulated), whereas SMAD4 is categorized as a Co-SMAD.  A different set of R-SMADS, SMAD1, 5 and 8, are activated by distinct receptors for BMPs, which otherwise function in the same manner as TGFβ receptors. SMAD factors are not very potent transcriptional transactivators. Therefore, their activity and target gene spectrum depend on the interaction with other transcription factors that bind in their vicinity. This may partly explain why their action is often context-dependent and differs among various cell types. For instance, in conjunction with the TCF4 transcription factor SMAD3/4 heterodimers enhance the transcription of WNT pathway target genes (→6.9),

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whereas in conjunction with FOXO transcription factors they enhance the transcription of CDK inhibitor genes like CDKN1B and CDKN2B. The influence of TGFβ signaling on EMT is partly exerted through induction of transcription factors like SNAIL that subsequently bind together with SMAD proteins to the E-Cadherin gene (CDH1) promoter to repress the gene. Activation of the TGFβ pathway is counteracted by the inhibitory SMADs (I-SMADs) SMAD6 and SMAD7. They are induced by TGFβ as well as BMP signaling, leading to feedback inhibition of either pathway. Signaling through the pathways can be downregulated by ERK and JNK kinases that phosphorylate R-SMADs. Conversely, MAPK pathways are activated by some members of the TGFβ receptor family by noncanonical signaling mechanisms. Further crosstalk takes place between TGFβ signaling and the canonical WNT pathway. In general, these pathways tend to inhibit each other. Loss of responsiveness to TGFβ in the progression of carcinomas can be brought about by alterations at several steps of the pathway. They include mutations in receptor genes, especially TGFBR2, loss of R-SMADs or Co-SMADs, most often SMAD4, by deletions of their genes or their inactivation by mutation. Often, while canonical signaling is obliterated by these genetic changes, noncanonical signaling may be preserved. Such alterations are, e.g., observed in invasive colorectal carcinomas (→13.5) or in metastatic prostate carcinomas (→20.4).

6.7 Signaling Through NFkB The primary functions of NFκB signaling are the regulation of activation of lymphoid cells, and more broadly. the regulation of inflammation and apoptosis. In some cell types, NFκB signaling is also involved in the regulation of cell proliferation, specifically in response to cytokines. In carcinomas, the NFκB pathway may be most important in the actual cancer cells by influencing survival and communication with stromal cells, inflammation, and the tumor microenviron-

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ment. Modulation of apoptotic efficiency remains important in cancers as well. In principle, two NFκB pathways are distinguished, named “canonical” and “alternative.” The designation NFκB stands for “nuclear factor regulating the expression of the Ig kappa chain in B cells.” NFκB factors are heterodimeric transcriptional activators composed of a larger REL subunit and a smaller p50 or p52 subunit. The REL subunits comprise RELA, RELB, and c-REL.  RELA is the most abundant and widespread of these proteins and commonly known as p65. It is also the strongest transcriptional activator. In the canonical pathway RELA or c-REL form heterodimers with p50, which is derived from a larger p105 precursor. These heterodimers shuttle between the nucleus and cytoplasm. In the basal state, the dimers are retained in the cytoplasm by the protein inhibitor IκBα (Fig. 6.9). In the alternative pathway, RELB associates with the precursor of the p52 subunit, p100, which contains an inhibitory domain similar to IκB. The RELB-p100 complex is also retained in the cytoplasm. Activation of canonical and noncanonical NFκB signaling can be elicited by different signals, including various cellular stresses, reactive oxygen species (Box 1.1), or—in a more controlled fashion—from cytokine receptors. In lymphoid cells, these would be receptors for interleukins or co-receptors for lymphocyte activation by the TCR or immunoglobulin receptors. In macrophages, stimuli could be provided by receptors for cytokines or by bacterial lipopolysaccharides acting through toll-like-receptors (or other PAMPs, pathogen-associated molecular patterns). In B cells, the canonical pathway is also stimulated directly by activated B cell receptors. In many cell types, activation of canonical NFκB signaling counteracts apoptosis or influences its efficiency. In particular, it modulates the effects of TNFRSF family receptors (→7.4) on apoptosis and mediates their effects on induction of cytokines and other factors that influence immune responses and inflammation. The cytokine TNFα (Tumor necrosis factor α) is a prototypic activator of NFκB signaling in many cells.

6.7 Signaling Through NFΚB

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TNFSFRs

Toll-like receptors

IKKβ

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IKKα

Ub

MAPKs AKT

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IκB α

IKKβ

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Ub

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IκB α

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p65 NFκB-RE

Iκ

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Iκ

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RELB NFκB-RE

Fig. 6.9  An outline of the two NFκB pathways. The scheme emphasizes the activation of cytoplasmic NFκB proteins to nuclear transcriptional activators in the canonical (left) and alternative (right) pathways. The

events leading to their activation by a variety of signals are considerably more complicated than shown here. Moreover, they differ between cell types

Upon TNFα binding, its receptor TNFR associates at the cell membrane with a protein complex that becomes extensively ubiquitinated. In one outcome, this complex interacts with and activates the IKK kinase complex (composed of IKKα, β, and γ) by ubiquitination of its regulatory IKKγ subunit (more commonly known as NEMO) and by phosphorylation of the IKK kinases by the TAK1 protein kinase. Other components of the active TNFR complex may influence JNK, ERK, and p38α MAPK signaling. In some instances, moreover, RIP kinases in the complex may induce regulated necrotic cell death. Following their activation, the IKK protein kinases phosphorylate the inhibitory subunit IκBα, leading to rapid accumulation of the p50/ p65 dimer in the nucleus. Activation of the alternative pathway does not involve IKK and proceeds more slowly, but generates a more prolonged signal. This pathway

rather responds to regulators of immune cell differentiation and maturation. An important step consists of additional phosphorylation by the NIK protein kinase that initiates the proteolytic removal of the inhibitory domain of the p100 pre-­ protein to allow the active RELB/p52 dimer to enter the nucleus. At least four kinds of target genes are affected by NFκB factors. (1) NFκB induces its feedback inhibitors, especially IκB proteins. (2) Depending on the cell type, regulators of cell proliferation like MYC and Cyclin D1 may be induced. (3) A much larger set of induced proteins modulate apoptosis as activators or inhibitors. Most of these, like BCL-XL and FLIP, are antiapoptotic, while others, like CD95 (TNFSFR6) and its ligand (CD95L), are usually proapoptotic (→7.4). (4) An even wider set of activated genes relate to immune function and particularly influence inflammation; they are induced particularly strongly in immune cells,

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but also in other cell types. The gene products are often involved in cell-cell communication, e.g., as adhesion molecules, chemokines, and cytokines, but also comprise factors involved in specific or general immune responses. For instance, NFκB factors mediate induction of the inducible nitrogen oxide synthase (iNOS) and cyclooxygenase 2 (COX2), which contribute to the burst in reactive (oxygen) species in innate immune defenses and inflammation. The antiapoptotic effects of NFκB factors normally help to select, protect, and activate functional lymphoid cells, but can contribute to cancer development and to therapy resistance. Mutations in pathway components, including translocations and amplifications of REL genes, stimulate proliferation and support survival of the tumor cells in several human lymphomas. The retroviral ortholog of the REL genes, v-rel (→4.1), is a potent oncogene that causes leukemias and lymphomas. In comparison to its clearly oncogenic function in lymphomas, the effects of NFκB activation in carcinomas are less straightforward. Its effect on apoptosis may be relevant, especially in the survival of cancer cells during metastasis and during chemotherapy. During carcinogenesis, the pathway contributes to inflammation and specifically to chronic inflammation. It is therefore particularly crucial in the development of cancers associated with chronic inflammation. In some cases, activation is relatively specifically elicited by the pathogenic agent, such as Helicobacter pylori in the stomach (→18.2). In many carcinomas, NFκB signaling may be activated as a consequence of the prevalent aneuploidy and chromosomal instability, which lead to the liberation of genomic DNA into the cytosol. Cytosolic DNA activates cGAS/STING signaling and consequently stimulates—via the TBK1 kinase— canonical NFκB signaling. This mechanism ought to attract immune responses to damaged and infected cells, but during carcinogenesis often results in inflammation. Compounds blocking NFκB activation can therefore allay inflammation and prevent the development or slow the progression of cancers that are promoted by inflammation. These compounds include some commonly used nonsteroidal anti-inflammatory

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drugs (NSAIDs) like acetylsalicylic acid (aspirin) that inhibit IKK activity. At the primary site and in metastases, NFκB signaling mediates mutual interactions between carcinoma and stromal cells. Typically, in a vicious cycle, cytokines and growth factors act on the carcinoma cells via the NFκB pathway to promote the secretion of further secreted factors that stimulate the survival, activity, and cytokine production of stromal cells, which ultimately support survival, proliferation, and migration of the carcinoma cells (→9.6). For instance, breast and prostate cancers metastasize preferentially to bone by interacting with local osteoblasts or osteoclasts, through growth factors and cytokines acting partly through NFκB (→20.4). Inherited mutations in NFκB pathway components underlie a large variety of immune deficiencies and diseases, but the pathway is also implicated in at least one human dominantly inherited cancer syndrome. The rare familial cylindromatosis syndrome is caused by inherited mutations in the CYLD gene at chromosome 16q, which behaves as a classical tumor suppressor gene. Mutations in the second allele elicit tumors of the hair follicles and sweat glands. The protein encoded by the CYLD gene is a negative regulator of NFκB pathway activation that acts by deubiquitinating the TNF receptor complex.

6.8 Signaling Through STAT Factors Like the NFκB pathway, signaling through STAT factors is particularly important in immune cells, but is also relevant for regulating apoptosis and immune responses in epithelial cells. Genetic and epigenetic changes lead to overactivation of STAT signaling especially in cancers of the hematopoietic system, whereas in carcinomas STAT signaling contributes mostly to chronic inflammation and tumor-stroma interactions, in fact often in conjunction with the NFκB pathway. The actual STAT signaling pathway is quite straightforward (Fig. 6.10). Upon binding of their ligand, cytokine receptors recruit Janus kinases

6.8 Signaling Through Stat Factors Fig. 6.10  Activation of the JAK/STAT pathway by IL6. See the main text for details

141 IL-6 IL6R

P

P

JAK2

JAK1

STAT

STAT

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AS

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PI

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STAT

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STAT

(JAK1 or JAK2), which phosphorylate the receptor protein at tyrosine residues. STAT factors bind to these phosphotyrosines by their SH2 domains and subsequently become likewise phosphorylated. They then travel to the nucleus to act as transcriptional activators. A typical binding sequence for STAT factors is the GAS element mediating γ-interferon (IFNγ) responses; the slightly different ISRE elements respond ultimately to IFNα/β. Like SMAD factors, STAT proteins are not strong transcriptional activators and usually combine with other factors binding to adjacent sequences for efficient transcriptional activation. This provides one of several ways in which NFκB and STAT signaling interact in the regulation of gene expression. One set of STAT target genes encodes SOCS factors which act as JAK inhibitors and contribute to termination of STAT signals as feedback inhibitors. Termination of STAT signaling is supported by dephosphorylation of the transcription factors through SHP protein phosphatases. Many target genes of STAT3 and STAT5 encode negative regulators of apoptosis like BCL2, BCL-XL, and MCL-1 or cytokines, but also D-Cyclins.

The seven different STAT transcription factors relay signals to the nucleus in response to different cytokines. STAT1 and STAT2 induce a number of genes in response to interferons, while STAT4 and STA6 mediate the effects of specific interleukins, particularly in immune cells. STAT3 and the STAT5a and STAT5b proteins are activated by a broader range of cytokines. STAT3 in particular can be activated by growth factor receptors like EGFR and MET, but also by adhesion signals via SRC or the activated ABL kinase. All STAT factors are degraded by the proteasome following their poly-ubiquitination by PIAS proteins. Constitutive activation of STAT signaling is common in many cancer types. Typically, nuclear levels of (phosphorylated) STAT3 and STAT5 are increased, whereas STAT1 is rather downregulated. This downregulation may diminish the proapoptotic and immune-stimulatory effects of interferons and dampen the induction of CDKN1A. In hematological cancers, constitutive STAT activation may be caused by mutations in the pathway itself, for instance by oncogenic JAK2 mutations. These alterations include point

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mutations in the regulatory JH2 domain or in the C-terminal protein kinase domain as well as the formation of fusion genes retaining parts of the JAK protein, especially the protein kinase domain.5 Inactivation of SOCS genes, e.g., by promoter hypermethylation, may compound activation of STAT signaling. SOCS hypermethylation is accordingly also found in carcinomas with constitutive STAT activation resulting from receptor tyrosine kinase activation. More frequently and especially in carcinomas, sustained activation of STAT3 and STAT5 is the consequence of signaling by autocrine and paracrine factors from the tumor microenvironment. Since many STAT factors target genes encode cytokines, STAT signaling helps to maintain the fatal vicious cycle between tumor and stroma cells, especially in cancers promoted by chronic inflammation.

6.9 Developmental Regulatory Systems as Cancer Pathways Signaling through MAPK, PI3K, and other pathways is relevant for cell proliferation, differentiation, and apoptosis in adult tissues and of course also during ontogeny. Especially during ontogeny, additional pathways are required that direct morphogenesis through decisions on cell fate, cell differentiation, and on the expansion of cell populations. In particular, these “morphogenic” pathways define stem and precursor cells and support lineage choices. In a similar manner, morphogenic pathways remain particularly important for the maintenance of tissue stem and precursor cells and for the regulation of their proliferation and differentiation activities in adult tissues. Prominent morphogenic pathways are WNT (Fig. 6.11), Hedgehog/ SHH (Fig. 6.12), Notch (Fig. 6.13), and Hippo (Fig.  6.14) signaling. Morphogenic pathways are regulated by paracrine factors, which often In the nucleus JAK2 can additionally function as a histone kinase. 5 

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form gradients (like WNT and Hedgehog factors), or by cell-cell interactions (like Notch and Hippo signaling). Each of the pathways is activated in specific cancers by mutations in crucial regulatory components or—in the case of WNT and Hedgehog signaling—also through autocrine secretion of growth factors. Moreover, inactivation of Notch signaling and of parts of the Hippo pathway is found in specific cancers. WNT signaling: More than 20 factors constitute the WNT growth factor family in humans. All are ≈40 kDa proteins with over 30% homology towards each other. WNT factors are glycosylated and covalently linked to a lipophilic moiety, palmitoyl acid, before being secreted. Accordingly, the factors are poorly soluble and tend to stick to cell membranes, which restricts their diffusion. Therefore, they are limited to ­acting as paracrine or autocrine factors and tend to form gradients that emanate from their site of secretion. During development, WNT proteins drive the expansion and morphogenesis of many different tissues, e.g., in the gastrointestinal tract. To this end, WNT factors and pathways interact with others, e.g., with Hedgehog signaling in the development of the limbs. WNT factors remain involved in the homeostasis of adult tissues where they especially control stem cell compartments, e.g., in the gut. At the surface of their target cells, WNT factors are recognized by Frizzled receptor proteins (FZD) together with LRP proteins (LRP5 and LRP6) which appear to recognize rather the lipid part of the WNTs. The trimeric complex formed by ligands and receptor proteins is rapidly internalized and degraded following polyubiquitination by RNF43 or ZNRF3 transmembrane proteins. Another growth factor, R-Spondin, prolongs and intensifies WNT signaling by stimulating removal of RNF43 or ZNRF3 from the membrane by its receptors LGR4 or LGR5. Notably, some WNT factors, like WNT5A, bind and activate additional receptors, including the receptor tyrosine kinases RYK and ROR, and thereby activate alternative (noncanonical) WNT

6.9 Developmental Regulatory Systems as Cancer Pathways

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signaling pathways that regulate rather cell polarity and differentiation. The crucial step in the major, canonical WNT pathway is the stabilization and nuclear translocation of β-Catenin. It is therefore also commonly known as the WNT/β-Catenin pathway. From the FZD/LRP/WNT complex, it proceeds by tethering the β-Catenin destruction complex to the cell membrane. The destruction complex

comprises DVL (disheveled), AXIN, APC, and the protein kinases CK1 and GSK3β. In the absence of WNT, it binds β-Catenin, which is successively phosphorylated at several serine residues in its N-terminal region by the two kinases. Phosphorylated β-Catenin is then recognized by the E3 ligase β-TRCP and polyubiquitinated which initiates its proteasomal degradation. In this fashion, the concentration of β-Catenin in

Fig. 6.11  The core WNT/β-Catenin pathway. The inactive state (in the absence of WNT factor binding) is shown on the left and the active state on the right. This sketch of the central pathway does not incorporate several

additional modulators discussed in the main text and in Sect. 13.4. From: Nusse and Clevers (2017) l.c, with permission

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144

Fig. 6.12  The Hedgehog (SHH) pathway.

Left: inactivate state; right: active state. From Wu et al. Cell Chem. Biol. (2017) l.c. with permission See main text for further explanation

Receiving cell

Signaling cell

NOTCH receptor

Cleavage by ADAM

Cleavage by γ-secretase

NICD NRR NOTCH ligand

Fig. 6.13  Canonical Notch signaling. NRR: negative regulatory region; the NICD comprises the RAM (RBP-Jκ/CBF1-associated module, named for its interaction with CBF1/CSL), ankyrin (ANK), and PEST

MAML1 CSL CSL-BS

domains. The PEST domain is named for its high content of proline (P), glutamic acid (E), serine (S), and threonine (T). MAML1 is a coactivator synergizing with the NICD in gene activation

6.9 Developmental Regulatory Systems as Cancer Pathways hesion

GF sig

Cell ad Cytosol

naling

NF2 FAT1 P

MAPK RHO PDK

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SAV1

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P

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LATS1/2

MOB

14-

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TAZ/YAP

Nucleus

3-3

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Fig. 6.14  The core Hippo pathway. See main text for explanation

the cytosol is kept low.6 Upon active WNT ­signaling tethering of the destruction complex to the membrane leads to its—partial—dissociation and in turn to an increased level of β-Catenin in the cytosol. This enables translocation of β-Catenin into the nucleus, where it binds to TCF factors, especially TCF4. There, it replaces corepressors to act as coactivator in the induction of WNT target genes. Typical transcriptional targets of the WNT/β-­Catenin pathway include activators of the cell cycle like Cyclin D and MYC, but also growth factors, proteins mediating cell-cell interactions like ephrins, and secreted proteins acting on the ECM like Metalloproteinase 2. The transcription factor TCF1 (encoded by the TCF7 gene) and the AXIN1 paralog AXIN2 are likewise induced and serve as feedback inhibitors in the nucleus and the cytoplasm, respectively. Outside the cell, several secreted proteins modulate or inhibit WNT signals. Whereas R-Spondin augments WNT signaling through LGR4/5 as described above, several secreted Frizzled related proteins (SFRP) and WIF1 compete with FZD for WNT factors. DKK1 (Dickkopf 1) is another feedback inhibitor

Of note, β-Catenin is also a component of adhesion junctions, where it is stably bound to E-Cadherin and not available for WNT signaling. 6 

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induced by TCF/β-Catenin and interferes with WNT binding to the LRPs. Constitutive activation of the WNT/β-Catenin pathway in cancer can be brought about either by loss of inhibitory components like APC, the two Axins, or RNF43, or by oncogenic mutations activating β-Catenin. The relative contribution of these changes differs substantially among cancers of different tissues and even between subtypes of cancers in one tissue. Germline mutations in the classical tumor suppressor gene APC underlie the dominant familial adenomatous polyposis coli syndrome (for which the gene was named) which causes predominantly colorectal cancer. Mutations in APC are also the most frequent genetic alteration in sporadic colorectal cancers (→13.3). In other cancer types, e.g., hepatocellular carcinoma (→17), oncogenic mutations in β-Catenin predominate. Typically, these mutations destroy phosphorylation sites for GSK3β. As in the classical experiment where WNT1 activation by a retroviral insertion (→4.2) was discovered, autocrine stimulation by WNT factors is another mechanism increasing pathway activity in cancer and is enhanced by overexpression of FZD receptors. Genetic alterations affecting ligands and receptors predominate in breast and lung cancers, among others. Finally, epigenetic mechanisms, in particular hypermethylation of SFRP genes, exacerbate WNT overactivity in various cancers. Hedgehog signaling: In contrast to the large number of WNTs, there are only three hedgehog proteins in man, named Sonic hedgehog (SHH), Desert hedgehog (DHH), and Indian hedgehog (IHH). Like WNT factors, they act in a paracrine fashion and like WNTs, their diffusion is limited by a lipophilic modification, in this case the covalent attachment of cholesterol. Hedgehog factors were initially implicated particularly in limb development, but they are in fact crucial for the development of many other organs, as diverse as the skin, the prostate, and various structures in the brain. Like WNTs, they contribute to tissue homeostasis in adult humans and are likewise implicated in the maintenance of stem cell ­populations. The most broadly expressed factor in adult human tissues is SHH.

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The ultimate mediators of Hedgehog signals within the cell are the three GLI transcription factors. Of these GLI2 and (more strongly) GLI3 can act as transcriptional repressors, whereas GLI1 is a pure activator. Typically, GLI2 and GLI3 are expressed in the basal state, whereas GLI1 is mostly induced following activation of the pathway. Activation of Hedgehog signaling also involves a shift in the relative proportions of activating and repressing GLI2 and GLI3 isoforms. Notably, all three factors can bind the inhibitory protein SUFU. Hedgehog signaling at the cell membrane occurs via the transmembrane proteins PTCH1 (patched 1) and SMO (smoothened) at the primary cilium, a specialized organelle localized at one pole of a cell (Fig. 6.12). In the resting state, PTCH1 located at the base of the cilium inhibits accumulation of SMO at the cilium. The base and the length of the cilium are distinguished by ­different phosphorylated forms of phosphatidyl inositol. The mechanism by which PTCH1 inhibits SMO is indirect and is thought to involve transport of a yet unknown sterol compound by PTCH1. Binding of Hedgehog factors to PTCH1 leads to its internalization and degradation and liberates SMO which becomes phosphorylated and moves upwards on the cilium membrane. The ultimate effectors of Hedgehog signaling in the nucleus are processed GLI transcriptional activators. Outside the cilium, full-length Gli factors GLI2 and GLI3 are kept in a complex with SUFU.  Following their phosphorylation by the cAMP-dependent kinase (PKA), CK1 and GSK3, they are proteolytically processed to repressors (GLI2R and GlI3R), which can enter the nucleus to repress Hedgehog target genes. Notably, cAMP for PKA is in this case provided through GPR161, a G-protein-coupled receptor localized at the base of the cilium. Upon activation of Hedgehog signaling GPR161 is excluded from the cilium and residual cAMP is degraded by Phosphodiesterase 4. Accumulation of SMO at the cilium, where it associates with EVC proteins, allows the active transport of GLI proteins up into the cilium which is driven by kinesin motor proteins. There, the full-length proteins are turned into transcrip-

6  Cancer Pathways

tional activators (GLI2A and GlI3A), likely by phosphorylation, which replace their repressor isoforms in the nucleus and activate the target genes. One target gene is GLI1. As a pure transcriptional activator that is not phosphorylated by PKA GLI1 acts as a feed-forward amplifier of Hedgehog signaling. In contrast, HHIP and PTCH2 encode negative feedback regulators of the pathway. Other targets of Hedgehog signaling encode positive regulators of the cell cycle, antiapoptotic proteins, and growth factors, in a cell type-dependent fashion. Hedgehog signaling is overactive in many cancer types. However, overactivity is only caused by mutations in pathway components in specific cancer types, especially basal cell carcinoma (BCC) of the skin (→12.3) and a subtype of medulloblastoma (Box 11.1). PTCH1 is a classical tumor suppressor: germline mutations in the Gorlin syndrome cause BCC precursor lesions and PTCH1 mutations occur alternatively to SMO mutations in sporadic BCC.  A smaller number of BCCs arise through mutations in SUFU. PTCH1 and SUFU are therefore tumor suppressors and SMO is an oncogene in this pathway (Table 6.1). In a larger number of cancer types, Hedgehog pathway activation is caused by increased autocrine or paracrine signaling. Intriguingly, paracrine activation may involve reciprocal WNT/Hedgehog signaling between carcinoma and stromal cells. In this circuit, tumor cells secrete Hedgehog factors to receive WNT growth factors from stromal cells Notch signaling: Like the WNT and Hedgehog pathways, the Notch pathway controls stem and precursor cell compartments. Its most characteristic function is however the regulation of binary cell fate decisions, such as which daughter cell of a stem cell division remains a precursor or goes on to differentiate, e.g., in the basal layer of the epidermis. Notch signaling is also involved in choices between sub-lineages, e.g., whether a differentiating intestinal cell becomes an enterocyte or a goblet cell, or whether a cell committed to the lymphocyte lineage becomes a T cell or a B cell. In contrast to WNT and Hedgehog signaling, which employ secreted paracrine factors, Notch signaling is a juxtracrine pathway. Signals

6.9 Developmental Regulatory Systems as Cancer Pathways

are always relayed between adjacent cells, which may be of the same type, like basal and differentiating cells in a stratified epithelium, but also between different cell types, e.g., between carcinoma cells and endothelial cells in sprouting capillaries or between metastatic carcinoma cells and resident cells in bone metastases. The four different NOTCH receptors, NOTCH1-4, are expressed on the cell surface and are activated by ligands on the cell surface of neighboring cells (Fig. 6.13). Two different kinds of activating ligands comprise the three “delta-­ like” DLL1, DLL3, and DLL4 and the “jagged-­ like” JAG1 and JAG2 proteins. These ligands differ in particular in their sensitivity towards modification of NOTCH receptors by FRINGE glycosylases. These elongate glycosyl chains on NOTCH receptors that prevent binding of JAG, but not of DLL proteins. DLK1 is an example of an inhibitory ligand. Importantly, expression of NOTCH receptors and their ligands is each self-reinforcing and cross-inhibitory and therefore tends to become mutually exclusive. Thus, within an organized tissue, different types of cells express predominantly either receptors or specific ligands. A precursor cell population, e.g., may express a receptor, while cells that have taken a step towards differentiation express a ligand (or the other way round). The latter situation is found, e.g., in the epidermis, where basal cells express NOTCH ligands and cells in upper, differentiated layers express NOTCH1. The NOTCH receptors are heterodimers, each formed by the proteolytic cleavage of a single precursor protein by furin-like proteases during their biosynthesis. The N-domain (NEC) is extracellular and contains 11 or more EGF-like repeats and a negative regulator cysteine-rich domain. The C-terminal fragment remains bound to the NEC by its small extracellular domain and continues through the membrane into its larger intracellular part. This segment comprises a nuclear localization signal, ankyrin repeats, which mediate protein-protein interactions, a transcriptional transactivation domain, and a PEST sequence accelerating proteolytic degradation of the proteins.

