Title: Epigenetic Modifications in Breast Cancer and Their Role in Personalized Medicine
Abstract: In cancer, the overall patterns of epigenetic marks are severely distorted from the corresponding normal cell type. It is now well established that these changes can contribute to cancer development through inactivation of tumor suppressor genes and, conversely, through activation of oncogenes. Recent technological advances have enabled epigenome-wide analyses of cancers that are yielding unexpected findings. The study of cancer epigenetics holds great promise for expanding the range of therapeutic opportunities for personalized medicine. Here, we focus on DNA methylation in breast cancer and the potential implications for clinical management of patients. In cancer, the overall patterns of epigenetic marks are severely distorted from the corresponding normal cell type. It is now well established that these changes can contribute to cancer development through inactivation of tumor suppressor genes and, conversely, through activation of oncogenes. Recent technological advances have enabled epigenome-wide analyses of cancers that are yielding unexpected findings. The study of cancer epigenetics holds great promise for expanding the range of therapeutic opportunities for personalized medicine. Here, we focus on DNA methylation in breast cancer and the potential implications for clinical management of patients. CME Accreditation Statement: This activity ("ASIP 2013 AJP CME Program in Pathogenesis") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity ("ASIP 2013 AJP CME Program in Pathogenesis") for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity ("ASIP 2013 AJP CME Program in Pathogenesis") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity ("ASIP 2013 AJP CME Program in Pathogenesis") for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Breast cancer is the most common cancer among women and ranks among the top five leading causes of cancer-related deaths, according to the World Health Organization (http://www.who.int/mediacentre/factsheets/fs297/en; reviewed January 1, 2013). Inherited and acquired mutations in genetic material are known to be important contributors to the development of breast cancer. Indeed, family history is the strongest risk factor for developing breast cancer, for which germline mutations in the BRCA1, BRCA2, and TP53 (alias p53) genes are known to be high-risk factors.1Lalloo F. Evans D.G. Familial breast cancer.Clin Genet. 2012; 82: 105-114Crossref PubMed Scopus (76) Google Scholar Several other inherited mutations and genetic variations have been identified as risk factors confirmed by independent researchers, although their effects are estimated to be either moderate or low.2Fanale D. Amodeo V. Corsini L.R. Rizzo S. Bazan V. Russo A. Breast cancer genome-wide association studies: there is strength in numbers.Oncogene. 2012; 31: 2121-2128Crossref PubMed Scopus (0) Google Scholar Soon after the discovery that BRCA1 and BRCA2 are high-risk breast cancer susceptibility genes, functional studies consistently identified their gene products as components critical to the repair of double-stranded DNA breaks (DSBs).3Gudmundsdottir K. Ashworth A. 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Methylation of the BRCA1 promoter is associated with decreased BRCA1 mRNA levels in clinical breast cancer specimens.Carcinogenesis. 2000; 21: 1761-1765Crossref PubMed Google Scholar This finding has been confirmed in multiple independent studies and, given that BRCA1 is a well-known susceptibility gene, the finding strongly supports the hypothesis that epigenetic modifications, as well as genetic mutations, contribute to the development of breast cancer.14Esteller M. Silva J.M. Dominguez G. Bonilla F. Matias-Guiu X. Lerma E. Bussaglia E. Prat J. Harkes I.C. Repasky E.A. Gabrielson E. Schutte M. Baylin S.B. Herman J.G. Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors.J Natl Cancer Inst. 2000; 92: 564-569Crossref PubMed Google Scholar, 15Catteau A. Harris W.H. Xu C.F. Solomon E. 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In this review, we focus on DNA methylation in breast cancer and discuss potential implications for clinical practice. The genetic material is organized within the nucleus by the DNA helix wrapping around histone proteins. The structural organization of this DNA–histone complex, known as chromatin, is regulated by epigenetic factors involving DNA methylation and various types of histone marks and noncoding RNAs.21Baylin S.B. Jones P.A. A decade of exploring the cancer epigenome–biological and translational implications.Nat Rev Cancer. 