Cancer Epigenetics: Biomolecular Therapeutics in Human Cancer

Cancer Epigenetics: Biomolecular Therapeutics in Human Cancer is the only resource to focus on biomolecular approaches to cancer therapy. Its presentation of the latest research in cancer biology reflects the interdisciplinary nature of the field and aims to facilitate collaboration between the basic, translational, and clinical sciences.

• Language: English
• Publisher: Wiley
• Release date: Oct 7, 2011
• ISBN: 9781118005736

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Library of Congress Cataloging-in-Publication Data:

Cancer epigenetics: biomolecular therapeutics for human cancer / [edited by] Antonio Giordano Marcella Macaluso.

p. ; cm.

Includes bibliographical references.

ISBN 978-0-471-71096-7 (cloth)

1. Cancer-Genetic aspects. 2. Cancer-Treatment. 3. Epigenesis. 4. DNA-Methylation. I. Giordano, Antonio, MD. II. Macaluso, Marcella.

[DNLM: 1. Neoplasms-genetics. 2. Neoplasms-therapy. 3. DNA Methylation. 4. Drug Design. 5. Epigenesis, Genetic. 6. Histones-metabolism. Q7 266]

RC268.4C3492 2011

616.99–4042-dc22

2010041037

In loving memory of Professor Giovan Giacomo Giordano

September 12, 1925-July 29, 2010

A brilliant mind, passionate about the biology of life and its mysteries,

a prolific scientist, pathologist, and teacher of oncology,

a loving father, husband, and grandfather,

a man of integrity, and a true friend.

Contributors

Mayada Achour, Laboratoire de Biophotonique et de Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, Illkirch, France

Alfonso Bellacosa, Cancer Biology Program, Epigenetics and Progenitor Cells Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania

Wolfgang Bohn, Heinrich-Pette-Institute, Leibniz Institute for Experimental Virology, University of Hamburg, Hamburg, Germany

Christian Bronner, Laboratoire de Biophotonique et de Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, Illkirch, France

Serena Buontempo, Laboratory of Stem Cells Epigenetics, European Institute of Oncology, Milan, Italy

Thierry Chataigneau, Laboratoire de Biophotonique et de Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Illkirch, France

Roberta Ciarapica, Laboratory of Endothelial Cells and Angiogenesis, Ospedale Pediatrico Bambino Gesù, Rome, Italy

Marcella Cintorino, Department of Human Pathology and Oncology, University of Siena, Siena, Italy

Letizia Cito, Oncology Research Centre of Mercogliano (CROM), Avellino, Italy

Pier Paolo Claudio, Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania. Currently at Marshall University, Department of Biochemistry and Microbiology, Huntington, West Virginia

Sandra Coral, Cancer Bioimmunotherapy Unit, Department of Medical Oncology, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientifico, Aviano, Italy

Alessia Covre, Cancer Bioimmunotherapy Unit, Department of Medical Oncology, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientifico, Aviano, Italy

Riccardo Danielli, Division of Medical Oncology and Immunotherapy, Department of Oncology, Istituto Toscano Tumori, University Hospital of Siena, Siena, Italy

Wolfgang Deppert, Heinrich-Pette-Institute, Leibniz Institute for Experimental Virology, University of Hamburg, Hamburg, Germany

Claudia Esposito, INT–CROM, Pascale Foundation National Cancer Institute—Cancer Research Center, Mercogliano, Avellino, Italy

Elisabetta Fratta, Cancer Bioimmunotherapy Unit, Department of Medical Oncology, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientifico, Aviano, Italy

Cristina Giacinti, DAHFMO, University of Rome La Sapienza, Rome, Italy; Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania

Antonio Giordano, Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania; Department of Human Pathology and Oncology, University of Siena, Siena, Italy; Oncology Research Centre of Mercogliano (CROM), Avellino, Italy

Heike Helmbold, Heinrich-Pette-Institute, Leibniz Institute for Experimental Virology, University of Hamburg, Hamburg, Germany

Marcella Macaluso, Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania

Michele Maio, Cancer Bioimmunotherapy Unit, Department of Medical Oncology, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientifico, Aviano, Italy; Division of Medical Oncology and Immunotherapy, Department of Oncology, Istituto Toscano Tumori, University Hospital of Siena, Siena, Italy

Mario Mancino, INT–CROM, Pascale Foundation National Cancer Institute–Cancer Research Center, Mercogliano, Avellino, Italy

Francesco Masulli, Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania; DISI-Department of Computer and Information Sciences, University of Genoa, Genoa, Italy

