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DNA methylation and genomic imprinting: insights from cancer into epigenetic mechanisms
seminars in CANCER BIOLOGY, Vol. 12, 2002: pp. 389–398 doi:10.1016/S1044–579X(02)00059-7, available online at http://www.idealibrary.com on
DNA methylation and genomic imprinting: insights from cancer into epigenetic mechanisms Andrew P. Feinberg a,b,c,∗ , Hengmi Cui a,b and Rolf Ohlsson d general implication, not only in the understanding of cancer biology but chromatin in general. The first indication that epigenetics played a role in cancer was the discovery in 1983, by Feinberg and Vogelstein,1 of altered methylation of genes in colorectal tumors. These alterations were found at the time to occur in all cancers and adenomas,2 making them by far the most common type of genetic change in cancer, which is still true in light of current knowledge. While these first observations were of colorectal cancer, they have been generalized to virtually all types of neoplasia.3 Many genes in cancers lose methylation, many gain methylation, and at least in colon cancer, there is also a generalized loss of total methylation content in the genome.3–5 Both gains and losses are likely important. Gains of methylation include promoters of tumor suppressor genes, and it has been hypothesized that the methylation alteration itself is responsible for gene silencing.6 While that may be true, it is also possible that other chromatin alterations play a primary role in gene silencing. For example, it has recently been shown in Neurospora that methylation is dependent upon histone modification.7 Similarly, losses of methylation likely lead to chromosome instability. This has been shown directly by treatment with 5-aza-2 -deoxycytidine,8 and by direct observation of tumors.9 The epigenetic alteration that is the main focus of this chapter is loss of genomic imprinting, which is linked to alterations in methylation. Indeed epigenetic and genetic alterations are interrelated.10 One of the most important ideas we wish to communicate in this review is that the study of human disease is an extremely powerful tool to understand normal biology. This is an old idea in genetics, of course, first promulgated by Garrod in his studies of inborn errors of metabolism, and has been true from the studies of alkaptonuria, through the great insights in cholesterol metabolism.11 However, in the study of epigenetics this idea is particularly important, since outbred populations may reflect a more normal epigenetic milieu than an inbred laboratory strain. This
Since the discovery of epigenetic alterations in cancer 20 years ago by Feinberg and Vogelstein, a variety of such alterations have been found, including global hypomethylation, gene hypomethylation and hypermethylation, and loss of imprinting (LOI). LOI may precede the development of cancer and may thus serve as a common marker for risk, but also as a model for understanding the developmental mechanism for normal imprinting. Key words: epigenetics / DNA methylation / genomic imprinting © 2002 Published by Elsevier Science Ltd.
Introduction Epigenetics is defined as stable alterations in the genome, heritable through cell division, that do not involve the DNA sequence itself. Epigenetic alterations are reversible, at least in the germline, and they often act over a distance. The distance can be relatively small, in the order of kilobases, as in telomere silencing in yeast, or the distance can be large, in the order of megabases, as in position effect variegation in Drosophila. For these reasons, epigenetic alterations are all thought to involve modifications of chromatin, and one of the most intriguing questions in this field is whether common types of modification account for the diverse examples of epigenetic effects. As will be discussed in this review, some of the lessons gained from the study of imprinting in cancer do have From the a Institute of Genetic Medicine, b Departments of Medicine, of Molecular Biology & Genetics, and Oncology, Johns Hopkins University School of Medicine, 1064 Ross, 720 Rutland Avenue, Baltimore, MD 21205, USA and d Department of Animal Development and Genetics, Uppsala University, Uppsala, Sweden. *Corresponding author. E-mail: [email protected] © 2002 Published by Elsevier Science Ltd. 1044–579X / 02 / $ – see front matter c Departments
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idea was brought home acutely in a recent study that showed that the very first mutation every studied, by the Swedish botanist Carl von Linne (who of course is remembered as Linnaeus), a change in symmetry of the flower Linaria vulgaris. Vincent and Coen12 found that this mutation was caused by an epigenetic modification, namely methylation of the Lcyc gene, rather than by a conventional mutation, and this epigenetic alteration has been stably propagated for at least 200 years! In a striking discussion, the authors argue that this type of epigenetic alteration, while uncommon in laboratory strains, may be much more common in the natural world.12 This idea, that some of the most important insights come from studies of disease, is certainly true in the study of genomic imprinting.
allele is activated, but also includes silencing of a normally expressed allele. This is an important point, because it should clarify some confusion in the literature and it makes mechanistic sense. Thus, LOI can include activation of the normally silent copy of a growth-promoting gene, such as insulin-like growth factor II (IGF2),15 or silencing of the normally active copy of a growth-inhibitory gene, such as p57 KIP2 .16 The term ‘gain of imprinting’ has in our view been erroneously used by some authors to indicate silencing of an allele that is normally active.17 Since normal imprinting by definition arises in the gamete or zygote, if a gene shows aberrant parental origin-specific monoallelic silencing in a tumor, then a gametic mark must normally be present near that gene. Thus, aberrant silencing of an allele also falls within the scope of the definition of LOI, since it still involves disruption of an epigenetic mark. LOI was first discovered in Wilms tumor,15,18 an embryonal kidney cancer, and it is the most common molecular alteration in these tumors, occurring in 50–70% of tumors, compared to mutations in the WT1 gene, which occur in only 5% of tumors. These observations were first extended to other embryonal tumors of childhood, including hepatoblastoma and rhabdomyosarcoma.19–23 LOI was then also found commonly in most adult solid tumors, including colorectal, liver, and lung cancer,24–29,40 as well as in leukemia.30 The most commonly affected gene identified to date is IGF2, which shows relaxation of the normal silencing of the maternal allele in tumors. Other genes showing disrupted imprinting include p57 KIP2 , TSSC3, TSSC5, which are genes on 11p15, and fall within the same imprinted gene domain (reviewed in Reference 14). However, other imprinted genes are not immune to LOI. For example, ARHI, on 1p31, shows imprinting with silencing of the maternal allele and loss of expression of the paternal allele in ovarian and breast cancer.31 Likely, the number of imprinted genes altered in cancer is limited by the relatively small number of currently known imprinted genes, and our knowledge of the role of imprinting in cancer will be augmented greatly by the discovery of the much larger number of imprinted genes suspected to exist. Genomic imprinting need not require monoallelic expression. Indeed, it is more common in humans than in laboratory animals to observe allelic preference rather than monoallelic expression. This is likely due to the fact that outbred animals are quite different epigenetically from inbred strains. By extrapolation, the epigenetic background contributes to the differential
Genomic imprinting Genomic imprinting is a form of epigenetic inheritance that distinguishes maternal and paternal alleles. Imprinting, and epigenetic alterations in general, are commonly studied in the context of gene silencing, but the intrinsic definition refers to the heritable modification itself. This modification may have consequences beyond gene expression changes, such as pairing of homologous chromosomes, or organization of chromatin. It should be borne in mind that differential expression is not the original definition, which was introduced by Helen Crouse in 1961, to describe the preferential heterochromatinization and extrusion of paternal chromosomes.13 However, it is becoming increasingly clear that epigenetic alterations are often inter-related, and indeed modification of a chromosomal mark even in the absence of expression changes in a particular tissue might be biologically important. We suggest that genomic imprinting be defined broadly as an epigenetic modification that is parental origin specific, and/or preferential expression of a specific parental allele in somatic cells of the offspring.
Loss of imprinting (LOI) in cancer LOI is defined as a parental origin-specific epigenetic modification that is disrupted, and can include gain or loss of methylation or other chromosomal marks, or loss of the normal pattern of parental originspecific gene expression.14 The term was chosen because it does not require that a normally silenced 390
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penetrance of genetic diseases although the involved genetic lesions might be identical in the affected individuals. Accordingly, some genes might be imprinted in only a subpopulation of humans. Although the data are sparse and controversial, it has been argued that the WT1 and IGF2R genes are imprinted in only some individuals and tissues.32,33 While we need to analyze much larger cohorts, the various epigenetic backgrounds are likely to differentially influence the predisposition to cancer.
Table 1. A new view of the molecular biology of Wilms tumor
LOI is associated with a subclass of Wilms tumors
Developmental stage
Nephrogenic commitment
Nephrogenic expansion
Mechanism Age Molecular pathway Gene Frequency Pathology Hereditary syndrome
Genetic Younger Mutation, LOH WT1, WT2 1:15,000 ILNR Denys–Drash
Epigenetic Older Methylation, LOI IGF2 1:100 PLNR BWS
The bimodal age distribution of Wilms tumor can be explained by the mechanistic distinction between classical two-hit Knudsonian genetic alterations (WT1 and WT2), and epigenetic alterations with altered methylation and LOI.