147

Following engagement of the N-terminal domain by ligands, the extracellular domain is further cleaved by ADAM proteases to further allow proteolytic processing of the N-terminal subunit by γ-secretase7. This sets the intracellular NOTCH domain (NICD) free to move into the nucleus, where it replaces repressor proteins at the CSL transcription factor (also known as CBF1 or RBJ1) to activate transcription of its target genes together with another coactivator, MAML1. Transcriptional activation is normally transient because due to its C-terminal PEST domain, the NICD is rapidly polyubiquitinated by FBXW7, supported by phosphorylation by CDK8. The range of Notch target genes differs substantially between cell types. The most consistent targets encode HES and HEY transcription factors. In many cell types, Notch signaling induces MYC and consequently cell proliferation, whereas in others, such as keratinocytes, it induces differentiation markers and the p21CIP1 CDK inhibitor to elicit cell cycle arrest. The outcome of Notch signaling is therefore highly cell type-specific and depends on crosstalk with WNT/β-Catenin, Hedgehog, AP1, and NFκB signaling, among others. Depending on the tissue and even the particular cell type, either overactivity or inactivation of Notch signaling contributes to cancer development. One type of T cell acute leukemias (T-ALL) is characterized by a translocation between chromosome 7 and chromosome 9, t(7;9) (q34;q34.3), which leads to the overexpression of the cytoplasmic domain of NOTCH1 under the influence of the T cell receptor β (TCRβ) enhancer. In other cases of this cancer, point mutations activate NOTCH1 (see Fig. 5.11). In T-ALL, constitutively active Notch signaling appears to direct an inappropriately large fraction of lymphocyte precursors towards a T cell fate where they become malignant by further mutations. In a similar fashion, Notch overactivity appears to cooperate with The γ-secretase is a heteromeric complex composed of several proteins including Presenilin, known for its role in neurons and Alzheimer disease. Inhibitors of γ-secretase therefore do not specifically inhibit NOTCH signaling. 7 

148

the HPV viral oncoproteins E6 and E7 in some genital cancers. Here, the inhibitory effect of Notch signaling on the cell cycle is abrogated by the viral oncoproteins whereas its precursor cell maintenance function remains active and contributes to the expansion of the tumor. In many cancers, overactivity of Notch signaling through receptor mutations or deregulation of receptors, ligands or other components is thought to contribute to the maintenance of tumor stem cells. In contrast, NOTCH receptors appear to function as tumor suppressors in skin and head and neck squamous cell carcinomas (see Fig. 5.11). Also, clearly, increased expression of NOTCH ligands like JAG1 or DLL4 on carcinoma cells exerts effects on stromal cells, such as osteoblasts in bone metastases and endothelial cells at primary and metastatic sites. In many cancers however the function of the pathway remains unclear or is controversial. In addition to the general strong context-dependency of its effects, one specific reason is the difficulty of predicting the effect of mutations in the NOTCH genes. For instance, truncating mutations are normally typical of tumor suppressors, but in NOTCH genes they may remove mostly the PEST domain, stabilizing the NICD and enhancing its function. For that reason, inactivating mutations in FBXW7 also enhance Notch signaling. Predicting the effects of missense mutations in the various domains of NOTCH genes is even more difficult. They may variously enhance or impede interactions with ligands or proteolysis by ADAM proteases and γ-Secretase. The Hippo pathway: Like the Hedgehog and Notch pathways, the Hippo pathway derives its evocative name from the peculiar phenotype of Drosophila mutants. In mammals, its main function appears to reside in the regulation of cell numbers and organ size during development, but also during tissue regeneration. Hippo signaling furthermore mediates the phenomenon of “contact inhibition” in cultured cells and likely within tissues. The pathway responds to a variety of signals emanating mainly from the cell membrane, but also from crosstalk with other pathways. In turn, it influences the activity of other pathways. At its core are two transcriptional activators, TAZ and YAP, which, when phosphorylated, are

6  Cancer Pathways

retained in the cytoplasm by interaction with 14-3-3 proteins. Following subsequent polyubiquitination, they may become degraded. Upon dephosphorylation, they move to the nucleus, where they associate with the transcription factor TEAD to activate transcription. In particular, several genes activated by TCF4/β-Catenin also contain binding sites for TEAD/YAP.  Conversely, cytoplasmic TAZ inhibits the canonical WNT pathway. Moreover, YAP and TAZ influence the transport of BMP-dependent SMAD factors into the nucleus and serve as their coactivators. In addition to BMP and WNT target genes, YAP/ TAZ/TEAD induce cell adhesion proteins, regulators of cell death, and cytokines. The upstream Hippo module (Fig. 6.14) controlling YAP/TAZ phosphorylation consists of two kinase pairs. LATS1/2 phosphorylates YAP/ TAZ and is itself phosphorylated by MST1/2. MST1/2 is kept active by various signals from the cell membrane emerging, e.g., from FAT1 or Merlin. Merlin (also known as NF2) is a protein of the cortical cytoskeleton that is inactivated in the hereditary tumor syndrome neurofibromatosis type 2. This upper part of the Hippo pathway is kept active by interaction of cells with others and thereby mediates contact inhibition and the control of organ size following regeneration. Its inactivation, e.g., by loss of contact with neighboring cells, leads to dephosphorylation of YAP/ TAZ. Dephosphorylation is however also stimulated by mechanical stretching of cells or changes in cell shape during EMT.  The activity of the kinases in the Hippo pathway can also be overcome by growth factors and cytokines. Deregulation of the Hippo pathway in cancers is primarily brought about by genetic or epigenetic alterations that inactivate upstream regulators of YAP/TAZ.  Their inactivation results in enhanced nuclear localization of one or both coactivator proteins. Enhanced nuclear localization is however observed more broadly in different cancer types and is thought to result from loss of cell-cell contacts, cell polarity, or EMT.  In either case, the Hippo coactivators contribute to cell proliferation and survival especially during tumor progression and may serve to mediate resistance to molecular targeted and cytotoxic therapies.

149

6.10 Signaling Through Nuclear Receptors

6.10 Signaling Through Nuclear Receptors

(→19.4, →20.3) that encounter their ligands in the cytoplasm, translocate to the nucleus and bind to specific recognition sites (AREs and EREs, respectively) in the promoters or (more often) enhancers of their target genes. Although they may interact with other transcription factors in various ways, they typically bind to DNA as homodimers at their symmetric recognition sites. Other members of the superfamily are predominantly located in the nucleus or bind to their specific recognition sites as heterodimers. For instance, many nuclear receptors like the vitamin D receptor (VDR) and the peroxisomal proliferation receptors (PPAR) form heterodimers with RXR receptors. While their ligands are diverse, the mechanism of activation of nuclear receptor is rather

Different from most of the other pathways sketched in this chapter, nuclear receptors are not primarily regulated by signals from the cell membrane. Instead, they act as ligand-dependent transcriptional activators in the nucleus that respond to small-molecule signaling molecules, hormones or metabolites, which have entered the cell (Fig.  6.15). Their activity is however often modulated by crosstalk with signal transduction pathways that respond to growth factors, such as MAPK signaling. The nuclear receptor superfamily (Fig.  6.15) comprises steroid hormone receptors like the two estrogen receptors and the Androgen receptor

Fig. 6.15  Structures of some nuclear receptors. Nuclear receptors are composed of six domains labeled A–F, which differ in size between the individual members. Domains A and B (also termed AF-1 for activating function) can activate transcription, domain C is the DNA-binding domain, which contains crucial zinc fingers, domain D is the hinge region, domain E contains another transcription activation function, which is ligand-­dependent in most members of the family, domain F terminates the proteins. See Figs. 10.12, 19.5, and 20.8 for more detailed structures of specific nuclear receptors

AR

C D

A/B

E/F

913 aa

D ERα

ERβ

A/B

C

E/F

A/B C D

595 aa

E/F

530 aa

D A/B

PRβ

C

E/F

934 aa

RARα

A/B C D

E/F

462 aa

RARβ

A/B C D

E/F

448 aa

A/B VDR

C D

E/F

427 aa

150

uniform. Ligand binding to the eponymous domain results in a conformational change upon which corepressors dissociate and coactivators associate with the DNA-bound receptor dimer. In this fashion, the ligand-binding domain—often together with a second domain at the N-terminus of the protein—acquires the transactivating function required to stimulate transcription of the target genes. Some nuclear receptors like the Glucocorticoid receptor primarily contribute to metabolic regulation, while others like retinoic acid, androgen, and estrogen receptors influence proliferation, differentiation, and function of specific tissues. Some nuclear receptors, like the VDR and the peroxisomal proliferation receptors PPARγ, do both, depending on the tissue. Of note, the activities of many nuclear receptors are modulated by phosphorylation, allowing crosstalk with pathways relaying growth factor signals. Typically, nuclear receptors act as oncogenes in the same tissues in which they normally support proliferation, differentiation, and proper function. However, in the corresponding cancers, their action is shifted towards support of proliferation and survival as opposed to promotion of differentiation and physiological functions of the tissue. This shift is rarely brought about by mutations in the receptor genes, but rather by altered interactions with cotranscription factors and coactivators (→19.4, →20.3). Receptor gene mutations are more common at later stages of cancer development, often in response to therapy aiming at receptor inhibition. The retinoic acid receptor fusion proteins in acute promyelocytic leukemia that result from chromosomal translocations (→10.6) provide one prominent exception to this rule, mutations in PPARG and RXRA in bladder cancer (→14.4) another.

Further Reading Ak P, Levine AJ (2010) p53 and NFκB: different strategies for responding to stress lead to a functional antagonism. FASEB J. 24:3643–3652 Amakye D et al (2013) Unraveling the therapeutic potential of the hedgehog pathway in cancer. Nat Med 19:1410–1422

6  Cancer Pathways Belli S et al (2020) c-Src and EGFR inhibition in molecular cancer therapy: what else can we improve? Cancers 12:1489 Briscoe J, Therond PP (2013) The mechanisms of Hedgehog signalling and its roles in development and disease. Nat Rev Mol Cell Biol 14:416–429 Caunt CJ et  al (2015) MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road. Nat Rev Cancer 15:577–599 Chastney MR et al (2021). Integrin adhesion complexes. Curr Biol 31:R536–R542 Chaturvedi MM et  al (2011) NF-κB addiction and its role in cancer: ‘one size does not fit all’. Oncogene 30:1615–1630 Chen F (2012) JNK-induced apoptosis, compensatory growth and cancer stem cells. Cancer Res 72:379–386 David CJ, Massagué J (2018) Contextual determinants of TGFβ action in development, immunity and cancer. Nat Rev Mol Cell Biol 19:419–435 Davis JR, Tapos N (2019) Hippo signalling during development. Development 146:167106 Derynck R, Zhang YE (2003) Smad-dependent and Smad-independent pathways in TGF-β family signalling. Nature 425:577–584 Deschènes-Simard X et al (2014) ERKs in cancer: friend or foes? Cancer Res 7:412–419 Egloff AM, Grandis JR (2012) Molecular pathways: context-­dependent approaches to Notch targeting as cancer therapy. Clin Cancer Res 18:5188–5195 Fang JY, Richardson BC (2005) The MAPK signalling pathway and colorectal cancer. Lancet Oncol 6:322–327 Flanagan DJ et al (2019) Wnt signaling in cancer: not a binary ON:OFF switch. Cancer Res 79:5901–5906 Fruman DA et al (2017) The PI3K pathway in human disease. Cell 170:605–635 Georgescu MM (2011) PTEN tumor suppressor network in PI3K-Akt pathway control. Genes Cancer 1:1170–1177 Gerdes JM et  al (2009) The vertebrate primary cilium in development, homeostasis and disease. Cell 137:32–45 Hood JD, Cheresh DA (2002) Role of integrins in cell invasion and migration. Nat Rev Cancer 2:91–100 Hoppler S, Kavanagh CL (2009) Wnt signalling: variety at the core. J. Cell Sci 120:385–393 Igea A, Nebreda AR (2015) The stress kinase p38α as a target for cancer therapy. Cancer Res 75: 3997–4002 Jin H, Varner J (2004) Integrins: roles in cancer development and as treatment targets. Brit J Cancer 90:561–565 Korkut A et al (2018) A pan cancer analysis reveals high-­ frequency genetic alterations in mediators of signaling by the TGF-β superfamily. Cell Systems 7:422–437 Krauss G (2014) Biochemistry of signal transduction and regulation, 5th edn. Wiley-VCH Loh CY et al (2019) Signal Transducer and Activator of Transcription (STATs) proteins in cancer and inflammation: functions and therapeutic implication. Front Oncol 9:48

Further Reading Lum L, Beachy PA (2004) The hedgehog response network: sensors, switches and routers. Science 304:1755–1759 Manning BD, Toker A (2017) AKT/PKB signaling: navigating the network. Cell 169:381–405 Massagué J (2012) TGFβ signalling in context. Nat Rev Mol Cell Biol 13:616–630 Maurer G et  al (2011) Raf kinases in cancer  - roles and therapeutic applications. Oncogene 30: 3477–3488 Mauviel A et al (2012) Integrating developmental signals: a Hippo in the (path)way. Oncogene 31:1743–1756 Mendoza MC et  al (2011) The Ras-ERK and PI3K-­ mTOR pathways: cross-talk and compensation. TiBS 36:320–328 Meurette O, Mehlen P (2018) Notch signaling in the tumor microenvironment. Cancer Cell 34:536–548 Newton AC (2018) Protein kinase C as a tumor suppressor. Sem. Cancer Biol 48:18–26 Ntziachristos P et  al (2014) From fly wings to targeted cancer therapies: a centennial for Notch signaling. Cancer Cell 25:318–334 Nusse R, Clevers H (2017) Wnt/β-Catenin signaling, disease and emerging therapeutic modalities. Cell 169:985–999 Pak E, Segal RA (2016) Hedgehog signal transduction: key players, oncogenic drivers, and cancer therapy. Dev Cell 38:333–344 Parsons JT (2003) Focal adhesion kinase: the first ten years. J Cell Sci 116:1409–1416 Parsons MJ et al (2021) WNT as a driver and dependency in cancer. Cancer Disc 11:2413–2429 Philips RL et al (2022) The JAK-STAT pathway at 30: much learned, much more to do. Cell 185:3857–3876 Polakis P (2000) Wnt signaling and cancer. Genes Dev 14:1837–1851 Polakis P (2007) The many ways of Wnt in cancer. Curr Opin Genet Dev 17:45–51 Ranganathan P et  al (2011) Notch signalling in solid tumours: a little bit of everything but not all the time. Nat Rev Cancer 11:338–351 Ratner N, Miller SJ (2015) A RASopathy gene commonly mutated in cancer: the neurofibromatosis type I tumor suppressor. Nat Rev Cancer 15:290–301 Robbins DJ et al (2012) The Hedgehog signal transduction network. Sci Signal 5:1–13

151 Sanchez-Vega F et  al (2018) Oncogenic signaling pathways in The Cancer Genome Atlas. Cell 173:321–337 Sansone P, Bromberg J (2012) Targeting the interleukin-6/ Jak/Stat pathway in human malignancies. J Clin Oncol 30:1005–1013 Sears RC, Nevins JR (2002) Signaling networks that link cell proliferation and cell fate. JBC 277:11617–11620 Sethi N, Yang Y (2011) Notch signalling in cancer progression and bone metastasis. Brit J Cancer 105:1805–1810 Skoda AM et al (2018) The role of the hedgehog signaling pathway in cancer: a comprehensive review. Bosn J Basic Med Sci 18:8–20 Slee EA, O'Connor DJ, Lu X (2004) To die or not to die: how does p53 decide? Oncogene 23:2809–2818 Steinhart Z, Angers S (2018) Wnt signaling in development and tissue homeostasis. Development 145:1–8 Stern DF (2018) Keeping tumors out of the MAPK fitness zone. Cancer Discov 8:20–23 Taipale J, Beachy PA (2001) The Hedgehog and Wnt signalling pathways in cancer. Nature 411:349–354 Taniguchi K, Karin M (2018) NF-κB, inflammation, immunity and cancer: coming of age. Nat Rev Immunol 18:309–324 Van Veelen W et  al (2011) The long and winding road to rational treatment of cancer associated with LKB1/AMPK/TSC/mTORC1 signaling. Oncogene 30:2289–2303 Vogelstein B, Kinzler KW (2006) Cancer genes and the pathways they control. Nat Med 10:789–799 Wagener C, Stocking C, Müller O (2017) Cancer signaling: from molecular biology to targeted therapy. Wiley-VCH Wiese KE et al (2018) Wnt signalling: conquering complexity. Development 145:1–9 Wu F et al (2017) Hedgehog signaling: from basic biology to cancer therapy. Cell Chem Biol 24:252–280 Yu H et  al (2009) STATs in cancer inflammation and immunity: a leading role for STAT3. Nat Rev Cancer 9:798–809 Yu H, Jove R (2004) The STATs of cancer – new molecular targets come of age. Nat Rev Cancer 4:97 Zanconato F et al (2016) YAP/TAZ at the roots of cancer. Cancer Cell 29:783–803 Zhang Q et  al (2017) 30 years of NF-κB: a blossoming of relevance to human pathobiology. Cell 168:37–67

7

Cell Death and Replicative Senescence in Cancer

Key Points • In normal tissues, proliferation of precursor cells is counterbalanced by terminal differentiation and cell loss, often via apoptosis. Cell damage also elicits apoptosis or other forms of cell death like necrosis. Moreover, apoptosis can be induced in response to inappropriate proliferation or by cytotoxic immune cells. • Replicative senescence provides another limit to cell proliferation. Senescent cells survive but irreversibly exit from the cell cycle, as they do during terminal differentiation. Replicative senescence is established either after cells have undergone a large number of replicative cycles or more rapidly as a response to “inappropriate” proliferation signals. Senescent cells display distinctive changes in morphology and gene expression. While accumulation of senescent cells in aging tissues contributes to inflammation and aging, replicative senescence is an important fail-­ safe mechanism against inappropriate proliferation, alternatively to apoptosis. • Cancer cells must overcome the barriers posed by terminal differentiation, replicative senescence, and apoptosis. They need to avoid the permanent cell cycle arrest associated with terminal differentiation. Likewise, cancer cells are in general “immortalized” and capable of proliferating beyond the limits set by replicative senescence. Moreover, the apop-

totic response in cancers is often inadequate or blocked. • Telomeres in humans consist of several hundred repeats of the hexanucleotide TTAGGG.  They form a specialized T-loop structure which is protected by binding of the Shelterin protein complex. In most somatic cells, telomeres shorten during each division. In contrast, germ cells and tissue stem cells express the specialized reverse transcriptase Telomerase (encoded by TERT) and its RNA subunit TERC which serves as a template for telomere elongation by the Telomerase enzyme. • Replicative senescence is induced when telomere lengths have reached a critical minimum size after multiple cell doublings. Shortened telomeres possess an increased potential for recombination and fusion with each other. If replicative senescence cannot be established, short reactive telomeres contribute to chromosomal instability in cancer. Like DNA double-­ strand breaks, short telomeres activate checkpoints arresting the cell cycle via TP53, p21CIP1, and RB1. • Induction of replicative senescence can also be mediated by direct activation of RB1. Continuously proliferating cells gradually accumulate the CDK inhibitor p16INK4A that induces cell cycle arrest through RB1. More rapid induction of replicative senescence is

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 W. A. Schulz, Molecular Biology of Human Cancers, https://doi.org/10.1007/978-3-031-16286-2_7

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mediated by induction of p14ARF and p16INK4A by certain oncogenes or by viral infection and leads to activation of both TP53 and RB1. • Several mechanisms allow cancer cells to escape replicative senescence. Many cancers overexpress Telomerase as a consequence of mutations in the TERT promoter or amplification of the gene. An alternative (hence: ALT) mechanism for the stabilization of telomeres employs a DNA recombination mechanism; cancers with ALT regularly display inactivation of DAXX and ATRX.  Genetic inactivation of RB1, TP53, and CDK inhibitors can also prevent replicative senescence in cancer cells. Finally, cancers developing from tissue stem cells may retain stem cell properties, including sufficient Telomerase expression. • Apoptosis can be started via two largely separate signaling pathways which converge on a common executing cascade. Signaling and execution use specialized proteases called caspases. The intrinsic pathway, which is elicited, e.g., by strong DNA damage signals or by some activated oncogenes, generates the mitochondrial permeability transition that ultimately leads to the establishment of the “apoptosome” protein complex that then activates execution caspases. The extrinsic pathway is initiated prominently by certain cell surface receptors considered as “death receptors.” They are activated by cytokine ligands or surface proteins of cytotoxic immune cells. The intracellular death domains of activated death receptors associate with adaptor proteins in the DISC complex which puts distinct initiator caspases into action. These caspases initiate the execution cascade. In some cell types and instances, the extrinsic pathway requires support from the intrinsic pathway which is recruited through the BID protein. • Apoptosis is regulated at several steps in both pathways. BCL2 prohibits the related proapoptotic proteins BAX or BAK from inducing the mitochondrial permeability transition. Further BH3 proteins related to BCL2 likewise confer pro- or antiapoptotic signals. FLIP inhibits the extrinsic pathway at the receptors, whereas IAPs like XIAP act at the apopto-

7  Cell Death and Replicative Senescence in Cancer

some to prevent caspase activation. For efficient apoptosis, they are antagonized by SMAC/Diablo liberated during the mitochondrial permeability transition. • Apoptosis in tumor cells can be diminished by several means. Downregulation of death receptors or secretion of decoy receptors mutes the extrinsic pathway. Mutational inactivation of TP53 as well as silencing or mutation of its downstream proapoptotic mediators curb the response to signals that activate the intrinsic pathway. Moreover, the PI3K or NFκB pathways, which confer survival signals, are often overactive. Overexpression of inhibitory proteins, such as BCL2, BCL-XL, XIAP, and FLIP, occurs in many cancers. In some cancers, genes encoding components of the executing cascade are inactivated by mutations or repressed by epigenetic mechanisms.

7.1 Limits to Cell Proliferation The number of cells in normal tissues (as well as in tumors) is determined by the number of cells newly produced by cell proliferation and division and by the number of cells that die in a regular manner or are lost in other ways (e.g., by mechanical abrasion). The overall growth rate of a tissue is dependent on the proportion of cells that are actively traversing the cell cycle, i.e., the proliferative fraction. Terminal differentiation and replicative senescence irreversibly remove cells from the proliferative fraction without necessarily destroying them, at least in the short run. Cell death can occur by various mechanisms, especially apoptosis and necrosis (Fig. 7.1), each with many variants, and additional forms (see below). The specific functions of many tissues are carried out by cells that have irreversibly exited from the cell cycle in a process designated as “terminal differentiation.” Diverse strategies of terminal differentiation are used in various tissues. In some tissues, terminally differentiated cells are polyploid and/or multinuclear, e.g., the syncytia of the skeletal muscle or, less spectacularly, polyploid (tetraploid or octoploid) hepatocytes of the liver or umbrella cells of the

7.1 Limits to Cell Proliferation

155 Cell dehydration (shrinkage)

Apoptotic bodies

APOPTOSIS

Chromatin condensation Nuclear fragmentation Membrane integrity preserved

Cell and mitochondrial swelling

Plasma membrane rupture

NECROSIS

Fig. 7.1  A morphological comparison of apoptosis and necrosis. A standard representation of morphological changes in necrosis and apoptosis. From: Darżynkiewicz et  al. (1997) Cytometry 27, 1–20, with permission

urothelium. Robust terminal differentiation can also be efficiently achieved in diploid cells with normal, highly transcriptionally active nuclei, as impressively demonstrated by neurons. In some tissues, the nuclei of terminally differentiated cells shrink, are expelled, or dispersed. Cornified epithelia like the epidermis, the lens of the eye, and the erythrocyte lineage provide examples of this strategy. In some instances, the destruction of the nucleus resembles an apoptotic process and is indeed implemented by some of the same factors. In fact, in some tissues, terminal differentiation is a prelude to actual apoptosis, e.g., in the epithelia lining the gut. During differentiation, enterocytes gradually lose the ability to proliferate while moving from the crypts towards the tips of the villi, where they undergo apoptosis, their remnants being lost into the lumen of the gut.

While the mechanisms differ in detail between tissues, terminal differentiation in general involves a competition between cell type-specific transcription factors on the one hand and CDKs driving the cell cycle on the other hand. Differentiation usually requires a lengthening of the G1 phase of the cell cycle and the successive induction of CDK inhibitors like p21CIP1, p57KIP2, and p16INK4A which ultimately arrest the cell cycle. In the course of events, RB1 stably represses E2F-dependent gene expression. At other genes involved in cell cycle progression, the transcription-activating E2F factors 1–3 are replaced by the E2F4-6 repressors. This repression is associated with the remodeling of the E2F-dependent promoters towards an inactive epigenetic state (→8.3) that requires the SWI/ SNF chromatin remodeling complex. This com-

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plex as well supports the shift of the epigenetic state of the CDK inhibitor genes towards permanent expression, during which polycomb repressor complexes are displaced and active histone modification patterns are permanently established. In parallel, the expression of tissue-­ specific genes is activated by a combination of cell type-specific transcription factors and chromatin regulators (Fig.  7.2, see Sect. 8.5 for a more detailed discussion). A diminished rate of terminal differentiation is a fundamental requirement for tumor growth except in tissues where differentiated cells (such as plasma cells) remain proliferation-competent. Many cancers are in fact characterized by a complete lack of terminally differentiated cells, while others produce terminally differentiated cells at a diminished rate. Accordingly, in many cancers, proteins that are normally restricted to terminally differentiated cells are not detectable. In others, such proteins are expressed in the fraction of the tumor cells that still differentiate, or the cancer cells express some marker proteins of their differentiated tissue counterparts but do not exit from the cell cycle. Mechanistically, suppression of terminal differentiation can be achieved in various ways, including loss of cell type-specific transcription factors (e.g., CEBPα in myeloid cells), overexpression of factors promoting G1 progression (e.g., Cyclin D1), mutation or epigenetic repression of CDK inhibitors, loss of RB1 (→5.2), and in particular mutations inactiFig. 7.2  Interplay of cell cycle regulators, chromatin regulators, and cell type-specific transcription factors in the regulation of terminal differentiation. Compare Ruijtenberg S, van den Heuvel S (2016) l.c. See main text for further explanation

vating chromatin remodelers and histone-­ modifying enzymes required for differentiation or alternatively, overexpression of their antagonists, such as polycomb repressors (→8.3). Replicative senescence is likewise characterized by an irreversible exit from the cell cycle, after which cells survive for an extended period (weeks to months in vivo). Cultured cells undergoing replicative senescence often take on a characteristic morphology (Fig. 7.3) with a flattened appearance, enlarged nuclei with altered chromatin distribution (senescence-associated heterochromatin foci), and many small granules, reflecting, in particular, an increased content of lysosomes. They express characteristic proteins, such as SA-β-GAL, a lysosomal β-galactosidase with a comparatively acidic pH optimum, and high levels of CDK inhibitors like p21CIP1 or p16INK4A, but also of specific antiapoptotic proteins. Conversely, senescent cells express few or none of the markers that are diagnostic for terminally differentiated cells. There are overlaps however and senescent fibroblasts have been proposed to represent terminally differentiated cell. In cultured cells, replicative senescence arises in two very different instances. The classic instance was observed after the propagation of normal human cells over many passages, where replicative senescence sets in gradually. In fibroblasts, where the phenomenon (known as “Hayflick limit”) was first described, this phenomenon sets in after as many as 50–80 cell dou-

Proliferation

Positive cell cycle regulators

Differentiation

Negative cell cycle regulators

Precursor cell chromatin regulators

Differention transcription factors

Differentiation chromatin regulators

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Fig. 7.3  Morphological characteristics of cells with a senescent phenotype. Top: A tumor cell line containing cells with a typical senescent phenotype (right, arrowheads). The same cell

line prior to the induction of senescence is depicted on the left. Bottom: An epithelial tumor cell line undergoing EMT (see chapter 9) in response to epigenetic stress

blings. In cultured epithelial cells, it appears earlier. Thus, replicative senescence presents a limit to the lifespan of normal human somatic cells. Replicative senescence can experimentally be prevented by infection with certain DNA viruses, typically the SV40 tumor virus or specifically its large-T antigen (→5.3). Infection with SV40 or adenoviruses has been used to generate “immortalized” cell lines from many normal cell types; an alternative technique employs the Telomerase gene TERT (see below). Replicative senescence can alternatively be induced in a rapid mode, long before cells have exhausted their normal lifespan, by inappropriate

proliferation signals, like overexpressed mutant RAS proteins. Unlike terminal differentiation and apoptosis, replicative senescence does not seem to be employed in the human body for tissue homeostasis. Rather, it appears to act as a fail-safe mechanism. Interestingly, the number of senescent cells in human tissues has been shown to increase with age by the use of improved markers that allow the ascertainment of replicative senescence in vivo. Overall, senescence along with other mechanisms depleting stem and precursor cells contributes to human aging and may set a limit to the human lifespan (Box 7.1).

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Box 7.1 Human Aging and Cancer

Theories on the causes of human aging fall basically into two groups. The now predominant (damage accumulation) theory assumes that the phenotypic changes associated with aging are caused by the accumulation of unrepaired damage to tissues, cells (especially tissue stem cells), and macromolecules. A specific hypothesis of this type emphasizes oxidative damage by reactive oxygen species, but other endogenous processes certainly contribute. Many defects accumulate with age in extracellular tissue and in cells, including mutations in nuclear and mitochondrial genomes. For instance, the frequency of C→T transitions at CpG-dinucleotides clearly increases with age. Mitochondria with mutant genomes and deficient respiratory chain function likewise accumulate over time. Epigenetic changes contribute, affecting DNA methylation and histone modifications. Some DNA methylation changes occur in such a regular fashion that they can be used as an epigenetic clock of aging. The second group of theories (aging as a program) emphasizes that the regularity of the changes occurring with age resembles genetic programs controlling fetal development and maturation. The regular emergence of cellular senescence after a certain number of cell duplications caused by the gradual erosion of telomeres is one argument in favor of these theories. A minimum version of the programmed aging theory suggests that humans are “build” to last only for a certain period, with protective mechanisms holding out only as long as required for reproduction. This version is easily reconciled with theories of aging emphasizing the accumulation of damage as its prime cause. Replicative senescence is defined at the cell level. Senescent cells become more prevalent in aging humans, which may contribute to the aging of tissues and the entire organism. Whether “senolytic” drugs that promote

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the removal of senescent cells can hold up aging is yet to be proven. Note that replicative senescence can be straightforwardly integrated into theories of programmed aging, but it is not entirely incompatible with damage accumulation theories. The fact that the majority of human cancers arise in older people and the incidence, prevalence, and mortality of most cancers increase with age is compatible with both theories, if somewhat better with the damage theory. Many normal tissues of aging persons have been found to contain cell clones with mutant genomes, which ought to provide a source for cancers. In fact, there are indications that at very old age (beyond 85 years), the incidence and aggressiveness of cancers diminish. Again, both types of theories provide explanations for this (uncertain) effect, but the explanation by program theories seems more elegant, i.e., cancer cells, too, are affected by the programmed loss of “vigor.” Elucidation of the genetic basis of human premature aging syndromes might have decided the debate, as their very existence has traditionally been used as an argument in favor of programmed aging theories. Premature aging is observed in several inherited syndromes, especially some caused by defects in DNA repair and cell protection (→3.6). Two prototypic diseases are the Hutchison-Gilford and Werner syndromes, which differ in the age of onset and the range of symptoms. Hutchison-­Gilford syndrome is caused by mutations in the LAMA gene, which encodes a major structural protein of the nuclear membrane; the syndrome is therefore considered a “laminopathy.” While it remains puzzling why defects in the nuclear membrane should be associated with prepubertal aging, the mechanisms underlying the symptoms of the disease have become clearer through the recognition that the nuclear membrane is essential for the proper 3D structure of the genome and its epigenetic regulation. The

7.1 Limits to Cell Proliferation

Werner syndrome is caused by mutations inactivating the WRN helicase-exonuclease. The protein is involved in DNA repair, making a good case for damage accumulation theories. However, the WRN helicase may be particularly important for the maintenance of telomeres (→7.4), as one might postulate for a protein involved in programmed aging. Moreover, the syndrome sets in at puberty, apparently dependent on hormonal changes thereby fulfilling another postulate of program theories. Hayflick L (1994) How and why we age. Ballantine López-Otín C et  al. (2013) The hallmarks of aging. Cell 153:1194–1217 Bell CG et al. (2019) DNA methylation aging clocks: challenges and recommendations. Genome Biol 20:249 Evidently, cells in the germline must be exempt from replicative senescence. Moreover, tissue stem cells and the proliferating precursor cells should not undergo replicative senescence as easily as more differentiated somatic cells, since stem cells of a continuously replicating tissue may have to undergo more than 50–80 doublings during a human lifetime (→8.5). Replicative senescence is circumvented in many cancers. Cancer cells can often be apparently indefinitely propagated in culture or as xenografts in experimental animals, even without introducing additional viral genes. They are therefore regarded as immortalized. While immortalization is difficult to ascertain in humans in vivo, mechanisms that allow cancer cells to circumvent replicative senescence in culture can be demonstrated to be also active in many cancer tissues (see Sect. 7.3). In other cases, tumor cells may evade replicative senescence by retaining or acquiring properties of stem cells. This is evident in germ-cell cancers like testicular cancers. Likewise, certain cancers originating in somatic tissues may acquire or retain properties of the respective tissue stem cell or early precursor, e.g., many leukemias (→10), basal cell carcinoma of the skin (→12.3), and colorectal carcinoma (→13.4).

