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doi: 10.1111/joim.12224
Epigenetic stochasticity, nuclear structure and cancer: the implications for medicine A. P. Feinberg From the Center for Epigenetics and Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
Abstract. Feinberg AP (From the Center for Epigenetics and Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA). Epigenetic stochasticity, nuclear structure and cancer: the implications for medicine (Key Symposium). J Intern Med 2014; 276: 5–11. The aim of this review is to summarize an evolution of thinking about the epigenetic basis of human cancer, from the earliest studies of altered DNA methylation in cancer to the modern comprehensive epigenomic era. Converging data from epigenetic studies of primary cancers and from experimental studies of chromatin in development
Introduction This review follows from a lecture I presented at the 10th Key Symposium of the Journal of Internal Medicine and thus is written from a personal perspective with an emphasis on my own laboratory’s experiments and my personal motivations for designing them. Moreover, I wish here to present the ideas so that they may be of interest and value to a general internist. Therefore, cancer genetics specialists are referred to my recent more comprehensive scholarly review in Nature Reviews Cancer [1], which discusses the work of others comprehensively and provides much greater detail than is possible here. Epigenetics provides a molecular mechanism for phenotypic differences amongst organs of an individual, that are copied faithfully during cell division. Such information is transmitted almost entirely in a manner not involving the DNA sequence per se, as that is essentially the same across all cell types. Yet the phenotypic differences amongst organs are quite profound, and in many cases, more so than the differences between a given organ from species to species, at least within a given mammalian clade [1].
and epithelial–mesenchymal transition suggest a role for epigenetic stochasticity as a driving force of cancer, with Darwinian selection of tumour cells at the expense of the host. This increased epigenetic stochasticity appears to be mediated by large-scale changes in DNA methylation and chromatin in domains associated with the nuclear lamina. The implications for diagnosis include the potential to identify stochastically disrupted progenitor cells years before cancer develops, and to target drugs to epigenetic drivers of gene expression instability rather than to mean effects per se. Keywords: cancer, DNA methylation, heterochromatin, LOCKs, nuclear structure.
Moreover, epigenetic information is susceptible to changes in the environment, which can also have an effect on phenotype relevant to human disease. For example, increased intake of S-adenosylmethionine prenatally in a particular strain of an Agouti coat colour allele leads to variable silencing of an alternative promoter and repression of ectopic agouti expression, enhanced yellow coloration and obesity in the offspring [2]. Furthermore, the pesticide vinclozolin can act transgenerationally on sperm to affect DNA methylation and fertility [3]. The types of epigenetic change that mediate phenotypic differences amongst organs and potentially in disease include DNA methylation and histone modifications. DNA methylation is heritable during cell division in mammals only at the dinucleotide CpG. Although non-CpG methylation has been shown to exist, by definition the information is not heritable/epigenetic. DNA methylation is associated with gene silencing and is maintained by DNA methyltransferase I, which recognizes the hemimethylated mCpG/CpG after semiconservative DNA replication and adds a methyl group to the 5-position. There are over 200 known posttranslational modifications of histones, primarily
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on the tails of histones H3 and H4, but the core regions and the other histones are also modified. The canonical modifications most commonly studied are H3K27 and H3K9 methylation associated with silencing and H3K4 methylation and acetylation associated with activation. Epigenetics and human cancer The genetic model for cancer has been well established for three decades, based on identification of defining gene rearrangements for most leukaemias and lymphomas and gene mutations for most solid tumours. Recent advances in high-throughput sequencing reveal many mutations, but relatively few ‘drivers’ that are present early in the tumour and carry through to all descendant clones and metastases [4]. Furthermore, the mutations found commonly in cancer still appear to be quite rare in normal tissue, and thus genetically driven intervention before most cancers arise seems impractical, despite the fact that environmental exposure and even the effect of combined low penetrance genetic alleles act for years before the development of overt disease. Almost as old as the genetic hypothesis of cancer is the epigenetic hypothesis that changes in DNA methylation or chromatin could substitute for, complement and/or precede the development of genetic mutations, contributing to abnormal and heterogeneous gene expression in cancer. The idea arose from an attempt to relate changes in the genome to functional effects on gene activity. The original observation was the loss of methylation of approximately one-third of single-copy genes [5], suggesting a generalized epigenetic disruption of large regions of nuclear chromatin and, as described below, I think we now understand much of the mechanism of this disruption. Moreover, this large-scale epigenetic disruption appears to explain most of the other epigenetic findings in cancer, including hypermethylated CpG islands, hypomethylated CpG island shores and epigenetically driven tumour cell heterogeneity. Before discussing these epigenomic changes, it is worth noting that a strong argument for a causal epigenetic role in cancer came from the study of the rare disorder Beckwith–Wiedemann syndrome (BWS), which increases the risk of Wilms tumour, the most common solid tumour (a kidney cancer) of childhood. Previously, it had not been possible to ascertain whether epigenetic changes were causal 6
ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 5–11
Key Symposium: Epigenetics stochasticity and cancer
or consequential in cancer. BWS shows genetic and epigenetic heterogeneity, including the following mechanisms: causes, including: (i) genetic mutations of the cyclin-dependent kinase inhibitor p57KIP2, an imprinted gene showing expression of a specific (in this case maternal) allele; (ii) aberrant imprinting of the same gene and a related antisense long RNA, LIT1; and (iii) loss of imprinting (LOI) of the gene insulin-like growth factor-II (IGF2), which we and others discovered in sporadically occurring Wilms tumours [6]. The effect of LOI is a double dose of IGF2, leading to overexpansion of nephrogenic blastema cells in the developing kidney, and a high frequency of Wilms tumour [7]. LOI of IGF2 is also associated with an increased frequency of colorectal cancer in adults [8]. In the first epigenotype–phenotype study of any disorder (comparable to genotype–phenotype studies in classical genetics), it was found that LOI of IGF2 accounts almost entirely for the cancer risk in BWS [9] and also universally leads to expansion of the premalignant nephroblastemic progenitor cell compartment [7]. Thus, the epigenetic change (LOI) cannot result as a consequence of cancer; it precedes cancer, is associated with cancer risk and provides a mechanistic basis (expansion of the precursor population) for tumour development [6]. New insights from cancer epigenomics Most of the recent work from my laboratory described below comes from the development of new genome-scale approaches to epigenetics, or ‘epigenomics’, that began with a Center for Epigenetics awarded by the National Human Genome Research Institute. Tools from our center and others in the USA and Europe gave rise to the US Human Epigenome Roadmap and the International Human Epigenome Consortium. For example, approximately 80% of the locations on the now widely used Infinium Human Methylation 450 BeadChip were chosen from experimental data derived from the 2.5 million CpG CHARM methylation array platform developed at our centre and from epigenomic sites identified by 21 other research groups. We first applied these tools to examine the cancer epigenome comprehensively, using the same paradigm as that used in our original study 30 years ago [5], namely human colorectal cancer and matched colonic mucosa from the same patients, the same tissue and cell layer from which the cancers arise. This analysis led to the surprising result that most differentially methylated regions