2011; 11: 726-734Crossref PubMed Scopus (0) Google Scholar The term epigenetics refers to heritable states of gene expression that are not attributed to the DNA sequence. DNA methylation, a well-known epigenetic mark, occurs at cytosine residues where cytosine (C) precedes a guanine (G) residue, known as CpG dinucleotides (where p stands for the phosphodiester bond connecting cytosine and guanine).22Jones P.A. Functions of DNA methylation: islands, start sites, gene bodies and beyond.Nat Rev Genet. 2012; 13: 484-492Crossref PubMed Scopus (1503) Google Scholar The distribution of CpGs is not random; genomic regions enriched in CpGs, known as CpG islands, are often found at gene promoter sequences. CpG islands characterize the promoter region of more than half of all genes in the human genome.23Antequera F. Bird A. Number of CpG islands and genes in human and mouse.Proc Natl Acad Sci USA. 1993; 90: 11995-11999Crossref PubMed Scopus (0) Google Scholar It is thought that the overall reduction in genomic CpGs has occurred over evolutionary time and that it relates to the spontaneous or enzymatic deamination of methylated cytosine residues in the germline and thereby conversion to thymine. Transcriptionally active genes are depleted in DNA methylation at their gene promoter CpG islands and, in this case, the flanking nucleosomes are often marked with trimethylation at histone H3 on lysine residue K4, known as the H3K4Me3 mark, while also containing the histone variant H2A.Z and acetylated lysine residues on histones H3 and H4. These features are thought to reduce the formation of nucleosomes, thereby leading to a stable nucleosome-depleted region that is characteristic of actively transcribed genes (Figure 1). It is now becoming clear that DNA demethylation can be achieved through the activity of the TET enzymes (ten-eleven methylcytosine dioxygenases) which convert methylated cytosines into hydroxymethylated cytosines (5hmC).24Williams K. Christensen J. Helin K. 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Robert F. Alberdi A. Lécluse Y. Plo I. Dreyfus F.J. Marzac C. Casadevall N. Lacombe C. Romana S.P. Dessen P. Soulier J. Viguié F. Fontenay M. Vainchenker W. Bernard O.A. Mutation in TET2 in myeloid cancers.N Engl J Med. 2009; 360: 2289-2301Crossref PubMed Scopus (973) Google Scholar In normal cells, CpG island methylation occurs infrequently and affects only a small number of autosomal genes, of which most are involved in developmental processes. Our research group confirmed CpG island methylation by DNA methylation profiling of 424 normal human tissue samples of various types, and further showed that CpG sites located at the non-CpG island 5′ ends best discriminate tissue-specific differences in terms of DNA methylation.26Fernandez A.F. Assenov Y. Martin-Subero J.I. Balint B. Siebert R. Taniguchi H. et al.A DNA methylation fingerprint of 1628 human samples.Genome Res. 2012; 22: 407-419Crossref PubMed Scopus (146) Google Scholar Indeed, in that study the different tissue types in the human body were shown to have clear differences in their epigenome-wide DNA methylation patterns. DNA methylation, especially at CpG islands, is generally thought to involve long-term silencing of genes such as those on the inactive X chromosome, imprinted genes, and genes expressed only in germ cells.22Jones P.A. Functions of DNA methylation: islands, start sites, gene bodies and beyond.Nat Rev Genet. 2012; 13: 484-492Crossref PubMed Scopus (1503) Google Scholar It is thought that CpG island gene promoter methylation functions to stabilize gene silencing after histone modifications. In this sense, repressive histone marks are applied before CpG island methylation, in what has sometimes been referred to as the locking model of epigenetic gene silencing. The intragenic regions of transcribed genes (ie, gene bodies) exhibit CpG methylation but are marked with histone H3 trimethylation at K36. The differential methylation between promoter regions and gene bodies supports the emerging view that the position of CpG methylation within the promoter is critical to understanding the relationship of methylation and gene expression activity. For example, CpG island promoter methylation tends to block transcription initiation, whereas gene-body methylation does not.22Jones P.A. Functions of DNA methylation: islands, start sites, gene bodies and beyond.Nat Rev Genet. 