Alexander Mazo, Kimmel Cancer Center, Departments of Cancer Biology, Medical Oncology, Microbiology and Immunology, Philadelphia, Pennsylvania

Steven B. McMahon, Kimmel Cancer Center, Departments of Cancer Biology, Medical Oncology, Microbiology and Immunology, Philadelphia, Pennsylvania

Micaela Montanari, Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania

Debora Muresu, Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania

Hugues Jean Marie Nicolay, Cancer Bioimmunotherapy Unit, Department of Medical Oncology, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientifico, Aviano, Italy; Division of Medical Oncology and Immunotherapy, Department of Oncology, Istituto Toscano Tumori, University Hospital of Siena, Siena, Italy

Laszlo Otvos, Jr., PeptheRx, Inc., Audubon, Pennsylvania

Giulia Parisi, Cancer Bioimmunotherapy Unit, Department of Medical Oncology, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientifico, Aviano, Italy

Raffaella Pasquale, INT–CROM, Pascale Foundation National Cancer Institute—Cancer Research Center, Mercogliano, Avellino, Italy

Francesca Pentimalli, INT–CROM, Pascale Foundation National Cancer Institute–Cancer Research Center, Mercogliano, Avellino, Italy

Richard G. Pestell, Kimmel Cancer Center, Departments of Cancer Biology, Medical Oncology, Microbiology and Immunology, Philadelphia, Pennsylvania

Vladimir M. Popov, Kimmel Cancer Center, Departments of Cancer Biology, Medical Oncology, Microbiology and Immunology, Philadelphia, Pennsylvania

Michael J. Powell, Kimmel Cancer Center, Departments of Cancer Biology, Medical Oncology, Microbiology and Immunology, Philadelphia, Pennsylvania

Andrew Puca, Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania; Department of Human Pathology and Oncology, University of Siena, Siena, Italy

Lavinia Raimondi, Laboratory of Endothelial Cells and Angiogenesis, Ospedale Pediatrico Bambino Gesù, Rome, Italy

Flavio Rizzolio, Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania; Department of Human Pathology and Oncology, University of Siena, Siena, Italy

Gaetano Romano, Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania

Rossella Rota, Laboratory of Endothelial Cells and Angiogenesis, Ospedale Pediatrico Bambino Gesù, Rome, Italy

Stefano Rovetta, DISI–Department of Computer and Information Sciences, University of Genova, Genoa, Italy

Giuseppe Russo, Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania; DISI–Department of Computer and Information Sciences, University of Genova, Genoa, Italy

Mara Sannai, Laboratory of Genomic Stability, Leibniz Institute for Age Research, Jena, Germany

Maria Irene Scarano, Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, Pennsylvania

Valérie B. Schini-Kerth, Laboratoire de Biophotonique et de Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, Illkirch, France

Luca Sigalotti, Cancer Bioimmunotherapy Unit, Department of Medical Oncology, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientifico, Aviano, Italy

Federica Verginelli, Laboratory of Endothelial Cells and Angiogenesis, Ospedale Pediatrico Bambino Gesù, Rome, Italy

Immacolata Vocca, INT–CROM, Pascale Foundation National Cancer Institute—Cancer Research Center, Mercogliano, Avellino, Italy

Paraskevi Vogiatzi, Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, Pennsylvania; Department of Molecular Biology, University of Siena, Siena, Italy

Xiang Wang, Kimmel Cancer Center, Departments of Cancer Biology, Medical Oncology, Microbiology and Immunology, Philadelphia, Pennsylvania

Preface

Epigenetics refers to a variety of processes, such as DNA methylation, histone methylation, and deacetylation, that alter the heritable state of gene expression and chromatin organization without changes in the DNA sequence. Epigenetic mechanisms regulate all biological processes from conception to death by establishing epigenetic marks that modulate the expression of genes involved in the regulation of cellular growth, including genome reprogramming during early embryogenesis and gametogenesis, cell differentiation, apoptosis, survival, and genome integrity. However, although these epigenetic patterns are established early during development and differentiation, modifications occur all through life in response to a variety of intrinsic and environmental stimuli that may lead to disease and cancer.

Cancer Epigenetics: Biomolecular Therapeutics for Human Cancer not only discusses the basic principles of epigenetic mechanisms but also examines the contribution of the epigenetic marks to human health, as well as the clinical consequences of epigenetic errors. The contributors focus on the complex network of epigenetic pathways that control cell growth and on the aberrant epigenetic mechanisms that play an important role in cancer formation and progression. Moreover, they examine the interaction between epigenetic regulation and genetic regulation, and discuss new strategies in the anticancer treatment.