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Apoptosis is a rapid process by which cells are destroyed in a thoroughly controlled fashion within hours1. Cells develop multiple blebs on their surface, lose their connections to other cells and the ECM, and round off while the nucleus and later the entire cell are fragmented into small, membraneenclosed particles. DNA is first cut into large >50 kb fragments, and later into smaller fragments corresponding to multiples of the nucleosomal units (≈200 bp). When apoptosis occurs in normal tissues, all cell remnants are phagocytosed or lost from the tissue, e.g., into the lumen of an organ, so that no inflammatory reaction is evoked. Apoptosis can be elicited by external or internal signals. Normally, it is employed to shape tissues during development, to eliminate superfluous or autoreactive immune cells, to maintain homeostasis of tissues with rapid or cyclical cell turnover, and to destroy cells infected by viruses. An absolute or relative decrease in the apoptotic rate or a failure to respond properly to apoptotic signals is a characteristic property of human cancers. A detailed understanding of the mechanisms of apoptosis therefore provides insights into the development and progression of cancers and can provide new angles for therapy. Necrosis is a different form of cell death that is elicited, e.g., by mechanical, chemical, or thermal damage as well as by some infectious agents. Necrotic cells burst, usually after swelling, releasing their content into the surrounding tissue in an uncontrolled fashion (Fig. 7.1). Frequently, inflammation ensues as a consequence. Unlike apoptosis, necrosis may proceed in the absence of cellular energy. Thus, in a large solid tumor, hypoxic areas will typically show an enhanced rate of apoptosis, but the central core, which is often almost anoxic and devoid of nutrients, may be necrotic. Theoretically, the inflammatory reaction ensuing from necrotic tumor cells could be beneficial for the patient, since it attracts immune cells. In reality, it is ambiguous, because inflammation may not only eliminate tumor cells but also contributes to the destruction of normal tisDepending on the cell type and stimulus, apoptosis lasts between 1 h and 1 d. Its short duration and variability make the estimation of the apoptotic rate in a tissue difficult. 1 

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sue structures and facilitates invasion and metastasis. Several other forms of cell death are relevant in the context of cancer. Anoikis is a specialized form of apoptosis elicited by lack of attachment, especially of epithelial cells. Necroptosis, as the name implies, is a form of programmed cell death that ends in necrosis. It is often an alternative mode of cell death if apoptosis is blocked and is actually regulated by the same death receptor signaling complexes that usually initiate the extrinsic apoptosis pathway (→7.4). Necroptosis regularly promotes inflammation. Ferroptosis requires ferrous ions and leads to cell death by excessive oxidation of unsaturated fatty acids in cellular membrane lipids. It is antagonized by molecules and enzymes protecting against oxidative stress (→3.7), but especially by Glutathione peroxidase 4 (GPX4) via glutathione. Ferroptosis is therefore favored by insufficient cysteine supply, especially due to downregulation of the cystine transporter SLC7A11. Ferroptosis is particularly relevant as a response to cytotoxic chemotherapy. Pyroptosis is an important mode of cell death during inflammatory processes. It is activated by specialized protein complexes (termed “inflammasome”) and involves activation of a different set of caspases than in apoptosis. Autophagy is predominantly a survival mechanism by which organelles and cellular compartments are recycled and their biochemical constituents are used to ensure cell survival despite insufficient energy and nutrient supply. Excessive autophagy may however cause cell death. A pivotal regulator of autophagy is mTORC1 on which signals from several energy and nutrient sensors converge (→6.3).

7.2 Replicative Senescence Replicative senescence can be evoked by different kinds of signals which use overlapping pathways for execution. One type of signal emanates from short telomeres, and another from overproliferation. Telomeres in human cells are 5–30 kb long and made up of 1000–5000 repeats of TTAGGG

7  Cell Death and Replicative Senescence in Cancer

hexamers. The bulk of each telomere consists of double-stranded DNA wrapped around nucleosomes with core histones, but 75–150 nt at the telomere ends are single-stranded. In intact telomeres, these single strands are folded back into the double strand, forming a T-loop; alternatively, the G-rich telomere sequences are in principle capable of forming quadruplex DNA.  The protein complex Shelterin protects the T-loop structure (Fig.  7.4). It contains the proteins TRF1, TRF2, TPP1, POT1 (which binds the single strand DNA sequence), TIN2, and RAP1. This peculiar arrangement serves to hide the telomere ends, which would otherwise be recognized as DNA strand breaks or might fuse with one another. The Shelterin complex interacts with DNA repair factors, inhibiting DNA damage signaling under normal instances on the one hand, but helping to initiate DNA repair upon damage to telomeres on the other hand. Shelterin components also aid in DNA replication into the telomere sequence, as far as it is feasible. The complex furthermore regulates the access and the activity of the Telomerase holoenzyme to the telomeres and thus their extension (see below). In this regard, TRF2 and TRF1 can be considered as antagonists. Whereas TRF2 facilitates Telomerase access and therefore telomere elongation, its paralog TRF1 rather favors telomere shortening. TRF1 is regulated by Tankyrase, a poly-­adenosine diphosphate ribosylase. Poly-ADP-­ ribosylation of TRF1 leads to its degradation. Without Telomerase, telomeres would shorten with each DNA replication in somatic cells because of the end-replication problem. The top strand (with a 5’-end at the telomere) is replicated by the elongation of an RNA primer at or near its end. When the primer is removed by RNase H after DNA synthesis has proceeded, the resulting gap cannot be filled, since DNA polymerases work invariably in the 5’→3’ direction. This end-replication dilemma predicts a theoretical minimum size for the loss of telomere sequences during each replication. In reality, its extent can be larger and it is regulated by TRF1 and TRF2. In germline and stem cells, the decrease in telomere length is prevented by Telomerase

7.2 Replicative Senescence

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Fig. 7.4  Structure of human telomeres. The presumed T-loop structure of human telomeres with the Shelterin complex is depicted at the bottom.

Quadruplex DNA is shown in the center and a depiction of active Telomerase is at the top. From Fernandes SG et al. (2020) l.c. Originally published under the terms of CC BY

(Fig.  7.5). Accordingly, telomeres in germline cells are approximately twice as long as those in somatic cells. Telomerase is a specialized reverse transcriptase that uses an RNA template (AAUCCC) provided by its TERC (also short: TR) RNA subunit to elongate telomeres. While the TERC RNA is more widely expressed, the expression of the catalytic subunit (encoded by TERT gene, also known as hTERT) is restricted in humans to a selection of cells with high replicative potential, like germline cells, tissue stem cells, and memory immune cells. Expression of

TERT is induced by a number of proliferation-­ stimulating and stem cell-maintaining factors. In particular, its promoter is a target of MYC proteins. In addition to the Telomerase enzyme and the TERC RNA, the Telomerase holoenzyme contains the proteins Dyskerin (gene: DKC1), NHP2, NOP10, and GAR, which are involved in the processing of TERC and its stabilization. Mutations in DKC1 and TERC, or less frequently other components of this complex, cause the human disease dyskeratosis congenita. Patients with this rare inherited affliction do not only

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162 TT

hTERT LINE-RT

Telomerase motif

HIV-RT HBV-RT Reverse transcriptase protein motifs

1

2

A

B

C

D

E

Fig. 7.5  Structure of the human Telomerase catalytic subunit. The 127 kDa human Telomerase enzyme contains a specific motif (T) characteristic of telomerase as well as sev-

eral motifs shared by other reverse transcriptases (RT), including those of endogenous human LINE-1 retrotransposons and the viruses HIV and HBV

present with defects in skin, hair, and the ­hematopoietic system, but are also prone to cancer development. When telomeres are shortened to below a critical length, they are no longer protected by the Shelterin complex and are recognized as DNA double strand breaks. Two responses ensue: (1) DNA repair, especially by NHEJ, is activated, which can lead to chromosomal end-to-end fusions, starting BFB cycles (Fig. 2.7) that result in chromosomal instability. (2) Cell cycle checkpoints are activated, mainly through ATM and subsequently TP53 and RB1. Checkpoint activation in this case leads to an essentially irreversible arrest of the cell cycle. Consequently, obliteration of RB1 and TP53 function allows cells to tolerate telomere shortening to some extent and to continue proliferating. However, very short telomeres may lead to excessive chromosomal instability that is not well compatible with cell proliferation and eventually results in a state described as “crisis.” Shortening of telomeres occurs if cells that do not possess active Telomerase proliferate over many generations. This process is responsible for the Hayflick limit observed in cultured normal human cells. Loss of functional telomeres can moreover be caused by DNA damage close to the chromosome ends. For instance, the G-rich telomere sequence itself is quite vulnerable to oxidative stress that generates 8-oxoguanosine and strand breaks.

Whereas telomere shortening is in most cells a slow process occurring over many cell divisions, another protective mechanism allows a rapid arrest of the cell cycle in response to overproliferation (Fig. 7.6). It is designated as stress-­ induced or oncogene-induced senescence (abbreviated SIS or OIS) and is mediated by CDK inhibitors, in particular, p16INK4A. Since both p16INK4A mRNA and protein are quite long-­lived, they accumulate over time if cells proliferate continuously and rapidly until the CDK inhibitor reaches a concentration that inhibits CDK4/6 and activates RB1 to repress E2F1-­dependent genes required for S phase. Moreover, both promoters in the CDKN2A gene respond to hyperproliferation signals, e.g., from RAS and MYC oncogenes, leading to activation of both RB1 and TP53 via p16INK4A and p14ARF. In senescent cells, other CDK inhibitors like p21CIP1 are also strongly induced. Induction of specific antiapoptotic proteins like BCL2 confers resistance to cell death, so that senescent cells may survive for prolonged periods. Finally, the senescent state is not only characterized by lack of proliferation, but also by marked changes in nuclear structure with increased heterochromatinization. In addition, transcription is reprogrammed so that cells secrete a large number of inflammatory cytokines, chemokines, and matrix-remodeling proteins. This phenomenon is known as SASP

7.3 Mechanisms to Escape Replicative Senescence in Human Cancers Fig. 7.6 CDK inhibitors as regulators of replicative senescence. See main text for further explanations

Hyperproliferation

Telomere dysfunction

163

Replication stress

Severe DNA damage

p14ARF p16INK4A

TP53

p21CIPI

RB1

Replicative senescence

(senescence-associated secreted proteome). The Activation of Telomerase: Many human cansecreted factors act in autocrine or paracrine cers express the TERT gene and the Telomerase manners to enforce senescence but also promote holoenzyme can be shown to be enzymatically inflammation, an immune-suppressive microen- active in cancer tissue extracts. In some tissues vironment and in some cases epithelial-­ and body fluids, Telomerase expression or activmesenchymal transition. With regard to cancer, ity can therefore serve as a cancer biomarker. replicative senescence is therefore ambivalent, Telomerase activation stabilizes telomere lengths protecting against cellular transformation on the and may actually allow a rebound to intact one hand, but creating conditions favorable for ­structures from the unstable state that had caused tumor progression on the other hand. Through chromosomal instability. SASP, in particular, cellular senescence may conSince the other components of the Telomerase tribute to human aging, when senescent cells holoenzyme are expressed in most cells, the critiaccumulate in aging tissues. This phenomenon cal step of Telomerase reactivation is activation has inspired the development of “senolytic drugs” of the TERT gene. Of note, other components that are intended to improve tissue function, pre- such as TERC and DKC1 may as well be overexvent cancers, and reverse aging. pressed in tumors compared to the respective normal tissues. TERT activation is often achieved by genetic mechanisms. In many tissues with a normally low turnover, the corresponding tumors 7.3 Mechanisms to Escape contain mutations in the TERT promoter. These Replicative Senescence mutations at −228 and −250 bp generate new in Human Cancers binding sites for ETS transcription factors that Several mechanisms allow cancer cells to escape mediate reactivation of the silenced gene replicative senescence and perform an (at least in (Fig.  7.7). Across all cancers, TERT promoter mutations may be the most frequent mutations in theory) unlimited number of cell divisions.2 non-coding sequences of the genome; they are detected in ≈10% of all cancers. They are particularly frequent in gliomas, bladder cancers, 2  It has been calculated that millions of tons and more than 10,000 generations of the HeLa cancer cell line have been and hepatocellular carcinoma, but are conspicuproduced since its establishment in 1951. ously rare in some cancer types that are thought

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164 p-arm

q-arm

Chromosome 5 TERT

1,295,495

1,295,162

1,295,250

1,295,228

–146

–124

r2853669

C250T

C228T

WT

T

CCCCTCCCGG

CCCCCTCCGG

MT

C

CCCCTTCCGG

CCCCTTCCGG

–245 TERT promoter

Transcription start site Initiator sequence

1,295,104 ATG (+1) TERT coding sequence

novel ETS1 binding motif

Fig. 7.7 Activating mutations in the TERT gene promoter. Localization of the TERT gene and structure of its regulatory region. Note the mutations at −228 and −250 bp (or

in another annotation −146 and −124 bp) and the more distant polymorphism. From Powter B et  al. (2021) J Cancer Res Clin Oncol 147:1007–1017. See main text for more explanation

to arise from stem cell populations such as colorectal carcinoma. Another mechanism leading to TERT reactivation or overexpression is gene amplification. In some cases, TERT overexpression is caused by provirus insertion upstream of the gene, e.g., by HBV sequences in hepatocellular carcinoma. TERT reactivation may also occur as a consequence of epigenetic changes. Indeed, DNA methylation patterns at active and inactive TERT promoters differ with respect to which parts are more or less strongly methylated. Alternative lengthening of telomeres: This alternative mechanism for the stabilization of telomeres (acronym: ALT) employs a DNA recombination mechanism. Telomeres may associate with PML nuclear bodies (→10.6) in the nucleus in order to exchange and extend their sequences in a process that is still incompletely understood. It is likely initiated by DNA replication forks stalling at telomeres and requires the BLM helicase to unwind the telomeric DNA as well as components of homologous DNA recombination repair like RAD51, RAD52, and the

MRE11 exonuclease. A positive regulator of the process is the lncRNA TERRA, which is often overexpressed in cancers with ALT. Such cancers also regularly display loss of the DAXX and ATRX chaperones, which deposit the histone variant H3.3 at telomeres. Since ALT involves recombination between different telomeres to elongate some, while others may be shortened, telomeres in ALT+ cancers are very heterogeneous in length. ALT is more frequently detected in cancers of mesenchymal origin, but also emerges in other cancer types as a mechanism of resistance against therapies targeting Telomerase. Inactivation of RB1 and TP53: The two tumor suppressors interfere with cancer development and progression in various ways (→5.2, →5.3). Clearly, induction and establishment of replicative senescence in response to telomere erosion, replicative stress, or uncontrolled proliferation is one important facet of their tumor-suppressive action. The CDK inhibitors p21CIP1, which is particularly strongly induced by TP53, p16INK4A, which influences and is in turn regulated by RB1 activity, and p57KIP2, in the cell types that express

7.4 Mechanisms of Apoptosis

it, are involved in this response. Accordingly, inactivation of RB1, TP53, and CDKN2A contributes also to the evasion of replicative senescence, explaining (partly) the prevalence of deleterious mutations in these three genes across many cancers. Specifically, the function of p16INK4A of “counting” rapid cell divisions may explain why p16INK4A is the most important tumor suppressor of all INK4 proteins. Depending on the cancer type, inactivation of this mechanism for establishing replicative senescence may suffice to escape the ­consequences of telomere erosion and replicative stress. In other cases, inactivation of this pathway may add to the effects of Telomerase reactivation. Possibly, in such cancers, inactivation of the mechanisms executing replicative senescence may allow cells to maintain a proliferative state until Telomerase is reactivated. This sequence is supported by experiments in long-time cultured normal cells, which demonstrate two subsequent “crises” (termed M1 and M2) that are overcome by RB1/TP53 inactivation and Telomerase reactivation, respectively. Cancers from stem cells: Since tissue stem cells typically express Telomerase, other than their more differentiated progeny, cancers developing from tissue stem cells may retain stem cell properties including sufficient TERT and TERC expression. This would explain why genetic alterations of the TERT locus are essentially never observed in cancers like colorectal carcinoma. Cancers of this kind are usually sustained by inappropriate activation of pathways that support and maintain stem cells in normal tissues, like WNT/β-Catenin signaling in colorectal carcinoma. Telomerase is one target of this pathway. Its induction is mediated among others by MYC, a known positive regulator of TERT expression. TERT transcription is furthermore regulated by STAT and NFκB transcription factors and crucially, by the transcription factor KLF4. Consequently, activation of stem cell-­maintaining pathways in other cancer types associated with MYC overexpression may likewise contribute to Telomerase expression. Of note, there is good evidence that the Telomerase protein has functions beyond the maintenance of telomers,

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including even regulation of mitochondria. Overexpression of Telomerase may therefore benefit cancer cells in additional ways.

7.4 Mechanisms of Apoptosis The process of apoptosis can be divided into several stages, i.e., initiation, execution, and removal of the cell remnants. As a rule, initiation of apoptosis proceeds via two alternative pathways designated “intrinsic” and “extrinsic,” which converge on a common execution pathway. The intrinsic pathway responds to internal signals, e.g., from DNA damage, whereas the extrinsic pathway responds to external signals, e.g., cytokines and cytotoxic T cells. In some cell types, the extrinsic pathway can proceed towards execution on its own whereas in others it requires a contribution from the intrinsic pathway. Since all steps in these pathways are well-defined and controlled, apoptosis is considered a form of programmed cell death. The decisive step of the intrinsic pathway (Fig. 7.8) takes place at the mitochondrial membrane and is regulated by proteins of the BCL2 family. Around 20 members of this family are known, some of which are proapoptotic, whereas others are antiapoptotic (Table  7.1). They share common domains, termed BH1 to BH4 (BCL2 homology domains) and some contain a transmembrane domain (TM). The founding member BCL2 was discovered as the oncogene activated by the characteristic translocation t(14;18) in follicular B cell lymphoma. This translocation places the BCL2 gene under the control of the immunoglobulin heavy chain enhancer. Overexpression of BCL2 prevents apoptosis of follicular B cells and is the initiating event in this cancer. Apoptosis via the intrinsic pathway is crucially mediated by the BCL2 family members BAX and BAK, which contain multiple BH domains. Apoptosis at the mitochondrial membrane is initiated by BAX and BAK homodimers that subsequently aggregate to multimers containing both proteins. Antiapoptotic proteins like BCL2, BCL-XL, or MCL-1 form heterodimers

7  Cell Death and Replicative Senescence in Cancer

166 PUMA BAX

BCL2

BAX

SMAC

BID

IAPs

Cyt c Procaspase 9 Procaspase 3 APAF 1

Apoptosome Caspase 3

Fig. 7.8  The intrinsic pathway of apoptosis. The pathway starts in the upper left corner, leading to the processing of Procaspase 3 to Caspase 3 in the lower left corner. See main text for more detailed explanation Table 7.1  The BCL2 family of apoptotic regulators Subfamily Antiapoptotic regulator Proapoptotic effector BH3-only proapoptotic regulator

Domains BH4-BH3-BH1-­ BH2-TM BH3-BH1-BH2-TM BH3-TM or BH3

with BAX and BAK, preventing apoptosis. Conversely, proapoptotic members of the BCL2 family are distinguished by containing a single BH3 domain and no transmembrane domain (Table 7.1). They relieve inhibition by antiapoptotic proteins and stimulate BAK/BAX multimerization. The BCL2 family protein BID mediates the cooptation of the intrinsic pathway in those cells in which it is needed for support of the extrinsic pathway. To that end, the Caspase 8 or Caspase 10 proteases cleave the inactive BID

Representative members BCL2, BCL-XL BAK, BAX BID, BAD, NOXA, PUMA, NIX

Other members BCL-W, MCL-1, A1, NRF3 BOK BAD, BIK, BLK, BMF

precursor protein to generate two shorter forms of BID, p15, and p13. Various kinds of cellular stress can induce apoptosis. The precise pathway depends on the cell type and the kind of cellular stress. Prominently, TP53 activated in response to DNA double-strand breaks induces transcription of the proapoptotic BCL2-family proteins NOXA, PUMA, and BAX, while downregulating BCL2. Proapoptotic proteins override antiapoptotic ­signals to initiate the next step in the intrinsic

7.4 Mechanisms of Apoptosis

apoptotic pathway, the “mitochondrial outer membrane permeabilization” (MOMP) that leads to the formation of pores in the outer mitochondrial membrane. This transition in particular is mediated by BAX and BAK. At the contact sites between the outer and the inner mitochondrial membrane, a multiprotein complex is formed by proteins from all mitochondrial compartments. One crucial component is the Adenine nucleotide translocator which otherwise exchanges ADP + Pi for ATP across the normally tight inner mitochondrial membrane. Ultimately, the mitochondrial transmembrane potential breaks down. Following the mitochondrial permeability transition, mitochondria release several proteins, mostly from the intermembrane compartment, such as Cytochrome c and the SMAC/Diablo protein. The flavoprotein Apoptosis-inducing factor (AIF) is released from the mitochondrial matrix. In the cytoplasm, 8 molecules of Cytochrome c associate with the same number of APAF1 proteins to form a large structure (resembling the spokes of a wheel), also known as the “apoptosome.” The apoptosome binds a stochiometric number of the pro-protease Procaspase 9 and supports its autocatalytic activation in an ATP-dependent process. Caspases are cysteine proteases which cleave the peptide bond following an Asp in the consensus sequence QAD↓RG. The more than 10 caspases in humans are categorized into three groups, initiator caspases (including Caspase 9), executor caspases (including Caspase 3), and inflammatory caspases, which are not directly involved in apoptosis but rather process cytokines. The prototypic enzyme of that group is Caspase 1, or Interleukin-converting enzyme (ICE); it is also important for pyroptosis. Procaspase 9 is a homodimer containing two “CARD” domains through which it binds to the APAF1 adaptor proteins in the apoptosome. Following autocatalytic cleavage, the active Caspase 9 is a hetero-tetramer composed of two smaller and two larger subunits each. Active Caspase 9 goes on to process and activate the executioner Caspase 3 to initiate the execution phase of apoptosis.

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Activation of caspases is supported by the AIF protein released from the mitochondrial matrix. Another protein liberated from this compartment, SMAC/Diablo, has a distinct function (Fig. 7.8). Activation of initiator caspases (and in some instances even of executioner caspases) is often not sufficient to actually elicit apoptosis because a number of protein inhibitors designated IAPs (inhibitors of apoptosis) inhibit the proteases. IAPs are mostly small proteins characterized by one or several “BIR” domains. XIAP and other IAPs inhibit the intrinsic pathway at the step of activated Caspase 9 and even Caspase 3 by binding the enzymes through their BIR domains and inhibiting their protease activity. SMAC/Diablo binds and sequesters IAPs thereby removing a final obstacle to apoptosis. Several viruses— including oncoviruses—express their own IAPs to prevent cells from undergoing apoptosis while they multiply. The extrinsic pathway (Fig.  7.9) is initiated when certain cell surface receptors are engaged by their specific ligands. As a rule, these “death receptors” belong to the TNF receptor superfamily (Fig. 7.10). Tumor necrosis factor α (TNFα) is one of several cytokine ligands of this receptor superfamily. This peptide is secreted by monocytes, macrophages, and other cells of the immune system during inflammatory reactions and in response to cellular stress. It elicits various reactions in cells expressing the TNFRI receptor, which may include apoptosis. A second receptor for TNFα, TNFRII, is found in a more limited range of cell types compared to TNFRI. It can be activated only by membrane-bound TNFα. Other TNFRSF ligands are present mainly on the surface of immune cells, e.g., CD95L, and the ligand-receptor interaction is part of a cell-cell interaction that may result in apoptosis (→9.6). CD95L activates TNFRSF6 (alias CD95, FAS, or APO-1) and is also known as “FAS ligand.” The CD95/CD95L pair is an important component in the killing of infected and tumor cells by cytotoxic T cells. It is also employed in the selective elimination of autoreactive immune cells. Defects in CD95 function occur in autoimmune diseases as well as in cancers (→7.5).

7  Cell Death and Replicative Senescence in Cancer

168 Fig. 7.9  The extrinsic pathway of apoptosis. Caspase 8 and/or Caspase 10 may be activated depending on the cell type and receptor. Activation of BID to coopt the intrinsic pathway (green dotted line box) is not obligatory in all cell types

Ligand

TNFSFR

Procaspase 8/10

FADD/TRADD

FLIP Caspase 8/10 Procaspase 3

BID

Caspase 3

Execution

The distinction between membrane-bound and soluble ligands of TNFRs is in fact blurry. Some cytokines, including TNFα, are also present as active membrane-bound forms on the cell surface, and conversely, CD95L can also be secreted. Receptors can be cleaved by proteases (like the TACE enzymes) and interact with soluble and membrane-bound ligands. For instance, in addition to the membrane-bound form of CD95 (also known as tmFAS), a soluble form is generated (sFAS) which acts as a decoy receptor to decrease

Intrinsic pathway

the responsiveness to CD95L.  Apoptotic responses to the TNFα-related cytokine TRAIL are dependent on the relative expression of four different TNFRSF members, TRAIL-R1 through TRAIL-R4, two of which are true receptors and two are largely decoys. Like TNFRI, they feed into additional pathways upon activation. In some cell types, TRAIL therefore induces cell death, prominently in hepatocytes. Responses of tumor cells vary, some respond to TRAIL by apoptosis, but others by increased proliferation.

7.4 Mechanisms of Apoptosis TRAIL-R1

169 CD95/FAS

sFAS

TNF-R1

CRD

DED

Fig. 7.10  Some members of the TNFR superfamily. All members of the family share similar cysteine-rich domains (hatched) in their extracellular region, whose numbers vary. In the intracellular domain, they consistently contain a death domain (DED) and several cysteine-rich domains (CRD) indicated by rectangular boxes.