A surprising recent study of Ravenel et al.34 offers a model for understanding the role LOI may play in childhood tumors, and the study shows that the prior conception of Wilms tumor genetics was likely incomplete. Wilms tumor shows a bimodal age distribution, with tumors arising in very early infancy, or at several years of age. Knudson and Strong35 proposed that the earlier tumors arise in patients who have a germline mutation and a second arising postnatally, and that the later tumors require two hits in postnatal life. Knudson’s model, correct for retinoblastoma, has grown to embody a hypothesis of Comings that the two hits are allelic for a tumor suppressor gene.36,37 By Knudson’s model, the early tumors tend to be bilateral (because the first hit is in all cells) and the later tumors are predominantly unilateral.35 However, as Breslow and coworkers have pointed out, the epidemiology of Wilms tumor is more complex, as bilateral tumors are rare, accounting only for a small percentage of Wilms tumors, even though half of Wilms tumors arise early.38,39 Ravenel et al.34 resolved this conundrum, by showing that the late-arising tumors develop from an epigenetic mechanism involving LOI, and the early-arising tumors arise by a conventional genetic mechanism, including both the bilateral and unilateral Knudsonian cases, with mutations in the WT1 tumor suppressor gene, or LOH of a region harboring a WT2 gene on chromosome 11. These authors observed that essentially all of the tumors with LOI arise late, and show a distinct pathology associated with renal proliferation late in development, rather than the disrupted nephrogenesis characteristic of tumors with WT1 mutations and/or LOH.34 Consistent with these observations, these authors also showed for the first time with statistical power that tumors with LOI undergo increased expression of IGF2, a potent mitogenic factor in normal late nephrogenesis, as well
as an important autocrine growth factor in cancer.34 Intriguingly, p57 KIP2 expression, an imprinted tumor suppressor gene, showed reduced expression in these tumors, suggesting that IGF2 effects may be mediated through p57 KIP2 , and direct evidence for this idea comes from cultured cells in defined media exposed to increasing amounts of IGF2.34 This new mechanistic view of the biology of Wilms tumor is summarized in Tables 1 and 2, as the bimodal age distribution of Wilms tumor can be explained by the mechanistic distinction between classical two-hit Knudsonian genetic alterations (WT1 and WT2), and epigenetic alterations with altered methylation and LOI.
Table 2. Recent advances in understanding epigenetic mechanisms Insights into mechanism Identification of DNA methyltransferases and paralogues Proteins that bind methylated DNA (MBDs) Proteins that don’t bind methylated DNA (CTCF) Renaissance of histones (posttranslational modifications including histone methylation) Genome-wide identification of CpG islands (methylome) There is ample evidence that LOI involves a range of mechanisms. We should expect, therefore, that the repertoire of epigenetic dysregulation of ICRs and other pivotal cis elements will vary depending on which control mechanism is perturbed.
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syndrome, respectively. In particular, Feinberg and coworkers41 mapped BWS, a disorder of prenatal overgrowth, birth defects and predisposition to Wilms and other tumors, to 11p15, and Mannens et al.42 observed that the disorder showed parental origin-specific disease penetrance. Feinberg and coworkers then identified the first cluster of imprinted genes, distributed over a 1 Mb region, and found both genetic and epigenetic alterations of several of them in cancer and BWS.15,43–47 Furthermore, these investigators established that this 1 Mb domain is itself divided into two imprinted subdomains, with several nonimprinted genes between them, formulating a two-domain hypothesis for this region.47–49 p57 KIP2 is mutated in a minority of BWS patients,50–52 and the gene, which encodes a cyclin-dependent kinase inhibitor, is epigenetically silenced in some tumors.16,53 They also found that KV LQT1, which encodes a voltage-gated potassium channel, is imprinted and expressed from the maternal chromosome. It is a large gene (450 kb) and spans all of the balanced germline chromosomal rearrangement breakpoints within the BWSCR1 cluster in BWS patients.54 We have hypothesized that these breakpoints define a region that separates an enhancer from p57 KIP2 in these patients.49,55 Consistent with this idea, Lee et al.47 and Smilinich et al.56 discovered a CpG island upstream of a paternally expressed RNA antisense to KV LQT1, termed LIT1. This CpG island is methylated exclusively on the maternal allele, consistent with LIT1 silencing, and is thus a differentially methylated region (DMR). The LIT1 DMR undergoes loss of methylation with LOI of LIT1, consistent with the hypothesis of p57 KIP2 regulation, i.e. is now amenable to an insulator separating p57 KIP2 from its enhancer.47 Also consistent with this idea, knockout of this sequence leads to silencing of p57 KIP2 on the same chromosome,57 and rearrangement within this region in mice silences p57 KIP2 as well.58 In this case, studies of human disease predicted and led to mouse studies that confirmed them. Furthermore, most of the genes in this region were identified in humans before they were studied in the mouse. In fact, the comparison between human and mouse is particularly helpful in identifying the key regulatory sequences that control epigenetic silencing in this (and other) large gene domains. Onyango et al. 59 performed a comparison of the sequence of 1 Mb of both human and mouse. In order to identify cis-acting regulatory sequences that can mediate epigenetic alterations, e.g. methylation, over such a large region, they compared the 1 Mb imprinted gene domain on 11p15 with the orthologous region on mouse 7.
Epigenetic lesions occur early in colorectal cancer The analysis of LOI has also provided novel insights in the understanding of adult malignancies. Thus, Cui et al.40 found that about one-third of colorectal cancers undergo LOI, and that LOI is also found in the matched normal colon of the same patients. In order to prove that LOI was not simply a developmental alteration unrelated to cancer per se, these authors analyzed the normal colon of patients without colorectal cancer, and found that the frequency of LOI was threefold greater in cancer patients.40 If this observation is confirmed in larger series of patients, it will represent the first common abnormality in normal cells in the general population associated with cancer risk, and will thus afford the opportunity for more effective risk assessment than conventional family history information. In addition, in at least some of the patients these authors studied, LOI was a constitutional abnormality also present in lymphocytes of these individuals, and thus might lead to a simple blood test for cancer risk.40 Moreover, Cui et al.40 observed that LOI occurred in the very patients whose tumors exhibited microsatellite instability (MSI), even though MSI is not present in the normal colonic cells of these patients. This work indicates a link between LOI and other epigenetic alterations in cancer, as MSI is not normally associated with mismatch repair gene mutations. We propose, therefore, that epigenetic lesions in normal tissue set the stage for neoplasia. The IGF2 LOI could in these instances directly contribute to a modified epigenetic state that leads to a range of effects, including inactivation of mismatch repair genes. However, we cannot exclude the possibility that IGF2 LOI is diagnostic of a more general epigenetic disruption in the ‘normal’ colon of the affected patients. These epigenetic lesions may be themselves defined in a Mendelian nature in families, they may be acquired, they may depend on environmental factors, such as nutrition, or all of these mechanisms may be true. In this respect, the epigenetics of cancer faces the same challenges met by genetic studies in the previous decade.
Clusters of imprinted genes are differentially dysregulated in human diseases The identification of imprinted genes on chromosomes 11 and 15 led from studies of Beckwith– Wiedemann syndrome (BWS) and Prader–Willi 392
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By this approach, 33 CpG islands and 49 other orthologous nonexonic non-island but GC-rich sequences were identified. They also proposed a two-island rule for imprinted genes, which seems generally true for most or all imprinted genes.59 This imprinted domain hierarchy has subsequently been confirmed in the mouse.60 In human and mouse, the two domains are independently regulated, as LOI in one subdomain does not lead to LOI of the other. For example, LOI in Wilms tumor does not involve p57 KIP2 , KV LQT1 or LIT1.55 Similarly, disrupted imprinting of LIT1 in BWS is independent of LOI of Igf2.47 Similarly, in the mouse, the deletion of the H19 ICR results in LOI of the IGF2 gene, whereas the imprinted states of the other cluster members outside the Igf2/H19 domain are unaffected.61 Even within a domain, LOI can occur by multiple mechanisms, since LOI of Igf2 can occur independently of inactivation of the maternal H19 allele.19,20 Although it has been argued that the H19 gene transcript, which is not translated, has tumor suppressor functions, the generality of this observation remains to be determined. It is potentially interesting that H19 overexpression correlates with reduced translatability of IGF2 mRNAs. Since the transcription of both IGF2 and H19 genes are normally coordinated, the H19 gene might function as a negative feedback regulator of the expressivity of the IGF2 ligand.62 The crosstalk in trans between both within and outside the imprinting clusters is further exemplified by the observation that the IGF2 ligand has been demonstrated to suppress expression of the p57 KIP2 gene.63 Another insight that comes from the study of disease is a recent analysis of BWS that has led to the first epigenotype–phenotype study, determining which epigenetic alterations are associated with which phenotypes in these patients. By performing a case-cohort study of 92 patients, they found that epigenetic alterations of H19, linked to LOI of IGF2, were specifically associated with cancer risk.64 Conversely, epigenetic alterations of LIT1, linked (hypothetically) to p57 KIP2 , were specifically associated with midline abdominal wall closure defects in these patients.64 This makes perfect sense, since LOI of IGF2 is found in tumors, and deletion of p57 KIP2 leads to midline closure defects. It also suggests that the two regions of the imprinted gene domain (separated by 650 kb) have distinct effects, even though disruption of either region causes the syndrome recognized as BWS. Assuming p57 KIP2 is the target of LIT1 regulation, then p57 KIP2 and IGF2 are likely not part of a simple regulatory pathway, although this view does seem at variance
with experiments suggesting that exogenous IGF2 can modify p57 KIP2 expression.63
