Epigenetic Changes in Solid and Hematopoietic Tumors

Epigenetic Changes in Solid and Hematopoietic Tumors Minoru Toyotaa and Jean-Pierre J. Issab There are three connected molecular mechanisms of epigenetic cellular memory in mammalian cells: DNA methylation, histone modifications, and RNA interference. The first two have now been firmly linked to neoplastic transformation. Hypermethylation of CpG-rich promoters triggers local histone code modifications resulting in a cellular camouflage mechanism that sequesters gene promoters away from transcription factors and results in stable silencing. This normally restricted mechanism is ubiquitously used in cancer to silence hundreds of genes, among which some critically contribute to the neoplastic phenotype. Virtually every pathway important to cancer formation is affected by this process. Methylation profiling of human cancers reveals tissue-specific epigenetic signatures, as well as tumor-specific signatures, reflecting in particular the presence of epigenetic instability in a subset of cancers affected by the CpG island methylator phenotype. Generally, methylation patterns can be traced to a tissue-specific, proliferation-dependent accumulation of aberrant promoter methylation in aging tissues, a process that can be accelerated by chronic inflammation and less well-defined mechanisms including, possibly, diet and genetic predisposition. The epigenetic machinery can also be altered in cancer by specific lesions in epigenetic effector genes, or by aberrant recruitment of these genes by mutant transcription factors and coactivators. Epigenetic patterns are proving clinically useful in human oncology via risk assessment, early detection, and prognostic classification. Pharmacologic manipulation of these patterns— epigenetic therapy—is also poised to change the way we treat cancer in the clinic. Semin Oncol 32:521-531 © 2005 Elsevier Inc. All rights reserved.

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pigenetics has risen from a fascinating but obscure field driven by research in plants and model organisms to a major player in human development, physiology, and pathophysiology of disease.1,2 There has been substantial progress in unraveling the epigenetics of cancer, and the field has advanced to clinical applications, including the recent approval of an epigenetic-acting drug for the treatment of myelodysplastic syndrome.3 This review will summarize epigenetic changes in neoplastic cells and discuss potential underlying mechanisms.

Epigenetic Mechanisms Epigenetics refers to stable changes in gene expression that cannot be accounted for by changes in primary DNA coding sequence. There are both transcriptional and post-transcripaSapporo

Medical University, Sapporo, Japan. of Texas, M.D. Anderson Cancer Center, Houston, TX. Address correspondence to Jean-Pierre J. Issa, MD, Department of Leukemia, M.D. Anderson Cancer Center, Box 428, 1515 Holcombe, Houston, TX 77030. E-mail: [email protected]

bUniversity

0093-7754/05/$-see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1053/j.seminoncol.2005.07.003

tional mechanisms for epigenetics, and the best understood type of epigenetic regulation is silencing, whereby gene expression is lost.2

DNA Methylation DNA methylation refers to the enzymatic addition of a methyl group to the 5 position of cytosine incorporated into DNA.4,5 In mammals, methylation is largely limited to cytosines that are part of the symmetrical dinucleotide CpG. Most CpG sites in the human genome are normally methylated, except for clusters of such sites (CpG islands) that are present in about half of all human genes and are normally free of methylation, regardless of the expression status of the associated gene. A switch from unmethylated to methylated CpG island results in permanent loss of gene expression, as initially demonstrated on the inactive X-chromosome in women.6 Cancer cells usurp this mechanism and apply the switch to silence genes undesirable for the transformed phenotype.7 The mechanisms of DNA methylation associated gene silencing are not entirely known, although a leading current hypothesis is that it entails a cascade of chromatin modifications triggered by the methylated state of DNA.8,9 521

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Histone Changes Histones are proteins around which DNA is wrapped to form the nucleosome, which is the basic structure of DNA inside the nucleus.10 Post-transcriptional modifications of histones affect gene expression by targeting various protein complexes to DNA, which result in either an open chromatin structure ready for expression or a closed chromatin configuration that is impermeable to transcription factors and associated with gene silencing.11 Histone modifications can be triggered by local factors such as transcription factors and repressors, or by DNA methylation, which triggers sequential binding by methyl-binding proteins, histone deacetylases, histone methylases, and finally silencing proteins of the polycomb gene family. It has been proposed that histone changes could also represent an independent epigenetic mark (the histone code), although mechanisms of perpetuating this code in the absence of triggering factors are still being investigated.