Some members have additional signaling functions and domains. The sFAS protein is otherwise identical to CD95/FAS, but lacks transmembrane and intracellular domains and acts as a decoy receptor. The ligands for TRAIL-R1 and TNFR1 are TRAIL and TNFα, respectively. In the active state, TNFRs usually form trimers

Members of the TNFRSF family act upon several pathways, notably the NFκB pathway (→6.7). Family members that can activate the extrinsic apoptosis pathway differ from their homologs by the presence of an additional intracellular domain, named the “death domain” (Fig. 7.10). This domain is required for the activation of the extrinsic apoptotic pathway. The NFκB pathway as a rule counteracts apoptosis, e.g., by inducing FLIP and A20 which moderate or inhibit the response to death receptor activation. The actual cellular response will therefore often depend on the relative strengths of the pathways activated in parallel and the repertoire of pro- and antiapoptotic factors in the cell. In specific cell types, cytokine receptors may even stimulate cell proliferation. Following ligand binding to an active TNFR, such as TNFRI or TNFRSF6, the ligand/receptor complexes trimerize and the receptor death domains bind FADD by interaction with the

homologous domains in this adaptor protein (Fig.  7.9). In addition, FADD contains a death effector domain homologous to that in initiator caspases. By binding to the death receptor, this domain is exposed and binds an initiator procaspase, usually Procaspase 8 or Procaspase 10. The resulting complex is known as “death inducing signaling complex” (DISC). Its function in apoptosis consists in bringing procaspase molecules into close proximity so that they can dimerize and activate each other. The activated initiator caspases then activate executioner caspases like Caspase 3 setting the execution phase into motion. The FLIP protein acts as an inhibitor of the extrinsic pathway by interfering with initiator caspase dimerization. Of note, several components of the DISC complex, especially the RIPK1 kinase, are also present in another complex, the ripoptosome, which initiates necroptosis by activating the RIPK3 kinase. The relative activities and the outcome of DISC and ripoptosome sig-

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170

naling are dependent, in particular, on the cellular levels and distribution of certain IAP proteins like cIAP1, cIAP2, and XIAP.  For that reason, activation of TNF receptors can also result in necroptosis, especially if apoptosis should be blocked. In some cells, activation of the extrinsic pathway by certain death receptor ligands is sufficient to elicit apoptosis. In such cases, the expression levels of BCL2 and BCL-XL are quite irrelevant. In others, induction of apoptosis requires the participation of the intrinsic pathway. In response to external signals, the intrinsic pathway is typically coopted via the BID protein which is cleaved by Caspase 8 to an active form. Conversely, the intrinsic pathway also influences the extrinsic pathway. For instance, TP53 induces activators of the extrinsic pathway like PUMA, NOXA, BAX, and AIP, but also increases the expression of CD95 thereby sensitizing cells to proapoptotic external signals. The multiple biochemical and morphological changes that can be observed during the execution phase of apoptosis are caused by proteolytic cleavage of more than 300 cellular proteins by Caspase 3 and other executioner caspases like Caspase 6. The substrates comprise regulators of the cell cycle such as RB1, DNA repair proteins such as DNA-PK and Poly-ADP-ribosyl polymerase (PARP), and cytoskeletal proteins such as Actins, Lamins, and the cytokeratin KRT18. PARP cleavage is frequently used as an experimental means of detecting apoptosis. Another characteristic of apoptosis, a “nucleosomal ladder” DNA fragmentation pattern, is caused by several DNases, prominently CAD (Caspase activated DNase) that are liberated by cleavage of the inhibitory proteins to which they are normally bound. Cleavage of FAK, PAK2 (p21-associated kinase), and Gelsolin contributes to the loss of adhesion and the characteristic membrane changes such as blebbing and to the redistribution of membrane proteins and phospholipids. Exposure of specific phospholipids and proteins creates signals for the subsequent phase in which the apoptotic cells and their fragments are removed, mainly by phagocytosis (efferocytosis). In particular, phosphatidylserine is normally restricted strictly to the inner layer of the membrane bilayer. In apop-

totic cells it is flipped to the outer layer3 and recognized by receptors on phagocytosing macrophages that are attracted by further chemotactic signals diffusing out from the dying cell.

7.5 Mechanisms Diminishing Apoptosis in Cancer Diminished apoptosis of cancer cells is important for a number of reasons. (1) In some cancers diminished apoptosis is the primary cause of tumorous growth, e.g., in follicular B cell lymphoma. In tumors of this kind, cells that ought to undergo apoptosis in the course of normal tissue homeostasis survive, which leads to an oversized and progressively disorganized tissue mass. Typically, cancers driven primarily by impediments of apoptosis grow slowly, but relentlessly. (2) A diminished rate of apoptosis exacerbates hyperproliferation in many different cancers. Many tumors exhibit death rates that are distinctly higher than those in the normal tissue of origin, but the rate of proliferation is even higher resulting in a net increase in cell numbers. In such cancers, even small decreases in the rate of cell death can have disproportionate effects on tumor growth. (3) Apoptosis is (like replicative senescence) a fail-safe mechanism in response to “inappropriate” proliferation signals and to pronounced DNA damage, e.g., unrepaired double-strand breaks. Therefore, a decreased response to internal proapoptotic signals allows cells to proliferate in spite of inappropriate proliferation signals or persisting severe DNA damage. This occurs in many cancer types, particularly during progression. (4) Many viruses that promote cancer development, such as EBV (→10.3) or HHV8 (Box 7.2), express antiapoptotic factors to prevent apoptosis in response to both internal and external signals thereby creating cell populaDetection of exposed phosphatidylserine via Annexin V and PARP cleavage are commonly used techniques to follow apoptosis in lab experiments. 3 

7.5 Mechanisms Diminishing Apoptosis in Cancer

tions that are susceptible to further carcinogenesis. (5) Cytotoxic T cells that protect against cancer and infections induce apoptosis to kill virus-­ infected and cancer cells through cytokines like TNFα and death receptor ligands like CD95L via the extrinsic pathway, and via Granzyme, which activates the intrinsic apoptotic pathway following its transport into the target cell by Perforin. Decreased responsiveness to these proapoptotic signals is one mechanism by which tumor cells can evade immune responses by cytotoxic T cells and NK cells (→9.6). Damaged cancer cells may furthermore escape from phagocytosis by macrophages by expressing the CD47 “don’t-eat-me” signal protein on their surface and manage to survive. CD47 is accordingly upregulated in many cancers. (6) Many cytotoxic and targeted drugs employed in chemotherapy as well as radiotherapy act by inducing apoptosis. Decreased apoptotic responsiveness therefore contributes to primary and secondary resistance to chemoand radiotherapy. (7) Cancer cells occasionally recover from incomplete apoptosis. These survivors present with increased genomic instability and activated pro-survival pathways and may be largely resistant to current therapies.

Box 7.2 Carcinogenesis by HHV8

Human herpes virus 8 (HHV8 or KHSV) is a DNA virus with an ≈165 kb genome that contains over 90 open reading frames (ORFs). It is endemic in certain regions, where infections occur earlier in life, whereas transmission in non-endemic regions mainly takes place through sexual contact between males. HHV8 causes a number of rare cancers in immunosuppressed persons, including Kaposi sarcoma in men infected by HIV1, where HHV8 was first discovered. Like other herpes viruses, HHV8 can enter a latent state in persistently infected cells, in which the linear genome is circularized and replicated with the host genome.

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From the latent state, the virus can be reactivated by various stress conditions or specific compounds to produce infectious viral particles and lyse the host cells. In the latent state, only a fraction of the viral genes are expressed. Some persistently infected cells are transformed by viral gene products. Kaposi sarcoma in particular is derived from endothelial cells that assume a mesenchymal phenotype, over-­proliferate, and become invasive. These cells express a mixture of markers of endothelial and fibroblast differentiation. The malignant phenotype of the Kaposi tumor cells remains dependent on the viral gene products which act through several mechanisms. Some viral gene products activate signal transduction pathways that augment cell proliferation, survival, and migration either directly or indirectly by acting on cytokine receptors. One gene product, e.g., is homologous to the cytokine IL6, others mimic chemokines (→9.5), yet others induce production of cytokines by the infected cells. Other viral gene products inhibit cellular responses to viral infection and replication, in particular, TP53 stabilization and interferon signaling. The viral protein vCYC is homologous to D-Cyclins and promotes cell cycle progression through G1. Crucially, a number of viral products interfere specifically with the activation of apoptosis. For instance, a viral ortholog of FLIP, vFLIP, diverts death receptor signaling from apoptosis towards activation of NFκB. Other viral products downregulate or block proapoptotic proteins like BID and BIM.  Notably, HHV8 expresses a substantial number of miRNAs that regulate its interaction with the host cell; some of these contribute to carcinogenesis. Mariggiò G et  al. (2017) Kaposi sarcoma herpesvirus pathogenesis. Philos Trans R Soc Lond B Biol Sci 372:20160275 Jary A et  al. (2021) Kaposi's sarcoma-­ associated herpesvirus, the etiological agent of all epidemiological forms of Kaposi's sarcoma. Cancers 13:6208.

172 Table 7.2  Mechanisms causing diminished apoptosis in human cancers Mechanism Desensitization of death receptors (initiation and signaling of extrinsic pathway) Counterattack (avoidance of death receptor signaling) Loss of TP53 function Desensitization or inactivation of the intrinsic pathway Overexpression of IAPs Activation of antiapoptotic signaling pathways Expression of viral antiapoptotic factors

In human cancers, diminished apoptosis may originate from alterations in many different steps of apoptosis by a variety of mechanisms (Table 7.2). Proteins that relay internal or external proapoptotic signals can be inactivated, the extrinsic or the intrinsic pathway may become deactivated or desensitized, and even the execution stage can be impeded. In one and same cancer, several different steps can be affected. Moreover, diminished apoptosis in cancer cells can be caused by the overactivity of survival signal pathways rather than by primary genetic or epigenetic alterations in genes encoding apoptotic pathway components. Moreover, many oncogenic viruses express antiapoptotic proteins or regulatory RNAs (see Box 7.2). Thus, while apoptosis is rarely completely inactivated in human cancers, it does not occur at the same rate as in normal cells exposed to comparable external and internal signals. Typical changes that diminish the apoptotic rate in human cancer cells include the following. Desensitization of death receptors: The CD95/ CD95L system is incapacitated for induction of apoptosis in many human cancers, in hematological cancers as well as in carcinomas. While mutation of the TNFRSF6 gene encoding CD95 is occasionally observed, downregulation of receptor expression is more frequent. In some cancers, expression shifts from the transmembrane towards a soluble (decoy) receptor. As a rule however residual CD95 remains expressed at the membrane. Altered expression of FADD proteins, decreased expression of Caspase 8 and APAF1 (caused by epigenetic mechanisms), and overexpression of FLIP, which inhibits the activation of caspases at the DISC, have been identi-

7  Cell Death and Replicative Senescence in Cancer

fied as causes of post-receptor defects in some cancers. In each case, the overall consequence is a weaker response to cytotoxic immune cells, but also to chemotherapeutic agents, which induce apoptosis partly through increased expression of CD95 and its ligand. Other members of the TNFRSF family like the TRAIL receptors are inactivated by similar mechanisms. Notably, activation of signal transduction pathways by death receptors, especially of NFκB signaling, remains often intact or is even enhanced. Counterattack: Decreased expression of the CD95 receptor is accompanied by increased secretion of soluble CD95 ligand in some cancers. It is thought that secretion of CD95L normally contributes to the establishment of “immune-privileged” sites in the human body. Immune-privileged sites are established in organs such as the anterior eye chamber or the testes that could not function properly in the presence of lymphocytes and must therefore exclude them. It has therefore been postulated that increased production of CD95L may help cancers to prevent immune responses and may even destroy T cells and other cells expressing CD95. A different interpretation is that CD95 does not regularly induce apoptosis in cancer cells, especially if FLIP is overexpressed or Caspase 8 is inactivated. In those cases, activation of CD95 by (membrane-bound or soluble) CD95L may paradoxically stimulate pro-survival pathways like NFκB signaling. Loss of TP53: While changes of TNFR family receptors or DISC components and regulators inactivate external proapoptotic signaling, inactivation of TP53 may be the most common alteration that compromises internal proapoptotic signaling. TP53 mediates induction of apoptosis in response to DNA damage as well as to hyperproliferation (→5.3). Intrinsic pathway inactivation: A varied assortment of alterations affects the intrinsic apoptotic pathway in cancers. BCL2 was discovered as an oncogene protein activated by the most characteristic translocation in follicular lymphoma (→4.3). Overexpression of BCL2 is a driver in this cancer and essential for the survival of the cancer cells. Compounds like venetoclax

7.5 Mechanisms Diminishing Apoptosis in Cancer

173

and navitoclax inhibiting the interactions of BCL2 with other family members are now used in its treatment (Fig. 7.11). In another slowly but relentlessly growing hematological cancer, chronic lymphocytic leukemia (CLL), overexpression of BCL2 results from the loss of the miRNAs miR-15 and miR-16 that otherwise negatively regulate BCL2 expression. BCL2 is also overexpressed in some carcinomas, but in general, carcinomas rather overexpress BCL-XL or MCL-1. BCL-XL is induced a. o. by the NFκB pathway, and MCL-1 by MAPK signaling. Either of their genes (BCL2L1 and MCL1) are amplified in various carcinomas. Conversely, proapoptotic members of the BCL2 family such as BAD or NOXA (Table 7.2) are downregulated in a variety of cancers, in some cases by promoter hypermethylation, and are therefore not inducible by activated TP53 or other signals. The most generally downregulated member of the family may be BAX, perhaps because of its crucial effector function at the mitochondrial membrane. At a

later step, apoptosis may be blocked by downregulation of APAF1, likewise often by promoter hypermethylation (→8.2). In summary, in almost all cancers the balance between proapoptotic and antiapoptotic members of the BCL2 family is tilted towards the latter. The overall result of this imbalance is a lower sensitivity towards apoptotic signals, particularly those elicited by hyperproliferation and aneuploidy, but also by chemotherapy. IAP overexpression: Both the intrinsic and extrinsic pathways are impeded by overexpression of IAP proteins like XIAP which block signaling through caspases as well as the activity of the actual execution caspases, especially Caspase 3. IAPs may also be induced by tumor viruses such as EBV (→10.3) or HBV (→17.4). The overexpression of IAPs that block the execution phase can result in an unresolved state in a cancer cell, viz., partial activation of caspases that is not sufficient for execution of cell death. Possible consequences of such partial activation comprise

Venetoclax V

BCL-2 BIM

V

PUMA

V

BCL-2 O

Displacement of BH3only proteins from BCL-2

O O O S O

Activated BAX/BAK1 BIM PUMA

S

CF3 H N

O N

S

NH

BAX N

BAK1

P4

N

Increased MOMP and release of pro-apoptotic Cytochrome c proteins

P2 CI

IAPs

Apoptosome generation and activation of initiator caspase 9 Activation of effector caspases

Fig. 7.11  Mechanism of action of BCL2 inhibitor drugs. Left: Mechanism of action of venetoclax. From Klanova M, Klener P (2020) l.c. Originally published under the terms of CC BY; Right: Structure of navitoclax and its

interaction with BCL2. From Souers AJ et al. (2013) Nat Med 19:203–208. See Sect. 23.5 for more information on the drugs

174

altered cell morphology and adhesion as well as increased genomic instability. Overexpression of certain other IAPs or of FLIP impede primarily the signaling phase of the extrinsic pathway by inhibiting the signal from FADD to Caspase 8 or Caspase 10. The 16.5 kD protein Survivin (gene: BIRC5) is counted among the IAPs but is now thought to be actually more important for cell division. It is expressed at high levels during fetal development, but almost undetectable in normal resting tissues. Its expression increases during normal proliferation and during regenerative processes such as wound repair. In many human cancers, Survivin expression is so strongly and consistently increased that it may be used as a tumor biomarker. Activation of antiapoptotic pathways: The defects in the actual apoptotic signaling and execution cascades in cancer cells are almost regularly compounded by increased activity of cancer pathways that convey survival signals. Arguably, most important in this regard are the PI3K (→6.3) and the NFκB (→6.7) pathways. The PI3K pathway is activated in many cancers, indirectly or directly by a variety of mechanisms (→6.2). The pathway does stimulate proliferation and particularly the growth of cells, but in many cancers, a major contribution of its activation may lie in the ensuing inhibition of apoptosis. A crucial step in this inhibition is phosphorylation of BAX and BAD by AKT which prevents the proteins from activating the intrinsic apoptotic pathway. AKT also phosphorylates and activates the forkhead transcription factor FKHR-L1, which counteracts apoptosis at the level of transcription. Conversely, FOXO transcription factors that induce proapoptotic proteins are kept in an inactive state by AKT phosphorylation. PI3K pathway activity therefore tends to diminish the effect of cancer chemotherapy. Compared to the PI3K pathway, the NFκB pathway is less frequently subject to direct activation by genetic alterations in human cancers. However, in many cell types, it limits the extent of induction of apoptosis through activation of TNFRSF death receptors. Therefore, its

7  Cell Death and Replicative Senescence in Cancer

indirect activation in tumor cells, which can be achieved by a variety of cytokines and stress signals, contributes to the resistance towards induction of apoptosis by external signals such as TNFα or CD95L.

Further Reading Angrisani A et  al (2014) Human dyskerin: beyond telomeres. Biol Chem 395:593–610 Ben-Porath I, Weinberg RA (2005) The signals and pathways activating cellular senescence. Int J Biochem Cell Biol 37:961–976 Botchkarev VA (2015) Integration of the transcription factor-­regulated and epigenetic mechanisms in the control of keratinocyte differentiation. J Invest Dermatol 17:30–32 Calado RT, Young NS (2009) Telomere diseases. NEJM 361:2353–2365 Castedo M et al (2004) Cell death by mitotic catastrophe: a molecular definition. Oncogene 23:2825–2837 Chakravarti D (2021) Telomeres: history, health, and hallmarks of aging. Cell 184:306–322 Cheung TH, Rando TA (2013) Molecular regulation of stem cell quiescence. Nat Rev Mol Cell Biol 14:329–340 Cory S, Huang DC, Adams JM (2003) The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene 22:8590–8607 Criscione SW et  al (2016) The chromatin landscape of cellular senescence. Trends Genet 32:751–760 Davalos AR et  al (2010) Senescent cells as a source of inflammatory factors for tumor progression. Cancer Metastasis Rev 29:273–283 Debatin KM, Krammer PH (2004) Death receptors in chemotherapy and cancer. Oncogene 23:2950–2966 Eckhart L et al (2013) Cell death by cornification. BBA 1833:3471–3480 Feldser DM, Hackett JA, Greider CW (2003) Telomere dysfunction and the initiation of genomic instability. Nat Rev Cancer 3:623–627 Fernandes SG et  al (2020) Role of telomeres and telomeric proteins in human malignancies and their therapeutic potential. Cancers 12:1901 Finkel T et al (2007) The common biology of cancer and ageing. Nature 448:767–774 Galluzzi L et  al (2018) Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Diff 25:486–541 Gorgoulis V et al (2019) Cellular senescence: defining a path forward. Cell 179:813–827 Holmberg J, Perlman T (2012) Maintaining differentiated cell identity. Nat Rev Genet 13:429–439

Further Reading Klanova M, Klener P (2020) BCL-2 proteins in pathogenesis and therapy of B-cell non-Hodgkin lymphomas. Cancers 12:938 Koren E, Fuchs Y (2021) Modes of regulated cell death in cancer. Cancer Discov 11:245–265 Lanigan F et al (2011) Transcriptional regulation of cellular senescence. Oncogene 30:2901–2911 Lopez-Otin C (2013) The hallmarks of aging. Cell 153:1194–1217 Lowe SW et al (2004) Intrinsic tumor suppression. Nature 432:307–315 Ohtani N, Hara E (2013) Roles and mechanisms of cellular senescence in regulation of tissue homeostasis. Cancer Sci 104:525–530 Peter ME et al (2015) The role of CD95 and CD95 ligand in cancer. Cell Death Differ 22:549–559 Ramlee MK et  al (2016) Transcription regulation of the Human Telomerase Reverse Transcriptase (hTERT) gene. Genes 7:50 Ruijtenberg S, van den Heuvel S (2016) Coordinating cell proliferation and differentiation: antagonism between cell cycle regulators and cell type-specific gene expression. Cell Cycle 15:196–212 Ruis P, Boulton SJ (2021) The end protection problem—an unexpected twist in the tail. Genes Dev 35:1–21 Sell S, Pierce GB (1994) Maturation arrest of stem cell differentiation is a common pathway for the cellular origin

175 of teratocarcinomas and epithelial cancers. Lab Invest 70:6–22 Sharma S & Chowdhury S (2022) Emerging mechanisms of telomerase reactivation in cancer. Trends Cancer 8:632-641 Sharpless NE, DePinho RA (2004) Telomeres, stem cells, senescence, and cancer. J Clin Invest 113:160–168 Simons BD, Clevers H (2011) Strategies for homeostatic stem cell self-renewal in adult tissues. Cell 145:851–862 Soufi A, Dalton S (2016) Cycling through developmental decisions: how cell cycle dynamics control pluripotency, differentiation and reprogramming. Development 143:4301–4311 Sulli G et al (2012) Crosstalk between chromatin state and DNA damage response in cellular senescence and cancer. Nat Rev Cancer 12:709–720 Wang B, Demaria M (2021) The quest to define and target cellular senescence in cancer. Cancer Res 81:6087–6089 Wang L et al. (2022) Exploiting senescence for the treatment of cancer. Nat Rev Cancer 22:340–355 Wouters BG, Koritzinsky M (2008) Hypoxia signaling through mTOR and the unfolded protein response in cancer. Nat Rev Cancer 8:851–864 Zhang JM, Zou L (2020) Alternative lengthening of telomeres: from molecular mechanisms to therapeutic outlooks. Cell Biosci 10:30

8

Cancer Epigenetics

Key Points scriptional start sites of many human genes, • In humans, cell differentiation does not termed “CpG-islands,” are usually devoid of involve changes in the base sequence or in the methylation. amount of DNA per cell, with few, specific • In many human tumors, a (highly variable) exceptions. Rather, “epigenetic” mechanisms number of CpG-islands become aberrantly are employed to establish stable cell type-­ methylated. This “hypermethylation” is as a specific patterns of gene expression. rule associated with silencing of the hyperCollectively, epigenetic changes in tumor cells methylated promoter. In spite of such increases can therefore be understood as reflecting their in methylation at specific sites, the overall altered differentiation state, which is premethylcytosine content is often lower in tumor served by similar mechanisms as in normal cells, owing to partial demethylation of repeticells. tive sequences and gene bodies. This phenom• Epigenetic changes in tumor cells can arise by enon, designated as “global hypomethylation,” accidents, i.e., as epimutations. More commay be related to chromosomal instability. monly, they are secondary consequences of Both types of methylation changes can be changes in cancer pathways that drive the cells quite straightforwardly detected and monitowards aberrant states. Frequently, epigenetic tored and can be used for tumor diagnostics. regulators are inactivated or deregulated by • DNA methylation is one of several interacting mutations. Notably, some epigenetic changes mechanisms that downregulate gene expresmay be apparent only, because the epigenetic sion in a progressively stable fashion. The state of tumor cells resembles that of their parmost dynamical of these mechanisms is ticular cell of origin rather than that of an deacetylation of the histones in the nucleoentire normal tissue. somes of gene promoters and enhancers. • An important component of epigenetic regulaDeacetylation is catalyzed by histone deacetytion is DNA methylation at cytosine residues lases (HDACs) recruited by DNA-binding in the palindromic CpG dinucleotide sequence. repressors or corepressors. Conversely, acetyIn normal somatic human cells, the majority lation is established by coactivators with hisof CpG-dinucleotides are methylated, espetone acetyltransferase (HAT) activity acting in cially in repetitive sequences, in the body of concert with DNA-binding transcriptional genes and in the regulatory regions of non-­ activators. Methylation of histones at specific expressed genes. In contrast, comparatively sites, prominently at lysine 9 of H3 (H3K9), CpG-rich sequences overlapping the tranby histone methyltransferases (HMTs) is a © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 W. A. Schulz, Molecular Biology of Human Cancers, https://doi.org/10.1007/978-3-031-16286-2_8

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8  Cancer Epigenetics

178

•

•

•

•

further step towards silencing, while methylation at other sites, especially H3K4 stabilizes gene activation. Modification at H3K9 attracts repressor proteins, such as HP1 in heterochromatin, but also DNA methyltransferases (DNMTs), which together “lock in” gene silencing. DNA methylation on the one hand directly inhibits the binding of some transcriptional activators and on the other hand promotes the binding of repressor protein complexes recognizing methylcytosine via MBD proteins. MBD proteins and DNMTs further interact with HDACs and specific HMTs to reinforce silencing. Active genes carry acetylated histones, especially around their transcriptional start site, which is further accentuated by H3K4 trimethylation and often eviction of one or two nucleosomes. Actively transcribed regions are methylated at H3K36. Activation as well as repression of genes require chromatin remodeling complexes to open or close chromatin, respectively, by repositioning particularly nucleosomes. Chromatin remodeling complexes mutually interact with activators and repressors and recognize histone modifications. Remodeling complexes furthermore support DNA replication, repair, and other processes that require reorganization of chromatin. H3K27 trimethylation is introduced by the polycomb complex PRC2 and serves in principle to repress genes. This modification can however be reversed by dedicated demethylases acting as components of “trithorax-like” complexes like the COMPASS complex. H3K27me3 is employed during development and differentiation to repress genes in specific lineages or cell types. Bivalent modification of H3 by both K27 and K4 methylation marks promoters and enhancers that can be selected for either expression or repression during differentiation. The PRC1 complex intensifies gene repression by PRC2, among others by mono-ubiquitinating H2AK119, but can also repress genes independently of PRC2. Aberrant gene silencing by epigenetic mechanisms in tumor cells is often, but not always

•

•

•

•

accompanied by DNA hypermethylation. In many cases, it is instead caused by polycomb-­ mediated repression. Many cancers harbor mutations in genes encoding epigenetic regulators, including DNA and histone methyltransferases, histone demethylases, histone acetyltransferases, and chromatin remodeling factors. Most mutations in epigenetic regulators are deleterious, but for some factors gene amplification and specific mutations leading to oncogenic activation are also observed. Specific epigenetic mechanisms are involved in the inactivation of the second X chromosome in female cells and in genomic imprinting, the selective monoallelic expression of alleles inherited from the mother or father. Aberrant genomic imprinting is a cause of certain pediatric tumors, e.g., Wilms tumors. Loss of imprinting may also contribute to cancers in adults. Cell type-specific gene expression is crucially regulated by enhancers. Specific patterns of enhancer activity characterize individual cell types. Active enhancers carry H3K4me and histones acetylated by the HAT p300, whereas inactive enhancers lack these modifications and may further be marked by H3K27me3. DNA methylation varies with the enhancer activity state, from low to high with decreasing activity. These patterns, like cell differentiation at large, are established by cell type-specific transcription factors. These are usually organized in networks or cascades and interact with various chromatin regulators. Specific epigenetic states characterize pluripotent stem cells and tissue-specific multipotent precursors. Cancer cells need to acquire some of their properties, especially the ability for (largely) unlimited self-renewal. This may occur via Telomerase activation and inactivation of CDK inhibitors in some cancers; others may be derived from tissue stem cells that already possess the required abilities. While some cancers do maintain hierarchies similar to those in normal tissues, more generally, increased plasticity as a consequence of

8.1  Mechanisms of Epigenetic Inheritance

altered epigenetic regulation blurs the distinctions between stem cells and differentiated progeny in cancers.

8.1 Mechanisms of Epigenetic Inheritance While genetic alterations are crucial causes of almost all cancers (→2), the properties of cancer cells are ultimately determined by their pattern of gene expression. Altered gene expression patterns can be a consequence of genetic alterations, but genetic alterations are not absolutely required to generate aberrant phenotypes. Evidently, profoundly different gene expression patterns are established in the various normal cells of the body and these can be stably maintained during proliferation if needed or can be changed in a controlled fashion during development and differentiation. For instance, stem cells in some tissues retain their phenotype through the multiple divisions in a human lifetime. Likewise, cell differentiation in humans is in general achieved without alterations in the sequence and amount of DNA in the cells. Of note, there are a few exceptions. Differentiation of B and T lymphocytes involves gene rearrangements with loss of DNA segments from the immunoglobin and T cell receptor genes, respectively. In some tissues, terminally differentiated cells are polyploid and some have lost their genome. In general, mechanisms leading to a stably inherited cellular1 phenotype without changes in the DNA sequence and amount are designated as “epigenetic.” Theoretically, a cancer cell phenotype could be achieved by mechanisms similar to those that determine normal differentiated states. Instead, in most cancers, epigenetic alterations interact with genetic alterations to establish the altered phenotype of cancer cells. In principle, three scenarios can be envisioned and are indeed observed to various degrees in individual tumor types. (1) Cancer may be driven primarily by genetic changes which employ epigenetic mechanisms to establish the altered phenotype The definition extends to organisms in an analogous manner. 1 

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in an analogous manner as a normal phenotype is established in normal cells. (2) Accidental (stably transmitted) epigenetic alterations— “epimutations”—cause the tumor phenotype, e.g., by arresting cells in a permanently proliferative, undifferentiated state. Rare cases of pediatric cancers appear in fact to be devoid of identifiable genetic changes and may be caused by epigenetic mechanisms only. More typically, epimutations may contribute in a manner analogous to single gene (genetic) mutations, e.g., by inactivating tumor suppressors. (3) Genetic changes inactivate, deregulate, or structurally alter epigenetic regulators to disturb overall epigenetic patterns. The definition of what is considered an epigenetic phenomenon has undergone some changes over time. In addition to cell differentiation during development and tissue homeostasis, genomic imprinting and X-chromosome inactivation are certainly prime examples of epigenetic phenomena in mammals. 2 In both cases, (essentially) identical homologous DNA sequences in the genome are differentially expressed in a stably inherited fashion. The current definition of epigenetics focuses on nuclear mechanisms that establish, maintain, and change chromatin structure and the organization of the genome in the nucleus (Box 8.1). During development and in tissue homeostasis, these mechanisms integrate signals from cell-autonomous programs and from the tissue microenvironment. Active genes and their regulatory regions are in general associated with an open chromatin conformation, whereas chromatin assumes a less accessible conformation at inactive genes. Constitutive heterochromatin represents the most condensed state in interphase cells. Epigenetic mechanisms interact with each other. They include DNA methylation at cytosine residues, a large variety of histone modifications, deposition of variant histones, transcription factor networks, chromatin remodeling, and several means for large-scale organization of chromatin domains in the nucleus (Table 8.1). Disturbances of each of these mechanisms are found in human cancers. Several chapters in Allis et  al. l.c. present fascinating examples of epigenetic phenomena in other species. 2 

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Box 8.1 A Broader Definition of Epigenetics

The concept of “epigenetics” was developed in order to explain how very different phenotypes of cells (and even entire organisms) can emerge from the same genome and can be stably propagated across cell divisions. The currently prevailing concept of epigenetics emphasizes cell-autonomous mechanisms within the nucleus. It is however possible to extend the concept to include mechanisms and phenomena beyond the nucleus and further beyond a single cell. Undoubtedly, gene expression within a cell is influenced by mechanisms acting outside the nucleus notably through posttranscriptional regulation by miRNAs and other non-coding RNAs. The issue is rather to which extent these mechanisms contribute to the stable inheritance of gene expression patterns across cell divisions. Similarly, cellular phenotypes are established and maintained in normal tissues and even in tumors by—often mutual— signaling with other cells. In this fashion, stem cells are maintained by cell-­ autonomous mechanisms but also by signals from other cell types and the extracellular matrix in the niche. Mutual paracrine cell-cell interactions, especially between mesenchymal and epithelial cells, are essential for tissue homeostasis, lineage choices, and wound healing. In cancers, non-coding RNAs have been demonstrated to contribute substantially to phenotypic changes like the epithelial-­ mesenchymal transition. Stromal cells have been demonstrated to acquire a kind of epigenetic memory. For instance, cancer-­ associated fibroblasts maintain their altered phenotype across several generations after being separated from carcinoma cells.