Mechanisms of LOI and the role of imprinting control regions Imprinting control regions (ICRs) provide a key to our understanding of LOI, since their manifestation ensures that gametic marks will be interpreted in the soma to establish parent of origin-dependent expression domains. As discussed before, not only the methylated state, but also the unmethylated state constitutes an imprint, since it is the methylation difference that matters. This is exemplified by the H19 ICR, which represses the maternal IGF2 allele when unmethylated and the paternal H19 when methylated. The repression of the maternal IGF2 allele is likely to involve the chromatin insulator factor CTCF, which interacts with only the unmethylated H19 ICR to block communication between the upstream promoters and downstream enhancer elements.65 Conversely, the methylated H19 ICR does not interact with CTCF and has no insulator function, leading to an active paternal IGF2 allele.66 The methylation state constitutes, as it were, a binary switch that partitions the IGF2 into an active or inactive expression domain depending on the sex of the transmitting parent. A similar scenario has been documented for the LIT1 ICR,67 suggesting that CTCF-dependent and methylation-sensitive chromatin insulators are common themes for ICRs. For a more detailed treatise of the evidence that identifies CTCF as a key factor controlling the expression status of the IGF2 gene, see the article by Klenova et al. in this volume. Not surprisingly, Wilms tumors with LOI universally show aberrant methylation of the maternal H19 ICR,68,69 specifically involving CTCF binding sites,70 which in all likelihood abrogates CTCF binding and hence, allows IGF2 activation. What are the underlying mechanisms? Since point mutations of CTCF target sites within the H19 ICR lead to loss of methylation protection during mouse development (Pant, Kanduri and Ohlsson, unpublished observation), mechanisms that interfere with CTCF–DNA interactions are likely culprits. For example, mutations in CTCF zinc fingers that lead to loss of ability to interact with the H19 ICR71 are likely to lead to IGF2 LOI. However, CTCF mutations are very rare in Wilms tumors.71 Instead, overexpression of methyltransferases may by brute force overcome the methylation protection of the maternal H19 ICR allele, providing 393
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a more general explanation for LOI. Indeed, an episomal system transfected into cells overexpressing the de novo DNMT3B methyltransferase recapitulates the kinetics of both IGF2 and H19 LOI. Although the chromatin insulator function and the unique chromatin conformation of the H19 ICR are initially present, they eventually succumb to the methylation pressure mounted by an overexpressed DNMT3B. Intriguingly, the methylation spreading originates from the H19 gene body, which is rich in inverted repeats. In this system, reporter gene expression is initially silent due to the insulator function, while the gradual acquisition of a methylated state subsequently activates the reporter gene due to loss of insulator function. Finally, the methylation spreading process silences the reporter gene permanently (Kanduri et al., unpublished observation). Other mechanisms are also possible. The insulator domain of the maternal H19 ICR allele could be intact, but rendered superfluous by accessory factors. Indeed, our preliminary results show that the H19 ICR insulates the IGF2 promoter in one cell type, but not in another, notably the Hep3B cell line. The insulator function could be restored in the same cells by switching the IGF2 promoter to an H19 promoter (Pant and Ohlsson, unpublished observation). Since the IGF2 promoter-specific effect
does not depend on an independent activation of the IGF2 promoter, we postulate the existence of factors that are able to neutralize insulator functions without perturbing their chromatin contexts. In addition to gain of methylation disrupting imprinting, loss of methylation may also be important in some tumor types. While Wilms tumors with LOI universally show aberrant methylation of the maternal H19 ICR, specifically involving CTCF binding sites, abrogating CTCF binding and allows IGF2 activation, that is not the case for other tumors. It was first shown that hepatoblastoma does not undergo altered methylation of the H19 ICR or H19 silencing, even in the presence of LOI, indicating that there is a distinct mechanism not only for LOI, but for normal silencing of IGF2. More recently, Nakagawa et al.72 showed aberrant methylation of the H19 ICR in colorectal cancer, although the data of some of the present authors is not consistent with this finding, and that loss of methylation may be important in tumors with LOI (H. M. Cui and A. P. Feinberg, unpublished observation). Consistent with this idea, Cui and Feinberg have observed that engineered disruption of DNMT1 and DNMT3B together in colorectal cancer cells, but not either gene alone, causes loss of methylation of the H19 ICR and other nearby DMR sequences within 11p15
Figure 1. Inverse correlation between IGF2 and H19 gene expression in a BWS patient displaying hemohypertrophy. (A) Genotyping shows that the maternal alleles for D11S922 and INT2 markers for chromosome 11 are deleted specifically in the right-hand tongue specimen of the BWS patient. The left-hand tongue is trisomic for chromosome 11 while the right-hand tongue is predominantly UPD 11. (B) In situ hybridization analysis shows that H19 expression is much reduced in the right-hand tongue compared to the left-hand tongue, visualizing mosaicism of aneuploid correction. (C) Shows the spatial patterns of IGF2 and H19 expression in adjacent sections of right-hand tongue. The expression patterns of the H19 and IGF2 genes are strikingly reciprocal, reinforcing the previously observed inverse correlation in trans between cytoplasmic H19 and IGF2 transcript levels.62 (D) RNAase protection analysis20 to show that IGF2 is expressed from only the paternally derived allele(s) of IGF2 in the left-hand tongue of this BWS patient.
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(as discussed earlier), as well as LOI, i.e. abnormal activation of the normally silent maternal allele of IGF2 (H. M. Cui and A. P. Feinberg, unpublished observation). These studies, therefore, will likely elucidate an independent mechanism for LOI, but also demonstrate the presence of an independent epigenetic regulator for IGF2, distinct from the H19 ICR. Another potential mechanism for inhibition of IGF2 expression is a trans-acting effect of the H19 RNA itself. Loss of H19 may promote tumor growth.73 Moreover, a BWS case with pronounced hemihypertrophy showed mosaicism for paternal chromosome 11 triploidy and uniparental disomy in the tongue. Figure 1 shows the previously unpublished observation that loss of the maternal (expressed) copy of H19 was, at the cellular level, associated with increased expression of IGF2 in the right-hand tongue. This observation underscores the existence of a correlation in trans between cytoplasmic H19 transcripts and levels and translatability of cytoplasmic IGF2 mRNAs.62 Yet another mechanism to generate LOI might depend on the recently discovered paralogue of CTCF. This novel gene is termed BORIS for Brother of Regulator of Imprinted States. Although normally expressed in association with epigenetic reprogramming during male germline development,74 BORIS shows ectopic expression in a wide variety of tumors, including Wilms tumor. These intriguing observations suggest that BORIS can disrupt normal CTCF-mediated gene silencing in normal development and in tumors. We envisage that overexpression of BORIS will lead to down-regulation of CTCF expression. Since we also predict that the range of target sites for BORIS and CTCF are only overlapping, a subset of CTCF-specific target sites might be left unprotected leading to biallelic methylation and LOI. Taken together, there is ample evidence that LOI involves a range of mechanisms. We should expect therefore, that the repertoire of epigenetic dysregulation of ICRs and other pivotal cis elements will vary depending on which control mechanism is perturbed. These alternative mechanisms of LOI are depicted schematically in Figure 2.
Figure 2. Multiple mechanisms leading to LOI of IGF2. Normally IGF2 is expressed from the paternal allele, and H19 from the maternal allele. Several mechanisms can lead to LOI and biallelic expression of IGF2: (1) The H19 ICR is normally unmethylated on the maternal allele (open box), allowing binding to CTCF (red circle), which serves as an insulator separating an enhancer (diamond) from IGF2 on the maternally inherited chromosome. Aberrant methylation of the maternal H19 ICR (hatched box), possibly related to increased methyltransferase activity (green pentagon), abrogates CTCF binding, allowing activation of IGF2 by the enhancer, as on the paternal chromosome; (2) BORIS (green circle) may displace CTCF or disrupt H19 ICR methylation; (3) A DMR within IGF2 is normally methylated (hatched box) on the maternal chromosome, and it may undergo loss of methylation (open box), possibly due to decreased methyltransferase activity (red pentagon), allowing binding of a transcriptional activator (green triangle) and activation of the maternal IGF2 allele; (4) The H19 transcript may itself normally inhibit IGF2 expression, and loss of the transcript (by LOH, for example), may lead to activation of IGF2.
site, because ordinary sequencing does not reveal it. Nevertheless, it represents the fruitful target of epigenetic modifications in normal development and disease. Strichman-Almashanu et al.75 have recently made a jump toward identifying the methylome in a strategy designed to isolate normally methylated CpG islands from the human genome. In addition to finding many new DMRs, at least some of which clearly identify novel imprinted genes, they found a class of previously unrecognized CpG islands methylated on both maternal and paternal alleles in somatic cells. Some of these lie within the promoters of genes that show germline-restricted expression, e.g. MAGE, the melanoma and testis-specific antigen. Others appear to serve a global epigenetic role, as they are consistently located near the ends of chromosomes.75 Moreover, the identification of these sequences raises two additional intriguing possibilities: (1) They may
The methylome: a novel perspective of cancer epigenetics An important future direction for these studies is the definition of the components of the methylome, i.e. the total methylation content of the cell. The methylome cannot be found on the GenBank web395
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show loss of methylation in tumors, causing aberrant gene activation; (2) They may show polymorphic variation in the population, adding epigenetic diversity in both normal development and disease. Examples of both possibilities are forthcoming (R. S. Lee and A. P. Feinberg, unpublished observation). Finally, in the context of cancer it is important to bear in mind that epigenetic alterations are reversible, and possibly easily affected by the environment, unlike conventional genetic mutations. While it is difficult to conceive a therapeutic strategy to replace a mutated gene in patients with a normal copy, it is much less fantastic to imagine restoring a normal pattern of imprinting and methylation to cells. For example, 5aza-2-deoxycytidine has been shown to restore a normal pattern of imprinting to tumor cells with LOI, without disrupting imprinting on the normally marked allele.76 The studies of Cui et al.40 are particularly exciting in this regard, in that one may be able to identify an epigenetic alteration affecting the entire organ system, prior to the development of cancer. We conclude with a very hopeful idea. Our current approach to cancer is focused on established disease, and to a much lesser extent on early detection of malignancy. Instead of this (or in addition), we suggest that we will survey all individuals for epigenetic alterations, identify patients at risk for enhanced surveillance (e.g. colonoscopy), and indeed modify or eliminate their risk with dietary or therapeutic modifications targeted at these epigenetic alterations. In that respect, the practice of oncology will come more to resemble the practice of cardiology, and it will be the rare octogenarian who complains to his physician that these interventions were a waste of his time because he never developed cancer in the first place.
