Overview of Cancer Epigenetics Peter A. Jones Epigenetic mechanisms including DNA and histone modifications result in silencing of genes without changing the coding sequence of the gene. Even though these events are heritable, they are potentially reversible, thus opening up opportunities for therapeutic intervention. The importance of epigenetic changes in human cancer is only now being recognized in the medical community. A series of discoveries over the last four decades has thrust epigenetics into the forefront of new drug discoveries. Three systems—DNA methylation, RNA-associated silencing, and histone modification—are used to initiate and sustain epigenetic silencing. Current knowledge suggests that agents that intervene in this process by “turning back on” silenced genes may represent a significant advancement in treating many forms of cancer. In addition, changed patterns of methylation can be detected with a high degree of sensitivity thus providing clinicians with prognostic information. Semin Hematol 42:S3-S8 © 2005 Elsevier Inc. All rights reserved.
he term “epigenetics” refers to all meiotically and mitotically heritable changes in gene expression that are not coded in the DNA sequence. Three systems—DNA methylation, RNAassociated silencing, and histone modification—are used to initiate and sustain epigenetic silencing. There are interactions between these three systems and they may act to stabilize one another. A disruption in one of these systems can lead to inappropriate expression or silencing of genes, resulting in epigenetic diseases such as cancer. A distinguishing feature between epigenetic changes and genetic changes is that the former tend to occur in a gradual rather than abrupt fashion. Even though the events are heritable, they are potentially reversible and thus open up opportunities for therapeutic intervention. However, therapeutic interventions must target the multifaceted changes that are associated with epigenetic disease.
Historical Perspective of Epigenetics The importance of epigenetic changes in human cancer is only now being recognized in the medical community. A series of discoveries over the last four decades has thrust epigenetics into
Departments of Urology, Biochemisty, and Molecular Biology, University of Southern California/Norris Comprehensive Cancer Center, Keck School of Medicine, Los Angeles, CA. Conflict of Interest Statement: The author is a stockholder and consultant for Epigeneomics AG. Address correspondence to Peter A. Jones, PhD, Departments of Urology, Biochemisty, and Molecular Biology, University of Southern California/ Norris Comprehensive Cancer Center Keck School of Medicine, 1441 Eastlake Ave MS8302L, Los Angeles, CA 90089-9181. Email: [email protected]
0037-1963/05/$-see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1053/j.seminhematol.2005.05.001
the forefront of new drug discoveries. Srinivasan and Borek73 first introduced the hypothesis of methylases as oncogenic agents in 1964. Nearly 15 years later, decreased levels of 5-methylcytosine in animal tumors was reported.52 The first publication describing the use of DNA methylation inhibitors, 5-azacytidine (azacytidine; Vidaza, Pharmion Corp, Boulder, CO) and 5-aza-2=-deoxycytidine (decitabine; Dacogen™, MGI Pharma, Inc, Bloomington, MN) and their role in gene reactivation appeared a year later.44 Since then, numerous other landmark discoveries have been reported, including decreased genomic and gene-specific methylation in human tumors,23,28,29 methylation of a CpG island in cancer,3 hot spots for p53 mutations at methylated CpG sites,68 allele-specific methylation of the retinoblastoma tumor-suppressor gene,72 hypermethylation of CpG islands associated with aging,38,39 fewer tumors developing in mice with decreased methylation,51 and DNA repair gene (MLH1) methylation in somatic cells.33 Other important findings related to DNA methylation inhibitors came in 1984 when Frost et al30 reported that they alter tumorigenic phenotype and in 2002 when synergistic activity with histone deacetylase inhibitors for rapid isolation of tumor-suppressor genes was published. Finally in 2004, the US Food and Drug Administration approved azacytidine for treatment of myelodysplastic syndromes (MDS) and is currently reviewing the application for decitabine for MDS.