Hypoxia in carcinoma cells activates the proliferation of endothelial cells and hemangioblasts to influence their phenotype. Whether such phenomena in normal tissues and cancers are covered by the concept of epigenetics is evidently a matter of definition. Allis CD et al. (2015) Epigenetics. Cold Spring Harbor Press, 2nd edition.

Table 8.1  Selected epigenetic molecular mechanisms DNA methylation Histone acetylation Histone methylation(s) Histone variant deposition Chromatin remodeling Chromatin looping Transcription factor networks

Importantly, the epigenetic mechanisms establishing stably transmitted cellular states can also be applied to control other cellular processes, ranging from the basic organization of the nuclear chromatin into euchromatic and heterochromatic regions, DNA replication, mitosis, and DNA repair to the control of endogenous retroelements and invading viruses. It is therefore difficult to strictly distinguish between gene regulation and epigenetic inheritance as both make use of the same repertoire of molecular mechanisms. The defining characteristic is that epigenetic mechanisms are those that establish chromatin states that are stably transmitted to daughter cells. In this regard, dynamics is obviously crucial. Thus, DNA methylation is an example of a long-term acting mechanism, whereas histone acetylation and deacetylation are highly dynamic processes that occur in most genes. Nevertheless, not only DNA methyltransferases and demethylases but also histone acetyltransferases and histone deacetylases are required for epigenetic inheritance.

8.2  DNA Methylation

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8.2 DNA Methylation

unmethylated sites remain unmethylated, whereas methylated sites reacquire symmetrical methylaIn mammals, physiological methylation of DNA tion in both daughter strands. Its preference for is essentially restricted to the 5-position of cyto- hemimethylated DNA makes DNMT1 an ideal sine residues, and as a rule to cytosines in CpG-­ enzyme for stably propagating DNA methylation dinucleotides.3 Since CG is a palindromic states when cells proliferate. In keeping with its sequence, a CpG site can be non-methylated, function during replication, DNMT1 is tightly hemimethylated (i.e., in one strand only), or fully associated with the replisome, where it binds to methylated, symmetrically in both strands PCNA, and the enzyme is more strongly expressed (Fig. 8.1). during S phase and in proliferating cells. The default state of methylated CpG sites in Methylation at previously unmethylated sites, human DNA is symmetrical methylation. termed “de-novo-methylation” (Fig.  8.1), is usuReplication however creates hemimethylated sites, ally introduced by the de-novo-­methyltransferases because DNA polymerases insert unmodified cyti- DNMT3A or DNMT3B rather than DNMT1. dines. Very shortly following the duplication of Unlike DNMT1 they may act independent of DNA DNA, symmetrical methylation is reestablished by replication and outside S phase. As a rule, the maintenance DNA methyltransferase 1 DNMT3A methylates rather single loci, including (DNMT1); the methyl group is provided by gene bodies, whereas the highly processive SAM.  DNMT1 prefers hemimethylated over DNMT3B methylates heterochromatic sequences. unmethylated DNA by a large margin. Thus, Failure to reestablish fully methylated CpG sites during successive rounds of replication results in “passive” demethylation. Active demeth3  CpH methylation (methylation at cytosines followed by ylation—independent of replication—can be cataany base) is observed at some early developmental stages lyzed by the DNA demethylases TET1, TET2, or and especially in pluripotent cells.

MBD

Interpreting

Maintaining

M Maintenance Methylase DNMT1

CG GC

W

M

Replication Demethylase TET1-3

Changing

CG GC

Replication Demethylase

De-novoMethylase

TET1-3

DNMT3 CG GC

Fig. 8.1  Establishment and changes of methylation status at CpG sites. Hemimethylated DNA formed during DNA replication from symmetrically methylated DNA is reconverted by a maintenance methylase (normally DNMT1). Insufficient activity of the enzyme can lead to loss of methylation after two rounds of replication (passive demethylation). Unmethylated sites can be methylated by the successive action of de-novo-methylases

(normally, DNMT3A or DNMT3B) and the maintenance enzyme. All enzymes use S-adenosylmethionine (SAM) as the methyl group donor, converting it to S-adenosylhomocysteine (SAH). TET demethylases can actively remove methyl groups from methylcytosine (active demethylation). Methyl groups in DNA can be recognized among others by MBD-containing proteins

8  Cancer Epigenetics

GC content

182

Genome average

Gene A

0.5 - 2 kb

Gene B

Gene C

Normal methylation

Methylation in cancer

Fig. 8.2 CpG-islands in the human genome. Top: Schematic illustration of variations in GC content and distribution of CpG sites (stick and circle symbols) in the human genome. As customary, methylated sites are indi-

TET3. These dioxygenases use α-ketoglutarate as a co-substrate to oxidize the cytosine 5-methyl group to hydroxy-­methylcytosine; succinate and CO2 are the other products of the reaction. Hydroxy-methylcytosine may be relatively stable in some circumstances; it is enriched in postmitotic tissues such as the brain.4 Additional oxidation steps generate 5-­carboxycytosine, which is decarboxylated to restore unmethylated cytosine. Overall DNA methylation levels vary to limited extents among normal tissues, with more pronounced changes during germ cell and embryonic development. In typical somatic cells, 3.5– 4% of all cytosines are methylated. DNA methylation is however not equally distributed across the genome. Many methylcytosines are contained in repetitive sequences such as LINE and SINE retrotransposons which are interspersed in the genome and in CpG-rich satellite

cated by filled and unmethylated sites by open circles. Note that gene B does not possess a CpG-island, like up to 40% of human genes. Bottom: methylation patterns in normal and cancer cells. Gene C is hypermethylated

repeats concentrated in peri- and juxtacentromeric regions; most intergenic regions are also more strongly methylated. In addition to these transcriptionally rather inert regions, the transcribed parts of genes, gene bodies, are also relatively densely methylated. In contrast, regulatory regions of active genes are generally undermethylated. Specifically, in more than half of all human genes, 0.5–2 kb stretches around the transcriptional start site, including the basal promoter sequence, are richer in CpG-dinucleotides than the rest of the genome, with a frequency of >0.6 found/expected in a random sequence and a higher GC content than the rest of the genome (Fig.  8.2). These sequences are termed “CpG-islands.” Most CpG-islands remain unmethylated throughout development and in all tissues. A prominent exception is the CpG-islands of silenced genes on the inactive X chromosome which are mostly methylated (see Sect 8.4). Moreover, methylation of selected 4  Altered overall levels and patterns of hydroxy-­ methylcytosine can be indicative of cancers and may be CpG-islands helps to stabilize fundamental cell applied for diagnostics. fate decisions during development.

8.2  DNA Methylation

Apparently, the lack of methylation helps to mark CpG-islands as regions of potential transcription initiation in the genome. Typically, genes with CpG-island-type promoters can be transcribed in different cell types. DNA methylation also regulates the transcription of some gene promoters that do not comprise CpG-islands, including some with cell type-specific expression. Most of the differences in DNA methylation between individual cell types and tissues are however found at sequences abutting CpG-­ islands, creatively termed “CpG-shores,” and importantly, at enhancers. Both CpG-density and average DNA methylation levels at these sequences are typically intermediate between those of CpG-islands and intergenic regions in general. CpG-islands stand out from the rest of the genome by containing a higher proportion of CpG-dinucleotides. More precisely, the rest of the genome contains fewer CpGs, as a consequence of cytosine loss during the course of evolution. Hydrolytic deamination of cytosine occurs frequently, spontaneously or induced by chemicals, and yields uracil. Uracil in DNA is very efficiently recognized as incongruous and accordingly repaired (→3.1). In contrast, deamination of methylcytosine yields methyl-uracil, i.e., thymine, although in a G-T mismatch. Such mismatches are accordingly preferentially repaired towards GC by TDG (Thymidine-DNA glycosylase), with the help of the protein MBD4 which recognizes the methylcytosine in the opposite strand of the CpG palindrome (→3.1). In spite of this dedicated repair mechanism, over evolutionary periods, CpGs have become depleted from heavily methylated sequences by mutating to TpG (corresponding to CpA in the reverse strand). This depletion has not affected sequences that are exempt from methylation and in this fashion the process has sculpted CpG-­ islands out of the genome background. In fact, the mutation rate at methylated cytosines remains higher in extant genomes. Therefore, methylated

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CpGs are preferential sites of mutations in the human germline and in cancers. In cancers as well as in normal tissues, CpG>TpG mutations accrue with age. DNA methylation patterns undergo substantial changes during development. During germ cell development, DNA is first globally demethylated and then remethylated to yield distinctive patterns in oocytes and sperm. Differential methylation at imprinted genes (see Sect. 8.4) is also established during this period. Following fertilization, methylation again decreases across the genome, although some specific sites, e.g., in imprinted genes, are exempt. Extraembryonic tissues remain strongly demethylated, whereas in the cells of the actual fetus the genome is de-novo-methylated during gastrulation to an overall methylation level similar to that of somatic cells. Demethylation of genes expressed in a cell type-specific fashion and their regulatory regions then creates the DNA methylation patterns of the various cell types. Of note, CpG-­ islands are in general exempt from these changes and remain unmethylated throughout development. Likewise, methylation patterns of imprinted genes follow their own rules. These wide swings in overall methylation likely reflect the necessity to completely reprogram the transcription pattern of the genome, first during the development of germ cells and then again during embryonic development. A major reason why cloned embryos are often defective and generation of iPS (induced pluripotent stem) cells is inefficient appears to be a failure to achieve this reprogramming properly. Very low levels of DNA methylation in germ cells may moreover help to generate a chromatin state that facilitates recombination during meiosis. Given this background, it is not unexpected that the altered state of cancer cells is in general accompanied by alterations in DNA methylation. Basically, two types of alterations can be distinguished (Fig.  8.3), which can both occur in the

8  Cancer Epigenetics

184 Fig. 8.3  Alterations of DNA methylation in human cancers. See main text for more details

Normal methylation

Methylation in cancer

Repeats

Intergenic

same cell. In many cancers, overall DNA methylation levels are diminished by up to 70% compared to the corresponding normal cell type. This decrease is termed “global” or “genome-wide” “hypomethylation” and affects prominently, albeit not only the methylcytosine contained in repetitive sequences and gene bodies. In contrast, specific sites can become “hypermethylated.” Hypermethylation may occur in particular at CpG-islands that are unmethylated in all normal tissues. This property renders hypermethylated CpG-islands excellent markers for cancer diagnostics (→22.3). The extent of hypomethylation and the patterns of hypermethylation differ widely between cancers. In some cancers, only individual CpG-­ islands become hypermethylated, whereas hundreds are afflicted in others. This latter state is designated “CpG-island hypermethylated phenotype” (CIMP). A CIMP phenotype was originally defined in colorectal cancers (→13.3) but has also been detected in subsets of other cancers. Moreover, hypermethylation affects different genes in different cancers, although some genes are prone to hypermethylation in many cancer types (Table  8.2). Alterations of DNA methylation may also affect the expression of imprinted genes (see Sect. 8.4). In normal somatic cells, most of the genome is densely methylated, especially repetitive sequences. CpG-islands form a prominent class of sequences exempt from methylation. Intergenic regions and gene bodies are normally quite

Enhancer

CpG-island

Gene body

Table 8.2 A selection of hypermethylated genes in human cancers Gene APAF1 CDH1

Function Apoptosis Cell adhesion, modulation of WNT signaling CDKN1C Cell cycle regulation CDKN2A Cell cycle regulator CDKN2B Cell cycle regulator ESR2 Regulation of estrogen response GSTP1 Detoxification, cell protection Reversion of base alkylation in DNA MLH1 DNA mismatch repair RARB2 Retinoic acid signaling RASSF1A Hippo signaling RB1 Cell cycle regulator SFRP1 Modulation of WNT signaling SOCS1 Signal transduction VHL Regulation of response to hypoxia MGMT

Cancers with hypermethylation Selected cancers Many carcinomas

Selected carcinomas many Selected leukemias Breast cancer, others Prostate carcinoma and selected other carcinomas Glioblastoma, others Many carcinomas Many carcinomas Many carcinomas Retinoblastoma Colorectal cancers and other carcinomas Selected cancers Clear-cell renal cell carcinoma (one allele)

densely methylated. Enhancer methylation is typically intermediate, varying between cell types. Typical alterations in cancer involve hypomethylation of repetitive sequences, decreased methylation of gene bodies and intergenic

8.2  DNA Methylation

regions, changes at specific enhancers (up or down), and hypermethylation of up to several hundred CpG-islands. Hypermethylation of CpG-islands is almost invariably associated with stable silencing of the affected genes. Therefore, hypermethylation is a mechanism of gene inactivation in cancers and may inactivate tumor suppressor genes as efficiently as mutations and deletions. For instance, promoters in the CDKN2A locus are inactivated in a variety of human cancers. In almost every cancer type, mutation, deletion, as well as promoter hypermethylation are observed as mechanisms of inactivation, although their relative contributions vary. One important difference compared to mutation and deletion is however that hypermethylation is in principle reversible by DNA methylation inhibitors. Consider moreover that in cancers with hundreds of genes inactivated by hypermethylation, not each one is a tumor suppressor. By comparison, global hypomethylation would be expected to increase gene expression across the genome at large. There is indeed some evidence that hypomethylation causes inappropriate expression of certain sequences, especially retroelements and “cancer testis antigens,” i.e., genes with expression normally restricted to developing male germ cells. Moreover, global hypomethylation appears to be associated with enhanced chromosomal instability. The underlying mechanisms are not fully clear. Possibly, decreased methylation in pericentromeric repeats and interspersed repetitive sequences facilitates illegitimate recombination and chromosomal breakage. Such defects are also observed in a human hereditary syndrome caused by deleterious mutations in DNMT3B, which is especially important for methylation of repeat sequences. Altered DNA methylation patterns in cancer are in fact a combination of apparent and real changes. Tumor cells arise from a particular subpopulation of cells in a tissue. Their methylation patterns therefore resemble those of the respective cells of origin more than those of the entire tissue. For instance, different types of B cell lymphomas arise from B cells at different stages of maturation (→10.4). Their basic state of DNA

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methylation reflects that of the respective cells of origin rather than of B cells in general, least other lymphoid cells. This basic state is then overlaid with cancer-specific alterations. Several mechanisms may underlie CpGisland hypermethylation, to different extents in different cancers. (1) One category of genes is prone to hypermethylation because they are already expressed at low levels in the corresponding cell of origin. These genes are often marked by bivalent histone modifications, i.e., a combination of active histone marks like trimethylation at lysine 4 of histone 3 (H3K4me3) and the repressive h­istone mark H3K27me3, which is established by the polycomb complex 2 (PRC2). During carcinogenesis, these promoters become fully inactivated. In this process, H3K27me3 and polycomb factors occupying the promoter may be replaced by DNA hypermethylation. As genes affected in this fashion are often developmental regulators, this process may restrict cell fate and disturb proper differentiation. (2) In some cancers, an unusually large number of CpG-islands are hypermethylated. A “hypermethylator” phenotype is found, e.g., in certain subtypes of gliomas and AML. In these cancers, it is caused by a failure in DNA demethylation which protects CpG-­islands from gaining DNA methylation. In some cases, the failure results from the mutational or epigenetic inactivation of TET demethylases. In others, it is caused by mutations in Isocitrate dehydrogenases (IDH1 or IDH2) that lead to the production of the oncometabolite α-hydroxyglutarate. This metabolite inhibits TET DNA demethylases (as well as many histone demethylases) that require α-ketoglutarate as a co-substrate. (3) Cancer cells tend to overexpress DNA methyltransferases since DNMT1 expression and activity are regulated in parallel with DNA synthesis in normal cells. Increased proliferation is therefore associated with increased DNMT1 expression. In certain cancers, however expression and activity of DNA methyltransferases are enhanced through overactivity of the MAPK pathway. This overactivity may explain in particular the hypermethylator phenotype of colon cancers with activating mutations in KRAS and especially in BRAF.

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As for hypermethylation, there is probably no increase self-­renewal of precursor cells. These single explanation for global hypomethylation in mutations alter the specificity of DNMT3A so cancers. One generally important factor may be that genes maintaining stem cell properties are the availability of substrates for DNA methyla- not properly repressed, whereas other sites in the tion. Maintenance of correct levels and patterns genome are aberrantly methylated. of DNA methylation depends not only on DNA methyltransferases and chromatin regulators but also on the availability of the methyl group donor 8.3 Histone Modifications SAM. More precisely, the reactions catalyzed by and Regulation of Chromatin DNA methyltransferases and many other methylStructure transferases in the cell are influenced by the ratio of their substrate and product, SAM and DNA methylation often represents a final step in S-adenosylhomocysteine (SAH). SAM is synthe- a chain of events that leads to stable gene repressized from the essential amino acid methionine, sion (Fig. 8.4) and serves to “lock in” the state of which is recycled through several steps from a gene silenced by other mechanisms. To elaboSAH (Fig. 21.4). The efficiency of recycling is rate on the metaphor, the door seems already to also dependent on the supply of folic acid and have been locked by other mechanisms and DNA vitamin B12. In rapidly growing cells, any of these methylation acts as an additional bolt. Specific factors may become limiting and cause an overall combinations of histone modifications help to decrease in DNA methylation levels, which is maintain and mark repressed genes and other then perpetuated. Notably, methyl group metabo- specific chromatin states. Transcriptionally active lism is not only influenced by dietary factors but genes and active enhancers are characterized by it also varies among humans as a consequence of acetylation of histones H3 and H4 as well as by genetic polymorphisms in enzymes involved in methylation of H3 at Lys4 (H3K4). Transcriptional folate metabolism and the methyl cycle (Fig. start sites are in particular distinguished by H3K4 21.4). These prevalent polymorphisms may con- trimethylation (H3K4me3). Transcribed regions tribute to cancer predisposition in persons with (gene bodies) carry the H3K36me3 modification. folate- and methionine-deficient diets. Actively transcribed genes are moreover enriched Finally, a failure to change DNA methylation in certain variant histones, especially H3.3 and patterns appropriately during cell differentiation H2A.Z, but these variant histones are also may contribute to cancer development. For enriched at additional specific specialized regions instance, mutations in the de-novo-­of the genomes such as telomeres and pericentric methyltransferase DNMT3A found in acute repeats. The H3K27me3 modification is found myeloid leukemias are thought to impede differ- especially at genes and enhancers that are inacentiation towards mature myeloid cells and tive in a particular tissue or at a particular develFig. 8.4  Interaction of histone H3 modifications and DNA methylation in the establishment of heterochromatin. For simplicity, only some modifications of H3 and none at H4 are displayed. Some of many feedback and feed-forward interactions are indicated by arrows

KDM1 HDAC

H3K4me H3K9Ac

HP1 DNMT

KMT1

H3K4 H3K9

H3K4 H3K9me3

Heterochromatinization

H3K4 H3K9me3

8.3  Histone Modifications and Regulation of Chromatin Structure Fig. 8.5 Writers, readers, and erasers in histone acetylation. HATs introducing acetyl groups (yellow triangles) on histones can be considered writers, HDACs erasers, and proteins binding to acetylated histones via bromodomains readers, respectively

187

Reader

Writer

Eraser Bromo

HDAC

HAT

opmental stage. Promoters and enhancers carrying H3K27me3 together with H3K4me are considered bivalent. This state is frequent in undifferentiated and embryonic cells; once they develop along a particular lineage, it becomes resolved towards either an active or repressed state at most genes marked in this fashion. Histone modifications, and thereby chromatin states, are established, maintained, and removed by a variety of proteins and certain regulatory RNAs, which are collectively termed “chromatin regulators.” They interact with each other in feedback and feed-forward loops to enforce and stabilize specific chromatin states. Three different types of proteins are distinguished, namely “writers,” “erasers,” and “readers.” For DNA methylation, DNMTs would be considered writers, TET enzymes erasers and proteins binding specifically to methylated DNA, such as MBD2, readers, respectively. Histone acetylation at many different lysine residues in the four histones, especially in their N-terminal non-globular domains, is introduced by histone acetyltransferases (HAT) and removed by histone deacetylases (HDACs) (Fig.  8.5). Acetyl groups for HATs are provided as acetyl-S-­ CoA.  Many HATs are coactivator proteins that are recruited to active chromatin by DNA-binding transcriptional activators. Other HATs are primarily involved in other processes that require open chromatin, like DNA repair or DNA replication (with incorporation of new histones).

Nuclear HDACs are often components of repressor complexes. 5 Notably, acetylation can be highly dynamic and both types of enzymes may be active at the same gene. Acetylation opens up chromatin and increases the accessibility of DNA to various factors in general by decreasing the positive charge of histones. In addition, acetylated histones are recognized (“read”) by specific protein domains containing binding pockets for acetyl-lysine-containing peptides. Many chromatin proteins contain such bromodomains and PHD domains. They encompass many readers, but also many writers establishing active chromatin. This allows a feed-forward mechanism, whereby acetylation once established promotes further active modifications within one nucleosome and beyond. Whereas acetylation is generally associated with active chromatin, rather independently of the particular modified lysine residue, the functions of histone methylation are more diverse and depend on the residue and the number of methyl groups. Accordingly, a variety of histone methyltransferases establish histone methylation. In addition to lysine residues, arginine side chains can likewise be modified by (symmetrical or HATs and HDACs acetylate and deacetylase not only histones, but also many other proteins. The official HAT gene names recognize this by the designation KAT, for lysine (K) acetyltransferase. Among the 18 human HDACs moreover several are mostly cytosolic or are mitochondrial proteins. 5 

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asymmetrical) attachment of one or several methyl groups. A prominent characteristic of histone lysine methylases is the catalytic SET domain. Histone methyltransferases catalyzing lysine methylation are now systematically named as KMTs, with other names still in use. The KMT2A-D enzymes, e.g., are also known as MLL1-4. Together with other enzymes, they establish H3K4 methylation modifications at active enhancers (rather KMT2C/D) and genes (rather KMT2A/B). Trimethylation of H3K4 at active transcriptional start sites is catalyzed by KMT2A and SETD1B in particular. Additional KMTs methylate H4. KMT3 enzymes like NSD1 introduce H3K36me3 at the transcribed gene bodies. KMT1 enzymes like G9a generate H3K9me3, the characteristic modification of heterochromatin. H3K9me3 is recognized by HP1 proteins that attract DNMTs, which act as repressors and may additionally introduce DNA methylation. KMT1 enzymes recognize methylated DNA via MBD domains thereby reenforcing a repressed chromatin state. Histone methylation and acetylation are prominent regulators of transcriptional activity, while phosphorylation of histones at serine residues is rather involved in chromatin regulation during other nuclear processes. Thus, H3 phosphorylation is required for mitosis and is a marker of mitotic cells. It is accordingly catalyzed by cell cycle-regulated histone kinases and removed by likewise cell cycle-regulated phosphatases. Histone kinases like Aurora A are therefore upregulated in many highly proliferating cells and notably in tumor cells with high NMYC activity. Inhibitors of this enzyme and other mitotic histone kinases are tested as anticancer drugs. Following recognition of DNA double-­ strand breaks, the variant histone H2A.X, which is distributed across the genome, becomes phosphorylated by checkpoint kinases and serves as a mark for further repair. Of note, in experiments, phosphorylated H3 is useful to identify mitotic cells and phosphorylated H2A.X (also known as H2A.Xγ) to identify DNA double-strand breaks, respectively. Stable gene repression thus requires mutually enforcing processes and interactions between

8  Cancer Epigenetics

several involved proteins and in some cases regulatory RNAs, like during X-chromosome inactivation. DNA methylation, e.g., can be directed to specific DNA sequences by repressor proteins, including HP1, as well as by a heterochromatic chromatin state per se. Methylcytosine in DNA interferes with transcription in two ways. First, some transcriptional activators cannot recognize DNA, if CpG-­ dinucleotides in their binding site are methylated. Secondly, additional repressor proteins recognize methylated DNA. These include MeCP2, MBD2, and MBD3 which bind preferentially to methylated DNA.  They block access for other factors while recruiting chromatin remodeling complexes and repressor complexes, which contain HDACs that remove histone acetylation. This assembly of proteins then targets neighboring nucleosomes as well thereby spreading the repressed state. The important regulatory modification H3K27me3 is introduced by KMT6 histone methyltransferases, better known as EZH1 and EZH2, which usually act as components of the polycomb complex 2 (PRC2), although EZH2 has additional functions on its own (Box 8.2). Methyl modifications at H3K27, of course, preclude acetylation at this lysine. H3K27me3 is recognized also by a CBX subunit of a polycomb complex 1 (PRC1) that contains BMI1. The central catalytic subunit of PRC1, either RING1A or RING1B, then ubiquitinates H2A at K119 to further stabilize the repressed state. Notably, PRC1 and PRC2 may also act in the reverse order to repress genes. In that case, the composition of PRC1 differs, with CBX and BMI1 subunits not required. Intriguingly, PRC2 is repelled from actively transcribed genes by nascent RNAs, including non-coding RNAs transcribed in the opposite direction from the promoter of a protein-­coding gene. Conversely, specific lncRNAs direct PRC2 to specific genes or enhancers and bring the polycomb complex into the proximity of additional chromatin regulators. This guiding mechanism is best established for the XIST lncRNA that organizes X-chromosome inactivation (see next section) but also underlies the oncogenic effects of the HOTAIR lncRNA in some cancers.