RNA Interference RNA interference (RNAi) is a post-transcriptional mechanism whereby gene expression is suppressed by RNA degradation, triggered by short stretches of complementary RNA.12 This process is a prominent mechanism of epigenetic regulation in plants and other organisms, where it plays roles varying from genome defense to chromosomal structure. Mammalian cells are equipped for RNAi, although the physiologic role of this process in mammalian biology remains to be clarified. RNAi has become an important laboratory tool to interrogate gene function through downregulation of gene expression.

Epigenetics and Cancer Cancer is characterized by substantial changes in epigenetic marks, including a global loss of 5-methylcytosine content (affecting primarily repetitive elements and pericentromeric DNA) and concomitant gains of methylation in promoterassociated CpG islands with silencing of the associated genes, including tumor-suppressor genes.13 The histone code is also altered in various malignancies, either through targeting by DNA methylation7 or through primary defects in histone modification controlling genes such as histone acetylases14 and methylases.15 The integrity of RNAi in cancer has not been reported to date.

Needle in a Haystack: Tumor-Suppressor Gene Methylation in Cancer Aberrant promoter CpG island methylation was initially described for the CALCA gene almost 20 years ago,16 and the number of genes known to be methylated in cancer has grown from a trickle to a torrent, so much so that the identification of such silencing events has been referred to as an industry.17 Of course, the number of genes known to be mutated in cancer has similarly grown exponentially and there also is an industry devoted to identifying them as well.18 It is important to consider the global patterns of DNA meth-

M. Toyota and J.-P.J. Issa ylation in neoplasia to evaluate the significance of individual hypermethylation events.

Hypermethylation in Cancer: Some Numbers Following a flurry of initial reports of tumor-suppressor genes hypermethylated in cancer, a number of techniques were developed to identify genes specifically based on promoter methylation.19 –21 These have been fairly successful, and we now know of several hundred genes hypermethylated and silenced in cancer. There are no easily distinguishable marks of susceptibility to this process. Affected genes can have small or large CpG islands, and functional gene homology does not imply shared susceptibility to hypermethylation. For example, among the six known mismatch repair genes, only one (MLH1) is appreciably methylated in cancer.22 Of the seven known (major) cyclin-dependent kinase inhibitors,23 three (p16INK4a, P15INK4b, and p57Kip2) show hypermethylation in cancer, while the others are rarely if ever involved. It has been difficult to estimate precisely the number of genes silenced in association with promoter DNA hypermethylation in any given neoplasm. The difficulties are confounded by the use of longstanding cell lines in some cases, which may have substantially more methylation than primary tumors24 or early passage cell lines.25 In one early estimate, it was found that 50% of all CpG rich fragments derived from a transformed cell line were hypermethylated.26 This is clearly an overestimate because the method used may have been biased towards identification of nonpromoter regions. Restriction landmark genomic scanning (RLGS), a global methylation analysis method, estimated that 0% to 10% of genes were hypermethylated in cell lines derived from different tissues, with an average of about 600 hypermethylated genes per tumor. While this is a reasonable estimate, RLGS does not necessarily sample promoter CpG islands, and an appreciable rate of false positives and negatives are encountered by this method, in part because it requires fairly dense methylation to identify a gene as positive. Methylation microarrays have also been used to estimate the number of genes methylated in cancer, and the estimate there was 1%.27 Such data should also be considered mere estimates, because the CpG islands arrayed are not necessarily promoter-associated and, as in all microarray experiments, the high number of experimental points makes the technique prone to false positives, and its sensitivity is also limited. Despite some uncertainty, it appears clear that most cancers carry several hundred hypermethylation events and that the associated silencing sculpts the physiology and phenotype of the neoplastic cell. There has been interest in whether CpG island methylation shows chromosomal clustering. A quick analysis of many of the genes known to be hypermethylated in cancer shows no such clustering. In general, lists of genes recovered by methylation-based screening methods also show no chromosomal preference,20,28,29 with the exception of one study that suggested clustering of hypermethylation events on

Epigenetic changes in solid and hematopoietic tumors chromosome 11.30 A careful analysis of this issue using combined RLGS and comparative genomic hybridization (CGH) analysis also revealed no particular clustering of methylation events along a given chromosome. At present, the factors that determine hypermethylation in cancer appear to be genespecific rather than chromosome-specific.