3. Feinberg AP (2000) DNA methylation, genomic imprinting and cancer. Curr Top Microbiol Immunol 249:87–99 4. Feinberg AP, Gehrke CW, Kuo KC, Ehrlich M (1988) Reduced genomic 5-methylcytosine content in human colonic neoplasia. Cancer Res 48:1159–1161 5. Feinberg AP, Rainier S, DeBaun MR (1995) Genomic imprinting, DNA methylation, and cancer. J Natl Cancer Inst Monogr 17:21–26 6. Merlo A, Herman JG, Mao L, Lee DJ, Gabrielson E, Burger PC et al. (1995) 5 CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nat Med 1:686–692 7. Tamaru H, Selker EU (2001) A histone H3 methyltransferase controls DNA methylation in Neurospora crassa. Nature 414:277–283 8. Schmid M, Haaf T, Grunert D (1984) 5-Azacytidine-induced undercondensations in human chromosomes. Hum Genet 67:257–263 9. Lengauer C, Kinzler KW, Vogelstein B (1998) Genetic instabilities in human cancers. Nature 396:643–649 10. Feinberg A (2001) Cancer epigenetics takes center stage. Proc Natl Acad Sci USA 98:392–394 11. Goldstein J, Brown MS (1973) Familial hypercholesterolemia— identification of a defect in regulation of 3-hydroxy-3methylglutaryl coenzyme-A reductase-activity associated with overproduction of cholesterol. Proc Natl Acad Sci USA 70:2804–2808 12. Cubas P, Vincent C, Coen E (1999) An epigenetic mutation responsible for natural variation in floral symmetry. Nature 401:157–161 13. Crouse HV, Brown A, Mumford BC (1971) L-Chromosome inheritance and problem of chromosome imprinting in Sciara (Sciaridae, Diptera). Chromosoma 34:324 14. Feinberg AP (2001) Genomic imprinting and cancer, in The Metabolic and Molecular Bases of Inherited Disease (Scriver CR, Beaudet AL, Sly WS, Valle D, eds) pp 525–537. McGraw-Hill, New York 15. Rainier S, Johnson LA, Dobry CJ, Ping AJ, Grundy PE, Feinberg AP (1993) Relaxation of imprinted genes in human cancer. Nature 362:747–749 16. Thompson JS, Reese KJ, DeBaun MR, Perlman EJ, Feinberg AP (1996) Reduced expression of the cyclin-dependent kinase inhibitor p57 KIP2 in Wilms tumor. Cancer Res 56:5723–5727 17. Schwienbacher C, Gramantieri L, Scelfo R, Veronese A, Calin GA, Bolondi L et al. (2000) Gain of imprinting at chromosome 11p15: a pathogenetic mechanism identified in human hepatocarcinomas. Proc Natl Acad Sci USA 97:5445–5449 18. Ogawa O, Eccles MR, Szeto J, McNoe LA, Yun K, Maw MA et al. (1993) Relaxation of insulin-like growth factor II gene imprinting implicated in Wilms’ tumour. Nature 362:749–751 19. Rainier S, Dobry CJ, Feinberg AP (1995) Loss of imprinting in hepatoblastoma. Cancer Res 55:1836–1838 20. Li X, Adam G, Cui H, Sandstedt B, Ohlsson R, Ekstrom TJ (1995) Expression, promoter usage and parental imprinting status of insulin-like growth factor II (IGF2) in human hepatoblastoma: uncoupling of IGF2 and H19 imprinting. Oncogene 11:221–229 21. Zhan S, Shapiro DN, Helman LJ (1994) Activation of an imprinted allele of the insulin-like growth factor II gene implicated in rhabdomyosarcoma. J Clin Invest 94:445–448 22. Zhan S, Shapiro DN, Helman LJ (1995) Loss of imprinting of IGF2 in Ewing’s sarcoma. Oncogene 11:2503–2507 23. Vu TH, Yballe C, Boonyanit S, Hoffman AR (1995) Insulin-like growth factor II in uterine smooth-muscle tumors: maintenance of genomic imprinting in leiomyomata and loss of imprinting in leiomyosarcomata. J Clin Endocrinol Metab 80:1670–1676
Acknowledgements This work was supported by NIH grant CA65145 (APF) and grants from Swedish Cancer Foundation, Pediatric Cancer Foundation and Lundsberg Foundation (RO). We thank Melinda Graber for preparing the manuscript.
References 1. Feinberg AP, Vogelstein B (1983) Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature 301:89–92 2. Goelz SE, Vogelstein B, Hamilton SR, Feinberg AP (1985) Hypomethylation of DNA from benign and malignant human colon neoplasms. Science 228:187–190
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24. Douc-Rasy S, Barrois M, Fogel S, Ahomadegbe JC, Stehelin D, Coll J et al. (1996) High incidence of loss of heterozygosity and abnormal imprinting of H19 and IGF2 genes in invasive cervical carcinomas. Uncoupling of H19 and IGF2 expression and biallelic hypomethylation of H19. Oncogene 12:423–430 25. Hibi K, Nakamura H, Hirai A, Fujikake Y, Kasai Y, Akiyama S et al. (1996) Loss of H19 imprinting in esophageal cancer. Cancer Res 56:480–482 26. Takeda S, Kondo M, Kumada T, Koshikawa T, Ueda R, Nishio M et al. (1996) Allelic-expression imbalance of the insulin-like growth factor 2 gene in hepatocellular carcinoma and underlying disease. Oncogene 12:1589–1592 27. Hashimoto K, Azuma C, Koyama M, Ohashi K, Kamiura S, Nobunaga T et al. (1995) Loss of imprinting in choriocarcinoma. Nat Genet 9:109–110 28. van Gurp RJHLM, Oosterhuis JW, Kalscheuer V, Mariman ECM, Looijenga LHJ (1994) Biallelic expression of the H19 and IGF2 genes in human testicular germ cell tumors. J Natl Cancer Inst 86:1070–1075 29. Jarrard DF, Bussemakers MJ, Bova GS, Isaacs WB (1995) Regional loss of imprinting of the insulin-like growth factor II gene occurs in human prostate tissues. Clin Cancer Res 1:1471–1478 30. Randhawa GS, Cui H, Barletta JM, Strichman-Almashanu L, Talpaz M, Kantarjian H et al. (1998) Loss of imprinting in disease progression in chronic myelogenous leukemia. Blood 91:3144– 3147 31. Yu Y, Xu F, Peng H, Fang X, Zhao S, Li Y et al. (1999) NOEY2 (ARHI), an imprinted putative tumor suppressor gene in ovarian and breast carcinomas. Proc Natl Acad Sci USA 96:214–219 32. Jinno Y, Yun K, Nishiwaki K, Kubota T, Ogawa O, Reeve AE et al. (1994) Mosaic and polymorphic imprinting of the WT1 gene in humans. Nat Genet 6:305–309 33. Xu Y, Goodyer CG, Deal C, Polychronakos C (1993) Functional polymorphism in the parental imprinting of the human IGF2R gene. Biochem Biophys Res Commun 197:747–754 34. Ravenel JD, Broman KW, Perlman EJ, Niemitz EL, Jayawardena TM, Bell DW et al. (2001) Loss of imprinting of insulin-like growth factor-II (IGF2) gene in distinguishing specific biologic subtypes of Wilms tumor. J Natl Cancer Inst 93:1698–1703 35. Knudson AG, Strong LC (1972) Mutation and cancer: a model for Wilms tumor of the kidney. J Natl Cancer Inst 48:313–324 36. Knudson AG (1993) Antioncogenes and human cancer. Proc Natl Acad Sci USA 90:10914–10921 37. Comings DE (1973) A general theory of carcinogenesis. Proc Natl Acad Sci USA 70:3324–3328 38. Breslow NE, Beckwith JB (1982) Epidemiological features of Wilms tumor: results of the National Wilms Tumor Study. J Natl Cancer Inst 68:429–436 39. Breslow N, Beckwith JB, Ciol M, Sharples K (1988) Age distribution of Wilms tumor: report from the national Wilms’ tumor study. Cancer Res 48:1653–1657 40. Cui H, Horon IL, Ohlsson R, Hamilton SR, Feinberg AP (1998) Loss of imprinting in normal tissue of colorectal cancer patients with microsatellite instability. Nat Med 4:1276–1280 41. Ping AJ, Reeve AE, Law DJ, Young MR, Boehnke M, Feinberg AP (1989) Genetic linkage of Beckwith–Wiedemann syndrome to 11p15. Am J Hum Genet 44:720–723 42. Mannens M, Hoovers JMN, Redeker E, Verjaal M, Feinberg AP, Little P et al. (1995) Parental imprinting of human chromosome region 11p15.4-pter involved in the Beckwith–Wiedemann syndrome and various human neoplasia. Hum Genet 2:3–23 43. Matsuoka S, Thompson JS, Edwards MC, Barletta JM, Grundy P, Kalikin LM et al. (1996) Imprinting of the gene encoding a human cyclin-dependent kinase inhibitor, p57 KIP2 , on chromosome 11p15. Proc Natl Acad Sci USA 93:3026–3030