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(DMRs) that distinguish cancer from normal tissue, that is, c-DMRs, are themselves also t-DMRs, that is, they distinguish some other normal tissue from other normal tissues [10]. In other words, the epigenetic disruptions in cancer lead cells to acquire epigenetic signatures of other tissues, albeit in a random inconsistent way. This result can help to explain the long-known fact that the defining properties of cancer, such as invasion and metastasis, are indeed properties of some normal cells at some stage of normal development; for example, neural crest cells migrate during embryogenesis through epithelial-mesenchymal transition [11], a process that is pathologically activated in cancer. A conundrum was also revealed by our epigenomic data. In addition to the widespread epigenetic heterogeneity we observed in cancers, there was also a degree of epigenetic heterogeneity in normal tissue [10]. Whilst this could be explained in part by genetic heterogeneity in the human population, we observed a similar epigenetic heterogeneity in the normal tissues of inbred laboratory mice [10], suggesting that the epigenome is driven to epigenetic heterogeneity spontaneously, at some genetic loci and not others. This intrinsic normal epigenetic heterogeneity, under genetic control, offered a surprising potential explanation for one of the most important roles of epigenetics in development and disease: stochastic epigenetic variation could be a driving force of development and evolutionary adaptation [12] (Table 1). This idea directly addresses the limits of determinism, first espoused for epigenetics by Konrad Waddington in the 1940s and 1950s, that development is predetermined genetically and that perturbations caused by the environment are corrected developmentally by an increasingly strong pull towards an inevitable tissue outcome, which he termed canalization [13]. Waddington used ordinary differential equations to describe this deterministic approach to developmental biology. However, we observed epigenetic stochasticity in genetically identical mice raised in the same environment, even from the same litter or mice, at crucial developmental genes, such as members of the HOX family of homeodomain proteins that specify axial differences in organ development [12]. This stochastic epigenetic variation does not occur everywhere, only at key genes in which environmental effects can vary. Thus, an underlying genetic sequence determines the variability, which then occurs stochastically or in response to environmental pressure. Such a
Key Symposium: Epigenetics stochasticity and cancer
model introduces a stochastic component to embryology, mediated by epigenetics, similar to the stochastic properties necessary for a complete description of the atom in physics. As in physics, we believe that stochastic mathematics can best describe these epigenetic properties; however, this is ‘work in progress’ and beyond the scope of the present review. We term these inherently epigenetically variable regions variably methylated regions (VMRs) [12]. The stochastic epigenetic model of cancer The stochastic epigenetic model (Table 1) has important implications for evolutionary biology considering environmental effects that may be consistent for many generations but can then change stochastically, for example in response to an environmental crisis such as drought or famine. We could show by modelling that over time, in the presence of an occasionally and randomly switching environment, natural selection would favour genetic sequences promoting variability per se, in the same way that it favours sequences for mean effects if the selecting constant environment is present for a sufficiently long period of time [12]. How might this stochastic epigenetic model underlie disease? An obvious example, as suggested in our model, is cancer, which arises in part from repeated changes to the microenvironment of the tissue. Most cancers arise from cycles of repeated injury and repair. Hepatocellular cancer, the most common fatal human solid tumour, arises directly from this process (in this context cirrhosis, i.e. injury and regeneration of the liver), whether the injury is induced by hepatitis B or by aflatoxin. Similarly, skin cancer arises from repeated cycles of blistering sunburn and repair. Furthermore, inflammatory bowel disease is the most dramatic example of the environmental effect of injury and repair that is likely to underlie many cases of colorectal cancer. We tested the stochastic epigenetic model in cancer by examining DNA methylation comprehensively across the genome as described earlier, but in this case testing specifically for stochastic variability in the methylation signature using methods from econometrics for analysis of random market fluctuations, the formal term for which is heteroscedasticity. We examined several common solid tumours types of adulthood in the West – lung, colon, thyroid and breast – and Wilms tumour, the most common solid tumour of childhood. As predicted, the ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 5–11
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Table 1 The stochastic epigenetic model of human cancer. See text for details, and [1, 12] for a more comprehensive discussion of the relevant literature The stochastic epigenetic model of human cancer Original idea to address ‘missing heritability’ and role of environment in common diseases Limitations of determinism, including Waddington Stochastic variation plays a fundamental role during normal development (similar to quantum mechanics in physics) This stochastic variation is mediated epigenetically The degree of stochastic variation is inherited genetically (no problem with Darwin) Genetic variants that increase epigenetic plasticity could increase fitness over evolution, for traits affected by a fluctuating environment Cancer arises in part from repeated changes in the microenvironment Cycles of injury/repair: hepatocellular cancer, skin cancer, inflammatory bowel disease Epigenetic changes in progenitor cells can increase cancer risk
variability of DNA methylation was an even stronger signature of cancer than was the mean change at any given site of DNA methylation [14]. However, there were also two unexpected findings from this analysis. First, we found that the methylation sites that were most variable in colorectal cancer were also highly variable in the other tumour types. This suggests a universality of these VMRs, and that they might in fact be VMRs for normal development. Secondly, the cancer VMRs completely segregated normal tissue types from each other by cluster analysis. Thus, the sites that show the greatest epigenetic variability in cancer are the very sites that ‘need’ to show stochastic variation in the development of normal tissues during embryonic development [14]. Thus, the epigenetic plasticity model appears to be correct, at least for cancer, and the sites that show stochastic variation in normal development in the derivation of normal tissue types are again prone to epigenetic variability in cancer, across all solid tumour types. This epigenetic stochasticity, and the sites of its variability, could thus provide a mechanistic explanation for the tumour cell heterogeneity that characterizes all cancer and inspired the first experiments in cancer epigenetics three decades ago [5]. 8