Evidence for Selection: Familial Cancer Genes One of the most controversial issues in the early years of DNA methylation in cancer research was whether epigenetic silencing associated with methylation contributed significantly to the neoplastic phenotype and thus was equivalent in many respects to gene mutations.31 The most convincing arguments in favor of this came from the study of familial cancer genes such as RB1, VHL, MLH1, and others (Table 1). In each case, two key observations support the equivalence of mutations and silencing. (1) The tissue spectrum of mutations and methylation is identical. For example, VHL is mutated almost exclusively in renal tumors, and VHL methylation can be observed primarily in this tumor type.32 (2) There is an allelic exclusivity for methylation and mutations. In other words, an allele that carries an inactivating mutation is never found to have promoter methylation, and vice versa. This has been clearly demonstrated for each of P16,33 RB1,34 and CDH1.35 These two observations demonstrate that promoter hypermethylation does not simply follow neoplastic transformation. Rather, gene inactivation by mutations or by epigenetic silencing provides equivalent selective advantage to an affected tumor, thus explaining both the tissue distribution of the events and the inverse correlation between mutations and methylation.

Pathways Affected Beyond familial cancer genes, virtually every pathway hypothesized as playing a role in cancer biology has been shown to be affected by epigenetic silencing of one of its members in some sporadic cancers. In fact, searches for CpG islands hypermethylated in cancer uncovered novel genes previously unsuspected of playing a role in cancer, including genes involved in growth factor signaling (eg, RASSF136), apoptosis (eg, TMS1,37 and HRK38) and others. Table 2 provides examples of genes silenced in cancer grouped by pathways.

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Not All That Glitters Is Gold Hypermethylation-based silencing provided a novel potential way of identifying unknown tumor-suppressor genes (TSG). As mentioned above, a number of methods have been developed to find such silenced genes via DNA-based approaches or RNA/reactivation-based approaches. These methods have been successful, and the list of novel targets for epigenetic silencing is now in the hundreds. Is it plausible that all of those loci are tumor-suppressor genes that provide a selective advantage? Clearly, the answer must be no. Some oncogenes are actually hypermethylated in cancer (eg, COX239 and TERT40) and other genes that are hypermethylated are not actually expressed in the relevant normal tissues,41 thus making the matter of silencing moot. Given that hundreds of genes are hypermethylated in any given tumor21 (and perhaps more for tumors with hypermethylator phenotypes42), it is important not to rush to conclusions regarding the functional importance of the event simply based on the presence of methyl groups in the promoter. In fact, experiments based on a panel of cell lines suggest that up to 40% of randomly selected promoter-associated CpG islands can be found methylated in one tumor type or another (unpublished observations), making methylation-based tumor-suppressor gene (TSG) detection only slightly better than random guesses.

What About Hypomethylation? The first described changes in DNA methylation in cancer were actually loss of methylation, observed more than 25 years ago now.43 Despite almost three decades of research, the functional consequences of this hypomethylation remain mysterious. Early speculations that this hypomethylation (which averages 10% of all CpG sites44) activates gene expression, including oncogenes,45 have not been experimentally substantiated. The closest we are to a functional consequence of hypomethylation is through chromosomal alterations. In the ICF syndrome (immunodeficiency, centromeric instability, facial anomalies), loss of methylation is associated with specific chromosomal breaks, reminiscent of some genetic changes observed in cancer.46 In mice with severe methylation defects (vastly greater than the average 10% loss in cancer), chromosomal instability could be demonstrated.47 However, in colon cancer cells where the same methylating

Table 1 Shared Tissue Distribution and Allelic Exclusivity of Mutations and Methylation of Genes Involved in Familial Cancer Syndromes Gene

Familial Cancer Syndrome

Mutations in Sporadic Cancer

Methylation in Sporadic Cancer

RBI MLH1 CDH1 VHL BRCA1 LKB1

Retinoblastoma HNPCC Stomach Von-Hippel Lindau Breast and ovarian cancer Peutz-Jeghers syndrome

Retinoblastomas Colon, endometrium Stomach Kidney Rare Rare

Retinoblastomas Colon, endometrium Stomach, others Kidney Breast118 Same spectrum as Peutz-Jeghers syndrome119