44. Lee MP, Hu R-J, Johnson LA, Feinberg AP (1997) Human KV LQT1 shows tissue-specific imprinting and is physically disrupted by Beckwith–Wiedemann syndrome chromosomal rearrangements. Nat Genet 15:181–185 45. Lee MP, Feinberg AP (1998) Genomic imprinting of a human apoptosis gene homologue, TSSC3. Cancer Res 58:1052–1056 46. Lee MP, Reeves C, Schmitt A, Su K, Connors TD, Hu R-J et al. (1998) Somatic mutation of TSSC5, a novel imprinted gene from human chromosome 11p15.5. Cancer Res 58:4155–4159 47. Lee MP, DeBaun MR, Mitsuya K, Galonek HL, Brandenburg S, Oshimura M et al. (1999) Loss of imprinting of a paternally expressed transcript, with antisense orientation to KVLQT1, occurs frequently in Beckwith–Wiedemann syndrome and is independent of insulin-like growth factor II imprinting. Proc Natl Acad Sci USA 96:5203–5208 48. Lee MP, Brandenburg S, Landes GM, Adams M, Miller G, Feinberg AP (1999) Two novel genes in the center of the 11p15 imprinted domain escape genomic imprinting. Hum Mol Genet 8:683–690 49. Feinberg AP (2000) The two-domain hypothesis in Beckwith– Wiedemann syndrome. J Clin Invest 106:739–740 50. Hatada I, Ohashi H, Fukushima Y, Kaneko Y, Inoue M, Komoto Y (1996) An imprinted gene p57 KIP2 is mutated in Beckwith–Wiedemann syndrome. Nat Genet 14:171–173 51. Lee MP, DeBaun M, Randhawa GS, Reichard BA, Feinberg AP (1997) Low frequency of p57 KIP2 mutation in Beckwith– Wiedemann syndrome. Am J Hum Genet 61:304–309 52. O’Keefe D, Dao D, Zhao L, Sanderson R, Warburton D, Weiss L et al. (1997) Coding mutations in p57 KIP2 are present in some cases of Beckwith–Wiedemann syndrome but are rare or absent in Wilms tumors. Am J Hum Genet 61:295–303 53. Oya M, Schulz WA (2000) Decreased expression of p57 KIP2 mRNA in human bladder cancer. Br J Cancer 83:626–631 54. Lee MP, Hu RJ, Johnson LA, Feinberg AP (1997) Human KV LQT1 gene shows tissue-specific imprinting and encompasses Beckwith–Wiedemann syndrome chromosomal rearrangements. Nat Genet 15:181–185 55. Mitsuya K, Meguro M, Lee MP, Katoh M, Schulz TC, Kugoh H et al. (1999) LIT1, an imprinted antisense RNA in the human KV LQT1 locus identified by screening for differentially expressed transcripts using monochromosomal hybrids. Hum Mol Genet 8:1209–1217 56. Smilinich NJ, Day CD, Fitzpatrick GV, Caldwell GM, Lossie AC, Cooper PR et al. (1999) A maternally methylated CpG island in KV LQT1 is associated with an antisense paternal transcript and loss of imprinting in Beckwith–Wiedemann syndrome. Proc Natl Acad Sci USA 96:8064–8069 57. Horike S, Mitsuya K, Meguro M, Kotobuki N, Kashiwagi A, Notsu T et al. (2000) Targeted disruption of the human LIT1 locus defines a putative imprinting control element playing an essential role in Beckwith–Wiedemann syndrome. Hum Mol Genet 9:2075–2083 58. Cleary MA, van Raamsdonk CD, Levorse J, Zheng BH, Bradley A, Tilghman SM (2001) Disruption of an imprinted gene cluster by a targeted chromosomal translocation in mice. Nat Genet 29:78–82 59. Onyango P, Miller W, Lehoczky J, Leung C, Birren B, Wheelan S et al. (2000) Sequence and comparative analysis of the mouse 1 megabase region orthologous to the human 11p15 imprinted domain. Genome Res 10:1697–1710 60. Paulsen M, Davies KR, Bowden LM, Villar AJ, Franck O, Fuermann M et al. (1998) Syntenic organization of the mouse distal chromosome 7 imprinting cluster and the Beckwith– Wiedemann syndrome region in chromosome 11p15.5. Hum Mol Genet 7:1149–1159
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69. Moulton T, Crenshaw T, Hao Y, Moosikasuwan J, Lin N, Dembitzer F et al. (1994) Epigenetic lesions at the H19 locus in Wilms’ tumour patients. Nat Genet 7:440–447 70. Cui H, Niemitz EL, Ravenel JD, Onyango P, Brandenburg SA, Lobanenkov VV et al. (2001) Loss of imprinting of insulin-like growth factor-II in Wilms tumor commonly involves altered methylation but not mutations of CTCF or its binding site. Cancer Res 61:4947–4950 71. Filippova GN, Qi CF, Ulmer JE, Moore JM, Ward MD, Hu YJ et al. (2002) Tumor-associated zinc finger mutations in the CTCF transcription factor selectively alter its DNA-binding specificity. Cancer Res 62:48–52 72. Nakagawa H, Chadwick RB, Peltomaki P, Plass C, Nakamura Y, de la CA (2001) Loss of imprinting of the insulin-like growth factor II gene occurs by biallelic methylation in a core region of H19-associated CTCF-binding sites in colorectal cancer. Proc Natl Acad Sci USA 98:591–596 73. Zhang Y, Tycko B (1992) Monoallelic expression of the human H19 gene. Nat Genet 1:40–44 74. Loukinov D, Pugacheva E, Vatolin S, Moon H, Pack VP, Kanduri C et al. (2002) The germ cell-specific factors BORIS, a paralogue of the ubiquitously expressed chromatin insulator protein CTCF, is abnormally activated in multiple cancers. Proc Natl Acad Sci USA 99:6806–6811 75. Strichman-Almashanu, LZ, Lee R, Onyango P, Perlman E, Flam F et al. (2002) A genome-wide screen for normally methylated human CpG islands that can identify novel imprinted genes. Genome Res 12:543–554 76. Barletta JM, Rainier S, Feinberg AP (1997) Reversal of loss of imprinting in tumor cells by 5-aza-2 -deoxycytidine. Cancer Res 57:48–50
61. Caspary T, Cleary MA, Baker CC, Guan XJ, Tilghman SM (1998) Multiple mechanisms regulate imprinting of the mouse distal chromosome 7 gene cluster. Mol Cell Biol 18:3466– 3474 62. Li YM, Franklin G, Cui HM, Svensson K, He XB, Adam G et al. (1998) The H19 transcript is associated with polysomes and may regulate IGF2 expression in trans. J Biol Chem 273:28247– 28252 63. Grandjean V, Smith J, Schofield PN, Ferguson-Smith AC (2000) Increased IGF-II protein affects p57 KIP2 expression in vivo and in vitro: implications for Beckwith–Wiedemann syndrome. Proc Natl Acad Sci USA 97:5279–5284 64. DeBaun MR, Niemitz EL, Mcneil DE, Brandenburg SA, Lee MP, Feinberg AP (2002) Epigenetic alterations of H19 and LIT1 distinguish patients with Beckwith–Wiedemann syndrome with cancer and birth defects. Am J Hum Genet 70:604–611 65. Ohlsson R, Renkawitz R, Lobanenkov V (2001) CTCF is a uniquely versatile transcription regulator linked to epigenetics and disease. Trends Genet 17:520–527 66. Holmgren C, Kanduri C, Dell G, Ward A, Mukhopadhya R, Kanduri M et al. (2001) CpG methylation regulates the Igf2/H19 insulator. Curr Biol 11:1128–1130 67. Kanduri C, Fitzpatrick G, Mukhopadhyay R, Kanduri M, Lobanenkov V, Higgins M et al. (2002) A differentially methylated imprinting control region within the Kcnq1 locus harbours a methylation-sensitive chromatin insulator. J Biol Chem 277:18106–18110 68. Steenman MJC, Rainier S, Dobry CJ, Grundy P, Horon IL, Feinberg AP (1994) Loss of imprinting of IGF2 is linked to reduced expression and abnormal methylation of H19 in Wilms’ tumor. Nat Genet 7:433–439
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DNA methylation and genomic imprinting: insights from cancer into epigenetic mechanisms Andrew P. Feinberg a,b,c,∗ , Hengmi Cui a,b and Rolf Ohlsson d general implication, not only in the understanding of cancer biology but chromatin in general. The first indication that epigenetics played a role in cancer was the discovery in 1983, by Feinberg and Vogelstein,1 of altered methylation of genes in colorectal tumors. These alterations were found at the time to occur in all cancers and adenomas,2 making them by far the most common type of genetic change in cancer, which is still true in light of current knowledge. While these first observations were of colorectal cancer, they have been generalized to virtually all types of neoplasia.3 Many genes in cancers lose methylation, many gain methylation, and at least in colon cancer, there is also a generalized loss of total methylation content in the genome.3–5 Both gains and losses are likely important. Gains of methylation include promoters of tumor suppressor genes, and it has been hypothesized that the methylation alteration itself is responsible for gene silencing.6 While that may be true, it is also possible that other chromatin alterations play a primary role in gene silencing. For example, it has recently been shown in Neurospora that methylation is dependent upon histone modification.7 Similarly, losses of methylation likely lead to chromosome instability. This has been shown directly by treatment with 5-aza-2 -deoxycytidine,8 and by direct observation of tumors.9 The epigenetic alteration that is the main focus of this chapter is loss of genomic imprinting, which is linked to alterations in methylation. Indeed epigenetic and genetic alterations are interrelated.10 One of the most important ideas we wish to communicate in this review is that the study of human disease is an extremely powerful tool to understand normal biology. This is an old idea in genetics, of course, first promulgated by Garrod in his studies of inborn errors of metabolism, and has been true from the studies of alkaptonuria, through the great insights in cholesterol metabolism.11 However, in the study of epigenetics this idea is particularly important, since outbred populations may reflect a more normal epigenetic milieu than an inbred laboratory strain. This
Since the discovery of epigenetic alterations in cancer 20 years ago by Feinberg and Vogelstein, a variety of such alterations have been found, including global hypomethylation, gene hypomethylation and hypermethylation, and loss of imprinting (LOI). LOI may precede the development of cancer and may thus serve as a common marker for risk, but also as a model for understanding the developmental mechanism for normal imprinting. Key words: epigenetics / DNA methylation / genomic imprinting © 2002 Published by Elsevier Science Ltd.
