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There is compelling evidence to support the importance of DNA methylation alterations in cancer development. Both losses and gains of DNA methylation are observed, thought to contribute pathophysiologically by inactivating tumor suppressor genes, inducing chromosomal instability and ectopically activating gene expression. Lesser known are the causes of aberrant DNA methylation. Recent studies have pointed out that intrinsic gene susceptibility to DNA methylation, environmental factors and gene function all have an intertwined participation in this process. Overall, these data support a deterministic rather than a stochastic mechanism for de novo DNA methylation in cancer. In this review article, we discuss the technologies available to study DNA methylation and the endogenous and exogenous factors that influence the onset of de novo methylation in cancer.
Understanding what drives cancer has been profoundly affected by the detailed analysis of the genome of multiple neoplasias, in some cases in single-base pair resolution [1-3]. These recent projects were made possible by the Human Genome Project; indeed, as predicted, the quest to sequence the human genome supported the development of new sequencing and computational methods . As we learned, the results of the Human Genome Project and the new methods it promoted revolutionized the genomics field and supported the development of yet more advanced techniques, the most prominent of which at this moment are the massively parallel sequencing techniques . As result, the discovery of new mutations that explain particular diseases (not only cancer but also inherited pathological conditions, as well as genotypes predisposed to develop such conditions) is achieved with extraordinary reduction in cost and time. The study of the human epigenome, referring to specific chemical modification of DNA or chromatin proteins, is also benefiting from these new technologies. Of special interest in cancer biology is the study of DNA methylation. While there is no question that cataloguing abnormally methylated gene promoters (along with other regions with regulatory function) is very necessary and will likely reveal important players in tumor biology, another pressing question is what drives cells to gain or to lose such a mark. In this review, we will discuss recent advances in mapping DNA methylation, the genomic compartments where it occurs and their biological relevance, and the possible causes of the high incidence of aberrant methylation in certain tumors.
The epigenome in genome-wide scale is still understood to a lesser degree of detail than the genome sequence. One reason is that there are as many epigenomes as cell types; although the bulk of DNA methylation changes little from one tissue to another, the fraction that changes profoundly impacts cell differentiation and disease [6-11]. Another reason for the relative lack of detailed mapping of the epigenome is that it cannot be directly measured. DNA methylation status, for example, is not revealed by direct sequencing and thus depends on additional manipulation . This has resulted in studies of a small fraction of the genome per experiment, analyses that are qualitative rather than quantitative, and in some cases biased measurements of DNA methylation to CpG-rich or CpG-poor regions of the genome, according the chosen method [13,14]. In many instances such biases are desirable, since it is still beyond the capacity of most laboratories to assay every single CpG nucleotide. Fortunately, as for genome studies, the methods to study the epigenome are evolving at a fast pace. The decreasing cost of high-throughput sequencing promises to make genome-wide mapping of methylated cytosines using bisulphite-treated DNA as template [15,16] accessible to a larger number of research groups. Also, new sequencing technologies have reported the direct discrimination of modified dinucleotide bases with a certain degree of accuracy .
Our understanding of the human methylome (both in normal and cancerous cells) has evolved throughout the years, reflecting both the introduction of new technologies and the description of biochemical routes regulating the addition and removal of methylated CpG sites. As for technological advances, the single most extraordinary breakthrough was the introduction of bisulphite-treatment , which moved the field from inaccurate estimates based on Southern blot analysis  to accurate, quantitative analyses. Most important, DNA bisulphite-treatment finally allowed a positive identification of individual methylated CpG sites using the polymerase chain reaction (PCR). Thus, bisulfite-based methods also made possible the study of DNA available in small quantities or of poor quality, like those obtained from frozen or paraffin-embedded tissues. Throughout the years, the technology to study single genes moved from qualitative (methylation-specific PCR ), to semi-quantitative (COBRA, Q-MSP, among other methods [20,21]) and highly quantitative (pyroMeth [22,23]) assays. All these assays were designed to study discrete genomic regions, covering a few hundred base pairs at a time (bisulphite-sequencing) but mostly evaluating a few CpG sites of a single gene per experiment. As a consequence, the studied genes were selected a priori based on function or genomic location, and tumor suppressor genes were the prime candidates. This biased selection of genes resulted in the commonly accepted concept that DNA methylation is an alternative damage to genetic mutations in tumor suppressor genes; that DNA methylation has a result similar to inactivating mutations is true, but DNA methylation targets other molecular functional categories, as well. Also, not all tumor suppressor genes are targeted by DNA methylation. This topic is discussed in further detail in the next sections of this review.