8.3  Histone Modifications and Regulation of Chromatin Structure

Box 8.2 EZH2 Functions beyond PRC2

Together with SUZ12, EED, and RBBP4 (or RBBP7), EZH2 forms the core of the polycomb repressor complex (PRC2). As part of the complex, EZH2 methylates H3K27, catalyzing all three methyl group transfers from unmethylated H3K27 to yield the repressive modification H3K27me3. The other PRC2 core subunits mediate binding to nucleosomes and stimulate the otherwise relatively weak methyltransferase activity of EZH2. Accessory subunits like JARID2 modulate the activity of EZH2 as well and direct the PRC2 complex to specific sites in the genome. PRC2 activity within the genome is also directed by PRC1 (and vice versa) and certain lncRNAs. Conversely, dense DNA methylation as well as active transcription repel the PRC2 complex. In a feed-forward mechanism, EZH2 methylates JARID2 which in turn stimulates the activity of the PRC2 complex. Whereas JARID2 methylation by EZH2 takes place within the PRC2, EZH2 can also methylate lysine residues in a number of further proteins, especially transcriptional activators and cofactors, independently of PRC2 or in conjunction with other core subunits. Methylation may increase the activity or stability of the respective transcription factors, or, conversely, decrease their activity or promote their proteasomal degradation. For instance, methylation by EZH2 inactivates the transcriptional activator GATA4 but activates STAT3, whereas the orphan nuclear receptor RORα and the corepressor PLZF are marked for enhanced degradation. Mechanistically, methylation of lysine residues in transcription factors may interfere with acetylation, as in histones. A particularly interesting case is the interaction of EZH2 with the andro-

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gen receptor (AR). EZH2 is commonly upregulated in prostate cancers and in such cases contributes to the altered activity of the AR that is crucial for the development of this cancer type (→20.3). Interaction with the AR requires phosphorylation of EZH2 at a serine residue (Ser21) by AKT. Phosphorylated EZH2 then methylates and stabilizes the AR and further acts as a transcriptional coactivator for the nuclear receptor. The p38α MAPK phosphorylates EZH2 at a different residue (Thr372) stimulating the formation of a repressor complex with the DNA-binding (facultative) repressor protein YY1. Whereas the oncogenic activity of EZH2 in many cancer types results from its overexpression (in some cases due to gene amplification), EZH2 is activated to an oncogene by specific mutations, commonly at Tyr641, in specific cancers, especially B cell lymphomas. Inhibitors of EZH2 like tazemetostat are approved for the treatment of these cancers while being tested in a broader range of cancer types. Given the oncogenic functions of EZH2 in many cancer types, it may seem surprising that inactivation of EZH2 by deleterious mutations (truncations and point mutations, even biallelic) is observed in some myeloid malignancies. In these cancers, H3K37 methylation is decreased at PRC2 target genes, likely interfering with the proper differentiation of myeloid cells. Aubert Y et  al. (2019) The unexpected noncatalytic roles of histone modifiers in development and disease. Trends Genet 35:645–657 Rinke J et al. (2020) EZH2 in myeloid malignancies. Cells 9:1639 Park SH et  al. (2021) Going beyond polycomb: EZH2 functions in prostate cancer. Oncogene 40:5788–5798

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Histone methylation at lysine residues is reversed by KDM enzymes, each of which is likewise specific for one or several modified residues and states. Most of these enzymes contain a catalytic jumonji domain and (like TET DNA demethylases) use oxygen and α-ketoglutarate as co-substrates for oxidative demethylation reactions. As a flavin-dependent monoamine oxidase KMT1A (also known as LSD1) is one exception to the rule. The enzyme is likewise exceptional by its (context-dependent) specificity for both (activating) K4 and (repressive) K9 methylation. KDM6A, also known as UTX, removes di- and trimethylation from H3K27 thereby antagonizing EZH2 and repression by the PRC2 complex. In fact, the enzyme acts in concert with KMT2C or KMT2D methyltransferases and the histone acetyltransferases p300 (gene: EP300) or CBP (gene: CREBBP) to activate enhancers and genes. KMT2C or KMT2D associate with a core complex known as WARD (or WRAD) and KDM6A to form the COMPASS complex, which is directed by cell type-specific transcription factors to selected enhancers. The catalytic activity of the COMPASS components together with that of the associated HATs then establishes the characteristic histone modification pattern of active enhancers, with H3K4 mono-methylation and broad histone acetylation. Once established, this chromatin state allows access to additional transcription factors that further stabilize and spread the active state through their coactivators. During development and cell differentiation, the COMPASS complex often acts antagonistically to polycomb complexes; it is therefore also considered as trithorax-like, in reference to the antagonistic regulators known from Drosophila developmental biology. In addition to specific histone modifications, activation of promoters or enhancers from a repressed state requires chromatin remodeling, namely the repositioning and appropriate spacing of nucleosomes to enhance accessibility to transcription activators and—at the transcriptional start site—to RNA polymerase II and its basal cofactors. In particular, one or two nucleosomes are evicted from the transcriptional start site of many active genes to mark the initiation

8  Cancer Epigenetics

site and facilitate access of basal transcription factors and the Pol II holoenzyme. During development and cell differentiation, predominantly the SWI/SNF (switch/sucrose non-fermentable) protein complex supports gene activation in this manner. However, chromatin remodeling is also required for gene repression and every other process that involves opening or closing of chromatin, like DNA replication, repair, and recombination. In addition, newly synthesized nucleosomes need to be loaded onto DNA during replication and histone variants are substituted at specific regions in the genome (independent of replication). These processes are supported by different remodeling complexes named ISWI (imitation switch), CHD/NuRD (chromodomain helicase DNA-­ binding), and INO80. ISWI and CHD are particularly important for nucleosome assembly and organization following DNA replication, whereas INO80 primarily organizes the exchange of standard histones for variants. In summary, the transition from a repressed to an active gene state requires a combination of removing and establishing histone modifications and repositioning of nucleosomes and other chromatin proteins (Fig.  8.6). Importantly, this process is initiated and driven by DNA-binding transcription factors (or in some cases by regulatory RNAs). Transcriptional activators differ in their ability to bind to closed chromatin. Thus, pioneer factors will be able to initiate gene activation, recruiting coactivators and excluding corepressor complexes. This allows binding of additional transcription factors, coactivators, and chromatin remodeling until a fully active state is stably attained. Many proteins involved in chromatin modification are mutated or aberrantly expressed in human cancers (Table 8.3). In particular, components of the COMPASS complex and its accessory proteins are recurrently inactivated in a variety of cancers by truncating mutations, deletions, and missense mutations interfering with their catalytic function. Since the major function of this complex is the establishment of cell typespecific active enhancers, the consequence of these alterations is a change in the pattern of

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8.3  Histone Modifications and Regulation of Chromatin Structure

Fig. 8.6  Establishment of active enhancer states by the COMPASS complex. Activation of enhancers occurs in response to transcription factor binding (not shown) and involves changes in histone modification and nucleosome positioning by the coordinated action of the COMPASS

complex with KMT2C or KMT2D and KDM demethylases like KDM6A, HATs (often p300) and the SWI/SNF remodeling complex. For simplicity, only some modifications of H3 and none at H4 are displayed

Table 8.3  A selection of mutated chromatin regulators in human cancers Gene DNMT3A KMT2A KMT2B KMT2C KMT2D KDM6A CREBBP EP300 ARID1A ARID2 PBRM1 SMARCA4 SMARCB2 EZH2 NSD2 SETD2 BAP1

Function DNA methylation H3K4 methylation H3K4 methylation COMPASS COMPASS COMPASS HAT HAT SWI/SNF SWI/SNF SWI/SNF SWI/SNF SWI/SNF PRC2 H3K36me2 H3K36me3 H2AK119 deubiquitylation

Cancers with mutations AML Fusions in leukemia Various Various, bladder, renal Various, bladder, renal, lymphomas Various, bladder, renal, T-ALL Various, bladder Various, bladder Various, bladder Various, breast, Mostly renal cancer Various, lung AC Rhabdoid tumors, sarcomas Various ALL, multiple myeloma Brain, renal Renal

active enhancers in the tumor cells, which impedes differentiation or leads to an aberrantly differentiated state. Notably, the WRAD core components are very rarely affected by genetic changes, presumably because the core complex also associates with other proteins and is essential for cell function. Likewise, the genetic changes in the COMPASS components do not regularly lead to their complete (biallelic) inactivation. Thus, although their mutational pattern resembles that of tumor suppressors by the pre-

Oncogenic Oncogenic Inactivation Inactivation Inactivation Inactivation Inactivation Inactivation Inactivation Inactivation Inactivation Inactivation Inactivation Overexpression, mutation Overexpression, mutation Inactivation Inactivation

dominance of deleterious changes, epigenetic regulators appear to differ from classical tumor suppressors in that respect (cf. 5.4). Moreover, the COMPASS complex is required for the activity of many transcription activators, including steroid hormone receptors like the ERα and the AR. In cancers driven by such transcription factors, some activity of the complex may be essential. Mutations inactivating SWI/SNF components (especially in the ARID1A, ARID2, PBRM1, and

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192

SMARCA4 genes) are likewise observed in a large variety of human cancers, at a frequency estimated as up to 20% across all tumors. One specific component of the SWI/SNF remodeling complex, encoded by the SMARCB2 gene at chromosome 22q11, behaves as a tumor suppressor in rare pediatric cancers of the nervous system. These cancers are clearly caused by a failure in cell differentiation. Conversely, overexpression of specific chromatin regulators is also observed in many ­cancers; these include particularly HDAC1 and HDAC2 among the HDACs, specific HATs like GCN5 and some histone methyltransferases. Overexpression of the polycomb repressor protein EZH2 has been observed among others in prostate and breast cancers (→20.2), and oncogenic mutations in EZH2 have been observed in certain hematological cancers. These alterations may affect the activity of the PRC2 polycomb complex or affect gene regulation processes where EZH2 acts independently of PRC2  in combination with other factors (Box 8.2). The PRC1 protein BMI1 is also overexpressed in some cancers, prominently in acute leukemias arising from hematopoietic stem cells. Maintenance of the stem cell population in these cancers requires the repressive function of the PRC1 complex. In particular, the combined activity of PRC1 and PRC2, directed by a lncRNA, ANRIL, transcribed from a gene near the main locus, represses CDKN2A transcription. This mechanism prohibits the accumulation of p16INK4A in response to continuous proliferation of stem cells and cancer (stem) cells, that would otherwise induce replicative senescence (→7.2). In addition, loss of repressors can also contribute to cancer development. With regard to cancer, a prominent chromatin-regulating repressor protein is RB1 (→5.2) which directs deacetylation of promoters by binding to E2F factors and can elicit further remodeling at E2F-dependent promoters towards a permanently repressed chromatin state. RB1 may therefore cause more persistent and in some cases irreversible inactivation of such promoters, effectively establishing replicative senescence or enforcing terminal differentia-

tion. These relationships further explain why loss of RB1 function constitutes such a widespread alteration in cancer. Importantly, HATs, HDAC, and HMTs have all been found as parts of oncogenic fusion proteins in leukemias and lymphomas (→10). A common mechanism underlying the action of these fusion oncogenes may be misdirection of corepressor proteins as in, e.g., acute promyelocytic leukemia (→10.6). An unexpected addition to the large repertoire of genetic alterations affecting regulators of chromatin structure and activity are missense mutations in histones, predominantly in H3 and its major variant H3.3, but also less commonly in H2A, H2B, and H4, i.e., in all core histones. These mutations were initially discovered in rare entities like pediatric brain tumors, but have now been identified in many common cancers of adults as well. The best-understood alteration is the H3 K27M mutation. This amino acid exchange occurs at the target site of the EZH2 methyltransferase and consequently inhibits PRC2, resulting in a global decrease of H3K27 trimethylation and loss of transcriptional silencing. Other mutant histones are thought to act in analogous ways to prohibit specific modifications thereby inhibiting and redirecting chromatin-­ modifying and -remodeling complexes, resulting in global changes in chromatin structure and gene regulation that ultimately interfere with cell differentiation. Finally, like alterations in DNA methylation, changes in histone modifications in cancer may also be elicited or be aggravated by metabolic disturbances. Modification of histones relies on crucial substrates from intermediary metabolism like acetyl-CoA, SAM, and ATP and responds to changes in these metabolites.6 Most histone demethylases, like the TET DNA demethylases, require α-ketoglutarate and oxygen as co-­ substrates. Several histone demethylases are accordingly induced by hypoxia.

Even lactate can be used to modify histones; lactylation is actually employed to regulate cellular responses to enhanced glycolysis. 6 

8.4  Genomic Imprinting and X-Chromosome Inactivation

8.4 Genomic Imprinting and X-Chromosome Inactivation Genomic imprinting and the inactivation of the second X chromosome in females for dosage compensation are two profoundly epigenetic phenomena. In both cases, DNA sequences that do not (substantially) differ in sequence are strongly differentially expressed. Different epigenetic mechanisms, including DNA ­methylation, histone modification, and large-scale organization of chromatin cooperate in both processes. Most autosomal genes in humans are expressed equally strongly from both alleles. A minority is expressed in a monoallelic fashion. The expression of genomically imprinted genes in particular differs between the alleles inherited from the mother (“maternal”) and father (“paternal”). In humans, about 170 genes are subject to genomic imprinting. They often occur in clusters, i.e., several imprinted genes are located within one chromosomal region. Notably, each imprinted gene cluster contains a gene expressing a lncRNA which contributes to regulation of the cluster in cis but may have additional functions. Expression differences between the maternal and paternal alleles of individual imprinted genes may be ubiquitous or found only in selected tissues; they may be qualitative or quantitative. The most pronounced differences are found in fetal tissues, in the placenta, and in the brain. Imprinted genes influence particularly growth, metabolism, and Fig. 8.7  Regulation of the imprinted loci IGF2 and H19 A simplified illustration of the mechanism by which alternate activation of IGF2 and H19 is achieved at maternal (top) and paternal (bottom) alleles. E: enhancer. See main text for details

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neonatal behavior. A plausible explanation for the phenomenon is provided by the “battle of the sexes” hypothesis. It posits that genes preferentially expressed from the paternal allele promote growth of the fetus and the placenta thereby straining the mother’s resources. In contrast, genes expressed from the maternal allele tend to limit growth. Whether correct or not this hypothesis is helpful to memorize which genes are preferentially expressed from which allele. A well-studied example of imprinted genes is the mini-cluster comprising the IGF2 and H19 genes located near the tip of chromosome 11 at 11p15.5 (Fig. 8.7). They are imprinted in opposite ways. IGF2 encodes an insulin-like growth factor and is expressed from the paternally inherited allele. H19 is located telomeric to IGF2 and encodes a lncRNA that is expressed only from the maternal allele. Intriguingly, a miRNA gene is embedded in each gene, miR-675 in H19 and miR-483 in IGF2. H19 and IGF2 have their own promoters, but share an enhancer located telomeric to the H19 gene. On the paternal allele, the enhancer interacts with the IGF2 promoter; on the maternal allele it interacts with that of H19. The choice between them is imposed by a boundary element located in an intron of the IGF2 gene that constitutes the “imprinting center” of the cluster, commonly designated IC1. It can bind the chromatin protein CTCF that prohibits the interaction between enhancer and promoter across the boundary. More precisely, CTCF is the DNA-binding component of the cohesin complex

CTCF

E

E

E

E

H19

IGF2 IC1

ZFP57

IGF2

H19 IC1

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that organizes chromatin looping (in addition to the cohesion of chromatids during cell division). CTCF binding to the IC1 creates a shorter loop that contains only H19 and the enhancer(s), whereas lack of binding allows formation of a larger loop that includes IGF2 as well. This is a prime example of how differential chromatin loop formation can cause differences in gene expression. Differential loop formation is an important mechanism for cell type-specific gene expression, but also for the activation of oncogenes by overexpression via enhancer activation. Binding of CTCF is sensitive to methylation of cytosines within its recognition sequence. Methylation of the boundary element on the maternal allele therefore directs the enhancer towards the H19 promoter, diminishing expression of the IGF2 gene. Conversely, the CTCF binding site is unmethylated on the paternal allele, promoting expression of IGF2. The IC1 is further distinguished between the two alleles by differential histone modifications, namely H3K4 methylation on the maternal allele and H3K9me3 and H4K20me3 on the paternal allele. Moreover, CTCF binding to the maternal allele is supported by binding of further transcription factors, whereas inactivity of the IC1 on the paternal allele is supported by the zinc finger-containing repressor ZFP57. This elegant regulatory system is disturbed in many human cancers. Overexpression of the growth factor IGF2 is most frequent, due to expression from both alleles (or more, if gained) in the cancer cells. Expression of both alleles corresponds to a “loss of imprinting” (abbreviated as LOI). LOI can have several causes. In some pediatric cancers, notably in Wilms tumors (→11.3) and in germ cell cancers, imprinting may be lacking because it was never properly established during development. In some cancers of adults, the maternal allele is lost by deletion or recombination. Alternatively, imprinting may be disturbed by loss of DNA methylation at the boundary site or by altered expression of chromatin proteins regulating maintenance of the boundary. In some cases, the regulation of the twin locus is so profoundly disturbed that both IGF2 and H19 become overexpressed. The issue is fur-

ther complicated by differential use of the four promoters in the IGF2 locus. In cancers, the P3 and P4 promoters are used preferentially, other than in adult normal tissues. In addition to the IGF2 locus, several other imprinted loci affect signaling through IGFs via the PI3K pathway (see Sect. 6.3) and thereby regulation of growth and metabolism, among them the INPP4B locus which encodes a phosphatidylinositol phosphatase. In mice, albeit not humans, the decoy receptor IGF2R, too, is expressed from an imprinted gene. The imprinted CDKN1C locus encodes a direct regulator of cell proliferation, the CDK inhibitor p57KIP2 (Box 8.3). In accord with the general rule, as a growth inhibitor, it is expressed from the maternal allele, albeit in a tissue-­specific fashion. The CDKN1C gene (Fig. 8.8) is likewise located on chromosome 11p15.5, centromeric of IGF2/H19, at a distance, in a separate, larger cluster of imprinted genes (Fig. 11.4). Its imprinting is regulated by the distinct “imprinting center” IC2, which is located in an intron of the KCNQ1 gene at the center of the cluster. Like the boundary element in the IGF2/H19 locus, IC2 is differentially methylated on maternal and paternal alleles. As at IC1, differential histone modifications and ZFP57 binding accompany the different epigenetic states. IC2 harbors a promoter from which a lncRNA (KCNQ1OT1, or more conveniently LIT1) is transcribed in opposite orientation to the KCNQ1 mRNA. The LIT1 lncRNA induces repression of CDKN1C and other loci in the cluster by mechanisms that are presently not fully understood. In any case, disruption of the physical proximity between the imprinting center and CDKN1C by translocations leads to LOI. Such translocations are one cause of the human Beckwith-Wiedemann syndrome 7 (→11.2), which is characterized by fetal overgrowth and a propensity to childhood tumors such as nephroblastoma and hepatoblastoma. Variants of the syndrome are caused by loss-of-­ function mutations in CDKN1C or by failure of The converse defect, i.e., LOI towards the maternal allele, leads to the opposite phenotype in the RussellSilver syndrome. 7 

8.4  Genomic Imprinting and X-Chromosome Inactivation

Box 8.3 The p57KIP2 Cell Cycle Inhibitor

The relatively compact CDKN1C gene encompasses four exons within 2 kb and encodes the p57KIP2 cell cycle inhibitor. The p57 protein is considerably bigger than the other CIP/KIP cell cycle inhibitors, p21CIP1 and p27KIP1, because it comprises two further domains in addition to its N-terminal CDK inhibitor domain. The central, polymorphic PAPA domain consists of proline-­ alanine repeats. The QT domain near the carboxy-terminus contains the NLS and sites for phosphorylation by CDK2 that initiate polyubiquitination by SKP2 (as for p27KIP1) and proteasomal degradation. The p57KIP2 CDK inhibitor domain can inhibit several CDKs, including CDK2 complexed with Cyclin E or Cyclin A. Like p21CIP1 and p27KIP1, p57 promotes the assembly of active CDK4/Cyclin D complexes. The N-terminal domain moreover allows binding to certain transcription factors, including MYOD. By stabilizing MYOD and enhancing its transcriptional activity as well as by arresting the cell cycle, p57 supports myocyte differentiation. Interactions of p57 with other transcription factors like MYB and the RNA Pol II co-factors CDK7 and CDK9 however rather inhibit their activity. The PAPA domain is not found in any other CDK inhibitor and is presumably responsible for unique functions of p57. It interacts in particular with LIM kinase 1, which relays effects of RHOA on the actin cytoskeleton. The QT domain can bind PCNA and interact with JNK1. Binding to PCNA inhibits cell proliferation, and binding to JNK1 contributes to inhibition of apoptosis. Moreover, cell cycle arrest following phosphorylation of p57 by p38MAPK protects against apoptosis. Both cell cycle arrest and inhibition of apoptosis by p57 are thus context-dependent. Compared to p21CIP1 and p27KIP1, expression of p57KIP2 is more tissue-specific.

195

Accordingly, p57 promotes differentiation not only in skeletal muscle, but also in several other tissues by interactions with tissue-­ specific transcription factors. Notably, not only lack of p57 can endanger proper differentiation. Too high levels of the protein (as in Silver-Russell syndrome caused by loss of imprinting at the maternal allele) may deplete precursor cells. Precocious activation of p57KIP2 is therefore suppressed in precursor cells, e.g., through HES repressor proteins acting downstream of Notch signaling. In some stem cells, e.g., in the hematopoietic system, p57KIP2 expression helps to maintain quiescence. Creff J, Besson A (2020) Functional versatility of the CDK inhibitor p57Kip2. Front Cell Dev Biol 8:584590

imprinting across the 11p15.5 domain. Downregulation of p57KIP2 is also observed in several cancer types of adults and may be accompanied by hypermethylation of the CDKN1C promoter. Inactivating mutations in CDKN1C are however rare. In addition to IGF2, H19, and CDKN1C, several other imprinted genes are frequently deregulated in cancers, including PLAGL1 (encoding a transcription factor also known as ZAC1) and two oppositely imprinted genes in a large cluster of imprinted genes at 14q32, DLK1 (encoding an inhibitory NOTCH ligand) and MEG3 (encoding a regulatory lncRNA). In fact, disturbances in one of these genes may influence the expression of others, since they are linked in a regulatory network termed the “imprinted gene network” (IGN). Notably, besides ZAC1, the lncRNAs H19 and MEG3 contribute substantially to the regulation within the IGN.  In addition, several miRNA loci located in imprinted gene clusters or—like miR-675  in the case of H19—within imprinted genes may participate in the IGN through posttranscriptional regulation. Mechanisms very similar to those responsible for genomic imprinting are employed in X-chromosome inactivation in females. As in

8  Cancer Epigenetics

196 Fig. 8.8  Regulation of the imprinted gene CDKN1C Mechanism of the regulation of CDKN1C imprinting, which are largely modeled on the better-understood mechanisms at the IGF2/H19 loci (cf. Fig. 8.7). Imprinting regulation is overlaid with tissue-specific regulation by unknown elements.

ZFP57

CDKN1C

KCNQ1

IC2

LITI

CDKN1C

KCNQ1

other mammals, the second X chromosome in inactivated and its promoter is methylated on the human females is largely inactivated and hetero- active X chromosome of males and females. 8 chromatic, except for the small “pseudo-­ Following the establishment of differential chroautosomal” region which is homologous to a matin states in the early embryo, the activity stretch of the Y chromosome and a (significant) states of the X chromosomes are then faithfully number of genes that are exempt from inactiva- maintained through many cell generations in tion. In humans, the choice of the X chromosome somatic cells. subject to inactivation is entirely random, even in In cancer research, X-chromosome inactivaextra-fetal tissues. Inactivation sets in during gas- tion was traditionally used to investigate whether trulation and is initiated by the increased expres- cancers (in females) are monoclonal or polysion of the non-coding XIST RNA from the X clonal based on the argument that if a cancer coninactivation center on the chromosome destined tains cells from only one clone, then the same X for inactivation. This increase is initially achieved chromosome should always be inactivated. predominantly by posttranscriptional stabiliza- Historically, enzyme variants such as the isotion and leads to coating of the chromosome with zymes A and B of glucose-6-phosphate dehydroXIST lncRNA. Chromatin is remodeled and his- genase were investigated or more recently, tones are hypoacetylated and hypermethylated at polymorphisms in genes located on the X chroH3K9 and H4K20. Heterochromatin proteins mosome, like those in the androgen receptor gene including HP1 bind and DNA methyltransferases (AR) at Xq12. This type of analysis has become methylate the promoter regions of many inacti- obsolete through the use of microsatellite analyvated genes to lock in the inactive state. In addi- ses, next-generation sequencing, and most tion to the histone modifications typically found recently, single-cell analysis sequencing in heterochromatin, trimethylation at H3K27 and techniques. ubiquitination at H2AK119 are observed at many Intriguingly, a disproportionate number of silenced genes; these modifications indicate that genes classified as tumor suppressors are located PRC2 and PRC1, respectively, contribute to on the X chromosome. Not all of these are subject X-chromosome inactivation. At a late step of inactivation, the histone variant macroH2A accu- 8  Deletion of the XIST locus is lethal in embryos and spemulates on the X chromosome, maintained by the cific deletion in hematopoietic cells causes hematological XIST RNA. Conversely, the XIST gene becomes cancers in mice.

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8.5  Epigenetics of Cell Differentiation

to inactivation on both X chromosomes in females. For instance, the KDM6A locus encoding a H3K27 demethylase (also known as UTX for the location of the gene on the X chromosome) is expressed from both X chromosomes in females. Males express a paralogous gene, UTY, located on the Y chromosome, in addition to UTX from the single X chromosome. The histone demethylase activity of UTY is however weak and UTY cannot substitute for UTX in all respects. Mutations inactivating a single KDM6A allele in females therefore have a less severe effect than in males. Complete inactivation of KDM6A in females requires deletion or mutation of the second allele as well, which is observed in many cancers. Interestingly, though, loss of chromosome Y and deleterious mutations in UTY are also observed and quite frequent in some cancer types. Nevertheless, the higher dosage of tumor suppressors in females may account, at least partly, for pronounced differences in the risk for certain cancers between the sexes. A case in point is urinary bladder cancer with an ≈3-fold higher incidence in males and a high frequency of mutations in KDM6A/UTY. Conversely, despite the prominence of tumor suppressors on the X chromosome, many cancers contain supernumerary X chromosomes in their aneuploid genomes. These may contribute to the stability of tumor cell metabolism and to survival overall, but also provide additional copies of specific genes. For instance, X-chromosome gain and amplification of the Xq12 region containing the AR gene contribute to resistance against anti-­ androgenic therapies in prostate cancer (→20.3). Furthermore, some cancers in males may be detected by the presence of transcriptionally active, unmethylated XIST sequences, and increased XIST levels in serum DNA (→22.3).

8.5 Epigenetics of Cell Differentiation Cell differentiation is directed and controlled by cell type-specific transcription factors, which interact with various chromatin regulators to establish the respective epigenetic states of tissue stem cells, precursor populations, and (terminally) differentiated cells. During differentiation, these

transcription factors are often organized in cascades, where one factor elicits the activation of successive others, or in networks, where several cell type-specific factors interact among themselves and with additional transcriptional activators and repressors to organize the expression of cell type-specific functional genes, but also control the expression of the other factors in the network. In appropriate experimental models and at appropriate stages of development, the expression of a single transcription factor (or a few) may be sufficient to initiate a differentiation cascade. One well-studied example is myoblast differentiation (Fig.  8.9). It is initiated and carried out by muscle-specific transcription factors (MSTFs or MRFs) that, like the MYC proteins (→4.5), belong to the basic helix-loop-helix family (bHLH). Accordingly, they bind to specific DNA sequences called E-boxes. E-boxes are present in genes encoding the typical proteins of muscle cells, but also in the enhancers of the genes encoding the MRFs. In appropriate cells, like myocyte precursors, but also fibroblasts, expression of the MYOD transcription factor beyond a threshold starts an autocatalytic cascade, in which several MRFs become expressed at increasing levels until full differentiation is achieved. Notably, myoblast differentiation is associated with proliferation arrest. To achieve terminal differentiation, the MRFs must compete with and overcome the effects of transcription factors like MYC that maintain cell proliferation. This competition is further modulated by specialized inhibitor proteins, called ID (inhibitors of differentiation, also inhibitors of DNA-­ binding). These small proteins belong to the same general class of proteins as MRFs and MYC proteins but lack a DNA-binding domain. Rather, they heterodimerize with and block the action of cell type-specific bHLH transcription factors. Overexpression of ID proteins, sometimes as a consequence of gene amplification, is not uncommon in human cancers, particularly in carcinomas. 9

There are four ID proteins, ID1–ID4. ID4 differs from the other three and may actually antagonize their function in some situations. 9 

8  Cancer Epigenetics

198 Intermediate differentiation stages

Myoblast

Myocyte

ID MYC

ID MYOD MYOD MYC

MTF

MYOD

E–Box

E–Box

MYC

MTF

E–Box

E–Box

Fig. 8.9  A transcription factor cascade during cell differentiation. A highly simplified depiction of the interaction of pro-proliferative (MYC, IDs) and cell type-specific (MYOD, MTF) basic helix-loop-­helix proteins during the

MYOD furthermore represses the transcription of the AP1 factors FOS and JUN in differentiated cells. In addition, RB1 is activated during muscle differentiation and permanently inactivates E2F-dependent promoters required for cell proliferation thereby supporting the effects of MRFs to arrest the cell cycle. Similar transcription factor networks act in the differentiation of other cell types. For instance, in the differentiation of hepatocytes in the liver, HNF1, FOXA2, HNF4, HNF6, and LRH1 transcriptional activators are organized in a mutually activating network establishing and stabilizing the differentiated phenotype. A particularly well-studied network comprises the transcriptional activators OCT4 (POU5F1), SOX2, and KLF4. Introduction of these factors into many different cell types can reprogram them to a pluripotent state; the resulting cells are known as iPSC (induced pluripotent stem cells). In addition to the three, MYC is

Proliferation genes

Musclespecific genes

terminal differentiation of myoblasts. The (likewise simplified) autocatalytic loop between MYOD and other myoblast-­specific transcription factors (MTF) at the center of the figure makes the process irreversible

OCT4 NANOG

KLF4

SOX2

Fig. 8.10  The pluripotency transcription factor network. See text for details

required to stimulate proliferation and enhance chromatin plasticity. In iPSC, as in normal pluripotent embryonic cells, OCT4, SOX2, and another transcription factor, NANOG, form a core network, where the three core factors positively regulate each other’s expression (Fig. 8.10). They interact physically with each other at the promoters of many other genes to stabilize and

8.5  Epigenetics of Cell Differentiation

maintain the pluripotent state. KLF4 activates its own transcription and further that of the three core factors, especially during the generation of iPSC. Conversely, exit from the pluripotent state, i.e., differentiation towards somatic cell lineages, requires downregulation of the three core factors. In the process, their promoters acquire repressive histone modifications and dense DNA methylation. The maintenance of the undifferentiated, pluripotent state requires in addition repression of genes encoding transcription factors that determine specific lineages or germ layers. This is accomplished by polycomb-mediated repression and histone modifications. Notably, reprogramming to iPSC is normally inefficient, because it requires large-scale opening of chromatin to the more open and plastic state typical of early embryonic cells. Intriguingly, reprogramming is supported by MYC but antagonized by TP53 (Fig. 8.10). Each of the “stem cell factors” OCT4 (POU5F1), SOX2, NANOG, and KLF4 is expressed in specific somatic cells as well. Likewise, increased expression of the individual factors is observed in various cancers. The regulation and function of the core pluripotency factors differ however in somatic cells. For instance, SOX2 is strongly expressed in the basal cells of many epithelia and accordingly in carcinomas with a basal cell phenotype, but also in cancers with a neuroendocrine phenotype like small cell lung cancer. KLF4 likewise supports oncogenesis in specific cancers, while impeding tumor development in others. In the gut and skin, KLF4 actually supports differentiation but also contributes to stem cell maintenance. OCT4 expression has been reported in many cancer types and postulated to sustain cancer stem cells. However, the levels of OCT4 expression in cancer are often minute compared to embryonic cells and the interpretation of the observations is complicated by splice variants and pseudogenes. The complete network is however only established in pluripotent cells and in cancers resembling early embryonic cells like embryonal carcinoma. Cell type-specific gene expression patterns are established through transcription factors that bind to gene promoters and especially enhancers.