Introduction Epigenetics is defined as stable alterations in the genome, heritable through cell division, that do not involve the DNA sequence itself. Epigenetic alterations are reversible, at least in the germline, and they often act over a distance. The distance can be relatively small, in the order of kilobases, as in telomere silencing in yeast, or the distance can be large, in the order of megabases, as in position effect variegation in Drosophila. For these reasons, epigenetic alterations are all thought to involve modifications of chromatin, and one of the most intriguing questions in this field is whether common types of modification account for the diverse examples of epigenetic effects. As will be discussed in this review, some of the lessons gained from the study of imprinting in cancer do have From the a Institute of Genetic Medicine, b Departments of Medicine, of Molecular Biology & Genetics, and Oncology, Johns Hopkins University School of Medicine, 1064 Ross, 720 Rutland Avenue, Baltimore, MD 21205, USA and d Department of Animal Development and Genetics, Uppsala University, Uppsala, Sweden. *Corresponding author. E-mail: [email protected] © 2002 Published by Elsevier Science Ltd. 1044–579X / 02 / $ – see front matter c Departments
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idea was brought home acutely in a recent study that showed that the very first mutation every studied, by the Swedish botanist Carl von Linne (who of course is remembered as Linnaeus), a change in symmetry of the flower Linaria vulgaris. Vincent and Coen12 found that this mutation was caused by an epigenetic modification, namely methylation of the Lcyc gene, rather than by a conventional mutation, and this epigenetic alteration has been stably propagated for at least 200 years! In a striking discussion, the authors argue that this type of epigenetic alteration, while uncommon in laboratory strains, may be much more common in the natural world.12 This idea, that some of the most important insights come from studies of disease, is certainly true in the study of genomic imprinting.
allele is activated, but also includes silencing of a normally expressed allele. This is an important point, because it should clarify some confusion in the literature and it makes mechanistic sense. Thus, LOI can include activation of the normally silent copy of a growth-promoting gene, such as insulin-like growth factor II (IGF2),15 or silencing of the normally active copy of a growth-inhibitory gene, such as p57 KIP2 .16 The term ‘gain of imprinting’ has in our view been erroneously used by some authors to indicate silencing of an allele that is normally active.17 Since normal imprinting by definition arises in the gamete or zygote, if a gene shows aberrant parental origin-specific monoallelic silencing in a tumor, then a gametic mark must normally be present near that gene. Thus, aberrant silencing of an allele also falls within the scope of the definition of LOI, since it still involves disruption of an epigenetic mark. LOI was first discovered in Wilms tumor,15,18 an embryonal kidney cancer, and it is the most common molecular alteration in these tumors, occurring in 50–70% of tumors, compared to mutations in the WT1 gene, which occur in only 5% of tumors. These observations were first extended to other embryonal tumors of childhood, including hepatoblastoma and rhabdomyosarcoma.19–23 LOI was then also found commonly in most adult solid tumors, including colorectal, liver, and lung cancer,24–29,40 as well as in leukemia.30 The most commonly affected gene identified to date is IGF2, which shows relaxation of the normal silencing of the maternal allele in tumors. Other genes showing disrupted imprinting include p57 KIP2 , TSSC3, TSSC5, which are genes on 11p15, and fall within the same imprinted gene domain (reviewed in Reference 14). However, other imprinted genes are not immune to LOI. For example, ARHI, on 1p31, shows imprinting with silencing of the maternal allele and loss of expression of the paternal allele in ovarian and breast cancer.31 Likely, the number of imprinted genes altered in cancer is limited by the relatively small number of currently known imprinted genes, and our knowledge of the role of imprinting in cancer will be augmented greatly by the discovery of the much larger number of imprinted genes suspected to exist. Genomic imprinting need not require monoallelic expression. Indeed, it is more common in humans than in laboratory animals to observe allelic preference rather than monoallelic expression. This is likely due to the fact that outbred animals are quite different epigenetically from inbred strains. By extrapolation, the epigenetic background contributes to the differential
Genomic imprinting Genomic imprinting is a form of epigenetic inheritance that distinguishes maternal and paternal alleles. Imprinting, and epigenetic alterations in general, are commonly studied in the context of gene silencing, but the intrinsic definition refers to the heritable modification itself. This modification may have consequences beyond gene expression changes, such as pairing of homologous chromosomes, or organization of chromatin. It should be borne in mind that differential expression is not the original definition, which was introduced by Helen Crouse in 1961, to describe the preferential heterochromatinization and extrusion of paternal chromosomes.13 However, it is becoming increasingly clear that epigenetic alterations are often inter-related, and indeed modification of a chromosomal mark even in the absence of expression changes in a particular tissue might be biologically important. We suggest that genomic imprinting be defined broadly as an epigenetic modification that is parental origin specific, and/or preferential expression of a specific parental allele in somatic cells of the offspring.
Loss of imprinting (LOI) in cancer LOI is defined as a parental origin-specific epigenetic modification that is disrupted, and can include gain or loss of methylation or other chromosomal marks, or loss of the normal pattern of parental originspecific gene expression.14 The term was chosen because it does not require that a normally silenced 390
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penetrance of genetic diseases although the involved genetic lesions might be identical in the affected individuals. Accordingly, some genes might be imprinted in only a subpopulation of humans. Although the data are sparse and controversial, it has been argued that the WT1 and IGF2R genes are imprinted in only some individuals and tissues.32,33 While we need to analyze much larger cohorts, the various epigenetic backgrounds are likely to differentially influence the predisposition to cancer.
Table 1. A new view of the molecular biology of Wilms tumor
LOI is associated with a subclass of Wilms tumors
Developmental stage
Nephrogenic commitment
Nephrogenic expansion
Mechanism Age Molecular pathway Gene Frequency Pathology Hereditary syndrome
Genetic Younger Mutation, LOH WT1, WT2 1:15,000 ILNR Denys–Drash
Epigenetic Older Methylation, LOI IGF2 1:100 PLNR BWS
The bimodal age distribution of Wilms tumor can be explained by the mechanistic distinction between classical two-hit Knudsonian genetic alterations (WT1 and WT2), and epigenetic alterations with altered methylation and LOI.
A surprising recent study of Ravenel et al.34 offers a model for understanding the role LOI may play in childhood tumors, and the study shows that the prior conception of Wilms tumor genetics was likely incomplete. Wilms tumor shows a bimodal age distribution, with tumors arising in very early infancy, or at several years of age. Knudson and Strong35 proposed that the earlier tumors arise in patients who have a germline mutation and a second arising postnatally, and that the later tumors require two hits in postnatal life. Knudson’s model, correct for retinoblastoma, has grown to embody a hypothesis of Comings that the two hits are allelic for a tumor suppressor gene.36,37 By Knudson’s model, the early tumors tend to be bilateral (because the first hit is in all cells) and the later tumors are predominantly unilateral.35 However, as Breslow and coworkers have pointed out, the epidemiology of Wilms tumor is more complex, as bilateral tumors are rare, accounting only for a small percentage of Wilms tumors, even though half of Wilms tumors arise early.38,39 Ravenel et al.34 resolved this conundrum, by showing that the late-arising tumors develop from an epigenetic mechanism involving LOI, and the early-arising tumors arise by a conventional genetic mechanism, including both the bilateral and unilateral Knudsonian cases, with mutations in the WT1 tumor suppressor gene, or LOH of a region harboring a WT2 gene on chromosome 11. These authors observed that essentially all of the tumors with LOI arise late, and show a distinct pathology associated with renal proliferation late in development, rather than the disrupted nephrogenesis characteristic of tumors with WT1 mutations and/or LOH.34 Consistent with these observations, these authors also showed for the first time with statistical power that tumors with LOI undergo increased expression of IGF2, a potent mitogenic factor in normal late nephrogenesis, as well
as an important autocrine growth factor in cancer.34 Intriguingly, p57 KIP2 expression, an imprinted tumor suppressor gene, showed reduced expression in these tumors, suggesting that IGF2 effects may be mediated through p57 KIP2 , and direct evidence for this idea comes from cultured cells in defined media exposed to increasing amounts of IGF2.34 This new mechanistic view of the biology of Wilms tumor is summarized in Tables 1 and 2, as the bimodal age distribution of Wilms tumor can be explained by the mechanistic distinction between classical two-hit Knudsonian genetic alterations (WT1 and WT2), and epigenetic alterations with altered methylation and LOI.