In the same way that global profiling of gene expression is a better strategy to identify molecular signatures of tumor subtypes and clinical outcome, large-scale DNA methylation is also more powerful, mainly when critical markers are unknown for the disease of interest. One of the first attempts of genome-wide (or at least large-scale) methylome analysis adapted a previously published method to detect copy number changes . In this method, after serial digestion of DNA with methylation-sensitive and insensitive restriction enzymes, methylated fragments could be detected as missing signals in two-dimensional gel analysis [25,26]. We learned from this method that a large number of genes (on average 5%) are hypermethylated in cancer, and more tumor-specific markers were identified in this process . The fact that assigning a genomic position to each identified differentially-methylated target is somewhat labor-intensive (the method required gel-extraction and cloning of fragments of interest) was an obstacle to its incorporation in routine analysis. Other methods with similar design become available and again were adopted by a few groups, sometimes limited to the original developers of the method [28-30]. In a short period of time, however, several groups independently developed DNA microarray-based methods to assay DNA methylation [31-33]. These have the advantage that targets are known in advance, so labor-intensive cloning and sequencing of differentially methylated loci is not necessary. With a greater interest in cancer epigenetics, companies developed arrays specifically designed to investigate promoter regions and CpG islands (actually, the first adopted arrays to study DNA methylation were designed for ChIP-on-Chip experiments, but the coverage of gene promoter made these arrays compatible with DNA methylation studies). A limitation of most methylation-microarray methods is that they are still qualitative. Nonetheless, hundreds to thousands of cancer-methylated genes were identified using these methods, and they further confirmed the tissue-specificity and age-related nature of DNA methylation [34,35].
The most recent developments in methylome analysis resulted from massively parallel sequencing techniques. All methods used to generate methylation libraries suitable for microarray analysis can be directly or with slight modifications applied to each of these platforms (thus meDIP has become meDIP-Seq, HELP has became HELP-Seq, and so on; Table 1). Due to the complexity of mammalian genomes and the high-cost associated with single-base mapping of methylated cytosine, most groups employ reduced-representation libraries to study the epigenome [36,37]. The only truly genome-wide mapping of methylated cytosines in humans has been performed for normal cells [16,38], but similar maps of adult cells and of the cancer epigenome will certainly become available soon. Once again, despite the increase in coverage, the results are in concordance with what has been previously reported: promoter CpG islands are in their vast majority unmethylated in normal cells, and gene body methylation correlates positively with gene expression. As novel findings, a difference in CpG methylation between exon and intron regions raises the possibility that gene body methylation participates in splicing regulation. Also, the presence of non-CG methylation (which occurs mostly in the CHG and CHH contexts) in the human genome was reported by Lister et al.  when profiling the methylome of ES (embryonic stem) cells. Non-CpG methylation is well documented in plants and although it has been previously reported for individual genes in mouse ES cells [39,40], the discovery of non-CG methylation mark in such high frequency in the human genome (up to 25% according to Lister et al.) was surprising. Supporting a possible functional role, non-CG methylation showed different distribution between gene bodies (enriched) and regulatory regions (depleted), and is virtually lost during differentiation. There is limited information about non-CG methylation in the cancer methylome [41,42] and more studies are necessary to resolve its prevalence and physiological consequence.
In terms of methods, as one of the biggest breakthroughs in methylation analysis was the development of bisulphite-treatment, the next breakthrough will be the acquisition of information of chemically modified nucleotide bases (not only 5-methylcytosine, but also the recently rediscovered 5-hydroxymethylcytosine) at the same time as collecting sequence information. New sequencing technology yet in development follows the dynamic of dinucleotide incorporation in real-time and records the brief stalling of DNA polymerase when it encounters modified DNA bases . Competing technology using nanopores is also in development . Both methods have only been validated for short synthetic pieces of DNA and need more optimization before routine use for DNA methylation analysis.