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Key Symposium: Epigenetics stochasticity and cancer
An important clue to the mechanism of this epigenetic stochasticity came from whole-genome bisulphite sequencing of three colorectal cancers and their matched colonic mucosa, as well as two premalignant colon adenomas [14]. There were three major results from this analysis. First, essentially all of the epigenetic change in cancer entails large blocks of hypomethylation, involving half of the genome and one-third of single-copy genes; that is, the original observation of cancer hypomethylation accounts for >98% of the affected genomic region in cancer. As a technical aside, these large blocks, up to a megabase in size, do not involve specific hypomethylation of repetitive elements (which are simply enriched about 1.5-fold in the blocks). Rather they involve the entire regions, including the genes therein [14]. Secondly, essentially all of the ‘hypermethylation’ of CpG islands is in fact due to instability at the boundary of these dense CG-rich regions within the large hypomethylated blocks [14, 15]. Thus, the presence of the islands within the blocks, undergoing loss of DNA methylation, causes disruption to the underlying architecture of the islands themselves. Note also that the island hypermethylation is in fact an erosion of a boundary of high methylation outside the island and low methylation within; in fact what occurs is a loss of methylation at the boundaries (shores) and gain of methylation in the interior (the change is not specific to the island element itself). Thirdly, and perhaps most importantly, these changes involve stochastic variability of DNA methylation in the cancer, consistent with a loss of regulation confined to these large blocks. Indeed, the genes within the blocks include the most hypervariably expressed genes in human cancer, and those genes are important mechanistically [14]. Of note, specific defining mutations are not associated with cancer invasion and metastasis, rather these seem to involve epigenetic mechanisms leading to aberrant expression in the absence of mutation [1]. In addition, these same genes, such as matrix metalloproteinases underlying tumour invasion, lie within the blocks, and show hypervariable methylation and expression in cancer [14]. We believe that these changes in block hypomethylation, hypervariability and disrupted CpG island boundaries occur early in cancer, as we found to be the case also in adenomas. To test this idea, in collaboration with Georg and Eva Klein and colleagues at the Karolinska Institutet, we examined
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the effect of cellular transformation by the oncogenic Epstein–Barr virus (EBV) on B lymphocytes, using as a control experimentally activated B cells from the same volunteers. We observed nearly identical hypomethylation of blocks following transformation (but not simply activation). Moreover, this hypomethylation led to almost identical hypervariability of gene expression within the blocks [16]. Thus, epigenetically cancer appears to be a genomic disease involving large blocks of DNA that become hypomethylated, leading to variability of methylation of smaller structures such as CpG islands within them, and hypervariability of gene expression, probably contributing to tumour cell heterogeneity. Epigenetic stochasticity and nuclear structure So what are these blocks? Herein is an example of converging observations in cancer epigenetics. In 2009, we first reported the existence of large heterochromatin blocks that we termed LOCKs [i.e. ‘large organized chromatin K (lysine)-modifications’] at H3K9Me2 [17]; these were later described by others also at H3K9Me2 as well as at H3K9Me3 and H3K27Me3 [18–20]. The precise modifications vary in nature and location between cell types and under different conditions, perhaps because the antibodies themselves are polyclonal and batch sensitive. However, their existence is well described, and they seem to colocalize with ‘lamin-associated domains’ observed indirectly using a transfected construct that promotes a DNA modification where they occur [18–20]. In our original report, we also described a reduction in LOCKs in human tumours [17], although this has not been followed up in detail by us or others to date. So if blocks are LOCKs, what is their normal developmental function? An important clue came from experiments on epithelial–mesenchymal transition, a defining feature of development and cancer, using transforming growth factor-beta treatment of normal cells. We found reversible widespread loss of H3K9Me2, corresponding to about one-third of LOCKs, and accompanied by a gain of the euchromatic modifications H3K4Me3 and H3K36Me3 [21]. Thus, LOCKs/blocks appear to play an important role in silencing and then reversibly activating a large portion of the genome in response to stress. We hypothesize that on repeated injury and repair, these changes become permanent, with the accompanying large-scale hypomethylation in blocks we found in cancer.
Key Symposium: Epigenetics stochasticity and cancer
Clinical implications of epigenetic stochasticity What are the implications for the prevailing genetic model of human cancer? Although this model is clearly correct, we believe strongly that it is only about 50% complete. We consider that epigenetics lies at the heart of tumour evolution and can explain why this process occurs over decades instead of days (Fig. 1). According to this model, epigenetic instability is the key defining feature of all (or perhaps all solid) cancers, involving the same mechanism and to a large extent the same target genes. Thus, mutations that promote cancer do so in part by inducing epigenetic plasticity, largely within these heterochromatin regions/ blocks/LOCKs. The tumour cell then evolves by natural selection within the host. We propose that the hallmarks of cancer, including inflammation, invasion, metabolic change and proliferation, are selected for rather than predetermined, depending on the microenvironment of the tumour at any given time, and due to the increased epigenetic heterogeneity in tumours that allows their selection at the expense of the host [1]. This hypothesis fits very well the evolutionary origin of the stochastic epigenetic model itself, which was conceived to explain the rapid adaptability of species to a fluctuating environment. Cancer would involve a similar mechanism of genetically selected epigenetic variation, but in this case of the diseased cell at the expense of the normal cells of the host. However, the epigenetic instability must also occur as a primary effect and at an early time-point, not only as a result of acquired mutations, because epigenetic changes appear to occur in the normal tissue of patients with cancer [1, 12] (Fig. 1). The model also has important translational implications for diagnosis and therapy. It is predicted that epigenetic variability per se would arise in the normal tissue of patients with cancer. Indeed Teschendorff and colleagues found that they could predict the development of cancer through epigenetic variability (but not mean), using a test for heteroscedasticity, years before cancer developed [22, 23]. The implications of their analysis are extremely important because we might be able to develop screening for cancer risk, using methylation and mathematics, for commonly occurring disease and then target conventional imaging and invasive screening to at-risk patients. It is noteworthy that the problem with colonoscopy, for example, besides cost and risk, is that the positive predictive value is very low because the prevalence ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 5–11