Table 2. Recent advances in understanding epigenetic mechanisms Insights into mechanism Identification of DNA methyltransferases and paralogues Proteins that bind methylated DNA (MBDs) Proteins that don’t bind methylated DNA (CTCF) Renaissance of histones (posttranslational modifications including histone methylation) Genome-wide identification of CpG islands (methylome) There is ample evidence that LOI involves a range of mechanisms. We should expect, therefore, that the repertoire of epigenetic dysregulation of ICRs and other pivotal cis elements will vary depending on which control mechanism is perturbed.
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syndrome, respectively. In particular, Feinberg and coworkers41 mapped BWS, a disorder of prenatal overgrowth, birth defects and predisposition to Wilms and other tumors, to 11p15, and Mannens et al.42 observed that the disorder showed parental origin-specific disease penetrance. Feinberg and coworkers then identified the first cluster of imprinted genes, distributed over a 1 Mb region, and found both genetic and epigenetic alterations of several of them in cancer and BWS.15,43–47 Furthermore, these investigators established that this 1 Mb domain is itself divided into two imprinted subdomains, with several nonimprinted genes between them, formulating a two-domain hypothesis for this region.47–49 p57 KIP2 is mutated in a minority of BWS patients,50–52 and the gene, which encodes a cyclin-dependent kinase inhibitor, is epigenetically silenced in some tumors.16,53 They also found that KV LQT1, which encodes a voltage-gated potassium channel, is imprinted and expressed from the maternal chromosome. It is a large gene (450 kb) and spans all of the balanced germline chromosomal rearrangement breakpoints within the BWSCR1 cluster in BWS patients.54 We have hypothesized that these breakpoints define a region that separates an enhancer from p57 KIP2 in these patients.49,55 Consistent with this idea, Lee et al.47 and Smilinich et al.56 discovered a CpG island upstream of a paternally expressed RNA antisense to KV LQT1, termed LIT1. This CpG island is methylated exclusively on the maternal allele, consistent with LIT1 silencing, and is thus a differentially methylated region (DMR). The LIT1 DMR undergoes loss of methylation with LOI of LIT1, consistent with the hypothesis of p57 KIP2 regulation, i.e. is now amenable to an insulator separating p57 KIP2 from its enhancer.47 Also consistent with this idea, knockout of this sequence leads to silencing of p57 KIP2 on the same chromosome,57 and rearrangement within this region in mice silences p57 KIP2 as well.58 In this case, studies of human disease predicted and led to mouse studies that confirmed them. Furthermore, most of the genes in this region were identified in humans before they were studied in the mouse. In fact, the comparison between human and mouse is particularly helpful in identifying the key regulatory sequences that control epigenetic silencing in this (and other) large gene domains. Onyango et al. 59 performed a comparison of the sequence of 1 Mb of both human and mouse. In order to identify cis-acting regulatory sequences that can mediate epigenetic alterations, e.g. methylation, over such a large region, they compared the 1 Mb imprinted gene domain on 11p15 with the orthologous region on mouse 7.
Epigenetic lesions occur early in colorectal cancer The analysis of LOI has also provided novel insights in the understanding of adult malignancies. Thus, Cui et al.40 found that about one-third of colorectal cancers undergo LOI, and that LOI is also found in the matched normal colon of the same patients. In order to prove that LOI was not simply a developmental alteration unrelated to cancer per se, these authors analyzed the normal colon of patients without colorectal cancer, and found that the frequency of LOI was threefold greater in cancer patients.40 If this observation is confirmed in larger series of patients, it will represent the first common abnormality in normal cells in the general population associated with cancer risk, and will thus afford the opportunity for more effective risk assessment than conventional family history information. In addition, in at least some of the patients these authors studied, LOI was a constitutional abnormality also present in lymphocytes of these individuals, and thus might lead to a simple blood test for cancer risk.40 Moreover, Cui et al.40 observed that LOI occurred in the very patients whose tumors exhibited microsatellite instability (MSI), even though MSI is not present in the normal colonic cells of these patients. This work indicates a link between LOI and other epigenetic alterations in cancer, as MSI is not normally associated with mismatch repair gene mutations. We propose, therefore, that epigenetic lesions in normal tissue set the stage for neoplasia. The IGF2 LOI could in these instances directly contribute to a modified epigenetic state that leads to a range of effects, including inactivation of mismatch repair genes. However, we cannot exclude the possibility that IGF2 LOI is diagnostic of a more general epigenetic disruption in the ‘normal’ colon of the affected patients. These epigenetic lesions may be themselves defined in a Mendelian nature in families, they may be acquired, they may depend on environmental factors, such as nutrition, or all of these mechanisms may be true. In this respect, the epigenetics of cancer faces the same challenges met by genetic studies in the previous decade.
Clusters of imprinted genes are differentially dysregulated in human diseases The identification of imprinted genes on chromosomes 11 and 15 led from studies of Beckwith– Wiedemann syndrome (BWS) and Prader–Willi 392
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By this approach, 33 CpG islands and 49 other orthologous nonexonic non-island but GC-rich sequences were identified. They also proposed a two-island rule for imprinted genes, which seems generally true for most or all imprinted genes.59 This imprinted domain hierarchy has subsequently been confirmed in the mouse.60 In human and mouse, the two domains are independently regulated, as LOI in one subdomain does not lead to LOI of the other. For example, LOI in Wilms tumor does not involve p57 KIP2 , KV LQT1 or LIT1.55 Similarly, disrupted imprinting of LIT1 in BWS is independent of LOI of Igf2.47 Similarly, in the mouse, the deletion of the H19 ICR results in LOI of the IGF2 gene, whereas the imprinted states of the other cluster members outside the Igf2/H19 domain are unaffected.61 Even within a domain, LOI can occur by multiple mechanisms, since LOI of Igf2 can occur independently of inactivation of the maternal H19 allele.19,20 Although it has been argued that the H19 gene transcript, which is not translated, has tumor suppressor functions, the generality of this observation remains to be determined. It is potentially interesting that H19 overexpression correlates with reduced translatability of IGF2 mRNAs. Since the transcription of both IGF2 and H19 genes are normally coordinated, the H19 gene might function as a negative feedback regulator of the expressivity of the IGF2 ligand.62 The crosstalk in trans between both within and outside the imprinting clusters is further exemplified by the observation that the IGF2 ligand has been demonstrated to suppress expression of the p57 KIP2 gene.63 Another insight that comes from the study of disease is a recent analysis of BWS that has led to the first epigenotype–phenotype study, determining which epigenetic alterations are associated with which phenotypes in these patients. By performing a case-cohort study of 92 patients, they found that epigenetic alterations of H19, linked to LOI of IGF2, were specifically associated with cancer risk.64 Conversely, epigenetic alterations of LIT1, linked (hypothetically) to p57 KIP2 , were specifically associated with midline abdominal wall closure defects in these patients.64 This makes perfect sense, since LOI of IGF2 is found in tumors, and deletion of p57 KIP2 leads to midline closure defects. It also suggests that the two regions of the imprinted gene domain (separated by 650 kb) have distinct effects, even though disruption of either region causes the syndrome recognized as BWS. Assuming p57 KIP2 is the target of LIT1 regulation, then p57 KIP2 and IGF2 are likely not part of a simple regulatory pathway, although this view does seem at variance
with experiments suggesting that exogenous IGF2 can modify p57 KIP2 expression.63
Mechanisms of LOI and the role of imprinting control regions Imprinting control regions (ICRs) provide a key to our understanding of LOI, since their manifestation ensures that gametic marks will be interpreted in the soma to establish parent of origin-dependent expression domains. As discussed before, not only the methylated state, but also the unmethylated state constitutes an imprint, since it is the methylation difference that matters. This is exemplified by the H19 ICR, which represses the maternal IGF2 allele when unmethylated and the paternal H19 when methylated. The repression of the maternal IGF2 allele is likely to involve the chromatin insulator factor CTCF, which interacts with only the unmethylated H19 ICR to block communication between the upstream promoters and downstream enhancer elements.65 Conversely, the methylated H19 ICR does not interact with CTCF and has no insulator function, leading to an active paternal IGF2 allele.66 The methylation state constitutes, as it were, a binary switch that partitions the IGF2 into an active or inactive expression domain depending on the sex of the transmitting parent. A similar scenario has been documented for the LIT1 ICR,67 suggesting that CTCF-dependent and methylation-sensitive chromatin insulators are common themes for ICRs. For a more detailed treatise of the evidence that identifies CTCF as a key factor controlling the expression status of the IGF2 gene, see the article by Klenova et al. in this volume. Not surprisingly, Wilms tumors with LOI universally show aberrant methylation of the maternal H19 ICR,68,69 specifically involving CTCF binding sites,70 which in all likelihood abrogates CTCF binding and hence, allows IGF2 activation. What are the underlying mechanisms? Since point mutations of CTCF target sites within the H19 ICR lead to loss of methylation protection during mouse development (Pant, Kanduri and Ohlsson, unpublished observation), mechanisms that interfere with CTCF–DNA interactions are likely culprits. For example, mutations in CTCF zinc fingers that lead to loss of ability to interact with the H19 ICR71 are likely to lead to IGF2 LOI. However, CTCF mutations are very rare in Wilms tumors.71 Instead, overexpression of methyltransferases may by brute force overcome the methylation protection of the maternal H19 ICR allele, providing 393