Depending on where it is located, DNA methylation causes (or correlates with) different biological outcomes. In a very simplistic way, the genome of most species can be divided into two major compartments based on the DNA composition: single-copy DNA (represented by exons, introns and other non-repetitive DNA) and repetitive DNA (whose main components in humans are retrotransposons of the SINE and LINE classes) [44,45]. This natural division of the genome was described well before more detailed DNA sequencing maps were available [46,47]. Genes, or transcriptional units, are also better studied regarding the effect of DNA methylation on their transcriptional capability when divided into promoter region and gene body. Finally, gene promoters must be categorized as CpG island and non-CpG island promoters. Fig. 1 graphically represents these genomic locations, and how DNA methylation relates to function is summarized in Table 2.
Starting this discussion with repetitive DNA of transposon and retrotransposon kinds, we can appreciate the important role of DNA methylation in maintaining the genome stability. Repetitive elements of SINE (Small Interspersed Nuclear Element), LINE (Long Interspersed Nuclear Elements), LTR (Long Terminal Repeat) and DNA classes originate from DNA or RNA viruses, and also from our own genome mRNA and tRNA molecules which acquired the capacity to replicate independently of the host genome, and to move freely with the help of specialized proteins with endonuclease and ligase functions. Our discussion here will be limited to these kinds of repeats. Evolutionary and molecular studies of multiple species are concordant with an initially explosive activity of these elements, resulting in both an increase in genome size and reshaping both in cis (by introducing new sequences of few bases to several kilobases in length) and in trans (by promoting recombination of different loci intra- and inter-chromosomes) of the genome . Clearly, the result of their activity was increased diversity, the prime material of selective events . Indeed, it is well accepted that the current pattern of gene expression is strongly influenced by past reshaping of the genome by transposition events. However, deleterious effects make it impossible for any cell type or organism to endure massive activity of mobile elements, and these elements were selectively repressed by DNA methylation and other epigenetic modifications. Genome-wide and repeat-specific studies show that the vast majority of SINE, LINE, LTR and other repetitive elements are DNA methylated. Thus, due to their high coverage of the human genome, the bulk of methylated CpGs are in this compartment . It is not entirely clear how the cell machinery recognizes repetitive from non-repetitive DNA. The enzyme LSH (Lymphoid-Specific Helicase) was identified as important in the maintenance of DNA methylation of repetitive elements and thus dubbed a “heterochromatin guardian” [51,52], but further evaluation of knockout models of this enzyme showed an action not only in repetitive DNA, but also single-copy genes . Extensive reports have highlighted that DNA methylation of repetitive elements is frequently disrupted in cancer [54,55], causing genomic instability  and activation of oncogenes .
Gene bodies represent another methylation-rich compartment of the human genome. Contradicting the dogma that DNA methylation correlates with repressed states, gene body marking is found in actively transcribed genes, as measured by mRNA expression [58-60]. This so-called DNA methylation paradox was elegantly addressed in the late nineties , and the proposed explanation implied that the sliding of RNA polymerase over the gene body attracted DNA methyltransferase enzymes. Notably, this insightful proposal was done before the currently well-documented elongation-dependent marking of H3K36me3 in gene bodies . So far, it appears that DNA methylation in a gene body is a consequence of transcription, rather than an active agent in promoting it. However, not much work has been done to mechanistically address this question. Thus the conclusion that intragenic non-promoter DNA methylation simply follows gene expression may change. A recently published study proposes that DNA methylation excludes Polycomb group (PcG) protein binding to DNA, maintaining histones free of H3K27me3 and thus facilitating transcription . Another feature of gene body DNA methylation is an apparent higher DNA methylation of exonic compared to intronic regions, leading to a discussion on whether this marking facilitates splicing .