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Key Symposium: Epigenetics stochasticity and cancer
Inflammation
Invasion
Metabolism Apoptosis LOI and Decreased others
Environmental factors and carcinogens
Proliferation Epigenome
Stochastic ageing
Increased Disruption and positive feedback (EZH2, MYC and KLF4)
Precursor lesion APC
Ageing
Epigenetic regulators (TET2, MLL and ARID1A)
Genome instability
p53 Canonical genes
Mutation
Fig. 1 Collaboration of epigenetic modification and mutation in the hallmarks of cancer. The epigenome sits at the intersection of the environment, genetic mutation and tumour cell growth. Environmental factors, such as carcinogens or diet, as well as injury and inflammation, cause epigenetic reprogramming. The epigenome also accumulates damage stochastically and through ageing. The machinery for maintaining epigenetic integrity can be stably disrupted in either of two ways: by mutation or by epigenetic change itself with positive feedback. Examples of mutation include epigenetic regulator mutations (Table 1), whereas examples of epigenetic change include loss of imprinting (LOI) of insulin-like growth factor 2 (IGF2) in colorectal carcinogenesis, enhancer of zeste homologue 2 (EZH2) silencing in prostate cancer (Table 2) and overexpression of reprogramming factors. The disruption of epigenetic integrity maintenance leads to the loss of epigenetic regulation and stochastic drift from a normal set point, followed by selection for cellular growth at the expense of other cells (Figs 1 and 2). Some epigenetic modifications, such as shifting methylation boundaries at CpG islands and shores, lead to metabolic change and enhanced proliferation. Others, such as hypomethylated blocks, lead to increased invasion. Still others, such as LOI, directly change the balance between apoptosis and proliferation. Canonical mutations, such as in adenomatous polyposis coli (APC) and TP53 (which encodes p53), directly affect cancer hallmarks but can also cause epigenetic dysregulation. Similarly, epigenetic disruption, such as regional hypomethylation or CpG hypermethy lation, can lead to increased chromosomal rearrangements and mutations, respectively. Instability of CpG island methylation boundaries also contributes to epigenetic dysregulation, allowing for selection in response to the cellular environment for cellular growth advantage at the expense of the host. ARID1A, AT-rich interactive domain-containing protein 1A; KLF4, Kr€ u ppel-like factor 4; MLL, mixed lineage leukaemia; TET2, tet methylcytosine dioxygenase 2. Reprinted by permission from Macmillan Publishers Ltd: NATURE REVIEWS CANCER, 13:7. Cancer as a dysregulated epigenome allowing cellular growth advantage at the expense of the host. copyright 2013
of the disease is low in the tested population. The same is even truer for mammography, leading to its abandonment as standard practice for younger women. However, if methylation variability testing could be used to identify the subgroup of the population with a 50% risk of a given cancer, the positive predictive value of the more expensive/ invasive testing would exceed 90%, and the cancer deaths of a substantial fraction of those patients could be avoided. This is an unconventional idea, particularly given the abysmal record of conventional single-gene mean methylation testing, and 10
ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 5–11
the advent of genetic screening. However, I believe that the barrier to the effectiveness of this new proposal is preconceptions, not the science behind the idea itself. Acknowledgement This work was supported by NIH Grant CA54358. Conflict of interest statement The author has no competing interests to declare.
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References 1 Timp W, Feinberg AP. Cancer as a dysregulated epigenome allowing cellular growth advantage at the expense of the host. Nat Rev Cancer 2013; 13: 497–510. 2 Waterland RA, Jirtle RL. Transposable elements: targets for early nutritional effects on epigenetic gene regulation. Mol Cell Biol 2003; 23: 5293–300. 3 Anway MD, Cupp AS, Uzumcu M, Skinner MK. Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science 2005; 308: 1466–9. 4 Yachida S, Jones S, Bozic I et al. Distant metastasis occurs late during the genetic evolution of pancreatic cancer. Nature 2010; 467: 1114–7. 5 Feinberg AP, Vogelstein B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature 1983; 301: 89–92. 6 Feinberg AP, Tycko B. The history of cancer epigenetics. Nat Rev Cancer 2004; 4: 143–53. 7 Ravenel JD, Broman KW, Perlman EJ et al. Loss of imprinting of insulin-like growth factor-II (IGF2) gene in distinguishing specific biologic subtypes of Wilms tumor. J Natl Cancer Inst 2001; 93: 1698–703. 8 Cui H, Cruz-Correa M, Giardiello FM et al. Loss of IGF2 imprinting: a potential marker of colorectal cancer risk. Science 2003; 299: 1753–5. 9 DeBaun MR, Niemitz EL, McNeil DE, Brandenburg SA, Lee MP, Feinberg AP. Epigenetic alterations of H19 and LIT1 distinguish patients with Beckwith-Wiedemann syndrome with cancer and birth defects. Am J Hum Genet 2002; 70: 604–11. 10 Irizarry RA, Ladd-Acosta C, Wen B et al. The human colon cancer methylome shows similar hypo- and hypermethylation at conserved tissue-specific CpG island shores. Nat Genet 2009; 41: 178–86. 11 Theveneau E, Mayor R. Neural crest delamination and migration: from epithelium-to-mesenchyme transition to collective cell migration. Dev Biol 2012; 366: 34–54. 12 Feinberg AP, Irizarry RA. Evolution in health and medicine Sackler colloquium: stochastic epigenetic variation as a driving force of development, evolutionary adaptation, and disease. Proc Natl Acad Sci U S A 2010; 107(Suppl. 1): 1757–64. 13 Waddington CH. The Strategy of the Genes; a Discussion of some Aspects of Theoretical Biology. London: Allen & Unwin, 1957.
Key Symposium: Epigenetics stochasticity and cancer
14 Hansen KD, Timp W, Bravo HC et al. Increased methylation variation in epigenetic domains across cancer types. Nat Genet 2011; 43: 768–75. 15 Berman BP, Weisenberger DJ, Aman JF et al. Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina-associated domains. Nat Genet 2012; 44: 40–6. 16 Hansen KD, Sabunciyan S, Langmead B et al. Large-scale hypomethylated blocks associated with Epstein-Barr virus-induced B-cell immortalization. Genome Res 2013; 24: 177–84. 17 Wen B, Wu H, Shinkai Y, Irizarry RA, Feinberg AP. Large histone H3 lysine 9 dimethylated chromatin blocks distinguish differentiated from embryonic stem cells. Nat Genet 2009; 41: 246–50. 18 Hawkins RD, Hon GC, Lee LK et al. Distinct epigenomic landscapes of pluripotent and lineage-committed human cells. Cell Stem Cell 2010; 6: 479–91. 19 Hon GC, Hawkins RD, Caballero OL et al. Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer. Genome Res 2012; 22: 246–58. 20 Chen X, Skutt-Kakaria K, Davison J et al. G9a/GLP-dependent histone H3K9me2 patterning during human hematopoietic stem cell lineage commitment. Genes Dev 2012; 26: 2499–511. 21 McDonald OG, Wu H, Timp W, Doi A, Feinberg AP. Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition. Nat Struct Mol Biol 2011; 18: 867–74. 22 Teschendorff A, Jones A, Fiegl H et al. Epigenetic variability in cells of normal cytology is associated with the risk of future morphological transformation. Genome Med 2012; 4: 24. 23 Teschendorff AE, Widschwendter M. Differential variability improves the identification of cancer risk markers in DNA methylation studies profiling precursor cancer lesions. Bioinformatics 2012; 28: 1487–94. Correspondence: Andrew P. Feinberg, Center for Epigenetics and Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA. (fax: 410-614-9819; e-mail: [email protected]).