Epigenetic Mechanisms DNA Methylation CpG Island Methylation DNA methylation is one of the most common epigenetic events taking place in the human genome. DNA methylation S3
S4 is a complex process where DNA methyltransferases (DNMTs) catalyze the addition of a methyl group to the 5-carbon position of the cytosine. DNA methylation takes place only when a guanine base follows the cytosine, so only the dinucleotide CpG is methylated. CpG dinucleotides are underrepresented in DNA and are not uniformly distributed. They occur about once per 80 dinucleotides. Clusters of CpGs are called CpG islands and are found in association with genes, most often in the promoters and first exons but also in regions toward the 3= end, and are unmethylated in normal cells.7 Approximately 60% to 90% of CpG sequences are methylated, while unmethylated CpG dinucleotides are located primarily in the CpG islands.60 If the CpG island remains unmethylated with an open chromatic configuration in association with hyperacetylated histones, then the gene can be transcribed. CpG island methylation is associated with changes in chromatin structure and subsequent repression of gene transcription. Methylation of CpG promoters prevents transcriptional initiation and ensures the silencing of genes on the inactive X chromosome, imprinted genes, and parasitic DNAs. Gene silencing can spread, a finding developed from studies in the field of heterochromatinization in Drosophila and in X-inactivation. Gene silencing is a result of the spreading of methylation, thus is not a single discrete event but is a series of events that begins with a drop in transcription potential and ends with its complete cessation.78 CpG island methylation also may be detected in normal tissue before the onset of cancer, as in, for example, in nondysplastic tissue in patients with Barrett’s esophagus and adenocarcinoma.16,20 Molecular mechanisms for the significance of this promoter hypermethylation have been investigated. The presence of the 5= methyl-cytosine in the DNA helix may prevent binding of transcription factors40,47 or may bind with less affinity.40,55,75 Preferential binding of proteins to methylated promoters may prevent binding of transcriptional factors to the sequence. An example of such proteins are the methylcytosine-binding proteins, which bind to DNA at methylated sites and recruit histone deacetylases to methylated DNA in regions of transcriptional silencing.8,14,43,60 Therefore, DNA methylation enables the conversion of histones and other proteins into a nonacetylated state, which changes the configuration of chromatin to where it is refractory to transcription. DNA Methyltransferases The mechanisms by which DNA methylation is regulated or how patterns are established are poorly understood. To date, three DNMTs (DNMT1, DNMT3a, and DNMT3b) have been identified for “de novo” and “maintenance” methylation.62 No defects in these enzymes have been found in human tumors; however, upregulation of DNMT1 and DNMT3b has been described.25,57,70 The first DNMT to be discovered was DNMT1, which acts to maintain pre-existing methylation and preferentially acts on hemimethylated DNA, thus copying methylation patterns in newly synthesized DNA strands. DNMT1 can also form a transcriptional repressive complex with histone deacetylase 2 at replication foci.69 The DNMT3
Figure 1 Interaction between RNA, histone modification, and DNA methylation in gene silencing. Histone deacetylation and other modifications cause chromatin condensation and block transcriptional initiation. Histone modification can also attract DNA methyltransferases to initiate cytosine methylation, which can reinforce histone modification patterns conducive to silencing. Experiments in yeast and plants have shown the involvement of RNA interference in the establishment of heterochromatic states and silencing. RNA triggering of heritable quiescence might therefore also be involved in higher organisms. Adapted with permission.21 © Nature Publishing Group (http://www.nature.com/).
family of methyltransferases is tissue-specific and responsible for the de novo methylation that occurs during embryonic development.62 The DNMT3 enzymes add a methyl group to unmethylated CpG base pairs, leading to a hemimethylated then fully methylated CpG. The role of DNMTs may be more complex than originally appreciated. In cell culture models, each DNMT can directly repress transcription in reporter gene systems by interacting with histone deacetylases and by binding with other proteins with transcriptional-repression activities2,31,32,69,71 (Fig 1). Therefore DNMTs may participate in gene silencing with or without DNA methylation, which raises questions as to what comes first, methylation or gene silencing?
RNA-Associated Silencing The role of RNA in post-transcriptional silencing has also been studied and can lead to mitotically heritable transcriptional silencing by the formation of heterochromatin. This can occur in the form of antisense transcripts, noncoding RNAs (Xist), or RNA interference (RNAi). RNAi-directed silencing has not been described in mammals; however, antisense RNAs that are involved in silencing of some genes have been reported in mammals.64 An example is in a case of ␣-thalassaemia where antisense transcription led to DNA methylation and silencing of a globin gene.77 RNA modifications may be a trigger for histone modifications and DNA methylation to specific loci.
Histone Modification Post-transcriptional modifications of histones including acetylation and methylation of conserved lysine residues on the amino-terminal tail domains also have been implicated in
Overview of cancer epigenetics
Figure 2 Knudson’s two-hit model hypothesis. The model predicts that a phenotypic consequence of tumor-suppressor gene loss is not seen unless both alleles of a gene are inactivated in a tumor. The first hit is the point mutation or deletion and the second hit is methylation of CpG islands in gene promoters. Reprinted with permission.43 © Nature Publishing Group (http://www.nature.com/).