Proximal gene promoters comprehend in general the positions −1 kb to +0.5 kb from transcription start sites. Nearly 70% of the proximal promoters overlap with a CpG island; structurally, these promoters differ by being typically TATA-less. As discussed before, CpG island promoters are also typically methylation-free in normal cells, with less than 3% of them being methylated. An absence of methylation does not imply promoter activity, and consequently it does not explain tissue-specific differences in gene activity. Thus, gene promoter methylation has been ruled out as the main force behind cell lineage differentiation. However, methylated promoters are always repressed, at least in natural conditions. Non-CpG island promoters (or CpG-poor promoters) are found methylated in normal tissues, and the link between transcriptional repression and DNA methylation is less clear in this class of promoters: in many examples, active genes have methylated CpG-poor promoters. Right there lays the main difference between CpG island and non-CpG island promoters: The different regulatory potential of DNA methylation in promoter activity. Thus, CpG island promoters are the most straightforward compartment to evaluate when searching for aberrant DNA methylation in cancer and speculating as to its involvement in disease initiation and progression, and the knowledge acquired from these studies were summarized in the previous section. Naturally, similarly to genetic alterations, most of de novo DNA methylation in cancer occurs as passenger alterations. However, in many cases DNA methylation affects genes with an important role in tumorigenesis and thus is a driver force in the disease [65,66]. Once established, DNA methylation in CpG islands is stably transmitted to daughter cells. The only well-documented natural reversion to a demethylated status occurs after fertilization, where active erasure of DNA methylation is observed in the paternal DNA, and passive demethylation of maternal DNA . It is still unknown whether the hypomethylation of repetitive elements in cancer occurs through an active or passive mechanism. Inhibition of DNA methylation by drug treatment can reduce the levels of DNA methylation genome-wide, but never to complete erasure of this mark . This has proved a powerful therapeutic approach, particularly in hematopoietic malignances, which have a high rate of disease remission [69,70]. Upon drug withdrawal, re-methylation of repetitive elements and other normally methylated regions occurs relatively fast, suggesting that DNA methylation is closely monitored by cellular mechanisms of homeostasis. Again, CpG-poor promoters behave differently: in the cases where DNA methylation occurs together with a repressed status, forced gene re-expression followed by demethylation can be achieved by increasing the concentration of transcription factors .
CpG islands are also found outside gene promoters, occurring both in inter- and intragenic regions. The function of these CpG islands is less well known, but their overlap with conserved regions among different species suggests that they may be part of distal regulatory regions and enhancers. Their pattern of DNA methylation in normal and cancer resemble that observed for promoter CpG islands, but with a higher frequency of methylation in normal cells. Also, a fraction of inter- and intragenic CpG islands may represent promoter regions of unknown transcripts, or an alternative transcription start site of tissue-specific (or developmental stage-specific) gene variants. In a comprehensive analysis to determine the characteristics of intragenic CpG islands, it was found that they frequently overlap with capped mRNAs and, in further validation steps, novel gene transcripts were discovered to initiate in these islands .
Despite the lack of a large collection of samples from normal or diseased tissues profiled in genome-wide scale for DNA methylation, we have learned from single-gene studies that de novo DNA methylation of promoter CpG islands is a frequent alteration in cancer, resulting in transcriptional silencing of dozens to hundreds of genes per tumor [73,74]. Even with a biased selection of genes, DNA methylation profiling in normal and tumor tissues revealed some interesting patterns. Except for imprinted and X-chromosome inactivated genes, promoter CpG islands are very rarely methylated in normal tissues, but many show tissue-specific gains of methylation in cancer [75,76]. In a given tissue type, some tumors show very little DNA hypermethylation while others display concordant hypermethylation of a subgroup of genes that cannot be explained by chance and thus represent a hypermethylation phenomenon, termed CIMP (CpG island methylator phenotype) . It has also been reported that aging is an important factor in epigenetic instability, as a linear increase in DNA methylation in promoter CpG islands has been observed when comparing normal tissue specimens from young and old donors . Paradoxically, global levels of DNA methylation appear to be reduced in cancer and older individuals . When studied in patients with different disease outcomes, DNA methylation was also found to be a strong predictor of survival. This relationship was not oneway, though: an increased frequency of methylated genes was found to be a marker of poor prognosis in colorectal cancers and in some leukemia subtypes [77,80-83], but highly methylated cases showed better prognosis in acute myeloid leukemia .