We are grateful to the contributors for their extraordinary and tremendous contribution in writing this book.

Antonio Giordano

Marcella Macaluso

Part I

EPIGENETICS AND CELL CYCLE

Chapter 1

Epigenetic Modulation of Cell Cycle: an Overview

Micaela Montanari and Marcella Macaluso

Sbarro Institute for Cancer Research and Molecular Medicine, College of Science and Technology, Temple University Philadelphia, Pennsylvania

Antonio Giordano

Sbarro Institute for Cancer Research and Molecular Medicine, College of Science and Technology, Temple University Philadelphia, Pennsylvania; Department of Human Pathology and Oncology, University of Siena, Siena, Italy

Marcella Cintorino

Department of Human Pathology and Oncology, University of Siena, Siena, Italy

1.1 Introduction

The progression of the cell cycle is a very finely tuned process that responds to the specific needs of any specific tissue or cell, and is strictly controlled by intrinsic and extrinsic surveillance mechanisms (Giacinti and Giordano, 2006; Montanari et al., 2006; Satyanarayana and Kaldis, 2009). The intrinsic mechanisms appear at every cycle whereas the extrinsic mechanisms only act when defects are detected (Macaluso and Giordano, 2004; Johnson, 2009). The loss of these control mechanisms by genetic and epigenetic alterations leads to genomic instability, accumulation of DNA damage, uncontrolled cell proliferation, and eventually, tumor development. While genetic abnormalities are associated with changes in DNA sequence, epigenetic events alter the heritable state of gene expression and chromatin organization without change in DNA sequence. The most studied epigenetic modifications of DNA in mammals are methylation of cytosine in CpG dinucleotides (DNA methylation), imprinting, posttranslational modification of histones (principally changes in phosphorylation, acetylation, and ubiquitination status), and small RNA-mediated control, specifically miRNAs (Garzon et al., 2009; Kampranis and Tsichlis, 2009; Mendez, 2009; Simon and Kingston, 2009). Important biological processes are regulated by epigenetic mechanisms, including gene reprogramming during early embryogenesis and gametogenesis, cellular differentiation, and maintenance of a committed lineage. Epigenetic marks are established early during development and differentiation; however, modifications occur all through the life in response to a variety of intrinsic and environmental stimuli, which may lead to disease and cancer (Delcuve et al., 2009; Maccani and Marsit, 2009). Although the importance of genetic alterations in cancer has been long recognized, the appreciation of epigenetic changes is more recent. Numerous studies have provided evidence that aberrant epigenetic mechanisms affect the transcription of genes involved in cell proliferation, differentiation, survival, apoptosis, and genome integrity, and play an important role in cancer formation and progression (Humeniuk et al., 2009; Lopez et al., 2009; Toyota et al., 2009).

1.2 Epigenetic and Genetic Alterations of pRb and p53 Pathways

The progression of the cell cycle is tightly monitored by surveillance mechanisms, or cell cycle checkpoints, which ensure that the initiation of a later event is coupled with the completion of an early cell cycle event. The pRb (pRb/p16INK4/Cyclin D1) and p53 (p14ARF/mdm2/p53) pathways are the two main cell cycle control pathways (Fig. 1.1). The importance of these pathways in controlling cellular growth and apoptosis is underscored by many studies, indicating that mutations of the components of these pathways in all human cancers. Almost all human cancers show deregulation of either the pRb or p53 pathway, and often both pathways simultaneously (Macaluso et al., 2006; Yamasaki, 2006; Polager and Ginsberg, 2009).

Figure 1.1 A combinatorial signaling network between pRb (pRb/p16INK4/cyclin D1) and p53 (p14ARF/mdm2/p53) pathways control cell cycle progression through an array of autoregulatory feedback loops where pRb and p53 signals exhibit very intricate interactions with other proteins involved in the determination of cell fate. Loss of cell cycle control by genetic and epigenetic alterations leads to genomic instability, accumulation of DNA damage, uncontrolled cell proliferation, and eventually tumor development. (See insert for color representation of the figure.)

1.1

A combinatorial signaling network between pRb and p53 pathways controls cell cycle progression through an array of autoregulatory feedback loops where pRb and p53 signals exhibit very intricate interactions with other proteins involved in the determination of cell fate (Hallstrom and Nevins, 2009; Polager and Ginsberg, 2009).

Alterations in the pRb and/or p53 pathway converge to reach a common goal: uncontrolled cell cycle progression, cell growth, and proliferation. Then, loss of cell cycle control may lead to hyperplasia and eventually to tumor formation and progression (Sun et al., 2007; Lapenna and Giordano, 2009; Polager and Ginsberg, 2009).

