review article T h e new engl and journal o f medicine n engl j med 358 11 www.nejm.org march 13, 2008 1148 Molecular Origins of Cancer Epigenetics in Cancer Manel Esteller, M.D., Ph.D. From the Cancer Epigenetics Laboratory, Spanish National Cancer Research Center, Madrid. Address reprint requests to Dr. Esteller at the Cancer Epigenetics Labo- ratory, Spanish National Cancer Research Center, Melchor Fernandez Almagro 3, 28029 Madrid, Spain, or at mesteller@ cnio.es. N Engl J Med 2008 358:1148-59. Copyright �� 2008 Massachusetts Medical Society. Cidentical lassic genetics alone cannot explain the diversity of pheno- types within a population. Nor does classic genetics explain how, despite their DNA sequences, monozygotic twins1 or cloned animals2 can have different phenotypes and different susceptibilities to a disease. The concept of epi- genetics offers a partial explanation of these phenomena. First introduced by C.H. Waddington in 1939 to name ���the causal interactions between genes and their prod- ucts, which bring the phenotype into being,���3 epigenetics was later defined as heri- table changes in gene expression that are not due to any alteration in the DNA se- quence.4 The best-known epigenetic marker is DNA methylation. The initial finding of global hypomethylation of DNA in human tumors5 was soon followed by the identifi- cation of hypermethylated tumor-suppressor genes,6-11 and then, more recently, the discovery of inactivation of microRNA (miRNA) genes by DNA methylation.12,13 These and other demonstrations of how epigenetic changes can modify gene expression have led to human epigenome projects14 and epigenetic therapies.15 Moreover, we now know that DNA methylation occurs in a complex chromatin network and is influ- enced by the modifications in histone structure that are commonly disrupted in cancer cells.16-19 Epigenetic research uses powerful techniques for the study of DNA methylation, such as sodium bisulfite modification associated with polymerase-chain-reaction pro- cedures.20,21 Terms used in epigenetic research are defined in the Glossary. Compre- hensive epigenomic techniques22 have yielded preliminary descriptions of the epi- genomes of human cancer cells.23-25 This review summarizes new developments concerning hypermethylation of the promoter regions of tumor-suppressor genes26 and describes possible applications of epigenetics to the treatment of patients with cancer. Epigenetic Features of a Normal Cell DNA methylation has critical roles in the control of gene activity and the architecture of the nucleus of the cell. In humans, DNA methylation occurs in cytosines that pre- cede guanines these are called dinucleotide CpGs.26,27 CpG sites are not randomly distributed in the genome instead, there are CpG-rich regions known as CpG islands, which span the 5��� end of the regulatory region of many genes. These islands are usu- ally not methylated in normal cells.26,27 The methylation of particular subgroups of promoter CpG islands can, however, be detected in normal tissues. DNA methylation is one of the layers of control of certain tissue-specific genes, such as MASPIN, a member of the serum protease inhibitor family,28 and germ-line genes such as the MAGE genes, which are silent in almost all tissues except malig- nant tumors.29 Genomic imprinting also requires DNA hypermethylation at one of the two parental alleles of a gene to ensure monoallelic expression,30 and a similar gene-dosage reduction is involved in X-chromosome inactivation in females.31 The Copyright �� 2008 Massachusetts Medical Society. All rights reserved. Downloaded from www.nejm.org at CAUL on August 26, 2009 .

Molecular Origins of Cancer n engl j med 358 11 www.nejm.org march 13, 2008 1149 hypermethylation of repetitive genomic se- quences probably prevents chromosomal insta- bility, translocations, and gene disruption caused by the reactivation of transposable DNA sequenc- es.32 Cells that lack the stabilizing effect of DNA methylation because they have spontaneous de- fects in DNA methyltransferases (DNMTs)33 or experimentally disrupted DNMTs34 have promi- nent nuclear abnormalities. DNA methylation occurs in the context of chemical modifications of histone proteins.35 His- tones are not merely DNA-packaging proteins, but molecular structures that participate in the regu- lation of gene expression. They store epigenetic information through such post-translational mod- ifications as lysine acetylation, arginine and lysine methylation, and serine phosphorylation. These modifications affect gene transcription and DNA repair. It has been proposed that distinct histone modifications form a ���histone code.���36 Acetylation of histone lysines, for example, is generally associ- ated with transcriptional activation.15,16 The func- tional consequences of the methylation of histones depends on the type of residue ��� lysine (K) or arginine ��� and the specific site that the methyla- tion modifies (e.g., K4, K9, or K20).15,16 Methyla- tion of H3 at K4 is closely linked to transcriptional activation,37 whereas methylation of H3 at K9 or K27 and of H4 at K20 is associated with transcrip- tional repression. What emerges from these find- ings is a flexible but precise pattern of DNA meth- ylation and histone modification that is essential for the physiologic activities of cells and tissues. DNA Hypomethylation in Tumors The low level of DNA methylation in tumors as compared with the level of DNA methylation in their normal-tissue counterparts was one of the first epigenetic alterations to be found in human cancer.5 The loss of methylation is mainly due to hypomethylation of repetitive DNA sequences and demethylation of coding regions and introns ��� regions of DNA that allow alternative versions of the messenger RNA (mRNA) that is transcribed from a gene.38 A recent large-scale study of DNA methylation with the use of genomic microarrays has detected extensive hypomethylated genomic re- gions in gene-poor areas.24 During the develop- ment of a neoplasm, the degree of hypomethylation of genomic DNA increases as the lesion progresses from a benign proliferation of cells to an invasive cancer39 (Fig. 1). Three mechanisms have been proposed to ex- Glossary Acetylation: A reaction that introduces a functional acetyl group into an organic compound. Deacetylation is the remov- al of the acetyl group. Acetylation is a post-translational chemical modification of histones, tubulins, and the tumor suppressor p53. Bisulfite sequencing: The bisulfite treatment of DNA in order to determine its pattern of methylation. Treatment of DNA with bisulfite converts cytosine residues to uracil but leaves 5-methylcytosine residues unaffected. Chromatin: The complex of DNA and protein that composes chromosomes. Chromatin packages DNA into a volume that fits into the nucleus, allows mitosis and meiosis, and controls gene expression. Changes in chromatin struc- ture are affected by DNA methylation and histone modifications. CpG islands: Regions in DNA that contain many adjacent cytosine and guanine nucleotides. The ���p��� in CpG refers to the phosphodiester bond between the cytosine and the guanine. These islands occur in approximately 40% of the promoters of human genes. DNA methylation: The addition of a methyl group to DNA at the 5-carbon of the cytosine pyrimidine ring that precedes a guanine. DNA methyltransferases: Family of enzymes that catalyze the transfer of a methyl group to DNA, using S-adenosyl- methionine as the methyl donor. Epigenome: The overall epigenetic state of a cell. Genomic imprinting: The epigenetic marking of a locus on the basis of parental origin, which results in monoallelic gene expression. Histone: The main protein components of chromatin. The core histones ��� H2A, H2B, H3, and H4 ��� assemble to form the nucleosome each nucleosome winds around 146 base pairs of DNA. The linker histone H1 locks the DNA into place and allows the formation of a higher-order structure. Histone deacetylase: A class of enzymes that remove acetyl groups from an N-acetyl-lysine amino acid on a histone. Transposons: Sequences of DNA that can move around within the genome of a single cell. In this process, called trans- position, the sequences can cause mutations and change the organization of DNA in the genome. Copyright �� 2008 Massachusetts Medical Society. All rights reserved. Downloaded from www.nejm.org at CAUL on August 26, 2009 .