Allelic Exclusion Yes34 Yes116 Yes35 Yes117 ND ND

M. Toyota and J.-P.J. Issa

524 Table 2 Examples of Pathways and Genes Hypermethylated in Cancer Pathway Cell cycle

Apoptosis

DNA repair/DNA damage response Angiogenesis/hypoxia

Chromatin regulation and transcription Signal transduction

Hormone receptor

Cell attachment and invasion

Gene

RB134 p16INK4A120 p15INK4B121 p57KIP2122 p73123 CHFR124 DAPK125 TMS137 HRK38 hMLH122 MGMT126 BRCA1118 THBS1127 VHL32 BNIP3128 HLTF97 RIZ196 HIC1129 RASSF136 NORE1130 RUNX3131 SOCS3132 PTPRO133 SFRP28 ER52 RAR-beta134 PGR135 CDH1136 TSLC1137 Claudin-7138

Function

Tumors affected (examples)

Inhibitor of cyclin-dependent kinase Inhibitor of cyclin-dependent kinase Inhibitor of cyclin-dependent kinase Inhibitor of cyclin-dependent kinase Cell cycle checkpoint Spindle checkpoint Pro-apoptotic serine/threonine kinase Pro-apoptotic CARD domain family Pro-apoptotic BH3-only subfamily Mismatch repair DNA alkylation repair DNA damage response Angiogenesis inhibitor Ubiquitin ligase Hypoxia-mediated apoptosis Helicase-like transcription factor Histone methyltransferase Transcriptional repressor RAS effector RAS effector TGF-beta pathway Inhibitor of JAK/STAT pathway Tyrosine phosphatase inhibitor WNT signaling Estrogen receptor Retinoic acid receptor Progesterone receptor Cell adhesion Cell adhesion Tight junction protein

Retinoblastoma Colon, lung, breast Leukemia, lymphoma Leukemia, stomach Leukemia, lymphoma, neuroblastoma Colon, stomach, lung, breast Lymphoma, lung, colon, stomach Breast, ovary, neuroblastoma Colon, stomach Colon, stomach, endometrium Colon, lung Breast Colon, neuroblastoma Kidney Pancreas, colon, stomach Colon Breast, stomach, colon Breast Lung, colon, breast, ovary Lung Stomach Lung Lung Colon, stomach, breast Colon, breast Colon, breast, leukemias Breast Breast, stomach, lung Lung, stomach, Breast

enzymes were deleted resulting in profound hypomethylation as well, the chromosomes remained stable.48 There also appear to be no interactions between global hypomethylation and gene-specific promoter hypermethylation.49

Nonrandom Mosaics: Tissue-Specific and TumorSpecific Methylation Patterns The field of methylation analysis in cancer has moved from studying individual genes to focusing on an ever-growing list of potentially affected loci. This has been made simpler with the advent of more global methylation approaches such as RLGS or methylation microarrays. Overall, these studies have revealed remarkable tissue and tumor specificity for the methylation patterns.

Tissue-Specific Methylation— Normal and Neoplastic An inkling that methylation patterns in cancer depended on the tissue of origin of tumors came from the earliest genes studied—CALCA,16 MYOD,41 HIC1,50 and ER.51,52 In each case, methylation was specific to particular tumor types and suggested from the outset that tissue-specific factors influ-

enced the process. This has been confirmed in a large RLGS study in which thousands of loci were studied, and which clearly showed nonrandom distribution of methylation patterns, with specific genes methylated in specific tumors.21 Indeed, it has been suggested that methylation profiling could be sufficient to determine the tissue of origin of a given tumor type. The roots of tissue-specific methylation patterns are not completely resolved. Early work on HIC1, a gene on the frequently deleted portion of chromosome 17p, showed that methylation tracked with loss of heterozygosity.50 It was proposed then that tissue-specific methylation patterns are related to differential selective advantage for inactivation of a given gene in different tissues. However, as argued above, it is likely that many methylation events in cancer do not necessarily carry a selective advantage and still demonstrate tissue specificity. What then causes this distribution? A telling clue comes from analysis of screening methods to identify genes hypermethylated in cancer. In many cases, validation studies showed that many of the genes recovered also showed a low but clearly detectable degree of methylation in normal-appearing tissues of the same origin.28,42,53,54 It appears therefore that tissues differ in their susceptibility to hypermethylation specific genes, initially in normal appearing tissues and

Epigenetic changes in solid and hematopoietic tumors eventually in malignant cells. The origin of this methylation in normal appearing cells is discussed below.