1.2.1 pRb (pRb/p16INK4/Cyclin D1) Pathway

Several studies have documented the role of the pRb pathway, and its family members pRb2/p130 and p107, in regulating the progression through the G1 phase of the mammalian cell cycle (Giacinti and Giordano, 2006; Johnson, 2009; Poznic, 2009). In addition to pRb family proteins, key components of this pathway include the G1 cyclins, the cyclin-dependent kinases (CDKs), and the CDK inhibitors (Lapenna and Giordano, 2009; Poznic, 2009).

Alterations in the signaling network in which pRb, p107, and pRb2/p130 act have been reported in most human cancers. Genetic changes, such as mutations, insertions, and deletions, and also epigenetic alterations, such as promoter hypermethylation, are the most common molecular alterations affecting the function of pRb family proteins. Moreover, it has been reported that inherited allelic loss of pRb confers increased susceptibility to cancer formation (Mastrangelo et al., 2008; Sabado Alvarez, 2008; Poznic, 2009).

Numerous observations have indicated that pRb family proteins interact with a variety of transcription factors and chromatin-modifying enzymes (Brehm et al., 1998; Harbour et al., 1999; Macaluso et al., 2007). Nevertheless, the binding of pRb family proteins with the E2F family of transcription factors appears to be crucial in governing the progression of the cell cycle and the DNA replication by controlling the expression of cell cycle E2F-dependent genes. These genes include CCNE1 (cyclin E1), CCNA2 (cyclin A2), and CDC25A, which are all essential for the entry into the S phase of the cell cycle, and genes that are involved in the regulation of DNA replication, such as CDC6, DHFR, and TK1 (thymidine kinase) (Attwooll et al., 2004; Polager and Ginsberg, 2009). The INK4a/ARF locus (9p21) encodes two unique and unrelated proteins, p16INK4a and p14ARF, which act as tumor suppressors by modulating the responses to hyperproliferative signals (Quelle et al., 1995). One of the most frequent alterations affecting the pRb pathway regulation in cancer involves p16INK4a. Loss of p16INK4a occurs more frequently than loss of pRb, suggesting that p16INK4a suppresses cancer by regulating pRb as well as p107 and pRb2/p130. Loss of function of p16INK4a by gene deletion, promoter methylation, and mutation within the reading frame has frequently been found in human cancers (Sherr and McCormic, 2002). Different studies have indicated that p16INK4A can modulate the activity of pRb and it also seems to be under pRb regulatory control itself (Semczuk and Jacowicki, 2004). p16INK4a blocks cell cycle progression by binding Cdk4/6 and inhibiting the action of D-type cyclins. Moreover, p16INK4a controls cell proliferation through inhibition of pRb phosphorylation, then promotes the formation of pRb-E2Fs repressing complexes, which blocks the G1–S-phase progression of the cell cycle (Zhang et al., 1999). It has been reported that pRb forms a repressor containing histone deacetylase (HDAC) and the hSWI/SNF nucleosome remodeling complex, which inhibits transcription of genes for cyclins E and A, and arrests cells in the G1 phase of the cell cycle (Zhang et al., 2000). Both cyclin D1 overexpression and p16INK4a protein alteration produce persistent hyperphosphorylation of pRb, resulting in evasion of cell cycle arrest. Phosphorylation of pRb by cyclin D/cdk4 disrupts the association of the HDAC-Rb-hSWI/SNF complex, relieving repression of the cyclin E gene and G1 arrest. However, the persistence of Rb-hSWI/SNF complex appears to be sufficient to maintain the repression of the cyclin A and cdc2 genes, inhibiting exit from S phase (Zhang et al., 2000; Beasley et al., 2003). Interestingly, there is evidence that suppression of pRb2/p130, perhaps due to epigenetic alterations, abolishes the G1–S phase block, leads to cyclin A expression, and extends S-phase activity. In addition, it has also been reported that overexpression of p16INK4a or p21 causes accumulation of pRb2/p130 and senescence (Helmbold et al., 2009; Fiorentino et al., 2011). While p16INK4a mutations are not commonly reported, small homozygous deletions are the major mechanism of p16INK4a inactivation in different primary tumors such as glial tumors and mesotheliomas. The INK4a/ARF locus on 9p21 is deleted or rearranged in a large number of human cancers, and germline mutations in the gene have been shown to confer an inherited susceptibility to malignant melanoma and pancreatic carcinoma (Meyle and Guldberg, 2009; Scaini et al., 2009). Interestingly, it has been reported an increased risk of breast cancer in melanoma prone kindreds, owing to the inactivation of p16INK4a, p14ARF or both genes (Prowse et al., 2003). Aberrant methylation of p16INK4a has been reported in a wide variety of human tumors including tumors of the head and neck, colon, lung, breast, bladder, and esophagus (Blanco et al., 2007; Gold and Kim, 2009; Goto et al., 2009; Phe et al., 2009; Xu et al., 2010). Inactivation of the p16INK4a gene by promoter hypermethylation has been frequently reported in approximately 50% of human, non-small-cell lung cancer (NSCLC) (Zhu et al., 2006). Moreover, p16INK4a loss in preneoplastic lesions occurred exclusively in patients who also showed loss of p16INK4a expression in their related invasive carcinoma, indicating that p16INK4a may constitute a new biomarker for early diagnosis of this disease (Brambilla et al., 1999; Beasley et al., 2003).