A very important conclusion from single-gene analysis was that many silencing events have a driver function in cancer, or at least significantly modulate the tumor biology. Promoter DNA methylation of the CDKN2A gene, likely the most famous example of an epigenetically silenced gene in cancer, also has biological consequences with a tumorigenic potential. The protein product of the short isoform coded by the cdkn2a locus is a potent tumor suppressor gene, and it was known to be deleted in many solid tumors. The report that DNA methylation was the cause of its inactivation in a relatively large fraction of solid tumors  gave credence to the idea of hypermethylation as the “second hit” in Knudson’s tumor suppressor gene inactivation model. In practice, however, finding one allele inactivated by DNA mutation and the other allele hypermethylated is not as frequent as finding both alleles hypermethylated. Another good example of a hypermethylated driver gene is MLH1. Inactivation of this gene by promoter DNA methylation is observed in virtually all sporadic microsatellite unstable colorectal tumors, and further investigation confirmed MLH1 loss as the cause of microsatellite instability in these tumors [86,87]. These and many other findings suggest that, similar to genetic mutation, many DNA methylation events are drivers in tumorigenesis.
As mentioned in the previous sections, methylation errors in cancer have been extensively documented, and the introduction of genome-wide analytical methods has confirmed that both gain and loss in DNA methylation are frequent events in cancer. Also, genome-wide analysis of DNA methylation has confirmed that particular profiles are associated with clinico-pathological states [88-92]. More elusive is the origin of such alterations. What has become clear is that aberrant DNA methylation is not a random event, since only a subset of genes is affected by this modification. Several factors (both endogenous and exogenous) have been linked to abnormal DNA methylation – some discovered in correlative and epidemiological studies, others in well-controlled in vitro experiments (Fig. 2). Some of these factors are summarized here.
Initial reports of silencing of tumor suppressor genes by DNA methylation generated the perception that silencing mechanisms target preferentially certain functional categories. DNA methylation was understood as a random process, and growth advantage and clonal selection of cells with certain hits dictate the final observed methylome. The discrepancy in methylation-predisposition among genes with virtually identical cellular functions (e.g. MLH1 vs. MSH2) clearly indicates that selection does not universally explain all promoter hypermethylation. A deterministic model gained force with the description of genetic and chromatin signatures associated with gene predisposition to DNA methylation in cancer. In a comprehensive search for DNA signatures associated with gene predisposition to DNA methylation, Feltus et al.  described seven short DNA motifs which discriminate frequently methylated genes from others that are predominantly resistant to de novo methylation in cancer. Short DNA motifs were also shown to occur in different frequency between genes methylated in the cancer cell lines CaCo2 (colon) and PC-3 (prostate) .
Although the mechanism by which short DNA motifs are associated with protection from DNA methylation is not revealed in the previous works, it is possible that they are binding sites for proteins with insulator functions. A large part of the genome is constitutively maintained in a repressed state, not only by DNA methylation but also by histone modifications, for example H3K9me3 and H3K27me3. Repressed chromatin is confined in part due to the presence of insulator and blocking elements, for example CTCF and USF1/2 proteins [95,96]. Genes located close to euchromatin/heterochromatin boundaries are subject to position-effect variegation, and it has been proposed that, upon weakening or elimination of boundaries, heterochromatin would spread and cause silencing of adjacent genes . Thus, pockets of methylated DNA (or condensed chromatin) would act as methylation/nucleation centers. Indeed, heterochromatin spreading is a well-described phenomenon in yeast and plants, and it involves the participation of inverted repeats and non-coding RNA [98,99]. In mammalian cells, the involvement of non-coding RNA in DNA methylation has not been confirmed. However, DNA methylation spreading from repetitive DNA was seen in mouse cells, using engineered plasmids to mimic the mouse aprt loci . Paradoxically, we recently found that promoter CpG islands located in genomic regions rich in retrotransposons are resistant to de novo methylation in cancer , and that this marking is both complementary to and independent of other genetic and chromatin signatures, like the presence of short DNA motifs and PcG protein marking. It is unlikely that a simple unifying signature of methylation exists; rather, various predictors need to be reconciled in a multivariate mode with weighting scores. Also, a differentiation should be made between models that explain DNA methylation across tissues types, and those that will clarify, for example, why the p15 gene promoter is methylated in hematological malignancies but very rarely in solid tumors. Finally, these signatures must be understood as the basis upon which Darwinian selection works. In other words, selective events modify gene predisposition; indeed, methylation-prone oncogenes identified in by our predictive model are not found methylated in cancer (unpublished observations).