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At both proximal and distal regulatory sequences, proper chromatin states must be established and maintained. This is achieved by interactions of the transcription factors with various chromatin regulators. At promoters, transcription factors activate transcription through coactivators which stimulate Pol II but also modify chromatin, especially through acetylation. At enhancers, interaction with COMPASS-like complexes and histone acetyltransferases establishes an active state. At both regions, chromatin remodeling, e.g., by SWI/SNF complexes, is required. Opening of chromatin facilitates access of the basic transcription machinery, RNA polymerase II and its cofactors, but also of additional transcriptional activators at promoters and enhancers. Many transcription factors can only access enhancers after pioneer factors have initiated chromatin remodeling. For instance, the pioneer factor FOXA1 provides access to steroid hormone receptors like the ERα and the AR in this manner (cf. 19.4). Maintenance of the active enhancer state is then supported by Pol II-mediated transcription that generates (short) non-coding RNAs termed eRNAs. Finally, cell differentiation requires changes in chromatin looping to adapt enhancer–promoter interactions to the respective cell type. In addition to the activation of specific enhancers and thereby their interacting genes, cell differentiation requires conversely the (permanent) suppression of alternative cell fates. Lineage choices during development are often fixed by DNA methylation and repressive histone modification at genes encoding transcription factors that would direct cells to other lineages. For instance, in the differentiation of hematopoietic stem cells, DNMT3A is thought to contribute to the choice of myeloid over erythroid cell fate by methylating erythroid-specific genes; in addition, it contributes to diminishing the self-renewal ability of differentiating hematopoietic precursor cells. Overall, however changes of DNA methylation during differentiation of tissue precursor cells are minor in extent compared to those that take place during early development. Rather than DNA methylation, in normal tissues, histone modifications and chromatin remodeling are

200

employed to inactivate genes involved in precursor cell maintenance and in the differentiation towards alternate cell types. Notably, bivalent chromatin states at many promoters and enhancers are usually resolved in the course of lineage choices and cell differentiation. The resolution of bivalent states likewise requires not only the removal of the repressive H3K27me3 modification from the activated genes but also the removal of the active H3K4me3 (or H3K4me) modification by the respective histone demethylases from genes that become silenced. Polycomb repressors regulate the balance between precursor cell maintenance and further differentiation as well as choices between cell lineages. Thus, PRC2 is required to maintain the basal differentiation state of epidermal precursor cells and its activity must be overcome during differentiation by the (trithorax-like) action of transcription factors interacting with COMPASS and SWI/SNF complexes. In particular, PRC2 prevents AP1 factors from binding to a gene cluster on chromosome 1q21 named the epidermal differentiation complex (EDC) that contains a large number of genes expressed specifically in differentiated keratinocytes. Differentiation towards a specifically epidermal phenotype however requires PRC1 activity to suppress alternative fates. Similarly, the PRC2 complex, with EZH1 rather than EZH2 in this case, maintains hematopoietic stem cells (HSC), preventing senescence and differentiation, and preserving multipotency. A PRC1 complex with BMI1 and CBX7 maintains HSC, whereas PRC1 complexes with a different composition later support differentiation into the various cell types of the hematopoietic lineage. In summary, therefore polycomb complexes tend to maintain stem and precursor phenotypes and to inhibit differentiation, whereas trithorax-­ like complexes like COMPASS are definitely required for differentiation. However, this dichotomy is simplistic, as in fact both types of chromatin regulators are required for normal tissue homeostasis to strike a balance between (proliferating) precursor cells and differentiation to the (diverse) cell types. For instance, PRC2 prevents premature epidermal differentiation, but the basal cell phenotype of keratinocyte precursors is con-

8  Cancer Epigenetics

trolled by the transcription factor TP63  in ­conjunction with the COMPASS methyltransferase KMT2D. Specific and global alterations in enhancer activity and enhancer-gene interactions establish and maintain cancer cell phenotypes. These alterations can be caused by a variety of mechanisms which include (but are not restricted to) the following. (1) Mutations in specific transcription factors, especially pioneer factors like FOXA1, or loss of COMPASS or SWI/SNF components through mutations (see Sect. 8.3 and Table  8.3) can underlie blocked or aberrant differentiation. (2) Activation of oncogenes like MYC can be caused by structural chromosome alterations that place strong enhancers in the vicinity of the oncogene, but also by reorganization of enhancers that create super-enhancers leading to overexpression of the oncogene. (3) Point mutations in enhancer sequences may contribute to cancer development. For instance, in acute lymphoid leukemia (ALL), a point mutation in an enhancer sequence was found to generate a new binding site for the MYB transcription factor. MYB binding increases enhancer activity that induces overexpression of TAL1 to an oncogenic transcription factor. (4) Mutations at boundary sites may alter CTCF binding and accordingly chromatin looping and enhancer-gene interactions. Mutations in components of the cohesin complex, which affect especially frequently STAG2, may have similar consequences, but in a more genome-wide manner. A final example of epigenetic mechanisms that is relevant to both normal tissues and cancer concerns stem cells. Stem cells are in general defined as cells with unlimited proliferation potential, self-renewal ability as well as the ability to generate differentiated derivatives, usually with various phenotypes, and the ability to accomplish both tasks by asymmetric divisions that generate another stem cell and a more differentiated daughter cell (Fig.  8.11). Stem cells which can give rise to any somatic cell type are called pluripotent; cells in the early embryo that can additionally generate germline cells are termed totipotent. In adult humans, two distinct types of stem cells are present, namely germline

201

8.5  Epigenetics of Cell Differentiation Fig. 8.11  Properties of stem cells. Stem cells are characterized by the ability to self-renew, divide asymmetrically, and give rise to diverse differentiated cell types. Depending on the tissue, stem cells may divide frequently or rarely. Precursor cell populations (as a transit-amplifying fraction) often make the greatest contribution to the expansion of cell numbers

Differentiated cell types Stem cell

Precursors

and tissue stem cells. Tissue stem cells are not pluripotent but can give rise to a limited number of diverse cell types. They are therefore considered as “multi- or oligopotent.” As the DNA of tissue stem cells does not differ from that in other somatic cells, the stem cell state must be defined by epigenetic mechanisms. Actually, cell-autonomous epigenetic mechanisms that maintain the stem cell state are usually complemented by paracrine signals from other cells and emanating from cell adhesion in stem cell niches. For instance, stem cells at the bottom of the intestinal crypts require attachment to the basement membrane and WNT signals from neighboring cells and mesenchymal cells in the underlying tissue layer. In the intestine, as in other tissues, juxtacrine signaling through the Notch pathway supports the choice between stem cells and their progeny. Stem cells may be largely quiescent and the expansion of the cell population may be carried out largely by precursor cells at early differentiation stages, transit 10 amplifying cells. Other tissue stem cells proliferate continuously. Tissues may contain both types of stem cells, where the quiescent population may replete the proliferating stem cell pool when required. Notably, regulation of the stem cell pool size can also be “ transient” is also in use.

10 

achieved by increased self-renewal compared to asymmetric divisions and lacking tissue stem cells may be replaced by precursor cells that revert to a stem cell state. In this manner, the number of stem cells in normal tissues is maintained within a narrow range. Intracellular mechanisms that maintain stem cells include active transcription of TERT and expression of active Telomerase, which allows the escape from replicative senescence (→7.4). Telomerase expression can be stimulated by WNT or SHH-dependent pathways via MYC. Conversely, mechanisms inducing replicative senescence upon continued cell proliferation by induction of CDK inhibitors (→7.2) are repressed. Specifically, accumulation of p16INK4A appears to be prevented by polycomb repressor complexes, especially PRC1 with BMI1 as its crucial component, with the help of the lncRNA ANRIL. In quiescent stem cells, both RB1 and TP53 may contribute to limiting proliferation in addition to the CIP/KIP inhibitors p27 and p57. Cancers contain at least a fraction of cells with a stem cell phenotype. In some cancers, a differentiation hierarchy can be clearly discerned, resembling that in the tissue of origin and encompassing cancer stem cells at its apex. These cancer stem cells can generate entire tumors in appropriate experimental models, e.g., following transplantation or in cell culture, whereas more

8  Cancer Epigenetics

202

differentiated cancer cells cannot or at least can much less efficiently. In other cancers, it is difficult to detect specific cancer stem cells. This may be due to biological factors, e.g., because the hierarchy in the tumor is flatter or due to technical problems because appropriate experimental models are lacking. Notably, even in normal tissues, the stem cell phenotype is “plastic” in the sense that precursor cells (usually at early stages of differentiation) may replete the stem cell population if required. In cancers, plasticity is often more pronounced so that many cells in a tumor can acquire a stem cell phenotype under appropriate conditions. Several mechanisms may contribute to the generation of cancer stem cells (Fig.  8.12). (1) Some cancers are evidently generated by transformed tissue stem cells. Chronic myelocytic leukemia (CML) is clearly a stem cell disease of this kind (→10.5). In cancers of this type, increased self-renewal of the stem cells or of differentiated derivates, due to impeded or blocked differentiation, causes the increased cell numbers. Notably, the actual carcinogenic event in such cancers may have occurred in the actual stem cells or in precursor cells that have subsequently dedifferentiated back to the stem cell stage. (2) In several

Stem cell

Precursors

Fig. 8.12  Relationship of cancer cells to stem cells. Cancer (stem) cells can be derived directly from stem cells retaining their characteristics, but with at least partially blocked differentiation (left), from transit amplifying precursor cells which do not terminally differentiate

cancers signal transduction pathways involved in the maintenance of tissue stem cells are constitutively activated by mutations in one of their components. This overactivity allows the transformed stem cells or their derivatives to proliferate (relatively) independent of niche signals and outside of the stem cell niche. This mechanism is likely at work in colorectal cancers (→13.4) and basal cell carcinoma of the skin (→12.3). (3) Some cancers may need to acquire the relevant stem cell properties secondarily, most importantly the ability to continuously proliferate and self-renew. This may be achieved by activation of Telomerase through TERT mutations, loss of CDKN2A or epigenetic alterations that prohibit the expression of p16INK4A, and further inactivation of RB1 and TP53. While cells in such cancers may express markers of tissue-specific differentiation, they do not (or rarely) terminally differentiate. Therefore, genetic or epigenetic alterations may maintain such cancers in a precursor-like or intermediately differentiated state. Self-renewal ability may thus result simply from the inability to generate differentiated progeny during cell division. (4) A tumor stem cell phenotype may be conferred by placing cells into a specific environment through purely epigenetic mechanisms. Primordial germ

Differentiated cells

and/or secondarily acquire a stem cell phenotype (center), or from differentiated cell types that fail to turn off proliferation and/or revert to a less differentiated precursor stage (right)

Further Reading

cells reside in an environment that allows their maintenance and organized differentiation towards mature oocytes and spermatozoa. Stem cells in the epithelia of the seminiferous tubules of the testes (spermatogonia) divide asymmetrically to give rise to spermatozoa through several differentiation steps during which meiosis takes place. Their location within the testicular epithelia is crucial for the survival of these cells, as they tend to undergo apoptosis outside this environment. Primordial germ cells that do not reach this niche during fetal development can however develop into tumors at extragonadal sites. This type of tumorigenesis can be experimentally modeled in mice. The ensuing experimental teratocarcinomas appear to arise by purely epigenetic mechanisms.

Further Reading Allis CD et al (2015) Epigenetics, 2nd edn. Cold Spring Harbor Press Audia JE, Campbell RM (2016) Histone modifications and cancer. Cold Spring Harb Perspect Biol. 8:a019521 Baylin SB, Jones PA (2016) Epigenetic determinants of cancer. Cold Spring Harb Perspect Biol. 8:a019505 Brockdorff N et  al (2020) Progress toward understanding chromosome silencing by Xist RNA. Genes Dev 34:733–744 Buschbeck M, Hake SB (2017) Variants of core histones and their roles in cell fate decisions, development and cancer. Nat Rev Mol Cell Biol. 18m:299–314 Carlberg C, Molnár F (2018) Human epigenomics. Springer Cavalli G, Heard E (2019) Advances in epigenetics link genetics to the environment and disease. Nature 571:489–499 Cenik BK, Shilatifard A (2021) COMPASS and SWI/SNF complexes in development and disease. Nat Rev Genet 22:38–58 Centore RC et al (2020) Mammalian SWI/SNF chromatin remodeling complexes: emerging mechanisms and therapeutic strategies. Trends Genet. 39:936–950 Challen GA et al (2012) Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet 44:23–31 Chase A, Cross NCP (2011) Aberrations of EZH2 in cancer. Clin Cancer Res 17:2613–2618 Chen T, Dent SY (2014) Chromatin modifiers and remodellers: regulators of cellular differentiation. Nat Rev Genet 15:93–106 Costa RH et al (2003) Transcription factors in liver development, differentiation, and regeneration. Hepatology 38:1331–1347

203 Dawson MA (2017) The cancer epigenome: concepts, challenges, and therapeutic opportunities. Science 355:1147–1152 Egger G et  al (2004) Epigenetics in human disease and prospects for epigenetic therapy. Nature 429:457–463 Eggermann T et al (2014) CDKN1C mutations: two sides of the same coin. Trends Mol Med 20:614–622 Ehrlich M (2002) DNA methylation in cancer: too much, but also too little. Oncogene 21:5400–5413 Ezponda T, Licht JD (2014) Molecular pathways: deregulation of histone H3 lysine 27 methylation in cancer – different paths, same destination. Clin Cancer Res 20:5001–5008 Farria A et al (2015) KATs in cancer: functions and therapies. Oncogene 34:4901–4913 Feinberg AP et al (2016) Epigenetic modulators, modifiers and mediators in cancer aetiology and progression. Nat Rev Genet 17:284–299 Filipp FV (2017) Crosstalk between epigenetics and metabolism  – Yin and Yang of histone demethylases and methyltransferases in cancer. Brief Funct Genomics 16:320–325 Ghaleb AM, Yang VW (2017) Krüppel-like factor 4 (KLF4): What we currently know. Gene 611:27–37 Ghiraldini FG et al (2021) Solid tumours hijack the histone variant network. Nat Rev Cancer 21:257–275 Hanahan D (2022) Hallmarks of cancer: new dimensions. Cancer Discov 12:31–46 Jaffe LF (2003) Epigenetic theories of cancer initiation. Adv Cancer Res 90:209–230 Johnstone RW (2002) Histone-deacetylase inhibitors: novel drugs for the treatment of cancer. Nat Rev Drug Discov 1:287–299 Kadoch C et  al (2016) PRC2 and SWI/SNF chromatin remodeling complexes in health and disease. Biochemistry 55:1600–1614 Kazanets A et  al (2016) Epigenetic silencing of tumor suppressor genes: paradigms, puzzles and potential. Biochim Biophys Acta 1865:275–288 Klemm SL et  al (2019) Chromatin accessibility and the regulatory epigenome. Nat Rev Genet 20:207–220 Koschmann C et  al (2017) Mutated chromatin regulatory factors as tumor drivers in cancer. Cancer Res 77:227–233 Lawrence M et  al (2016) Lateral thinking: how histone modifications regulate gene expression. Trends Genet 32:42–56 Mensah IK et al (2022) Misregulation of the expression and activity of DNA methyltransferases in cancer. NAR Cancer 3:045 Miroshnikova YA et al (2019) Epigenetic gene regulation, chromatin structure, and force-induced chromatin remodelling in epidermal development and homeostasis. Curr Opin Genet Dev 55:46–51 Mohammad HP et al (2019) Targeting epigenetic modifications in cancer therapy: erasing the roadmap to cancer. Nat Med 25:403–418 Molenaar RJ et  al (2018) Wild-type and mutated IDH1/2 enzymes and therapy responses. Oncogene 37:1949–1960

204 Monk D et al (2019) Genomic imprinting disorders: lessons on how genome, epigenome and environment interact. Nat Rev Genet 20:235–248 Montenegro MF et  al (2015) Targeting the epigenetic machinery of cancer cells. Oncogene 34:135–143 Nacev BA et  al (2019) The expanding landscape of ‘oncohistone’ mutations in human cancers. Nature 567:473–478 Nakayama M et al (2004) GSTP1 CpG island hypermethylation as a molecular biomarker for prostate cancer. J Cell Biochem 91:540–552 Nishiyama A, Nakanishi M (2021) Navigating the DNA methylation landscape of cancer. Trends Genet 37:1012–1027 Ohlsson R et al (2003) Epigenetic variability and the evolution of human cancer. Adv Cancer Res 88:145–168 Passaguè E et  al (2003) Normal and leukemic hematopoiesis: are leukemias a stem cell disorder or a reacquisition of stem cell characteristics. PNAS 100:11842–11849 Piunti A, Shilatifard A (2016) Epigenetic balance of gene expression by Polycomb and COMPASS families. Science 352:1188–1203 Plass C et al (2013) Mutations in regulators of the epigenome and their connections to global chromatin patterns in cancer. Nat Rev Genet 14:765–780 Rao RJ, Dou Y (2015) Hijacked in cancer: the KMT2 (MLL) family of methyltransferases. Nat Rev Cancer 15:334–346 Roberts CWM, Orkin SH (2004) The SWI/SNF complex – chromatin and cancer. Nat Rev Cancer 4:133–142 Rodrigues CP et  al (2021) Epigenetic regulators as the gatekeepers of hematopoiesis. Trends Genet 37:125–142 Roy DM et  al (2014) Driver mutations of cancer epigenomes. Protein Cell 5:265–294

8  Cancer Epigenetics Saghafinia S et al (2018) Pan-cancer landscape of aberrant DNA methylation across human tumors. Cell Rep. 25:1066–1080 Schuettengruber B et  al (2017) Genome regulation by Polycomb and Trithorax: 70 years and counting. Cell 171:34–57 Sheikh BN, Akhtar A (2019) The many lives of KATs – detectors, integrators and modulators of the cellular environment. Nat. Rev. Genet. 20:7–23 Shen H, Laird PW (2013) Interplay between the cancer genome and epigenome. Cell 153:38–55 Sims RJ III, Nishioka K, Reinberg D (2003) Histone lysine methylation: a signature for chromatin function. Trends Genet. 19:629–639 Steffen PA, Ringrose L (2014) What are memories made of? How polycomb and trithorax proteins mediate epigenetic memory. Nat Rev Mol Cell Biol 15:340–356 Suelves M et  al (2016) DNA methylation dynamics in cellular commitment and differentiation. Brief Funct Genom 15:443–453 Suva ML et al (2013) Epigenetic reprogramming in cancer. Science 339:1567–1570 Torborg SR et al (2022) Cellular and molecular mechanisms of plasticity in cancer. Trends Cancer 8:735–746 Tucci V et al (2019) Genomic imprinting and physiological processes in mammals. Cell 176:952–965 Waitkus MS et  al (2018) Biological role and therapeutic potential of IDH mutations in cancer. Cancer Cell 34:186–195 Wood KH, Zhou Z (2016) Emerging molecular and biological functions of MBD2, a reader of DNA methylation. Front Genet 7:93 Zhao S et al (2021) The language of chromatin modification in human cancers. Nat Rev Cancer 21:413–430 Zhao Z, Shilatifard A (2019) Epigenetic modifications of histones in cancer. Genome Biol 20:245

9

Invasion and Metastasis

Key Points • The spread of solid cancers beyond the confinements of their tissue compartment into other parts of the same tissue and subsequently into neighboring tissues (invasion) and to distant organs (metastasis) is the most defining property of malignancy. Invasion and metastasis are decisive for the clinical course of most cancers. • Invasion and metastasis are complex processes, particularly in carcinomas, since normal epithelia are composed of strongly adherent cells and are confined by a basement membrane. Before or during invasion, carcinomas activate the surrounding connective tissue, eliciting inflammation and angiogenesis. Actual invasion by carcinoma cells requires diminished cell adhesion and increased cell motility as well as destruction of the basement membrane and remodeling of the extracellular matrix (ECM). Metastasis requires cancer cells, in addition, to enter blood or lymph vessels, to survive the passage, to extravasate, and to reestablish proliferation in a new, differing tissue microenvironment. Furthermore, during invasion and metastasis, cancer cells need to evade recognition and killing by immune cells such as cytotoxic T cells and natural killer (NK) cells. • Invasion and metastasis require extensive structural reorganization of carcinoma cells, particularly of their cytoskeleton and their sur-

face. This reorganization involves coordinated changes of gene expression and cell structure that impress as “programs” for invasion or metastasis. They resemble programs involved in embryonic and tissue development like the epithelial-mesenchymal transition (EMT) and its reversion (MET). In cancer cells, activation of these coordinated changes occurs predominantly secondary to mutations in oncogenes or tumor suppressor genes and is mediated by epigenetic regulation. Specific mutations, especially in cell adhesion molecules, contribute in some cancers. • Changes in cell surface molecules accompany altered cell-cell and cell-matrix interactions during invasion and metastasis. Molecules mediating strong homotypic interactions such as E-Cadherin are downregulated, replaced, or mutated. Expression patterns of proteins mediating interactions with the ECM, such as integrins, are likewise changed. Various other proteins on the cell surface including adhesion molecules, antigenic glycoproteins, and recognition proteins for immune cells are expressed at altered levels, alternatively spliced or processed, or are mutated. • The destruction of the basement membrane and other ECM components in connective tissues surrounding a solid cancer is predominantly accomplished by proteases secreted from tumor and stromal cells, like metalloproteinases and plasmin. Various members of the

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 W. A. Schulz, Molecular Biology of Human Cancers, https://doi.org/10.1007/978-3-031-16286-2_9

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matrix metalloproteinase (MMP) family are overexpressed in tumor tissues and their inhibitors (TIMP) may be downregulated. Proteases moreover activate latent growth factors from their storage sites in the ECM that act on carcinoma and stromal cells. At the same time, new ECM components are also produced and deposited by stromal and carcinoma cells. Neoangiogenesis is a prerequisite for the progression of many solid tumors. Migration and proliferation of endothelial cells which form new blood capillaries and lymph vessels is stimulated by several factors secreted by tumor cells and reactive stroma, including VEGFs, PDGF, and certain FGFs. Angiogenesis is limited by protein inhibitors, some ­ generated by proteolytic cleavage of ECM proteins. Successful invasion depends crucially on interactions with non-cancerous mesenchymal, endothelial, and inflammatory cells in the tissue. In cancers, these “stromal cells” likewise change their gene expression and behavior. The emergence of “activated stroma” is a characteristic of highly malignant carcinomas. Paracrine interactions between cancerous and non-cancerous stromal cells are copious in carcinomas. At least during early progression stages, carcinoma cells may still depend to a large degree on growth factors provided by stromal cells for their survival and proliferation. Carcinoma cells stimulate the production of growth factors and cytokines from stromal cells that consequently act on carcinoma cells as well as other cells in the tumor microenvironment. For instance, tumor cells secrete TGFβ which stimulates the proliferation and activity of connective tissue cells but inhibits lymphocytes. Interactions with stromal cells are arguably even more crucial in the establishment of metastases. Tissues with microcapillary systems, such as liver, lung, and bone, are preferred targets for metastases for mechanical reasons, but the actual pattern of metastases does not only depend on mechanical and anatomical factors. Rather, metastatic cancer cells

9  Invasion and Metastasis

need to set up productive mutual interactions with local stromal cells and the ECM in order to survive and expand in the target tissue. These relationships constitute the molecular basis of the “seed-and-soil” hypothesis. • Primary tumors and metastasizing tumor cells are potential targets of the immune system. Multiple mechanisms limit its ability to eliminate tumor cells. These comprise production of inhibitory cytokines, depletion of critical metabolites, downregulation of antigen presentation and recognition molecules, rewiring of signaling from death receptors used by cytotoxic immune cells, and conversely upregulation of proteins inhibiting cytotoxic T cells. In addition to the actual cancer cells, other cell types in the tumor environment, such as myeloid-derived suppressor cells (MDSC), M2 type macrophages, and regulatory T cells may limit or divert cytotoxic immune responses against the cancer cells.

9.1 Invasion and Metastasis as Multistep Processes In solid cancers like carcinomas, the extent of local invasion and distant metastasis determine the clinical outcome, even more than tumor size per se. More than 90% of all cancer deaths from carcinomas are brought about by metastases. Invasion and metastasis can be regarded as multistep processes, where invasion constitutes a prerequisite for metastasis. Each step requires and selects for certain properties of the tumor cells. For that reason, overall, metastasis may be a very inefficient process. While details vary, invasion and metastasis of a carcinoma can in general be roughly described by the following sequence of steps (Fig. 9.1): (1) While the carcinoma proliferates and extends laterally and vertically within the epithelium, the tumor cells become less adherent to each other and to adjacent normal epithelial cells (see Sect. 9.2). (2) The underlying stroma is activated and inflammation may set in or be exacerbated.