Deregulated tumor expression of p16INK4a has been described in association with clinical progression in sporadic colorectal cancer (CRC) patients (McCloud et al., 2004). p16INK4a hypermethylation has been shown to occur in advanced colorectal tumors and has been associated with patient survival (Cui et al., 2004). Significant correlation has also been reported between aberrant p16INK4a methylation and Dukes' stage and lymphatic invasion in colorectal carcinoma (Goto et al., 2009). Although the inactivation of p16INK4a seems to be a crucial event in the development of several human tumors, the relevance of this alteration in mammary carcinogenesis remains unclear. For example, p16INK4a homozygous deletions have been reported in 40–60% of breast cancer cell lines, while both homozygous deletions and point mutations are not frequently observed in primary breast carcinoma, suggesting that these alterations might have been acquired in culture (Silva et al., 2003). In addition, p16INK4a hypermethylation has been reported in breast carcinoma, although the relevance of this p16INK4a alteration is discordant among different studies (Lehmann et al., 2002; Tlsty et al., 2004). Interestingly, although methylation of p16INK4a promoter is common in cancer cells, it has been reported that epithelial cells from histologically normal-appearing mammary tissue of a significant fraction of healthy women show p16 promoter methylation as well (Holst et al., 2003; Bean et al., 2007). However, a recent study indicates a strong association between aberrant p16INK4a methylation and breast-cancer-specific mortality (Xu et al., 2010). Cyclin alteration represents one of the major factors leading to cancer formation and progression. Evidence indicates that a combination of cyclin/cdks, rather than a single kinase, executes pRb phosphorylation and at specific pRb-phosphorylation sites (Mittnacht, 2005). Moreover, it has been reported that the activation of the mitogen-activated protein kinase (MAPK) leads to pRb inactivation by sustaining cyclin levels and consequently activating CDKs (Hansen et al., 2009). Constitutive cell surface kinase receptors and persistent phosphorylation/inactivation of pRb, p107, and pRb2/p130 proteins have been implicated in conferring uncontrolled growth to melanoma cells (von Willbrand et al., 2003). A statistically significant difference has been reported in the expression profiles of p16, cyclin D1, and pRb between naevi and melanomas, with decreased, increased, and increased expression in the melanomas, respectively, supporting the hypothesis that cell cycle checkpoint proteins of G1/S transition are critical in the pathogenesis of melanoma (Karim et al., 2009). Moreover, overexpression of cyclin D1 has been found in a wide variety of cancers, including breast carcinoma, endocrine pancreatic tumors, multiple myeloma, mantle cell lymphoma, colon cancer, and various sarcomas (Kim and Diehl, 2009). The mechanisms altering the pRb pathway converge to reach a common goal: uncontrolled expression of key regulators that trigger, even in the absence of growth signals, an irreversible transition into the S phase and cell cycle progression. It is important to underscore that alterations affecting the components of pRb pathway often occur in a mutually exclusive manner, in that one alteration is unaccompanied by others. Moreover, the frequency of particular genetic and epigenetic events varies among tumor types.

1.2.2 p53 (p14ARF/mdm2/p53) Pathway

The tumor suppressor gene p53 is a key regulator of cell cycle checkpoints, which is activated in response to virtually all cancer-associated stress signals, including DNA damage and oncogene activation. Once activated, p53 can trigger several cellular responses, including growth arrest, apoptosis, and senescence. (Junttila and Evan, 2009; Menendez et al., 2009). The key role of p53 in tumor suppression is demonstrated by the prevalence of TP53 gene mutations in cancer: mutations of this gene occur in more than 50% of all human cancers (Vousden and Prives, 2009). Moreover, because p53 is the most frequently mutated gene in human cancer, it appears to be a crucial target for therapy with respect to tumor formation and elimination of the tumor cell (Portugal et al., 2009).