A distinctive feature of DNA methylation in cancer is its relative tissue-specificity. Thus, features other than DNA sequence must play a role in predisposition or resistance to silencing by methylation in cancer. Transcriptional programming evidenced by basal levels of gene expression and/or chromatin patterns in normal tissues may be revealing in this case. The prototypical example is promoter marking by PcG proteins such as Eed and Suz12, and the resulting H3K27me3 in embryonic stem (ES) cells. After the initial description of genome-wide targets of these proteins in human embryonic stem cells , three groups independently reported that genes targeted by PcG proteins are more frequently targeted by DNA methylation in cancer [103-105]. Further dissection of PcG marking and DNA methylation revealed that these two distinct silencing events occur more frequently alone than in combination in cancer , a finding confirmed by others and named “epigenetic switch” to describe the exchange of H3K27me3 mark in gene promoters in normal tissues by DNA methylation in cancer . The mechanism behind epigenetic switching is still unknown. However, the fact that chromatin marks in ES cells predict DNA methylation in cancer supports the idea that aberrant DNA methylation does not occur randomly, and can be linked to tissue-specific epigenetic states in normal tissues.
Gene specificity and transcriptional programs account for some of the fundamental patterns of aberrant DNA methylation in cancer, but cannot explain patient to patient variability in the process within a given tumor type. Thus, there must be some cellular and host factors (in addition to selection) that shape the eventual cancer methylome. Known mediators of epigenetic variability include aging, inflammation and exposures, and there are likely several as yet unknown factors as well.
Aging is undoubtedly the most important risk factor associated with adult human malignancies. The classical interpretation is that association occurs due to increased exposure to environmental carcinogens and accumulation of genetic mutations. From this point of view, it seems logical that the longer one survives, the higher the chance of occurrence of deleterious mutations affecting key regulatory genes. Both the DNA sequence and the epigenome change with aging. A decrease in DNA methylation content during aging was reported more than two decades ago , and such alteration occurs mostly through a loss of methylation in intergenic and interspersed retroelements. The lack of convincing data supporting a correlation between decreased methylation in aging and increased gene expression has undermined the oncogenic potential of such alterations. However, some recent evidence in colon cancer supports a role for global demethylation in the increased rate of chromosomal abnormalities, and in the activation of oncogenes [57,109]. Historically, the surprising discovery that a gain in methylation of single-copy CpG islands also occurs during aging gave a fresh view on this subject . In this case, increased methylation in gene promoters can account for reduced gene expression. This process was thought to be far less widespread than hypomethylation, with a few genes identified after much search. However, the use of genome-wide methylation profiling of young and old mice revealed that more than 700 genes show age-related methylation . Notably, according to similar studies done in humans, there is a large overlap among genes hypermethylated as a consequence of aging and tumorigenesis, suggesting a mechanistic link between these processes [110-112]. It is important to note that aging is partially due to the accumulative number of cell replications and, from this angle, cancer cells are the ultimate old cell, either in vivo or in vitro. Thus, aging accounts for a large fraction of epigenetic variation in normal tissues, and can also explain some of the variation seen in cancerous tissues as well.