9.1 Invasion and Metastasis as Multistep Processes

Angiogenesis

INVASION

Intraepithelial growth

Stroma activation

Intravasation

Continued local growth

Spread (via blood) Extravasation Micrometastasis

Angiogenesis

Stroma activation

Metastatic growth

Fig. 9.1  Steps in invasion and metastasis. See main text for a detailed description of the processes

The basement membrane which separates the epithelium from the underlying mesenchymal connective tissue is partly or completely destroyed. (3) The tumor continues its growth into the underlying connective tissue. This is one of the more variable steps. Some carcinomas continue to grow as solid, cohesive masses compressing the neighboring connective tissue or develop processes that spread into it, breaking up the extracellular matrix (ECM). From other carcinomas, small groups of cells or single cells split off and migrate into the underlying tissue, in a single-­file pattern or as adherent clusters, in one kind of collective migration. Invasive migration, even by single tumors cells, involves remodeling of the ECM (see Sect. 9.3). Invasive, migratory carcinoma cells often undergo changes in gene expression and phenotype to become more similar to

207

mesenchymal cells. This process is termed epithelial-mesenchymal transition (EMT) and may be complete or partial. (4) In fact, invasion of stroma by the tumor cells is not a one-sided affair. It is accompanied by altered gene activity in stromal cells, with some changes promoting and others inhibiting invasion (see Sect. 9.5). Prominently, quiescent resident fibroblasts are activated to proliferate, change their morphology, become motile and secrete cytokines, ECM remodeling enzymes, but also ECM components, and release extracellular vesicles like exosomes. These cells are known as cancer-associated fibroblasts (CAFs). The type of stromal reaction is an important factor determining the ability of a tumor to metastasize. Often, immune cells—leukocytes as well as lymphocytes— are attracted to the tumor, by signals emanating from stromal and tumor cells. Macrophages (or monocyte precursors) are attracted or activated and often polarized to an M2 phenotype that rather supports tumor growth and invasion. One outcome of the stromal reaction can be pronounced inflammation. In certain cancers, inflammation may precede and promote tumor development. These include lung, skin, esophageal, gastric, colorectal, and pancreatic cancers and hepatocellular carcinoma. Like the stromal reaction, the effect of inflammation is in general ambiguous: it may impede or promote invasion. As a rule, acute inflammation tends to suppress tumor development, whereas chronic inflammation tends to promote tumor progression. (5) A critical step in invasion is reached when the growing tumor mass or migrating cancer cells encounter blood or lymph vessels and invade them. Like the previous steps of invasion, this can occur by a tumor mass growing through the vessel wall into the lumen or by single cancer cells squeezing through the vessel lining. Through this “intravasation” step tumor cells gain access to the circulation and can reach distal organs by “lymphogenous” or “hematogenous”

208

routes. Gaining access to blood and lymph vessels is not necessarily a one-directional process either. Since many tumors induce angiogenesis, capillaries sprout from blood and lymph vessels in the direction of the tumor mass (see Sect. 9.4). Since neoangiogenic vessels are often leakier than normal vessels, they may facilitate access of cancer cells to the circulation. (6) Independent of whether metastasis occurs, invasion may continue into further layers of the organ from which the carcinoma arises, through a tissue capsule, into surrounding adipose tissue and into neighboring organs. An important spreading route for some cancers, like those of the ovary, kidney, ­ liver, and pancreas, is through the lumen of the peritoneum or retroperitoneum (“transcoelomic metastasis”). (7) Tumor cells having entered lymph vessels are transported to the filtering system of the local lymph nodes, where some may survive and initiate lymph node metastases. Cells from nodal metastases may eventually penetrate towards the main lymph vessels and eventually enter the blood by this route. Tumor cells or debris and signal molecules from tumor and stromal cells transported to the lymph nodes influence the immune reaction towards the primary tumor. (8) Tumor cells that have entered blood vessels can theoretically spread to any part of the body. However, they are larger than normal blood cells and are not well adapted for survival in a moving liquid. In fact, carcinoma cells are often observed in the bloodstream as small clusters which may also contain various leukocytes and aggregated platelets. Attachment to other cells can serve to suppress anoikis through the activation of antiapoptotic pathways. Survival of the passage through the bloodstream to reach distant tissues is a particularly important limiting factor for metastasis. The actual site of metastasis is determined by a mix of mechanical and biological factors. On the one hand, tumor cells are likely to get arrested in the closest capillary

9  Invasion and Metastasis

system that they have to pass. On the other hand, tumor cells must encounter a favorable pattern of growth conditions in a fitting “metastatic niche” to survive. In fact, they may even respond to attractants to find sites that provide such conditions. Favorable conditions to metastasis seeding can be generated by inflammation, among others, primarily by cytokines from neutrophils, or by other conditions leading to activation of the local stroma. Moreover, metastatic niches may be prepared by systemic signals emanating from tumor cells, through soluble factors or extracellular vesicles (EV, containing proteins and regulatory RNAs), or from bone marrow-derived cells (BMDCs) mobilized by the tumor. (9) To form metastases, carcinoma cells usually have to exit from the circulation by “extravasation.” Most often, extravasation takes place in organs with microcapillary systems, such as the liver, the lung, the kidney, and bone. Due to their size, single carcinoma cells and of course tumor cell clusters get stuck in capillaries. They may disintegrate there, persist, or pass through the capillary wall into the underlying tissue to establish micrometastases. (10) In the new target tissue environment, carcinoma cells have to reattach to the matrix, survive, and eventually start to establish micrometastases. Micrometastases may remain in a state of “dormancy” for many years before they cause recurrences. Importantly, dormant tumor cells are usually resistant to (cytotoxic) chemotherapies targeting proliferating cells. After all the complicated previous steps, it may be surprising to learn that reactivation of dormant cells to micrometastases is often considered the most critical, i.e., the least efficient step in metastasis. (11) Dormancy comes in two modes. “Cellular dormancy” involves quiescence, i.e., exit from the cell cycle to a (reversible) G0 state. “Population (or mass) dormancy” results if the rate of cell death equals the rate of cell proliferation by actively cycling

9.1 Invasion and Metastasis as Multistep Processes

cancer cells. In either case, it is unclear what causes “awakening,” either as reactivation of cancer cells from cellular dormancy or as a shift in the balance towards survival in population dormancy. (12) The final step in metastasis is the expansion of micrometastases to actively growing tumors, shortly after seeding or after emergence from dormancy. This step requires— among others—establishment of a sufficient nutrient and oxygen supply and interaction with a new, different type of stroma, often including once more stromal activation, induction of angiogenesis, and further local invasion. Multiple distinct metastases may originate from one or several clones in the primary cancer, but in some cases, a single metastasis may contain cells from more than one clone. Furthermore, new cascades of invasion and metastasis may start from established metastases. In some cancers, the majority of metastases are in fact generated by cross-seeding from a few initial metastases or even a single one. Although invasion and metastasis are such important processes in the course of cancer progression, they are incompletely understood. This owes largely to their complexity since almost every step involves complex interactions between different cell types and various extracellular tissue components. Moreover, which mode of spread hematogenous, lymphogenous, or transcoelomic—predominates, differs between tumor types. In fact, invasion and metastasis can proceed by additional routes, e.g., by growth and migration along nerves or the outer side of vessels. Evidently, (especially) the early steps of metastasis are difficult to observe directly in humans. The time course is also highly variable. In some cancers, tumor cell dissemination may begin long before the primary tumor becomes evident, in others it may start only many years after the primary tumor has formed. Moreover, metastases specimens are not as regularly available for investigation as primary tumors, since they are often not treated by surgery. New windows into metastases have been opened by the

209

analysis of circulating tumor cells (CTC), usually isolated from peripheral blood, and disseminated tumor cells (DTC), usually isolated from bone marrow biopsies (→22). Such cells are few however and it is of course unclear whether the isolated cells would eventually be capable of successfully establishing metastases. At this time, therefore many individual factors involved in invasion and metastasis have been identified and selected cellular processes and interactions have been pinpointed (see below). However, a sufficiently full picture that would allow to therapeutically target metastasis is not yet available. One particularly important issue is what drives the overall process of invasion and metastasis. A straightforward explanation would be that tumor cells acquire one property after another as they proceed through the steps outlined above. In this explanation, at each step, the best-adapted tumor cells are selected from the numerous variants continuously created by inherent genomic instability. If this were the case, metastases would show enrichment in those genetic alterations that favor metastasis and the presence of such alterations in primary tumors would be prognostic, indicating a higher risk of metastasis. This is not generally so, although some such associations have been observed, e.g., in the comparison of breast cancer subtypes (→19.3). In general, metastases contain the same spectrum of driver mutations as at least one clone in the primary tumor, even though the overall number of genetic alterations is typically higher in metastases. Moreover, metastases arise more often from cancer cells that have undergone whole-genome duplication. Genomic instability is thus undoubtedly a factor driving tumor progression and generating intratumoral heterogeneity, but specific mutations that drive specifically metastasis are probably rare. Rather, the same mutations appear to be responsible for local tumor growth and metastatic spread. An important argument in this respect is that different steps of metastasis select for different properties of the cancer cells. For instance, a switch to a more mesenchymal phenotype facilitates invasion and survival outside epithelia. However, it is often incompatible with maintaining self-renewal

9  Invasion and Metastasis

210

properties and expansion at the target site. Thus, EMT has to be followed by MET (mesenchymal-­ epithelial transition), 1 i.e., a reversal to an epithelial state, which is indeed characteristic of most metastases from carcinomas. The predominant explanation today is therefore that invasion and metastasis proceed via epigenetic “programs” that are otherwise employed in wound healing and tissue regeneration, but especially during embryonic development. While execution of these programs may be favored by certain genetic alterations selected from the genetically heterogeneous cancer cell p­ opulations at the primary site, they require most of all “plasticity” of the cellular phenotype and may rather involve reversible epigenetic changes than a succession of genetic alterations. 2 Moreover, these programs may be directed not only by autonomous signaling through cancer pathways in the tumor cells, but even more by factors from the tumor microenvironment and by interactions with stromal cells. Changing micro-environments at the primary site, during migration, and at the target site could explain how different programs can be chosen by sufficiently responsive and epigenetically malleable cancer cells. Moreover, in theory, metastases could only be formed by cells with a (cancer) stem cell phenotype. The enhanced epigenetic plasticity of cancer cells (see Sect. 8.5) may alleviate this theoretical requirement as well. Thus, while certain genetic alterations in primary cancer enable metastasis, survival of the strong and varied selection pressures during metastasis is favored not only by genomic heterogeneity but also by phenotypic variation of the cancer cells conferred by their epigenetic plasticity. In order to characterize the process in its entirety, four hallmarks have been proposed that enable metastasis. Tumor cells must (1) acquire invasiveness and motility, (2) the ability to modu-

Not to be confused with the HGF receptor tyrosine kinase MET. 2  In fact, some changes, like during the journey through the bloodstream, have to take place within minutes and require faster adaptation than achievable by transcriptional changes with subsequent protein biosynthesis. 1 

late the tissue microenvironment at the metastatic site as well, (3) sufficient plasticity to undergo changes like EMT and MET, and (4) the ability to invade and grow in a different tissue.

9.2 Genes and Proteins Involved in Cell-Cell and Cell-Matrix Adhesion during Invasion and Metastasis Epithelial cells adhere to each other and interact with each other through several types of contacts (Fig.  9.2), which need to be broken or reorganized during invasion and metastasis. Morphologically distinct and functionally important contacts include adherens junctions, gap junctions, and tight junctions (also known as occluding junctions). Occluding junctions seal epithelia and define the apical and lateral membrane compartments of an epithelial cell. Therefore, their loss in a carcinoma cell is associated with a loss of cell polarity as well. Typical components of occluding junctions like claudins and Occludin are therefore often downregulated during carcinoma progression. Adherens junctions are arranged in a belt-like configuration (hence also: belt desmosomes) between adjacent epithelial cells and are intracellularly connected to actin filaments. In adherens junctions, cadherins mediate homotypic interactions between adjacent epithelial cells, in epithelial cells this is typically done by E-Cadherin (Fig. 9.3). The interaction between the E-Cadherin proteins on neighboring cells is Ca2+-dependent. On the cytoplasmic surface of the cell membrane, E-Cadherin is linked to actin cytoskeletal filaments via α-Catenin and β-Catenin. Functional adherens junctions protect epithelial cells from apoptosis via PI3K signaling, inhibit receptor tyrosine kinases, activate Hippo signaling via Merlin (also known as NF2, the tumor suppressor inactivated in neurofibromatosis type II) and— through the associated p120-Catenin 3—inhibit NFκB and MAPK signaling. Furthermore, by

p120 catenin is also known as Catenin δ1.

3 

9.2 Genes and Proteins Involved in Cell-Cell and Cell-Matrix Adhesion during Invasion and Metastasis

211

Claudins Occludins Tight junctions

E-Cadherin

Adherens junction

Other cadherins

Desmosome Connexins Gap junction

Integrins

Focal adhesion Hemidesmosome

Fig. 9.2  Cell-cell- and cell-matrix-contacts of epithelial cells. From apical to basal, tight junctions, adherens junctions, desmosomes, gap junctions, and hemidesmosomes (focal adhesions) are depicted along with the central proteins Fig. 9.3  Structure and main interactions of E-Cadherin. See main text for further explanation

constituting these structures. Note that different integrins prefer different matrix proteins. Not shown are nonclassical interactions mediated, e.g., by selectins or Ig-superfamily CAMs like EpCAM

E-Cadherin

Catenins

20

p1

Actin

β

α

β α γ

20

p1

sequestering β-Catenin, E-Cadherin dampens canonical WNT signaling. Downregulation or mutation of E-Cadherin (gene: CDH1) is frequent in human cancers and often occurs during tumor progression. During tumor invasion, E-Cadherin is often replaced by other members of the family like N-Cadherin that are better suited to cell migration. This change is known as “cadherin switch.” Spot desmosomes are connected to cytokeratin filaments by Desmoplakin and Plakoglobin in

γ

α α

epithelial cells. The actual contact between cells is again made by specialized cadherins in these structures. Plakoglobin remains important in cancer for the adhesion of migrating tumor cell clusters. Gap junctions also contribute to adhesion between cells, but are primarily communication channels that connect cells of the same type in epithelia or other tissues, like the nervous system. They allow the passage of small molecular weight substances (70 μm as well as systemic anemia. Operationally, hypoxia is commonly defined as pO2 3N

i(12p) i(12p)

TCGT

> 3N

> 3N

Teratoma

< 3N

Yolk sac tumor

< 3N Embryonal carcinoma

Seminoma

< 3N

KIT expression KRAS overexpression

Fig. 11.6  Genomics of TGCT. The sequence of major genomic changes leading to the development of GCNIS, seminomas, and embryonal car-

cinomas as postulated by the TCGA consortium (compare: Shen et al. (2018) l.c.)

somatic cells. Furthermore, the balance between proapoptotic and antiapoptotic BH3 proteins (→7.4) at the mitochondria tends towards the former, resulting in a state termed “mitochondrial priming.” In particular, the proapoptotic protein NOXA is strongly expressed, since it is induced by OCT4 (see below). For these reasons and presumably others still to be elucidated, TGCT cells are prone to apoptosis, especially following DNA damage by cytotoxic chemotherapy drugs. Indeed, resistance to cytotoxic chemotherapy in TGCT is associated with TP53 inactivation, by mutations or MDM2 amplification, but also with MYCN amplification. TGCT are aneuploid, usually with hypertriploid genomes. Most likely, this state develops through whole genome duplication followed by chromosome losses (Fig.  11.6). Whereas some chromosomes tend to be preferentially lost, chromosome 12 is retained or often gained. Many

tumors contain an isochromosome 12p (i[12p]). These gains lead in particular to an increased dosage of a gene cluster at 12p13.1, containing NANOG, DPPA3, GDF3, and EDR1, that is involved in the regulation of pluripotency and germ cell development. In addition, KRAS at 12p12.1 and CCND2 (encoding Cyclin D2) at 12p13.3 are often gained or even amplified. In some seminomas, the region containing the gene cluster is amplified. The products of the cluster genes are all involved in the maintenance of pluripotency in early embryonic cells. The transcription factor NANOG in particular interacts with two others, OCT4 (also known as OCT3/4, gene: POU5F1)2 and SOX2 to establish pluripotency. These three factors form a stable network in OCT4 immunohistochemical staining is an excellent biomarker to identify TGCT cells in histopathological diagnostics. 2 

11.5  Testicular Germ Cell Tumors

which each one binds to the promoters of the genes encoding the two others in a feed-forward manner. In various combinations, the three factors then regulate the expression of other genes that maintain pluripotency while contributing to repression of differentiation factors (cf. Fig. 8.10). Inactivation of this network is required to exit the pluripotent state. Conceivably, therefore the increased dosage of genes at 12p may be responsible for the inability of seminoma and EC cells to exit the pluripotent state. Of note, the increased dosage of several other genes at 12p may support proliferation and survival of TGCT cells, especially KRAS and CCND2, but also GAPDH. Whereas all TGCT are aneuploid, point mutations are rare (with a frequency around 0.5/Mbp), and only a few genes are recurrently mutated. Moreover, recurrent mutations are found exclusively in seminomas and can be arranged in one pathway, namely KIT signaling. The KIT receptor tyrosine kinase on gonocytes responds to KIT ligand (also: Stem Cell Factor; gene: KITLG) from Sertoli cells to stimulate proliferation. Following autophosphorylation, KIT induces RAS and PI3K signaling through the SHC and GRB2 adaptor proteins. SRC and PLCγ are also activated. Oncogenic mutations in seminoma affect KIT itself or alternatively KRAS, NRAS, or PIK3CA, in about half of the cases. In addition, gene dosage and expression of the RTK ubiquitin ligase CBL are often diminished. In non-­ seminomas, neither of these genetic changes are observed and KIT is in fact often downregulated, presumably as a consequence of overexpression of the miR-222-3p. While a significant number of seminomas do not contain genetic alterations in the KIT pathway, if they occur, these alterations direct aneuploid primordial germ cells towards seminoma development, blocking embryonal carcinoma fate. The importance of KIT signaling in TGCT is underlined by the observation that genetic variants in KIT and KITLG are associated with increased predisposition to these cancers. Apart from the mutations activating the KIT pathway in seminomas, TGCT appear to be caused predominantly by gene dosage changes, typically arising from loss or gains of entire chro-

273

mosome arms. Very likely, however these are complemented by epigenetic changes. It is an open question which epigenetic alterations precisely are relevant for the development of TGCT, but distinctive changes in DNA methylation stand out. Overall methylation patterns differ strongly between seminomas and non-seminomas. Seminomas have extremely low DNA methylation levels across their entire genomes. DNA methylation in non-seminomas is overall higher, but the pattern of DNA methylation is aberrant. Correspondingly, imprinted genes and control regions are completely unmethylated in seminomas and methylated in various aberrant patterns in non-seminomas. EC in particular display not only CpG, but also CpH methylation, i.e., some cytosines followed by bases other than guanine are also methylated. This type of methylation also occurs in ESC and is ascribed to the high expression and activity of the de-novo-­ methyltransferases DNMT3A and DNMT3B, which can also be observed in embryonal carcinomas. In non-seminomas, moreover specific genes and sites are hypermethylated, which may contribute to tumor development and progression. Different TGCT subtypes can thus be distinguished by their DNA methylation patterns. Like DNA methylation patterns, expression patterns of miRNAs are distinctive for TGCT at large and differentiate between the histological subtypes. Whereas miR-22-3p expression distinguishes non-seminomas from seminoma, miR-­ 375 expression is particularly high in teratomas and yolk sac tumors. Conversely, miRNAs of the 371, 372, and 373 families are strongly expressed in seminomas, EC, and mixed-type non-­ seminomas. Detection of these miRNAs in serum therefore offers several options for diagnostics. An assay quantifying serum levels of miR-371-3 can be used for detection of the disease as well as monitoring for therapy success and recurrences. It provides better sensitivity and specificity than the established serum markers like β-Human chorionic gonadotropin (β-hCG), α-Fetoprotein (AFP), and Lactate dehydrogenase (LDH). TGCT may therefore represent one of the first cancers for which miRNA assays are routinely applied in clinical diagnostics.

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Further Reading Ackermann S et  al (2018) A mechanistic classification of clinical phenotypes in neuroblastoma. Science 362:1165–1170 Coorens THH et  al (2019) Embryonal precursors of Wilms tumor. Science 366:1247–1251 Creff J, Besson A (2020) Functional versatility of the CDK inhibitor p57Kip2. Front Cell Dev Biol 8:584590 Davidoff AD (2009) Wilms’ tumor. Curr Opin Pediatr 21:357–364 De Toni L et al (2019) Testicular cancer: genes, environment, hormones. Front Endocrin 10:408 de Vries G et al (2020) Testicular cancer: Determinants of cisplatin sensitivity and novel therapeutic opportunities. Cancer Treat Rev 88:102054 Dieckmann KP et  al (2012) MicroRNAs miR-371-3  in serum as diagnostic tools in the management of testicular germ cell tumors. Brit J Cancer 107: 1754–1760 Francis JH et al (2021) Molecular changes in retinoblastoma beyond RB1: findings from next-generation sequencing. Genes 13:149 Hanna NH, Einhorn LH (2014) Testicular cancer—discoveries and updates. NEJM 371:2005–2016 Hastie ND (2017) Wilms‘ tumour 1 (WT1) in development, homeostasis and disease. Development 144:2862–2872 Hovestadt V et  al (2020) Medulloblastoma revisited: biological and clinical insights from thousands of patients. Nat Rev Cancer 20:42–56 King J et al (2021) Testicular cancer: biology to bedside. Cancer Res 81:5369–5376 Laetsch TW et al (2021) Opportunities and challenges in drug development for pediatric cancers. Cancer Disc 11:545–559

11  Pediatric Cancers Lee SB, Haber DA (2001) Wilms tumor and the WT1 gene. Exp Cell Res 264:74–99 Li H et  al (2021) Embryonic kidney development, stem cells and the origin of Wilms tumor. Genes 12:318 Martinez-Sanchez M et al (2022) Retinoblastoma: from discovery to clinical management. FEBS J 289:4371–4382 Menke AL, Schedl A (2003) WT1 and glomerular function. Semin Cell Dev Biol 14:233–240 Newman S et  al (2021) Genomes for kids: the scope of pathogenic mutations in pediatric cancer revealed by comprehensive DNA and RNA sequencing. Cancer Disc 11:3008–3027 Rijlaarsdam MA et al (2015) Genome wide DNA methylation profiles provide clues to the origin and pathogenesis of germ cell tumors. PLoS ONE 10:e0122146 Shah MM et al (2004) Branching morphogenesis and kidney disease. Development 131:1449–1462 Shen H et al (2018) Integrated molecular characterization of testicular germ cell tumors. Cell Rep 23:3392–3406 Sweet-Cordero EA, Biegel JA (2019) The genomic landscape of pediatric cancers: implications for diagnosis and treatment. Science 363:1170–1175 Tang WWC et  al (2016) Specification and epigenetic programming of the human germ line. Nat Rev Genet 17:585–600 Taylor-Weiner A et  al (2016) Genomic evolution and chemoresistance in germ-cell tumours. Nature 540:114–118 Thériault BL et al (2014) The genomic landscape of retinoblastoma: a review. Clin Exp Opthamol 42:33–52 Treger TD et  al (2019) The genetic changes of Wilms tumour. Nat Rev Nephrol 15:240–251 Ulbright TM et al (2016) Germ cell tumors. In: Moch H et al (eds) WHO classification of tumours of the urinary system and male genital organs, 4th edn. IARC, Lyon, pp 189–226

Cancers of the Skin

Key Points • The skin is the largest organ and the most frequent site of cancers in humans. The lifetime risk of skin cancer may now add up to 40% for fair-skinned individuals. Fortunately, the two most frequent subtypes, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), are not usually life-threatening. Moderately frequent melanoma is more lethal and its incidence appears to increase alarmingly. • The most important carcinogen in the skin is UV radiation, specifically UVB (280–315 nm). It is incriminated by a clear correlation of incidence with exposure and by typical mutations found in skin cancers, especially transition mutations at dipyrimidine sequences, a. o. in TP53. This relationship is a paradigmatic example for molecular epidemiology, which aims at determining the causes of cancer from mutational signatures. In skin carcinogenesis, UV radiation acts not only as a mutagen, but also alters gene expression in epidermal keratinocytes and mesenchymal dermal cells and modulates immune responses. • The incidence of skin cancer is strongly influenced by inherited genetic variation. At the population level, skin pigmentation is the most obvious risk-modulating factor. Individuals with inherited deficiencies in the repair of UV-damaged DNA are at even greater risk, prominently xeroderma pigmentosum patients. In a different manner, inher-

12

ited high-­risk mutations in PTCH1 predispose to BCC and mutations in CDKN2A predispose to melanoma. • Cutaneous squamous cell carcinomas (SCC) contain terminally differentiated keratinocytes, but also a fraction of incessantly proliferating and invasive precursor cells, in a disordered tissue. SCC often contain UV-­induced TP53 mutations. Other recurrent changes include loss of CDKN2A/p16INK4A, inactivating mutations in NOTCH genes, and Telomerase activation. Together, these changes account for dysregulated cell differentiation, compromised cell cycle exit, and the escape from replicative senescence in this cancer type. • Many genomic alterations characteristic of cutaneous SCC are also found in the SCC of other tissues. In addition to recurrent mutations in TP53, CDKN2A and NOTCH genes and Telomerase activation, 3q gains and amplification lead to overactivity of PI3K and two transcription factors, ΔNp63 and SOX2, that together determine the differentiation state of basal cells in squamous epithelia. Hippo signaling is often impeded whereas EGFR signaling is enhanced. NRF2 signaling is activated, in some SCC by genetic changes. Human papilloma viruses (HPV) cause SCC in several tissues, especially the cervix and the oropharynx, with the viral oncogenes E6 and E7 obliterating the requirement for mutations inactivating TP53 and CDKN2A/RB1.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 W. A. Schulz, Molecular Biology of Human Cancers, https://doi.org/10.1007/978-3-031-16286-2_12

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• Basal cell carcinoma consists of undifferentiated keratinocytes resembling basal cells of the epidermis. They also often carry TP53 mutations with the UVB mutational signature. Other recurrent mutations lead to constitutive activation of the Hedgehog pathway; they occur either in an inhibitory or an activatory component. Most inactivating mutations affect PTCH1, a classical tumor suppressor. Germline mutations in PTCH1 cause basal cell nevus syndrome (BCNS or Gorlin syndrome) that predisposes to BCC and selected other cancers. Hedgehog pathway overactivity in sporadic BCC is alternatively caused by mutations activating SMO to an oncogene. In normal skin, Hedgehog signaling helps to maintain basal cells in a precursor state. Its constitutive activation thus explains the ­phenotype of BCC and offers options for targeted therapy. • Melanoma, the most lethal common skin cancer, develops from melanocytes which provide the protective pigments of the skin. Melanocytes originate from neural crest cells that migrate to the epidermis during fetal development. Melanomas are often highly invasive and form metastases much more readily than BCC and SCC. Mutations in melanomas also show the UVB signature, albeit not as consistently as in BCC and SCC. In all melanomas, MAPK signaling is activated through oncogenic mutations in either BRAF or NRAS or through inactivation of NF1, a negative regulator of the pathway. This activation likely stimulates cell proliferation and cell migration. MAPK activation may be complemented by mutations in TP53, CDKN2A, CDK4, and PTEN that interfere with the mechanisms by which inappropriate proliferation induces apoptosis or replicative senescence. • Survival of melanoma cells may moreover be promoted by alterations in the pathway mediating signaling by αMSH, a melanocyte-­ specific growth factor. This pathway proceeds via cAMP and PKA to activate the transcription factor MITF, which induces melanocyte-­ specific proteins as well as antiapoptotic proteins.

12  Cancers of the Skin

• Melanoma cells retain the expression of differentiation antigens specific for melanocytes such as gp100 and Tyrosinase. In addition, they express “cancer-testis antigens” which are otherwise found only in male germ cells. As in other skin cancers, the overall high rate of point mutations induces a variety of aberrant proteins and therefore generates neoantigens. All these antigens may become targets of host anti-tumor immune responses and are exploited in immunotherapy, which is a promising option for the treatment of metastatic melanoma.

12.1 Carcinogenesis in the Skin The skin is the largest organ in humans. As it covers and protects our external surface, it is subject to mechanical damage and is exposed to a variety of potential carcinogens, chemicals, radiation, and infectious agents alike. Thus, a strong capacity for repair and regeneration is mandatory for its maintenance. This requirement is met by continuous turnover of the epidermis in a structural arrangement that minimizes the impact of carcinogenic agents and protects the body as a whole and the skin itself from cancer development (Fig. 12.1). The outmost layer of the epidermis (stratum corneum) is composed of crosslinked dead keratinocytes filled with filament proteins. This layer forms a barrier that excludes many infectious agents, reacts with chemicals, and absorbs radiation to keep them from penetrating into living tissue. The underlying layers (stratum granulosum, stratum spinosum) are formed by living cells committed to terminal differentiation (→7.1). Therefore, genetic changes in these cells become rarely permanent, because the cells are destined to become incapable of proliferation, lose their nuclei, and are eventually eliminated by shedding. Even the basal layer of epithelial cells in the skin, to which proliferation activity is largely confined, consists mostly of cells with a limited replicative potential. They form the transit amplification compartment. The actual stem cells of the skin are rare and are thought to proliferate normally very

12.1  Carcinogenesis in the Skin Fig. 12.1 The protective organization of the skin. See main text for further details

277

Stratum corneum

Cell death Envelope formation

Stratum lucidum KRT2/9 Stratum granulosum

Stratum spinosum

KRT1/10

Keratin crosslinking Dissolution of nuclei Differentiation Keratin systhesis Proliferation

KRT5/14

Stratum basale Dermis

slowly, except during wound repair. Nevertheless, even in that event, the brunt of expansion is borne by the transit-amplifying fraction. The (facultative) stem cells in the basal layer of the epidermis may be repleted by stem cells from the hair bulge, which can generate other structures in the skin like hairs and glands as well. Another factor protecting against infection and carcinogenesis is the immune system of the skin. Langerhans cells are skin-specific dendritic cells which present antigens for recognition by T cells to elicit immune responses against infectious agents and cancer cells. Melanocytes help to protect specifically against sunlight by producing melanin pigments that are deposited in the keratinocytes. As these differentiate, they carry the pigments to the upper layers of the skin. The extent of pigmentation is the most obvious factor modulating skin cancer risk. In spite of its intricate protective system, the skin is the most frequent site of cancers in humans. The combined lifetime risk for all skin cancers is estimated as 30–40% for lightly pigmented Northern Europeans; it is lower in other populations with more intense pigmentation. Three different types of skin cancer are prevalent, in decreasing order of frequency, basal cell carcinoma (BCC), squamous cell carcinoma (SCC),

and melanoma (Fig. 12.2). Melanoma has by far the highest mortality of these cancers; it is lethal in about one-fifth of all cases. SCC and especially BCC metastasize rarely but can expand locally if left untreated. The incidences of all three skin cancer types have increased over the last decades. This is particularly worrying in the case of life-threatening melanoma. The presumed cause of the increase is an increased exposure to UV-rich sunlight (aggravated by increased use of tanning beds). While the risk of skin cancers is modulated by a variety of genetic factors, short wavelength light is the most important exogenous carcinogen in the skin. Accordingly, skin cancers, SCC and BCC, and to a lesser degree melanoma, develop most often in light-exposed areas of the skin. UV light is categorized by wavelength as UVC (200–280 nm), UVB (280–315 nm), and UVA (315–400 nm). UVC cannot penetrate the upper layer of the skin (and UVC from sunlight is absorbed in the atmosphere), but ≈0.4% of UVB and a few percent of UVA radiation reach the basal layer of the epidermis (Fig.  12.3). Some UVA photons even penetrate into the dermis, like most light in the visible wavelength range. How much UVA and visible light reach the deeper layers of the skin depends on the intensity of pigmentation.

12  Cancers of the Skin

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Fig. 12.2  Histologies of skin cancers. Histological aspects of left: squamous cell carcinoma, center: basal cell carcinoma, right: melanoma. Courtesy: R. Engers

Fig. 12.3  Penetrance of different wavelength UV radiation in the skin. See main text for detailed explanation. Courtesy: V. Kolb-Bachofen