The p53 (p14ARF/mdm2/p53) pathway appears to play a major role in mediating oncogene-induced apoptosis; therefore, the suppression of apoptosis by inactivation of this pathway has an important role in tumor development (Menendez et al., 2009). The check and balance existing between the pRb (pRb/p16INK4/Cyclin D1) and p53 (p14ARF/mdm2/p53) pathways involves the regulation of the G1 to S transition and its checkpoints. This network consists of, but is not limited to, an array of autoregulatory feedback loops, where pRb and p53 signals exhibit very intricate interactions with other proteins known to exert important roles in the determination of cell fate (Junttila and Evan, 2009; Polager and Ginsberg, 2009). p53 is activated in response to DNA damage, cellular stress, and ultraviolet irradiation, and the turnover of this protein is regulated by ubiquitination through mdm2 binding, which leads to p53 degradation by proteosomes. Moreover, p53 activates E3 ubiquitin ligase mdm2 transcription, ensuring a negative feedback regulation (Xin, 2005). Furthermore, it has been recently reported that mdm2 inhibits TP53 mRNA translation (Ofir-Rosenfeld et al., 2008). In tumors lacking p53 gene mutations, p53 function is often abrogated indirectly through the overexpression of mdm2 or the inactivation of the cell cycle inhibitor p14ARF (also known as p19 in rodents). p14ARF interferes with all the known functions of mdm2 and it has been shown that p14ARF binds to the mdm2–p53 complex, resulting in a stabilization of both proteins (Moule et al., 2004).

Significantly, p14ARF expression is positively regulated by members of the E2F family of transcription factors. This observation provides a link between the pRb (pRb/p16INK4/Cyclin D1) and p53 (p14ARF/mdm2/p53) pathways, suggesting a mechanism whereby the loss of function of pRb proteins leads to deregulation or hyperactivation of E2Fs, resulting in the functional inactivation of p53. These concurrent alterations have been observed in a wide range of human tumors, highlighting the crucial role of pRb (pRb/p16INK4/Cyclin D1) and p53 (p14ARF/mdm2/p53) pathways in oncogenesis in general (Polager and Ginsberg, 2009). p53 also activates the transcription of p21Cip/Kip, which is largely responsible for the p53-dependent G1 arrest in response to different cellular stress and DNA damage (Sherr, 2004). p21Cip/Kip regulates cyclin E/Cdk2 and cyclin A/Cdk2 complexes, both of which phosphorylate pRb, contributing to an irreversible transition into the S phase and cell cycle progression even in the absence of growth signals. Deletion inactivation of p14ARF has been reported in human cancers, but in these studies p16INK4a was always codeleted (Fulci et al., 2000; Newcomb et al., 2000; Sarkar et al., 2000). Only germline deletion of p14ARF-specific exon 1b in a family characterized by multiple melanoma and neural cell tumors has been reported (Randerson-Moor et al., 2001). Different studies have reported that epigenetic alterations such as CpG hypermethylation may be the first cause of p14ARF gene silencing, followed by p14ARF loss of heterozygosity (LOH) and homozygous deletions. p14ARF hypermethylation has been detected in several tumors including primary colorectal, breast, gastric, and lung tumors (Furonaka et al., 2004; Sharma et al., 2007; Zhao et al., 2007; Kominami et al., 2009).

1.3 Conclusion

The intricate crosstalk of signals connecting pRb (pRb/p16INK4/cyclin D1) and p53 (p14ARF/mdm2/p53) pathways is crucial in regulating cell cycle progression and viability. Genetic and epigenetic alterations disturbing this crosstalk appear to be a common part of the life history of human cancers, independent of age or tumor type. Data accumulated over the past years clearly indicate that although pRb and p53 pathways are each typically deregulated in human cancer, they do not function independently but through a complex network of communicating signals. Understanding the complex molecular mechanisms that regulate cell cycle progression and are involved in tumor formation and progression still remains the most important goal in cancer research. Indeed, an increased knowledge of the alterations in pRb and p53 pathways will be useful in improving anticancer treatments. Importantly, progress over the past years has greatly enhanced our understanding of the epigenetic mechanisms affecting the action of cell cycle key regulators and leading to cancer formation and progression, thus offering important tools for the diagnosis and prevention of this disease.

Acknowledgments

We would like to thank the Sbarro Health Research Organization (SHRO) for its support, and our colleagues who have done studies in this field. We apologize to those whose work were not directly cited in this chapter due to space limitation.