2012; 13: 484-492Crossref PubMed Scopus (1503) Google Scholar The function of gene-body methylation is not clear, although it may perhaps protect against major sources of mutagens such as endogenous reactive oxygen species and suppress intragenic retrotransposons such as LINE1 or Alu elements. Nevertheless, the presence of gene-body methylation comes at the price of the aforementioned deamination of methylated CpGs, which increases the risk of genetic mutations from cytosine to thymine residues. This type of mutation is commonly seen in known cancer genes such as TP53 and is therefore thought to be an important mechanism by which mutations arise and sometimes contribute to the development of cancer.27Nik-Zainal S. Alexandrov L.B. Wedge D.C. Van Loo P. Greenman C.D. Raine K. et al.Breast Cancer Working Group of the International Cancer Genome ConsortiumMutational processes molding the genomes of 21 breast cancers.Cell. 2012; 149: 979-993Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar Recent data have demonstrated that the H3K9Me3 repressive histone marker, a marker of constitutive heterochromatin, is associated with increased mutation density in various types of cancers.28Schuster-Böckler B. Lehner B. Chromatin organization is a major influence on regional mutation rates in human cancer cells.Nature. 2012; 488: 504-507Crossref PubMed Scopus (211) Google Scholar This finding reinforces the notion that the epigenetic organization of the human genome can influence the rate of acquired mutations relevant to the development of cancer. In the development of cancer, epigenetic mechanisms are important in terms of both silencing of tumor suppressor genes and activation of oncogenes.4Esteller M. Epigenetics in cancer.N Engl J Med. 2008; 358: 1148-1159Crossref PubMed Scopus (2034) Google Scholar Both silencing and activation occur through changes in chromatin configuration by which the accessibility of transcription factors is affected, with consequences for gene expression. In breast cancer, tumor suppressor genes such as BRCA1, CDKN2A, and PTEN undergo CpG island promoter methylation, but in normal cells the promoter region is unmethylated. The functional roles of genes inactivated by epigenetic mechanisms in breast cancer and other types of cancers are diverse and reflect various cancer hallmarks (Table 1). Hon et al40Hon G.C. Hawkins R.D. Caballero O.L. Lo C. Lister R. Pelizzola M. Valsesia A. Ye Z. Kuan S. Edsall L.E. Camargo A.A. Stevenson B.J. Ecker J.R. Bafna V. Strausberg R.L. Simpson A.J. Ren B. Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer.Genome Res. 2012; 22: 246-258Crossref PubMed Scopus (229) Google Scholar recently described a novel mechanism of epigenetic gene inactivation through hypomethylation of gene bodies without involving CpG island promoter hypermethylation (discussed below).Table 1Selected Examples of CpG Island Promoter Hypermethylated Genes in Breast Cancer, Demonstrating Diverse Biological ImplicationsGene symbolGene nameKnown functionReferencesBRCA1Breast cancer 1, early onsetDNA damage response13Rice J.C. Ozcelik H. Maxeiner P. Andrulis I. Futscher B.W. Methylation of the BRCA1 promoter is associated with decreased BRCA1 mRNA levels in clinical breast cancer specimens.Carcinogenesis. 2000; 21: 1761-1765Crossref PubMed Google Scholar, 14Esteller M. Silva J.M. Dominguez G. Bonilla F. Matias-Guiu X. Lerma E. Bussaglia E. Prat J. Harkes I.C. Repasky E.A. Gabrielson E. Schutte M. Baylin S.B. Herman J.G. Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors.J Natl Cancer Inst. 2000; 92: 564-569Crossref PubMed Google Scholar, 16Birgisdottir V. Stefansson O.A. Bodvarsdottir S.K. Hilmarsdottir H. Jonasson J.G. Eyfjord J.E. Epigenetic silencing and deletion of the BRCA1 gene in sporadic breast cancer.Breast Cancer Res. 2006; 8: R38Crossref PubMed Scopus (156) Google ScholarCDH1∗CDH1 has been reclassified as fizzy/cell division cycle 20 related 1 (Drosophila) (FZR1).Cadherin 1, type 1, E-cadherin (epithelial)Cell-to-cell adhesion29Esteller M. Fraga M.F. Guo M. Garcia-Foncillas J. 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