Tumor-Specific Methylation: The CpG Island Methylator Phenotype Methylation profiling of multiple genes within a given tumor type has uncovered a fascinating process whereby some cancers have substantially more frequent (three- to sixfold) CpG island methylation than others.55 This phenomenon, termed the “CpG island methylator phenotype” (CIMP) was first described in colorectal cancer,42 but appears to be a fairly universal feature of methylation patterns in malignancies, with clear evidence of the process in multiple other tumor types.55 Fundamentally, this phenotype provides strong evidence for a specific defect that leads to aberrant methylation in some cancers and provides additional evidence that hypermethylation is selected for (and thus contributes to) neoplasia. Moreover, tumors affected by this phenotype have distinct clinical and molecular features. In colon cancer, for example, CIMP⫹ cancers tend to be proximal and to occur more frequently in women and older patients.42,56 They have a distinct pathology,56 a distinct molecular genetic profile with frequent KRAS and BRAF mutations and infrequent P53 mutations,57,58 and a unique clinical course with a good prognosis if MLH1 is affected and a poor prognosis if it is not. The causes of CIMP are unknown at present. Association of the phenotype with a positive family history of cancer59 and the description of pedigrees with multiple cancers and frequent methylation all raise the hope that classical genetic linkage studies will lead to gene(s) that accelerate methylation and can account for CIMP.

The Age of Methylation: Causes of Aberrant Methylation in Cancer A largely overlooked issue in current research into the epigenetics of cancer is arguably the most important one: what causes these anomalies? On top of tissue-specific and tumorspecific patterns described above, there are intriguing genespecific patterns that cannot be attributed to gene function and/or selection. For example, mutational inactivation of MSH2 or MLH1, two DNA mismatch repair genes, results in indistinguishable colon cancer susceptibility phenotypes characterized by microsatellite instability. Yet, while both genes are targeted by mutations and loss of heterozygosity, only MLH1 is a target for gene silencing and aberrant promoter methylation in cancer. BRCA1 and BRCA2 represent another such puzzling pair concordant for mutations yet discordant for methylation. Finally, a number of frequently mutated tumor-suppressor genes such as P53 are also never hypermethylated in sporadic neoplasms. These arguments invalidate pure random events and selection as a cause of aberrant methylation in cancer. Rather, the process has to be viewed as a combination of gene-specific susceptibility, accelerating factors and selective advantage.

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The Seed and Spread Model The main alternative to the random methylation model is the seed and spread model of methylation centers and spreading, proposed initially to explain formation of methylation patterns in the APRT gene on the X-chromosome.60 This hypothesis attributes initial methylation to “methylation centers” that attract DNMTs, presumably by virtue of local structural DNA features. Methylation then spreads from these centers bidirectionally, in-cis. Spreading of methylation into CpG islands and transcription start sites is counteracted by mysterious trans-acting factors that protect against the spread of silencing. In the APRT gene, the methylation center is a retrotransposon, and spreading is resisted by an unidentified protein that binds to an SP1 site61 but is dispensable for transcription.62 Is this model operative in cancer? Spreading of methylation from CpG island borders towards the transcription start sites has been suggested by several studies,63– 65 and it is likely that this model applies indeed to cancer. The nature of these methylation centers however remains elusive. While human retrotransposons do attract methylation, an analysis of local factors that determine gene-specific susceptibility to methylation in cancer did not reveal an unusual concentration of repeats around susceptible promoters.66 Instead, specific short sequences were found that are statistically associated with susceptibility, although the functional significance of these sequences remains unknown. Protection against methylation in cancer is equally poorly defined at present. It has been speculated that, if specific methylation protective factors existed, inactivation of one or more of those would result in a phenomenon akin to CIMP.55,67 The seed and spread model has strong albeit indirect support from the few studies that addressed this issue directly. For example, transfection of an unmethylated promoter associated CpG island results in no appreciable methylation of the transfected construct, even if it is done in a cell line with methylation of the gene under study.68,69 As convincingly demonstrated for both MLH169 and VHL,68 even cell fusion between unmethylated and methylated cell lines is incapable of disrupting the methylation equilibrium. Thus, methylation patterns likely evolve over long periods of time that cannot be recapitulated in cell culture, and the “chromosomal autonomy” of the process confirms the predominance of local factors, at least early on. Moreover, the observation that those genes that show some methylation in normal-appearing tissues are those that go on to frequent hypermethylation20,42 also supports this concept. Methylation can then be viewed as a continuous process that starts early on outside promoters and spreads slowly over time. In a direct testing of this model, it was demonstrated that, in order to see substantial methylation of a GSTp1-transfected promoter, both premethylation (seeding) and mutation of SP1 binding sites (protection) were required.70