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Part II

EPIGENETICS AND CELL DEVELOPMENT, SENESCENCE AND DIFFERENTIATION

Chapter 2

Epigenetics in Skeletal Muscle Development

Cristina Giacinti

DAHFMO, University of Rome La Sapienza, Rome, Italy; Sbarro Institute for Cancer Research and Molecular Medicine, Temple University Philadelphia, Pennsylvania

Antonio Giordano

Department of Human Pathology and Oncology, University of Siena, Siena, Italy; Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, Pennsylvania; Oncology Research Centre of Mercogliano (CROM), Avellino, Italy

2.1 Introduction

The genetic programs that control the processes by which the body plans of animals are built were invented, and shaped, by evolution. How these programs work is a matter of great curiosity. Because gene networks constitute the control systems for development, analysis of such networks explains both the process of development and the process by which development has evolved (1). Induction-dependent changes in gene expression patterns invariably determine the fate of cells. A question of general importance for embryonic induction arises from the pleiotropic nature of these signals: how do they specify the stage- and tissue-specific expression patterns of the myogenic genes, given their functional cooperation at many different times and places in the embryo?

2.2 Epigenetics

As cells inherit genes, they also inherit a set of instructions that tell the genes when to become active, in which tissue, and to what extent. Without this epigenetic instruction manual, multicellular organisms would be impossible (1). For most developmental biologists, the term epigenesis describes the sum of processes by which a multicellular organism develops from a zygote, including morphogenesis, cell diversification, and pattern formation. This process explicates through a coordinate expression of genes. The term epigenesis, in its restricted sense, refers to mechanisms that regulate activation and repression of gene transcription. We refer to epigenetic events, looking at such more detailed subcellular mechanisms that modulate transcriptional activity as histone modification through acetylation and deacetylation, methylation and demethylation of DNA, and chromatin remodeling phenomena (2, 3).

2.2.1 Myogenic Regulatory Factors

In the developing chordate, skeletal muscle differentiation begins shortly after gastrulation and persists, in some respects, through the entire life span of the animal (4). Most embryonic skeletal myogenic progenitors, with the exception of some craniofacial and esophageal muscles, are formed from somites, which are transient condensations of the paraxial mesoderm (5).

Segmentation of the paraxial mesoderm into ball-like structures, known as somites, occurs along the dorsal–ventral axis and in a rostral to caudal direction. In response to signals from the notochord and the neural tube, the somites differentiate and subdivide to give rise to the dorsally located epithelial dermomyotome and the ventrally located mesenchymal sclerotome. The dermomyotome gives rise to the dermis and the skeletal muscle of the trunk and limbs, whereas the sclerotome develops into the cartilage and bone of the vertebrae and ribs (4).

The specification, proliferation, and terminal differentiation of the skeletal muscle cell are controlled by the combinatorial activities of several transcription factors, the myogenic regulatory factors (MRFs)—MyoD, Myf5, myogenin, and Mrf4 (Myf6). The MRFs share a homologous bHLH domain that is required for DNA binding and dimerization with the E-protein family of transcription factors. MRF–E-protein heterodimers and MRF monomers bind to the consensus E-box sequence CANNTG, which is found in the promoters of many muscle-specific genes. The DNA binding and transcriptional activity of these dimers is highly regulated by several protein–protein interactions and extrinsic cues (2). Among these, the MRFs, the myocyte enhancer factor-2 (Mef2) family of transcription factors are involved in the activation of muscle-specific gene expression in the mouse (6) and in Drosophila.

Among the MRFs, MyoD and Myf5 are required for commitment to the myogenic lineage, whereas myogenin plays a critical role in the expression of the terminal muscle phenotype previously established by MyoD and Myf5, and MRF4 partly subserves both roles. Thus, MyoD and Myf5, and to an extent, MRF4, can be considered commitment or specification factors, whereas myogenin is a differentiation factor, and MRF4 has aspects of both functions.

2.2.1.1 Pax Functions Upstream of the MRFs

Although MyoD and Myf5 define the identity of the skeletal myoblast, somitic precursors might be precommitted to the myogenic lineage before MRF expression. In the embryo, the paired-box transcription factor Pax3 is expressed in the presomitic mesoderm and early epithelial somites (7). In Pax3-deficient splotch mice, the limb and diaphragm muscles are not formed because of defective lateral migration and reduced proliferation in the dermomyotome (8). Pax7, a paralogue of Pax3, is not expressed in the presomitic mesoderm but it is induced during somite maturation. Interestingly, Pax7 is dispensable during embryonic myogenesis; however, its activity is essential for postnatal muscle formation (9).