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doi: 10.1111/joim.12224
Epigenetic stochasticity, nuclear structure and cancer: the implications for medicine A. P. Feinberg From the Center for Epigenetics and Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
Abstract. Feinberg AP (From the Center for Epigenetics and Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA). Epigenetic stochasticity, nuclear structure and cancer: the implications for medicine (Key Symposium). J Intern Med 2014; 276: 5–11. The aim of this review is to summarize an evolution of thinking about the epigenetic basis of human cancer, from the earliest studies of altered DNA methylation in cancer to the modern comprehensive epigenomic era. Converging data from epigenetic studies of primary cancers and from experimental studies of chromatin in development
Introduction This review follows from a lecture I presented at the 10th Key Symposium of the Journal of Internal Medicine and thus is written from a personal perspective with an emphasis on my own laboratory’s experiments and my personal motivations for designing them. Moreover, I wish here to present the ideas so that they may be of interest and value to a general internist. Therefore, cancer genetics specialists are referred to my recent more comprehensive scholarly review in Nature Reviews Cancer [1], which discusses the work of others comprehensively and provides much greater detail than is possible here. Epigenetics provides a molecular mechanism for phenotypic differences amongst organs of an individual, that are copied faithfully during cell division. Such information is transmitted almost entirely in a manner not involving the DNA sequence per se, as that is essentially the same across all cell types. Yet the phenotypic differences amongst organs are quite profound, and in many cases, more so than the differences between a given organ from species to species, at least within a given mammalian clade [1].
and epithelial–mesenchymal transition suggest a role for epigenetic stochasticity as a driving force of cancer, with Darwinian selection of tumour cells at the expense of the host. This increased epigenetic stochasticity appears to be mediated by large-scale changes in DNA methylation and chromatin in domains associated with the nuclear lamina. The implications for diagnosis include the potential to identify stochastically disrupted progenitor cells years before cancer develops, and to target drugs to epigenetic drivers of gene expression instability rather than to mean effects per se. Keywords: cancer, DNA methylation, heterochromatin, LOCKs, nuclear structure.
Moreover, epigenetic information is susceptible to changes in the environment, which can also have an effect on phenotype relevant to human disease. For example, increased intake of S-adenosylmethionine prenatally in a particular strain of an Agouti coat colour allele leads to variable silencing of an alternative promoter and repression of ectopic agouti expression, enhanced yellow coloration and obesity in the offspring [2]. Furthermore, the pesticide vinclozolin can act transgenerationally on sperm to affect DNA methylation and fertility [3]. The types of epigenetic change that mediate phenotypic differences amongst organs and potentially in disease include DNA methylation and histone modifications. DNA methylation is heritable during cell division in mammals only at the dinucleotide CpG. Although non-CpG methylation has been shown to exist, by definition the information is not heritable/epigenetic. DNA methylation is associated with gene silencing and is maintained by DNA methyltransferase I, which recognizes the hemimethylated mCpG/CpG after semiconservative DNA replication and adds a methyl group to the 5-position. There are over 200 known posttranslational modifications of histones, primarily
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on the tails of histones H3 and H4, but the core regions and the other histones are also modified. The canonical modifications most commonly studied are H3K27 and H3K9 methylation associated with silencing and H3K4 methylation and acetylation associated with activation. Epigenetics and human cancer The genetic model for cancer has been well established for three decades, based on identification of defining gene rearrangements for most leukaemias and lymphomas and gene mutations for most solid tumours. Recent advances in high-throughput sequencing reveal many mutations, but relatively few ‘drivers’ that are present early in the tumour and carry through to all descendant clones and metastases [4]. Furthermore, the mutations found commonly in cancer still appear to be quite rare in normal tissue, and thus genetically driven intervention before most cancers arise seems impractical, despite the fact that environmental exposure and even the effect of combined low penetrance genetic alleles act for years before the development of overt disease. Almost as old as the genetic hypothesis of cancer is the epigenetic hypothesis that changes in DNA methylation or chromatin could substitute for, complement and/or precede the development of genetic mutations, contributing to abnormal and heterogeneous gene expression in cancer. The idea arose from an attempt to relate changes in the genome to functional effects on gene activity. The original observation was the loss of methylation of approximately one-third of single-copy genes [5], suggesting a generalized epigenetic disruption of large regions of nuclear chromatin and, as described below, I think we now understand much of the mechanism of this disruption. Moreover, this large-scale epigenetic disruption appears to explain most of the other epigenetic findings in cancer, including hypermethylated CpG islands, hypomethylated CpG island shores and epigenetically driven tumour cell heterogeneity. Before discussing these epigenomic changes, it is worth noting that a strong argument for a causal epigenetic role in cancer came from the study of the rare disorder Beckwith–Wiedemann syndrome (BWS), which increases the risk of Wilms tumour, the most common solid tumour (a kidney cancer) of childhood. Previously, it had not been possible to ascertain whether epigenetic changes were causal 6
ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 5–11
Key Symposium: Epigenetics stochasticity and cancer
or consequential in cancer. BWS shows genetic and epigenetic heterogeneity, including the following mechanisms: causes, including: (i) genetic mutations of the cyclin-dependent kinase inhibitor p57KIP2, an imprinted gene showing expression of a specific (in this case maternal) allele; (ii) aberrant imprinting of the same gene and a related antisense long RNA, LIT1; and (iii) loss of imprinting (LOI) of the gene insulin-like growth factor-II (IGF2), which we and others discovered in sporadically occurring Wilms tumours [6]. The effect of LOI is a double dose of IGF2, leading to overexpansion of nephrogenic blastema cells in the developing kidney, and a high frequency of Wilms tumour [7]. LOI of IGF2 is also associated with an increased frequency of colorectal cancer in adults [8]. In the first epigenotype–phenotype study of any disorder (comparable to genotype–phenotype studies in classical genetics), it was found that LOI of IGF2 accounts almost entirely for the cancer risk in BWS [9] and also universally leads to expansion of the premalignant nephroblastemic progenitor cell compartment [7]. Thus, the epigenetic change (LOI) cannot result as a consequence of cancer; it precedes cancer, is associated with cancer risk and provides a mechanistic basis (expansion of the precursor population) for tumour development [6]. New insights from cancer epigenomics Most of the recent work from my laboratory described below comes from the development of new genome-scale approaches to epigenetics, or ‘epigenomics’, that began with a Center for Epigenetics awarded by the National Human Genome Research Institute. Tools from our center and others in the USA and Europe gave rise to the US Human Epigenome Roadmap and the International Human Epigenome Consortium. For example, approximately 80% of the locations on the now widely used Infinium Human Methylation 450 BeadChip were chosen from experimental data derived from the 2.5 million CpG CHARM methylation array platform developed at our centre and from epigenomic sites identified by 21 other research groups. We first applied these tools to examine the cancer epigenome comprehensively, using the same paradigm as that used in our original study 30 years ago [5], namely human colorectal cancer and matched colonic mucosa from the same patients, the same tissue and cell layer from which the cancers arise. This analysis led to the surprising result that most differentially methylated regions