Accelerators—Aging and the Inflammation Link In the light of the seed and spread model proposed above, it is useful to consider what factors could and do accelerate

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526 methylation, thus contributing to the final patterns observed. In vitro, it has been consistently observed that methylation spreading is a replication-dependent phenomenon,71,72 and thus it is not surprising that the predominant factor accounting for methylation in normal-appearing tissues is aging.51,73 In addition, chronic inflammatory states are associated with markedly elevated methylation rates, as demonstrated for ulcerative colitis in the colon,74 chronic hepatitis in the liver,75 Barrett’s esophagitis,76 inflammatory lesions of the prostate,77 and even atherosclerosis.78 The mechanism of this association is unknown, but it is proposed that the common link is increased stem cell mobilization and proliferation, triggered by injury/repair cycles. This hypothesis could also explain the impact of carcinogens such as smoking79 and radiation exposure80 in increasing methylation. One factor that has not been consistently associated with increased methylation is expression of DNA methyltransferases (DNMTs). There are modest increases in DNMT activity in various neoplasms,13,81 but it has not been reproducibly shown to be a factor in explaining methylation patterns.42,82 It remains possible that factors such as DNMT localization/targeting play a role in the process, and one study found that DNMT1 protein expression by immunohistochemistry was distinctly aberrant in cancer.83 Still, DNMT1 overexpression by 50-fold or more results in modest increases in gene methylation,84 making it an unlikely candidate to explain cancer-specific methylation patterns.

The Gene Expression Factor A raging controversy in the field is whether aberrant methylation follows gene inactivation (and perhaps locks it in), rather than initiates silencing.31 Evidence often quoted in favor of silencing preceding methylation include the facts that (1) in Neurospora, silencing by histone code changes leads to promoter methylation85; (2) in cells lacking DNMT1 and DNMT3B, P16 is initially reactivated but then silenced in association with histone code changes and only later develops promoter hypermethylation86; and (3) silencing of an integrated vector begins with histone code changes and only later is accompanied by methylation.87 There are objections to each of these observations that leave the issue open. Thus, while histone code changes precede and do not require methylation for silencing in the filamentous fungus Neurospora, the situation is different in other systems, where in different circumstances DNA methylation can be upstream, downstream, or independent of histone changes.88 –91 Moreover, neither integrated transgenes nor cells that genetically lack DNMT1 and DNMT3b represent definitive models to study (endogenous) gene silencing in mammalian cells. Perhaps the strongest objection to the silencing first model is that, in normal tissues, genes that are silent in a tissue-specific manner do not in fact (usually) show promoter CpG island methylation. While the silencing first model does not explain all available data in mammalian cells, gene expression may well play an important modulator role in the ultimate patterns of DNA methylation in cancer. In the setting of the seed and spread