changes in chromatin structure and gene regulation and are known as the histone code.41 Histone acetylation marks active transcriptionally competent regions and histone methylation marks active and inactive regions of chromatin. The histone deacetylases help maintain the histones around the start site of the gene in the deacetylated state. This may also be linked to the DNA cytosine methylation patterns.59 DNMTs can recruit histone deacetylases and thus may work together to cause gene silencing. It was originally thought that histone modification was secondary to DNA methylation, but studies in Neurospora indicated that histone modification could commence the process of DNA methylation.42
Epigenetics in Carcinogenesis Changes in the DNA sequence leading to inactivation of tumorsuppressor genes are known to be a major contributor to human cancer.49 However, abnormal methylation of the promoters of regulatory genes can cause their silencing and subsequent cancer development.5,45 Hypermethylation of the CpG islands within or around the promoter region in tumor-suppressor genes is now the most well-recognized epigenetic change that occurs in virtually every type of human neoplasm. Neoplastic cells may also have overexpression of DNMTs.19,70 As compared to normal cells, cancer cells show major disruption in their DNA methylation patterns, resulting in blocked expression of a gene that would otherwise be expressed.4,18 However, it may be that methylation is not the initial triggering event in gene silencing associated with cancer, rather that methylation of CpG islands is a consequence of prior gene silencing. Only after proteins are added to the methylation site forming chromatin does transcriptional repression occur.35 The support for this theory lies in the fact that changes in the histone code are needed prior to cytosine methylation.13 The significance of hypermethylation of tumor-suppressor gene promoters can be seen when examining Knudson’s twohit model (Fig 2). This model predicts that a phenotypic consequence of tumor-suppressor gene loss is not seen unless both alleles of a gene are inactivated in a tumor.50 Meth-
ylation of CpG islands in gene promoters is considered to be the second-hit in addition to the first hit of point mutation or deletion. Hypermethylation of the promoters of both copies of the gene is more common in nonfamilial tumors but seldom accounts for both hits in inherited or somatic tumors.25 Epigenetic changes are also thought to play a role in tumor progression and metastasis. This was shown for the repair gene MLH1 (mutL homologue 1, colon cancer, nonpolyposis type 2).37,45 Methylation changes of MLH1 have been seen in normal colonic epithelium of patients with colonic cancer with microsatellite instability58 and in hyperplastic regions preceding the development of endometrial cancers.24 Cells with the highest metastatic properties are those that exist as a subpopulation in a heterogeneous cell population; once they metastasize, they regenerate at the distant site—a process that is likely epigenetically driven. This theory is supported by our knowledge of the gene CDH (E-cadherin); loss of this gene’s function due to methylation (?) favors tumor cells that acquire the invasive properties of metastatic tumor cells.15,34,56 DNA hypomethylation may play as large a role in cancer as DNA hypermethylation. Under-methylation commonly occurs in highly and moderately repeated DNA sequences, including heterochromatic DNA repeats, dispersed retrotransposons, and endogenous retroviral elements. Hypomethylation may directly affect karyotypic stability and might initiate altered heterochromatic-euchromatic interactions, which will in turn favor oncogenesis.22 It is not known if hypomethylation actually causes a transformation or whether it is a consequence of the transformation. Hypomethylation is seen more commonly in solid tumors such as cervical cancer,48 hepatocellular cancer,53 and prostate tumors.6 Hypermethylation and hypomethylation appear to be independent processes.22 In other words, the role of hypomethylation in cancer development and spread must not be overlooked.
Genes Commonly Methylated in Human Cancer There is a growing list of genes that are silenced in the CpG island regions. Susceptible genes are those involved in cell
S6 Table 1 Epigenetic Therapies Target
Azacytidine Decitabine FCDR Zebularine Procainamide EGCG Psammaplin A Antisense oligomers Histone deacetylase Phenylbutyric acid SAHA Depsipeptide Valproic acid
Clinical Trials FDA-approved Phase I/II/III
Phase I Phase Phase Phase Phase Phase
I I/II I/II I/II I/II
Abbreviations: EGCG, epigallpcatechin-3-gallate; FCDR, 5-fluro-2=deoxycytidine; SAHA, suberoylanlide hydroxamic acid. Adapted with permission.21 © Nature Publishing Group (http://www. nature.com/).