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(DMRs) that distinguish cancer from normal tissue, that is, c-DMRs, are themselves also t-DMRs, that is, they distinguish some other normal tissue from other normal tissues [10]. In other words, the epigenetic disruptions in cancer lead cells to acquire epigenetic signatures of other tissues, albeit in a random inconsistent way. This result can help to explain the long-known fact that the defining properties of cancer, such as invasion and metastasis, are indeed properties of some normal cells at some stage of normal development; for example, neural crest cells migrate during embryogenesis through epithelial-mesenchymal transition [11], a process that is pathologically activated in cancer. A conundrum was also revealed by our epigenomic data. In addition to the widespread epigenetic heterogeneity we observed in cancers, there was also a degree of epigenetic heterogeneity in normal tissue [10]. Whilst this could be explained in part by genetic heterogeneity in the human population, we observed a similar epigenetic heterogeneity in the normal tissues of inbred laboratory mice [10], suggesting that the epigenome is driven to epigenetic heterogeneity spontaneously, at some genetic loci and not others. This intrinsic normal epigenetic heterogeneity, under genetic control, offered a surprising potential explanation for one of the most important roles of epigenetics in development and disease: stochastic epigenetic variation could be a driving force of development and evolutionary adaptation [12] (Table 1). This idea directly addresses the limits of determinism, first espoused for epigenetics by Konrad Waddington in the 1940s and 1950s, that development is predetermined genetically and that perturbations caused by the environment are corrected developmentally by an increasingly strong pull towards an inevitable tissue outcome, which he termed canalization [13]. Waddington used ordinary differential equations to describe this deterministic approach to developmental biology. However, we observed epigenetic stochasticity in genetically identical mice raised in the same environment, even from the same litter or mice, at crucial developmental genes, such as members of the HOX family of homeodomain proteins that specify axial differences in organ development [12]. This stochastic epigenetic variation does not occur everywhere, only at key genes in which environmental effects can vary. Thus, an underlying genetic sequence determines the variability, which then occurs stochastically or in response to environmental pressure. Such a
Key Symposium: Epigenetics stochasticity and cancer
model introduces a stochastic component to embryology, mediated by epigenetics, similar to the stochastic properties necessary for a complete description of the atom in physics. As in physics, we believe that stochastic mathematics can best describe these epigenetic properties; however, this is ‘work in progress’ and beyond the scope of the present review. We term these inherently epigenetically variable regions variably methylated regions (VMRs) [12]. The stochastic epigenetic model of cancer The stochastic epigenetic model (Table 1) has important implications for evolutionary biology considering environmental effects that may be consistent for many generations but can then change stochastically, for example in response to an environmental crisis such as drought or famine. We could show by modelling that over time, in the presence of an occasionally and randomly switching environment, natural selection would favour genetic sequences promoting variability per se, in the same way that it favours sequences for mean effects if the selecting constant environment is present for a sufficiently long period of time [12]. How might this stochastic epigenetic model underlie disease? An obvious example, as suggested in our model, is cancer, which arises in part from repeated changes to the microenvironment of the tissue. Most cancers arise from cycles of repeated injury and repair. Hepatocellular cancer, the most common fatal human solid tumour, arises directly from this process (in this context cirrhosis, i.e. injury and regeneration of the liver), whether the injury is induced by hepatitis B or by aflatoxin. Similarly, skin cancer arises from repeated cycles of blistering sunburn and repair. Furthermore, inflammatory bowel disease is the most dramatic example of the environmental effect of injury and repair that is likely to underlie many cases of colorectal cancer. We tested the stochastic epigenetic model in cancer by examining DNA methylation comprehensively across the genome as described earlier, but in this case testing specifically for stochastic variability in the methylation signature using methods from econometrics for analysis of random market fluctuations, the formal term for which is heteroscedasticity. We examined several common solid tumours types of adulthood in the West – lung, colon, thyroid and breast – and Wilms tumour, the most common solid tumour of childhood. As predicted, the ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 5–11
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Table 1 The stochastic epigenetic model of human cancer. See text for details, and [1, 12] for a more comprehensive discussion of the relevant literature The stochastic epigenetic model of human cancer Original idea to address ‘missing heritability’ and role of environment in common diseases Limitations of determinism, including Waddington Stochastic variation plays a fundamental role during normal development (similar to quantum mechanics in physics) This stochastic variation is mediated epigenetically The degree of stochastic variation is inherited genetically (no problem with Darwin) Genetic variants that increase epigenetic plasticity could increase fitness over evolution, for traits affected by a fluctuating environment Cancer arises in part from repeated changes in the microenvironment Cycles of injury/repair: hepatocellular cancer, skin cancer, inflammatory bowel disease Epigenetic changes in progenitor cells can increase cancer risk
variability of DNA methylation was an even stronger signature of cancer than was the mean change at any given site of DNA methylation [14]. However, there were also two unexpected findings from this analysis. First, we found that the methylation sites that were most variable in colorectal cancer were also highly variable in the other tumour types. This suggests a universality of these VMRs, and that they might in fact be VMRs for normal development. Secondly, the cancer VMRs completely segregated normal tissue types from each other by cluster analysis. Thus, the sites that show the greatest epigenetic variability in cancer are the very sites that ‘need’ to show stochastic variation in the development of normal tissues during embryonic development [14]. Thus, the epigenetic plasticity model appears to be correct, at least for cancer, and the sites that show stochastic variation in normal development in the derivation of normal tissue types are again prone to epigenetic variability in cancer, across all solid tumour types. This epigenetic stochasticity, and the sites of its variability, could thus provide a mechanistic explanation for the tumour cell heterogeneity that characterizes all cancer and inspired the first experiments in cancer epigenetics three decades ago [5]. 8