model, transient gene silencing may in fact allow for a more efficient spreading that would then lock in the silenced state.60 In a direct testing of this issue, it was demonstrated that an exogenously transfected GSTp1 CpG island becomes densely methylated only in the situation where pre-methylation (ie, seeding) is accompanied by mutations that reduce the ability of transcription factors to bind and activate the gene.70 Indirect supporting evidence in neoplastic cells comes from experiments showing that loss of transcription factor expression promotes methylation of some (but not all) of the downstream transcriptional targets of the affected gene.92,93 If seed and spread of methylation requires transcriptional silencing by other mechanisms to proceed efficiently, it is puzzling that, in many cases, cells in which a gene is silenced by DNA methylation are capable of fully expressing a transfected unmethylated copy of that gene.69 To account for this, “transient” decreases in gene expression have been proposed as a contributing factor. The origin of such (hypothetical) transient downregulation is unknown. It is tempting to speculate that, for genes expressed at low levels, stochastic fluctuations in gene expression in normal cells may result in some cells having no detectable expression at times.94 Perhaps these stochastic fluctuations promote methylation spreading over time, and this might be central to the observed age-related methylation changes in mammals. Some support for this notion comes from the fact that many genes affected by hypermethylation in cancer do in fact have relatively low levels of expression overall, and that genes highly expressed (as revealed by microarrays), tend to be rarely methylated in cancer, even when they are downregulated (unpublished observations). A final issue to consider is the actual contribution of DNA methylation to gene silencing. If full methylation requires prior transient (or less transient) silencing, what then is the role of DNA methylation? It has been suggested that methylation essentially locks the silenced state in, such that what was transient is now permanent silencing. A convincing demonstration of this concept came from the aforementioned cell fusion experiments, where a methylated allele remains silenced, even if it is coexisting with a fully unmethylated and expressing allele. DNA methylation thus compartmentalizes the genome into different gene expression states that are independent of the presence (or absence) of transcription factors.

Selection Much of the discussion above has all but ignored the function of affected genes. Arguments were presented to account for this— gene function does not seem to explain initial susceptibility to aberrant methylation in cancer. However, it is equally clear that, once a set of genes is susceptible to methylation, gene function greatly influences final methylation profiles of cancer through the process of selection. As discussed earlier, there are compelling arguments suggesting that methylation of some tumor-suppressor genes is selected for in cancer. However, given that early methylation steps are

Epigenetic changes in solid and hematopoietic tumors independent of gene function, there also is a class of genes for which methylation would provide a selective disadvantage and would therefore be selected against in cancer. Indeed, oncogenes such as TERT40 and COX239 have been shown to be hypermethylated in some tumors. In those cases, the frequency of methylation tends to be low, and primary tumors often show heterogeneous methylation patterns suggesting an ongoing battle between pressures to methylate the gene and pressure from the selective disadvantage that results from this methylation. This battle is presumably solved when cancers adopt alternate pathways to replicate the function of that oncogene, in which (rare) case methylation is allowed to completely silence the gene.

Epigenetic Mechanisms Other Than DNA Methylation Epigenetic deregulation in cancer is best understood when related to aberrant promoter CpG island methylation. However, it is clear that multiple other epigenetic mechanisms can also be altered and contribute to the neoplastic phenotype. These include both direct and indirect mechanisms.

Direct Effects: Alterations in Epigenetic Effectors Perhaps the most straightforward way of altering epigenetic patterns is through genetic changes in epigenetic effector molecules. A number of such mutations have been described in cancer. For example, inactivating mutations in SNF5, a member of the chromatin regulating SWI/SNF family of genes are at the root of the development of round cell tumors of childhood, a particularly aggressive though rare pediatric cancer.95 Translocations in acute leukemias can affect histone modifying genes, including MLL, a histone H3K4 methyltransferase,15 and CBP, a histone acetyl-transferase.14 Interestingly, some epigenetic effectors can be abnormally regulated in cancer by virtue of aberrant methylation itself, such as the SET-domain protein RIZ196 and the SWI/SNF family protein HLTF.97 It is presumed that these genetic and epigenetic events contribute to tumorigenesis by inappropriate epigenetic activation or inactivation of critical molecules, although the precise downstream mediators of neoplasia are unknown in most such cases.

Indirect Effects: Recruitment Epigenetic effector genes can also be altered in cancer indirectly, via aberrant recruitment to promoters. Several such examples have been described, including the RAR-PML translocation in acute promyelocytic leukemia and the AML1-EVI1 translocation in chronic myelogenous leukemia, both of which result in recruitment of histone deacetylases and resultant inhibition of gene expression.98,99 These changes are notable for the possibility of restoring gene expression therapeutically either via inhibition of the fusion protein created by the translocation, or by direct intervention with epigenetic acting drugs (see below).