cycle regulation (p16INK4a, p15INK4a, Rb, p14ARF), DNA repair (BRCA1, MGMT),27 apoptosis (DAPK, TMS1), drug resistance, detoxification, differentiation, metastasis, and angiogenesis.18 Not every gene is methylated in every tumor type. Many of these genes are responsible for familial forms of cancers; however, they may also be hypermethylated in nonfamilial cancer. An example is the breast cancer 1 gene (BRCA1), which was once thought to be important only for familial breast cancer; however, it is now recognized that 10% to 15% of women with the nonfamilial form of breast cancer also have hypermethyled BRCA1.26 Some of the more well-described genes associated with cancer include p15, p14, and p16 on chromosome 9p21, but the reasons for these methylation changes are not understood.12,61 Colon tumors have a high degree of methylation in the p16 gene promoter and exon. The promoter methylation is confined to the p16 gene in colonic tumor tissue, which is different from leukemia, where the p16 gene promoter is rarely methylated. Therefore, exonic regions may be more easily methylated than promoter regions and may occur in the early phases of transformation, while further methylation events may occur only in specific gene pathways for the given cancer type (such as p16 for colon cancer and p15 for leukemia).10,67,79 Some tumor-suppressor genes are silenced by promoter hypermethylation in various cancers but are not mutated. Examples include O6-methylguanine-DNA methyl-transferase (MGMT),26 a DNA-repair gene; cyclin-dependent kinase inhibitor 2B (CDKN2B), which encodes p1536; and the ras-associated gene, RASSFIA.9,17
Epigenetic Therapies As the understanding of epigenetic processes has increased so has the development of agents that may be termed epigenetic therapy. These agents alter the methylation patterns of DNA or the modification of histones , and many are currently in clinical trials (Table 1). Inhibitors of DNA methylation reac-
tivate the expression of previously silenced genes. Prototype agents in this class include azacytidine and decitabine. Both are converted to the deoxynucleotide triphosphates and then incorporated in place of cytosine into replicating DNA. Histone deacetylase inhibitors can induce differentiation, growth arrest, and/or apoptosis in transformed cells in culture and in tumors. Accumulation of acetylated proteins results in induction of genes and upregulation of genes that have become silenced. The links between DNA methylation and histone modification open up opportunities for combination therapy in order to target both of these epigenetic mechanisms. However, DNA methylation appears to be dominant over histone deacetylase inhibitors. Histone deacetylase inhibitors alone cannot induce the expression of hypermethylated genes in cancer cells.35,74 Although synergy of demethylation and histone deacetylase inhibitors has been previously described,11,74,81 further investigation is warranted. Increased doses of the DNA methylation inhibitors were linked to significant cytotoxicity when originally studied many years ago, so the use of combination therapy may allow lower doses. Also it appears that chronic administration of these compounds is necessary for sustained gene reactivation, so limiting toxicity is important. Another approach to combination therapy is the use of epigenetic therapy pretreatment to sensitize cells and then treat with traditional chemotherapy, interferon, or immunotherapy.46,66,80
Role of DNA Methylation in Diagnosis and Prognosis DNA methylation changes can now be detected in various bodily fluids with a high degree of sensitivity. In many cancers, cancer cells can be obtained from plasma in addition to fluids such as sputum, urine, and saliva, or from biopsy specimens. Analyzing these changes may serve many purposes in the clinical setting. Genes that undergo hypermethlaytion reside in cancer cells and most normal cells possess unmethylated CpG islands, thus providing opportunities to assess malignant transformation. These techniques may also be used for early detection as hypermethylation changes precede malignant changes. p16 promoter hypermethylation is used as a biomarker for lung cancer and has been detected in the sputum of smokers up to 3 years before the diagnosis of cancer.16,63 In addition, several methylated genes are closely related to prognosis. The RASSF1A gene is associated with poor prognosis in stage I lung adenocarcinomas,76 while presence of the CDKN2A gene correlates with prognosis in colon cancer.54 Finally, the methylation profile may be useful for predicting response to chemotherapy; for example, methylation of the hMLH1 gene in colorectal cells is associated with increased resistance to fluorouracil.1 In addtion, methylation within the WIT1 gene correlates with chemoresistance in acute myelogenous leukemia.65 The various techniques for detection of methylation42 include cDNA microarray, restriction landmark genomic scanning (RLGS), methylated CpG island amplification-represen-
Overview of cancer epigenetics tational difference analysis (MCA-RDA), and differential methylation hybridization (DMH). RLGS allows researchers to detect large numbers of CpG islands; however, some of these islands may not be in the promoter regions and are thus not involved with transcriptional regulation. The advantages of MCA-RDA are very similar to those of RLGS; however, even though many of the CpG islands are associated with genes, defining the start site of a gene and the exact relationship of the island to the transcriptional regulation of a gene can be laborious. DMH is an array-based method in which genomic DNA is pre-cut with methylation-insensitive enzymes. cDNA microarray has the advantage that the detection of hypermethylation sites is linked to the transcriptional status of the genes; the disadvantage is that the CpG island that is hypermethylated is not always easy to identify in genomic databases. The development of polymerase chain reaction (PCR) techniques has offered a quick and sensitive method to detect hypermethylation in CpG islands of tumorsuppressor genes. The practicality of these methods for detection has not been demonstrated yet and might be compromised by the fact that some methylation changes occur in normal epithelial cells.
Summary The role of epigenetic changes in carcinogenesis is evolving. Since methylation of CpG islands increases with age (Issa, 2000), there could be an association to other chronic diseases in addition to cancer. Further, changed patterns of methylation can be detected with a high degree of sensitivity and thus can provide clinicians with prognostic information. Understanding how epigenetic states are established and maintained and then how to translate these ideas into therapeutic interventions lies in the forefront of intense research. Current knowledge suggests that agents that intervene in this process by “turning back on” silenced genes may represent a significant advancement in treating many forms of cancer.
13. 14. 15.
21. 22. 23.
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