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Key Symposium: Epigenetics stochasticity and cancer
An important clue to the mechanism of this epigenetic stochasticity came from whole-genome bisulphite sequencing of three colorectal cancers and their matched colonic mucosa, as well as two premalignant colon adenomas [14]. There were three major results from this analysis. First, essentially all of the epigenetic change in cancer entails large blocks of hypomethylation, involving half of the genome and one-third of single-copy genes; that is, the original observation of cancer hypomethylation accounts for >98% of the affected genomic region in cancer. As a technical aside, these large blocks, up to a megabase in size, do not involve specific hypomethylation of repetitive elements (which are simply enriched about 1.5-fold in the blocks). Rather they involve the entire regions, including the genes therein [14]. Secondly, essentially all of the ‘hypermethylation’ of CpG islands is in fact due to instability at the boundary of these dense CG-rich regions within the large hypomethylated blocks [14, 15]. Thus, the presence of the islands within the blocks, undergoing loss of DNA methylation, causes disruption to the underlying architecture of the islands themselves. Note also that the island hypermethylation is in fact an erosion of a boundary of high methylation outside the island and low methylation within; in fact what occurs is a loss of methylation at the boundaries (shores) and gain of methylation in the interior (the change is not specific to the island element itself). Thirdly, and perhaps most importantly, these changes involve stochastic variability of DNA methylation in the cancer, consistent with a loss of regulation confined to these large blocks. Indeed, the genes within the blocks include the most hypervariably expressed genes in human cancer, and those genes are important mechanistically [14]. Of note, specific defining mutations are not associated with cancer invasion and metastasis, rather these seem to involve epigenetic mechanisms leading to aberrant expression in the absence of mutation [1]. In addition, these same genes, such as matrix metalloproteinases underlying tumour invasion, lie within the blocks, and show hypervariable methylation and expression in cancer [14]. We believe that these changes in block hypomethylation, hypervariability and disrupted CpG island boundaries occur early in cancer, as we found to be the case also in adenomas. To test this idea, in collaboration with Georg and Eva Klein and colleagues at the Karolinska Institutet, we examined
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the effect of cellular transformation by the oncogenic Epstein–Barr virus (EBV) on B lymphocytes, using as a control experimentally activated B cells from the same volunteers. We observed nearly identical hypomethylation of blocks following transformation (but not simply activation). Moreover, this hypomethylation led to almost identical hypervariability of gene expression within the blocks [16]. Thus, epigenetically cancer appears to be a genomic disease involving large blocks of DNA that become hypomethylated, leading to variability of methylation of smaller structures such as CpG islands within them, and hypervariability of gene expression, probably contributing to tumour cell heterogeneity. Epigenetic stochasticity and nuclear structure So what are these blocks? Herein is an example of converging observations in cancer epigenetics. In 2009, we first reported the existence of large heterochromatin blocks that we termed LOCKs [i.e. ‘large organized chromatin K (lysine)-modifications’] at H3K9Me2 [17]; these were later described by others also at H3K9Me2 as well as at H3K9Me3 and H3K27Me3 [18–20]. The precise modifications vary in nature and location between cell types and under different conditions, perhaps because the antibodies themselves are polyclonal and batch sensitive. However, their existence is well described, and they seem to colocalize with ‘lamin-associated domains’ observed indirectly using a transfected construct that promotes a DNA modification where they occur [18–20]. In our original report, we also described a reduction in LOCKs in human tumours [17], although this has not been followed up in detail by us or others to date. So if blocks are LOCKs, what is their normal developmental function? An important clue came from experiments on epithelial–mesenchymal transition, a defining feature of development and cancer, using transforming growth factor-beta treatment of normal cells. We found reversible widespread loss of H3K9Me2, corresponding to about one-third of LOCKs, and accompanied by a gain of the euchromatic modifications H3K4Me3 and H3K36Me3 [21]. Thus, LOCKs/blocks appear to play an important role in silencing and then reversibly activating a large portion of the genome in response to stress. We hypothesize that on repeated injury and repair, these changes become permanent, with the accompanying large-scale hypomethylation in blocks we found in cancer.
Key Symposium: Epigenetics stochasticity and cancer
Clinical implications of epigenetic stochasticity What are the implications for the prevailing genetic model of human cancer? Although this model is clearly correct, we believe strongly that it is only about 50% complete. We consider that epigenetics lies at the heart of tumour evolution and can explain why this process occurs over decades instead of days (Fig. 1). According to this model, epigenetic instability is the key defining feature of all (or perhaps all solid) cancers, involving the same mechanism and to a large extent the same target genes. Thus, mutations that promote cancer do so in part by inducing epigenetic plasticity, largely within these heterochromatin regions/ blocks/LOCKs. The tumour cell then evolves by natural selection within the host. We propose that the hallmarks of cancer, including inflammation, invasion, metabolic change and proliferation, are selected for rather than predetermined, depending on the microenvironment of the tumour at any given time, and due to the increased epigenetic heterogeneity in tumours that allows their selection at the expense of the host [1]. This hypothesis fits very well the evolutionary origin of the stochastic epigenetic model itself, which was conceived to explain the rapid adaptability of species to a fluctuating environment. Cancer would involve a similar mechanism of genetically selected epigenetic variation, but in this case of the diseased cell at the expense of the normal cells of the host. However, the epigenetic instability must also occur as a primary effect and at an early time-point, not only as a result of acquired mutations, because epigenetic changes appear to occur in the normal tissue of patients with cancer [1, 12] (Fig. 1). The model also has important translational implications for diagnosis and therapy. It is predicted that epigenetic variability per se would arise in the normal tissue of patients with cancer. Indeed Teschendorff and colleagues found that they could predict the development of cancer through epigenetic variability (but not mean), using a test for heteroscedasticity, years before cancer developed [22, 23]. The implications of their analysis are extremely important because we might be able to develop screening for cancer risk, using methylation and mathematics, for commonly occurring disease and then target conventional imaging and invasive screening to at-risk patients. It is noteworthy that the problem with colonoscopy, for example, besides cost and risk, is that the positive predictive value is very low because the prevalence ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 5–11