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Histone Code and Other Epigenetic Mechanisms Post-translational modifications in histone tails have been associated with epigenetic patterns of gene expression,11 and the resulting histone “code” has gained attention recently as a mechanism of gene silencing in cancer. The mechanisms by which this code is established and maintained remain somewhat controversial,100 as is the potential of histone modifications to transmit epigenetic information in the absence of targeting by other molecules. Indeed, it recently has been argued that histone replacement by premodified histones (rather than modifications of established nucleososmes) is key to the observed associations.101 Nevertheless, it is well established that silenced states are associated with specific histone code patterns. Thus, heterochromatin DNA is characterized by H3K9 hypoacetylation and demethylation, while silencing on the X-chromosome and some imprinted loci is marked by H3K27 hypoacetylation and trimethylation.11 Genes silenced by DNA methylation in promoter-associated CpG islands have been described to have a heterochromatin-like histone code, which may be triggered by affixing of methyl-binding proteins to involved promoters, followed by serial recruitment of histone modifying proteins.102–104 It remains to be seen whether this heterochromatin pattern (characterized by H3K9 methylation) can exist in the absence of DNA methylation, as it does in Yeast and Drosophila. The H3K27 code has not been characterized in cancer, and may well represent an alternate mechanism of epigenetic silencing. Indeed, overexpression of the H3K27 histone methyltransferase EZH2 has been described in various malignancies, in association with a poor prognosis.105 Finally, RNAbased degradation mechanisms such as RNAi and microRNA are intriguing candidates for epigenetic regulation in mammalian cells and little is known about whether they are deregulated in cancer, although it would be surprising if they are not, given the inventiveness of cancer cells in exploiting cellular mechanisms towards their nefarious ends.

Clinical Implications The prodigious frequency of aberrant epigenetic events in cancer has raised the interest of translational researchers worldwide. Gene-specific hypermethylation can be detected using sensitive and/or quantitative methods and promises to help track cancer from early detection to follow-up after therapy.106 Prior to cancer formation, methylation patterns in normal tissues may clarify susceptibility to acquired neoplasia.107 For example, constitutional IGF2 loss of imprinting related to H19 hypermethylation has been consistently associated with risk of development of Wilm’s tumor.108 Methylation in body fluids has been shown to mark (and possibly precede) neoplasia, such as sputum for lung cancer109 or urine for bladder prostate cancer,110 raising the prospect of methylation-based early detection. Within cancers, a high degree of methylation consistently tracks with a more rapidly progressing natural history111 (and, consequently, an adverse

528 outcome), as described in neoplasms of the lung, esophagus, liver, or hematopoietic cells, though why that should be remains to be determined. These issues have been reviewed extensively recently and in other chapters of this issue. It is worth mentioning a few pitfalls on the road to realizing this clinical promise. In terms of risk assessment, appropriate markers, tissues to be examined and methods of methylation analysis remain to be clarified. In the case of IGF2, for example, proliferating lymphocytes lack imprinting of this gene,112 making a peripheral blood– based detection assay for this event possibly problematic. For early detection, the promise of these markers is tempered by the realization that agerelated methylation could introduce a substantial number of “false positives,” all the more so when studying patients with chronic inflammatory states (including smokers). Thus, studies reporting positive findings should be carefully evaluated for the appropriateness of controls. Moreover, the fact that some cancers have substantially more methylation than others (see the discussion on CIMP above) suggests that the sensitivity of methylation markers for cancer detection may not be uniform for all cancers of a given tissue type. Perhaps the most pressing clinical implication to consider is the use of drugs that affect epigenetic patterns (epigenetic therapy) for the treatment of cancer.113 These drugs (DNA methylation inhibitors as well a histone deacetylase inhibitors) are now available for the treatment of hematologic malignancies, and are being tested in other neoplasms. Urgent research issues include whether they produce responses via epigenetic mechanisms,114,115 and whether cancers with specific molecular patterns are more likely to respond to these interventions. These issues are far from resolved at present.

Conclusions: Living in an Epigenetic World The data reviewed in this article establish that the molecular etiology of cancer rests in a combination of genetic and epigenetic events that work in concert towards defining the neoplastic phenotype. Epigenetic events are, for the most part, reset during embryogenesis. Therefore, the aberrant epigenetics of cancer primarily reflect interactions between genome structure and environmental/lifestyle factors, raising the idea that a major contribution of exposures to disease passes by epigenetics. Beyond issues of diagnosis and prognosis, epigenetic information may provide the key to exciting new doors in cancer prevention and treatment.

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