2.2.1.1.1 Epigentic Mechanisms in Muscle Differentiation

MyoD and Myf5, and E-proteins are expressed in undifferentiated myoblasts; yet, in this cellular context, they do not activate transcription. Undifferentiated muscle precursors indeed maintain the myogenic lineage while proliferating, and finally enter the differentiation program to give room to terminally differentiated cells. Terminal differentiation is characterized by the sequential activation of different subsets of muscle-specific genes (10) and the silencing of genes involved in cell cycle progression. Correlation of the genetic mechanisms of skeletal myogenesis with epigentic regulatory inputs reveals that muscle precursor cells need to modify the chromatin structure in many ways in order to become differentiated muscle cells.

2.2.1.1.2 MRFs Genes Transcription Repression

The finding that MyoD and MyF5 are expressed in undifferentiated muscle cells and that they transiently bound to the promoter of muscle genes.

During cell proliferation, all the E-proteins are sequestered from Id and they cannot heterodimerize with MyoD. Instead, Myod is transiently bound to a series of regulatory regions of muscle genes as homodimer, and it links on a noncanonical E-boxes sequence with low efficiency (11). MyoD seems to be recruited on the promoter of muscle genes through its particular ability to interact with the homeodomain protein PBX, which is constitutively bound to the chromatin of the myogenin promoter. This configuration of MyoD as homodimer facilitates the recruitment of the histone deacetylase protein on the promoter of muscle genes and facilitates gene transcription repression (12).

2.2.1.2 Methylation

The most abundant modification of vertebrate genomes is the methylation of cytosine at the CG dinucleotides. DNA methylation is dynamically regulated during embryonic development and plays a role in the stable repression of gene expression through nucleosomal histone deacetylation, silencing of transposable elements, and genomic imprinting in mammals (13).

A potential link between methylation and myogenesis was provided early on by the observation that 5-azacytidine treatment (which inhibits CpG methylation) converts 10T1/2 mouse embryonic fibroblasts at high frequency to muscle cells (13). Whether transcription of this master control gene is normally regulated by DNA methylation has remained controversial because the CpG island of the MyoD promoter is constitutively unmethylated in vivo (14). However, this possibility has been revived by the identification of a conserved distal enhancer element, which mediates the primary induction of human and mouse myoD genes in development, and by the subsequent finding that this enhancer undergoes a regulated demethylation in some somitic cells before the myoD gene is activated (14).

In undifferentiated myoblasts, the muscle regulatory regions contain the Polycomb group protein enhancer of zeste (Ezh2), a histone lysine methyltransferase (HKMT) that promotes transcriptional repression (15). Interestingly, Ezh2 is recruited to the chromatin of muscle regulatory regions via interaction with YY1, which recognizes the CarG-box motifs present in promoter regions of muscle genes (15). Further association with HDAC1 forms a repressive complex, which ensures repression of transcription and prevents MyoD–E complex binding. At the onset of differentiation, the simultaneous downregulation of Ezh2 and HDAC1 proteins and the replacement of YY1 with SRF allow the binding of MyoD–E12/47 and the recruitment of the positive coactivators to form an active myogenic transcriptosome (16).

2.2.1.3 Histone Deacetylation

Histone hypoacetylation is often associated to gene repression. In myoblasts, muscle-gene expression is silenced by the interaction between MRFs and MEF2 proteins with nuclear deacetylases (HDACs) (17, 18). Three distinct families of HDACs (19) play an important role in keeping the inactive state of muscle regulatory regions in proliferating myoblasts. Of them, class I HDACs associate with MyoD in undifferentiated myoblasts, and this association is disrupted upon induction of differentiation (17, 18, 20). HDAC1 acts also on MyoD so that MyoD, in its hypoacetylated form, is less efficient in linking DNA. In the myogenin promoter, MEF2 proteins associate with class II HDACs 4, 5, 7, and 9, leading to chromatin condensation via histone deacetylation and recruitment of corepressory complexes, such as heterochromatin protein 1 (HP1) and associated methyltransferases, which promote H3 lysine 9 methylation (18). Furthermore, class II HDACs potentiate SUMO2- and 3-dependent sumoylation at the C-terminal activation domain of MEF2D and MEF2C, leading to the inhibition of transcription (21).

An indirect action of mitogen-activated cyclin/cdks can be envisioned via hyperphosphorylation of pRb, which prevents interactions with class I HDACs, thereby favoring MyoD–HDAC1 association in myoblasts. It is still not known if all Rb in differentiated myotubes is inactive or in association with the histone deacetylase transcriptional