Inflammation and chronic diseases are also predisposition factors to cancer formation. For example, the risk of gastric cancer is higher in individuals with chronic gastritis compared to those without gastritis, and infection by Helicobacter pylori is the most common cause of gastric and duodenal ulcers . Other pathogens are also implicated in cancer development, for example HPV in cervical carcinomas, and HBV and HCV in hepatic cancer [114,115]. Inflammation is also the likely mediator of increased cancer risk in diseases such as Barret’s esophagus and inflammatory bowel disease . Also, chemical agents are also well-known carcinogens; tobacco smoking is the leading cause of lung cancer, and again inflammation of bronchial epithelium contributes to the carcinogenic potential of this agent . One common molecular link between all these exposures is aberrant DNA methylation. A positive relationship between H. pylori infection and DNA methylation has been seen before cellular transformation of the gastric mucosa [118-120], and similar observations have been reported for viral infection and liver cancer [121,122]. Chronic inflammatory states and smoking have both been linked to increased methylation in normal mucosa and/or cancer cells [123,124]. One possible explanation for these associations is that the higher proliferation of inflammatory tissues influences the onset of aberrant DNA methylation, in the same way that multiple cell divisions do during aging. Alternatively, epigenetic reprogramming as a direct consequence of viral infection is also possible: the adenovirus e1a protein has been shown to bind to gene promoters and cause the recruitment of cellular remodeling complexes, like p300/CBP . In addition, a link between HBV infection and down-regulation of mir152, resulting in up-regulation of DNMT1 and consequent hypermethylation of multiple gene promoters has been recently proposed . The association of inflammation and DNA methylation could provide another explanation for epigenetic variation in Humans.
The hundreds of reports on de novo methylated genes in cancer often attribute this to deregulation of the controlling mechanisms of epigenetic marking during tumorigenesis. However, as discussed earlier, much of this “deregulation” could be attributable to stochastic errors in replicating DNA methylation in a subset of the genome intrinsically susceptible to such errors. There is limited evidence or true ongoing deregulation of the DNA methylation machinery in most cancers. However, large scale studies have now identified subgroups of cancers that may in fact have such deregulation. A hypermethylation phenotype characterized by simultaneous DNA hypermethylation of multiple genes in the same tumor specimens was described by Toyota et al. , a phenomenon termed CIMP (CpG islands methylator phenotype). In colorectal tumors, this concomitant hypermethylation of selected loci (which did not show clear age-dependent methylation and were thus named cancer-specific) occurs in 10–40% of the patient samples, depending on how CIMP is defined. This phenotype is associated with specific clinico-pathological features, such as tumor location (predominance of right side lesions), prognosis (CIMP tumors have a poor outcome, except when affected by microsatellite instability) and molecular characteristics (BRAF and KRAS mutations are frequent in CIMP tumors) . After this initial report, CIMP has been confirmed in several solid and hematopoietic malignancies [128-131]. The causes of CIMP are unknown, but it is quite possible that a major regulator of epigenetic homeostasis is impaired in these tumors, thus causing true epigenetic instability. In addition, profound loss of methylation has been identified in a small subset of cancers , and it appears likely that an epigenetic regulator is abnormal in these cases as well. Recently, inactivating mutations in DNMT3a were described in some leukemias , and this gene is a candidate to explain profound hypomethylation in some cancers. Thus, in addition to aging, inflammation and exposures, there likely are specific defects in the epigenetic machinery that help account for the variation in DNA methylation observed in cancer.
Despite what has been learned in the past decade, there remain many unknowns in the field. For example, the relation between histone modifications and DNA methylation continues to be a rich avenue of research, particularly given the emerging data on a plethora of mutations in chromatin regulators in cancer. It will be interesting to see how these relate to DNA methylation. Similarly, the ideas of constitutional epigenetic defects predisposing to cancer or of genetic predisposition to epigenetic defects are beginning to be explored. Finally, the contribution to epigenetics of lifestyle factors such as diet, exercise or obesity, all of which play a role in cancer development, remains to be firmly established.
It is an exciting time for methylation studies. The current and prospective technologies allow for profiling of normal and tumor cells with unprecedented depth and accuracy. Methylome maps are being compared to maps of histone modifications, transcription factors and genetic variants. With correct bioinformatics tools to mine the meaningful data, we are poised to learn how gene microenvironments, transcription programs and host factors interact to influence the onset of DNA hypermethylation in cancer.
M.R.H.E. is supported by the Leukemia Specialized Program of Research Excellence Grant P50 CA100632. J.-P.J.I. is an American Cancer Society Clinical Research Professor.