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a more general explanation for LOI. Indeed, an episomal system transfected into cells overexpressing the de novo DNMT3B methyltransferase recapitulates the kinetics of both IGF2 and H19 LOI. Although the chromatin insulator function and the unique chromatin conformation of the H19 ICR are initially present, they eventually succumb to the methylation pressure mounted by an overexpressed DNMT3B. Intriguingly, the methylation spreading originates from the H19 gene body, which is rich in inverted repeats. In this system, reporter gene expression is initially silent due to the insulator function, while the gradual acquisition of a methylated state subsequently activates the reporter gene due to loss of insulator function. Finally, the methylation spreading process silences the reporter gene permanently (Kanduri et al., unpublished observation). Other mechanisms are also possible. The insulator domain of the maternal H19 ICR allele could be intact, but rendered superfluous by accessory factors. Indeed, our preliminary results show that the H19 ICR insulates the IGF2 promoter in one cell type, but not in another, notably the Hep3B cell line. The insulator function could be restored in the same cells by switching the IGF2 promoter to an H19 promoter (Pant and Ohlsson, unpublished observation). Since the IGF2 promoter-specific effect
does not depend on an independent activation of the IGF2 promoter, we postulate the existence of factors that are able to neutralize insulator functions without perturbing their chromatin contexts. In addition to gain of methylation disrupting imprinting, loss of methylation may also be important in some tumor types. While Wilms tumors with LOI universally show aberrant methylation of the maternal H19 ICR, specifically involving CTCF binding sites, abrogating CTCF binding and allows IGF2 activation, that is not the case for other tumors. It was first shown that hepatoblastoma does not undergo altered methylation of the H19 ICR or H19 silencing, even in the presence of LOI, indicating that there is a distinct mechanism not only for LOI, but for normal silencing of IGF2. More recently, Nakagawa et al.72 showed aberrant methylation of the H19 ICR in colorectal cancer, although the data of some of the present authors is not consistent with this finding, and that loss of methylation may be important in tumors with LOI (H. M. Cui and A. P. Feinberg, unpublished observation). Consistent with this idea, Cui and Feinberg have observed that engineered disruption of DNMT1 and DNMT3B together in colorectal cancer cells, but not either gene alone, causes loss of methylation of the H19 ICR and other nearby DMR sequences within 11p15
Figure 1. Inverse correlation between IGF2 and H19 gene expression in a BWS patient displaying hemohypertrophy. (A) Genotyping shows that the maternal alleles for D11S922 and INT2 markers for chromosome 11 are deleted specifically in the right-hand tongue specimen of the BWS patient. The left-hand tongue is trisomic for chromosome 11 while the right-hand tongue is predominantly UPD 11. (B) In situ hybridization analysis shows that H19 expression is much reduced in the right-hand tongue compared to the left-hand tongue, visualizing mosaicism of aneuploid correction. (C) Shows the spatial patterns of IGF2 and H19 expression in adjacent sections of right-hand tongue. The expression patterns of the H19 and IGF2 genes are strikingly reciprocal, reinforcing the previously observed inverse correlation in trans between cytoplasmic H19 and IGF2 transcript levels.62 (D) RNAase protection analysis20 to show that IGF2 is expressed from only the paternally derived allele(s) of IGF2 in the left-hand tongue of this BWS patient.
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(as discussed earlier), as well as LOI, i.e. abnormal activation of the normally silent maternal allele of IGF2 (H. M. Cui and A. P. Feinberg, unpublished observation). These studies, therefore, will likely elucidate an independent mechanism for LOI, but also demonstrate the presence of an independent epigenetic regulator for IGF2, distinct from the H19 ICR. Another potential mechanism for inhibition of IGF2 expression is a trans-acting effect of the H19 RNA itself. Loss of H19 may promote tumor growth.73 Moreover, a BWS case with pronounced hemihypertrophy showed mosaicism for paternal chromosome 11 triploidy and uniparental disomy in the tongue. Figure 1 shows the previously unpublished observation that loss of the maternal (expressed) copy of H19 was, at the cellular level, associated with increased expression of IGF2 in the right-hand tongue. This observation underscores the existence of a correlation in trans between cytoplasmic H19 transcripts and levels and translatability of cytoplasmic IGF2 mRNAs.62 Yet another mechanism to generate LOI might depend on the recently discovered paralogue of CTCF. This novel gene is termed BORIS for Brother of Regulator of Imprinted States. Although normally expressed in association with epigenetic reprogramming during male germline development,74 BORIS shows ectopic expression in a wide variety of tumors, including Wilms tumor. These intriguing observations suggest that BORIS can disrupt normal CTCF-mediated gene silencing in normal development and in tumors. We envisage that overexpression of BORIS will lead to down-regulation of CTCF expression. Since we also predict that the range of target sites for BORIS and CTCF are only overlapping, a subset of CTCF-specific target sites might be left unprotected leading to biallelic methylation and LOI. Taken together, there is ample evidence that LOI involves a range of mechanisms. We should expect therefore, that the repertoire of epigenetic dysregulation of ICRs and other pivotal cis elements will vary depending on which control mechanism is perturbed. These alternative mechanisms of LOI are depicted schematically in Figure 2.
Figure 2. Multiple mechanisms leading to LOI of IGF2. Normally IGF2 is expressed from the paternal allele, and H19 from the maternal allele. Several mechanisms can lead to LOI and biallelic expression of IGF2: (1) The H19 ICR is normally unmethylated on the maternal allele (open box), allowing binding to CTCF (red circle), which serves as an insulator separating an enhancer (diamond) from IGF2 on the maternally inherited chromosome. Aberrant methylation of the maternal H19 ICR (hatched box), possibly related to increased methyltransferase activity (green pentagon), abrogates CTCF binding, allowing activation of IGF2 by the enhancer, as on the paternal chromosome; (2) BORIS (green circle) may displace CTCF or disrupt H19 ICR methylation; (3) A DMR within IGF2 is normally methylated (hatched box) on the maternal chromosome, and it may undergo loss of methylation (open box), possibly due to decreased methyltransferase activity (red pentagon), allowing binding of a transcriptional activator (green triangle) and activation of the maternal IGF2 allele; (4) The H19 transcript may itself normally inhibit IGF2 expression, and loss of the transcript (by LOH, for example), may lead to activation of IGF2.
site, because ordinary sequencing does not reveal it. Nevertheless, it represents the fruitful target of epigenetic modifications in normal development and disease. Strichman-Almashanu et al.75 have recently made a jump toward identifying the methylome in a strategy designed to isolate normally methylated CpG islands from the human genome. In addition to finding many new DMRs, at least some of which clearly identify novel imprinted genes, they found a class of previously unrecognized CpG islands methylated on both maternal and paternal alleles in somatic cells. Some of these lie within the promoters of genes that show germline-restricted expression, e.g. MAGE, the melanoma and testis-specific antigen. Others appear to serve a global epigenetic role, as they are consistently located near the ends of chromosomes.75 Moreover, the identification of these sequences raises two additional intriguing possibilities: (1) They may
The methylome: a novel perspective of cancer epigenetics An important future direction for these studies is the definition of the components of the methylome, i.e. the total methylation content of the cell. The methylome cannot be found on the GenBank web395
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show loss of methylation in tumors, causing aberrant gene activation; (2) They may show polymorphic variation in the population, adding epigenetic diversity in both normal development and disease. Examples of both possibilities are forthcoming (R. S. Lee and A. P. Feinberg, unpublished observation). Finally, in the context of cancer it is important to bear in mind that epigenetic alterations are reversible, and possibly easily affected by the environment, unlike conventional genetic mutations. While it is difficult to conceive a therapeutic strategy to replace a mutated gene in patients with a normal copy, it is much less fantastic to imagine restoring a normal pattern of imprinting and methylation to cells. For example, 5aza-2-deoxycytidine has been shown to restore a normal pattern of imprinting to tumor cells with LOI, without disrupting imprinting on the normally marked allele.76 The studies of Cui et al.40 are particularly exciting in this regard, in that one may be able to identify an epigenetic alteration affecting the entire organ system, prior to the development of cancer. We conclude with a very hopeful idea. Our current approach to cancer is focused on established disease, and to a much lesser extent on early detection of malignancy. Instead of this (or in addition), we suggest that we will survey all individuals for epigenetic alterations, identify patients at risk for enhanced surveillance (e.g. colonoscopy), and indeed modify or eliminate their risk with dietary or therapeutic modifications targeted at these epigenetic alterations. In that respect, the practice of oncology will come more to resemble the practice of cardiology, and it will be the rare octogenarian who complains to his physician that these interventions were a waste of his time because he never developed cancer in the first place.
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Acknowledgements This work was supported by NIH grant CA65145 (APF) and grants from Swedish Cancer Foundation, Pediatric Cancer Foundation and Lundsberg Foundation (RO). We thank Melinda Graber for preparing the manuscript.
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