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Key Symposium: Epigenetics stochasticity and cancer
Inflammation
Invasion
Metabolism Apoptosis LOI and Decreased others
Environmental factors and carcinogens
Proliferation Epigenome
Stochastic ageing
Increased Disruption and positive feedback (EZH2, MYC and KLF4)
Precursor lesion APC
Ageing
Epigenetic regulators (TET2, MLL and ARID1A)
Genome instability
p53 Canonical genes
Mutation
Fig. 1 Collaboration of epigenetic modification and mutation in the hallmarks of cancer. The epigenome sits at the intersection of the environment, genetic mutation and tumour cell growth. Environmental factors, such as carcinogens or diet, as well as injury and inflammation, cause epigenetic reprogramming. The epigenome also accumulates damage stochastically and through ageing. The machinery for maintaining epigenetic integrity can be stably disrupted in either of two ways: by mutation or by epigenetic change itself with positive feedback. Examples of mutation include epigenetic regulator mutations (Table 1), whereas examples of epigenetic change include loss of imprinting (LOI) of insulin-like growth factor 2 (IGF2) in colorectal carcinogenesis, enhancer of zeste homologue 2 (EZH2) silencing in prostate cancer (Table 2) and overexpression of reprogramming factors. The disruption of epigenetic integrity maintenance leads to the loss of epigenetic regulation and stochastic drift from a normal set point, followed by selection for cellular growth at the expense of other cells (Figs 1 and 2). Some epigenetic modifications, such as shifting methylation boundaries at CpG islands and shores, lead to metabolic change and enhanced proliferation. Others, such as hypomethylated blocks, lead to increased invasion. Still others, such as LOI, directly change the balance between apoptosis and proliferation. Canonical mutations, such as in adenomatous polyposis coli (APC) and TP53 (which encodes p53), directly affect cancer hallmarks but can also cause epigenetic dysregulation. Similarly, epigenetic disruption, such as regional hypomethylation or CpG hypermethy lation, can lead to increased chromosomal rearrangements and mutations, respectively. Instability of CpG island methylation boundaries also contributes to epigenetic dysregulation, allowing for selection in response to the cellular environment for cellular growth advantage at the expense of the host. ARID1A, AT-rich interactive domain-containing protein 1A; KLF4, Kr€ u ppel-like factor 4; MLL, mixed lineage leukaemia; TET2, tet methylcytosine dioxygenase 2. Reprinted by permission from Macmillan Publishers Ltd: NATURE REVIEWS CANCER, 13:7. Cancer as a dysregulated epigenome allowing cellular growth advantage at the expense of the host. copyright 2013
of the disease is low in the tested population. The same is even truer for mammography, leading to its abandonment as standard practice for younger women. However, if methylation variability testing could be used to identify the subgroup of the population with a 50% risk of a given cancer, the positive predictive value of the more expensive/ invasive testing would exceed 90%, and the cancer deaths of a substantial fraction of those patients could be avoided. This is an unconventional idea, particularly given the abysmal record of conventional single-gene mean methylation testing, and 10
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the advent of genetic screening. However, I believe that the barrier to the effectiveness of this new proposal is preconceptions, not the science behind the idea itself. Acknowledgement This work was supported by NIH Grant CA54358. Conflict of interest statement The author has no competing interests to declare.
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References 1 Timp W, Feinberg AP. Cancer as a dysregulated epigenome allowing cellular growth advantage at the expense of the host. Nat Rev Cancer 2013; 13: 497–510. 2 Waterland RA, Jirtle RL. Transposable elements: targets for early nutritional effects on epigenetic gene regulation. Mol Cell Biol 2003; 23: 5293–300. 3 Anway MD, Cupp AS, Uzumcu M, Skinner MK. Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science 2005; 308: 1466–9. 4 Yachida S, Jones S, Bozic I et al. Distant metastasis occurs late during the genetic evolution of pancreatic cancer. Nature 2010; 467: 1114–7. 5 Feinberg AP, Vogelstein B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature 1983; 301: 89–92. 6 Feinberg AP, Tycko B. The history of cancer epigenetics. Nat Rev Cancer 2004; 4: 143–53. 7 Ravenel JD, Broman KW, Perlman EJ et al. Loss of imprinting of insulin-like growth factor-II (IGF2) gene in distinguishing specific biologic subtypes of Wilms tumor. J Natl Cancer Inst 2001; 93: 1698–703. 8 Cui H, Cruz-Correa M, Giardiello FM et al. Loss of IGF2 imprinting: a potential marker of colorectal cancer risk. Science 2003; 299: 1753–5. 9 DeBaun MR, Niemitz EL, McNeil DE, Brandenburg SA, Lee MP, Feinberg AP. Epigenetic alterations of H19 and LIT1 distinguish patients with Beckwith-Wiedemann syndrome with cancer and birth defects. Am J Hum Genet 2002; 70: 604–11. 10 Irizarry RA, Ladd-Acosta C, Wen B et al. The human colon cancer methylome shows similar hypo- and hypermethylation at conserved tissue-specific CpG island shores. Nat Genet 2009; 41: 178–86. 11 Theveneau E, Mayor R. Neural crest delamination and migration: from epithelium-to-mesenchyme transition to collective cell migration. Dev Biol 2012; 366: 34–54. 12 Feinberg AP, Irizarry RA. Evolution in health and medicine Sackler colloquium: stochastic epigenetic variation as a driving force of development, evolutionary adaptation, and disease. Proc Natl Acad Sci U S A 2010; 107(Suppl. 1): 1757–64. 13 Waddington CH. The Strategy of the Genes; a Discussion of some Aspects of Theoretical Biology. London: Allen & Unwin, 1957.
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14 Hansen KD, Timp W, Bravo HC et al. Increased methylation variation in epigenetic domains across cancer types. Nat Genet 2011; 43: 768–75. 15 Berman BP, Weisenberger DJ, Aman JF et al. Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina-associated domains. Nat Genet 2012; 44: 40–6. 16 Hansen KD, Sabunciyan S, Langmead B et al. Large-scale hypomethylated blocks associated with Epstein-Barr virus-induced B-cell immortalization. Genome Res 2013; 24: 177–84. 17 Wen B, Wu H, Shinkai Y, Irizarry RA, Feinberg AP. Large histone H3 lysine 9 dimethylated chromatin blocks distinguish differentiated from embryonic stem cells. Nat Genet 2009; 41: 246–50. 18 Hawkins RD, Hon GC, Lee LK et al. Distinct epigenomic landscapes of pluripotent and lineage-committed human cells. Cell Stem Cell 2010; 6: 479–91. 19 Hon GC, Hawkins RD, Caballero OL et al. Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer. Genome Res 2012; 22: 246–58. 20 Chen X, Skutt-Kakaria K, Davison J et al. G9a/GLP-dependent histone H3K9me2 patterning during human hematopoietic stem cell lineage commitment. Genes Dev 2012; 26: 2499–511. 21 McDonald OG, Wu H, Timp W, Doi A, Feinberg AP. Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition. Nat Struct Mol Biol 2011; 18: 867–74. 22 Teschendorff A, Jones A, Fiegl H et al. Epigenetic variability in cells of normal cytology is associated with the risk of future morphological transformation. Genome Med 2012; 4: 24. 23 Teschendorff AE, Widschwendter M. Differential variability improves the identification of cancer risk markers in DNA methylation studies profiling precursor cancer lesions. Bioinformatics 2012; 28: 1487–94. Correspondence: Andrew P. Feinberg, Center for Epigenetics and Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA. (fax: 410-614-9819; e-mail: [email protected]).
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