Dig Dis 2012;30:310–315 DOI: 10.1159/000337004
Role of DNA Methylation in Colorectal Carcinogenesis Árpád V. Patai a Béla Molnár a, b Alexandra Kalmár a Andrea Schöller a Kinga Tóth a Zsolt Tulassay a, b a 2nd Department of Internal Medicine, Semmelweis University, and b Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest, Hungary
Key Words Colorectal cancer ⴢ DNA methylation ⴢ Field effect ⴢ Aging ⴢ Ulcerative colitis ⴢ CpG island methylator phenotype ⴢ Prescreening
Abstract Colorectal cancer is the most common malignancy of the gastrointestinal tract and a leading cause of cancer-related deaths worldwide. In order to detect early precursor lesions, colonoscopy is widely used. Unfortunately, patient adherence to colonoscopy is poor, which is partially due to the modest performance of currently used prescreening tests. Recently, epigenetics added an additional layer to the understanding of colorectal carcinogenesis. DNA methylation as part of the epigenetic gene-silencing complex is a universally occurring change in colorectal cancer and arises prior to the onset of recognizable preneoplastic changes, which may have huge preventive implications. Herein we discuss the major developments in the field of colorectal carcinogenesis and DNA methylation, including alterations in nonneoplastic conditions such as aging and ulcerative colitis. We try to demonstrate how this epigenetic modification can be harnessed to address some of the key issues impeding the successful clinical management of colorectal cancer. Copyright © 2012 S. Karger AG, Basel
Á.V. Patai and B. Molnár contributed equally to this work.
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Introduction
Colorectal cancer (CRC) is the second most common cancer in the non-smoking population worldwide. It is estimated that 1,200,000 new cases are diagnosed annually and approximately 608,700 people die from it globally each year, that makes it a leading cause of cancerrelated deaths. Although improvements in the field of endoscopy have led to a decline in mortality in the Western world, incidence rates continue to increase in economically transitioning countries [1]. CRC is still often recognized at advanced stages when the tumor has enlarged enough to cause symptoms, and the prognosis is quite poor despite recent advances in the therapy. This scenario is even worse when the tumor occurs in the right side of the colon, where symptoms, mostly unspecific such as anemia or abdominal pain appear even later. However, if discovered at an early stage, there is a substantial chance to cure this otherwise deadly disease. Several approaches have tried to yield new biomarkers to detect CRC early; one of the most studied is DNA methylation. Recently, epigenetic changes including DNA methylation have been found to play an important role in carcinogenesis in addition to genetic changes. In this review we aim to discuss the most significant changes observed in DNA methylation during colorectal carcinogenesis. We will discuss how it emerges in healthy colonic mucosa, alters during aging and in ulcerative colitis (UC) and ultimately how it changed the classificaÁrpád V. Patai Semmelweis University, 2nd Department of Internal Medicine Laboratory of Cell Analytics, Szentkiralyi Street 46 HU–1088 Budapest (Hungary) Tel. +36 1 266 0926/55622, E-Mail arpad.patai @ gmail.com
tion of CRC. Finally we explore the connection of DNA methylation with environmental factors and examine potential translational implications in CRC.
DNMT inhibitors
N
DNA Methylation: Promoter Hypermethylation, Global Hypomethylation in Cancer
NH2
NH2
O
C N
C C
O SAM
Epigenetics is the study of heritable alterations of DNA that lead to changes in gene expression without underlying modifications in the actual genetic sequence. Epigenetic mechanisms include DNA methylation, histone modifications and small non-coding RNAs. DNA methylation, currently the best characterized epigenetic modulation, physiologically plays a fundamental role in embryogenesis, differentiation, imprinting and X chromosome inactivation in females. It is a covalent chemical modification resulting in the addition of a methyl group at the 5ⴕ position of the pyrimidine ring of cytosine in the setting of CpG dinucleotides. This reaction, using S-adenosyl methionine (SAM) as a methyl donor, is catalyzed by a family of enzymes called DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) during the S-phase (fig. 1) [2]. Generally, normal DNA methylation patterns in somatic cells are maintained by DNMT1, whereas de novo DNA methylation during embryonic development requires DNMT3A and DNMT3B; however, DNMT1 can also contribute to de novo DNA methylation and vice versa. In a normal differentiated cell, CpG dinucleotides are scattered throughout the genome and are generally underrepresented due to the spontaneous deamination of the methylated cytosine into thymine, and amount to 1% of the genome. However, certain CpG islands, predominantly located at the 5ⴕ promoter region of approximately 50% of genes remain unmethylated in normal tissues [3]. Methylation of cytosines within CpG islands leads to long-term transcriptional silencing through a series of events. This complicated process starts as nucleosomes insert at transcriptional start sites physically blocking the binding of transcriptional factors, followed by anchoring DNMT3A and DNMT3B, that serve as binding sites for the methylCpG-binding domain (MBD) proteins. DNA methylations act in concert with other epigenetic modifiers such as histone deacetylases (HDAC) and histone methyltransferases (HMT), leading to a compacted chromatin environment that is repressive for transcription [4]. During carcinogenesis, the faithful maintenance of normal DNA methylation patterns is disrupted and CpG islands become susceptible to methyltransferase activity and then become aberrantly hypermethylated, causing DNA Methylation in Colorectal Carcinogenesis
N
DNMTs
C N
C C
CH3
SAH
Fig. 1. DNA methylation is catalyzed by DNA methyltransferases
(DNMTs) using S-adenosyl methionine (SAM) as a methyl donor, resulting in the addition of a methyl group at the 5ⴕ carbon position of the pyrimidine ring of cytosine. This reaction can be reversed by several small synthetic and natural DNMT inhibitors. SAH = S-adenosylhomocysteine.
suppression of tumor suppressor and DNA repair genes, and an overall reduced epigenetic plasticity of critical developmental genes [4]. In the last decade hundreds of methylated genes have been described in various malignancies. First in CRC, a distinct group of cancers was identified based on the high frequency of CpG island methylation and termed CpG island methylator phenotype (CIMP). Complicating the already complex picture, according to recent results DNA methylation appears not exclusively at CpG islands and promoters, but 2 kb upstream, at the so-called CpG island shores [5]. On the other hand, CpG-poor regions undergo hypomethylation possible due to a transient depletion of DNMT1 leading to a global decrease of genomic 5-methylcytosine that may cause reactivation of transposable elements, increased recombination and mutation, overexpression of proto-oncogenes and activation of antigen-encoding CG genes increasing tumor immunogenicity [6].
DNA Methylation in the Normal Colorectal Mucosa
So far cancer research has been focused on primary tumors and their immediate nonmalignant precursor lesions. However, a growing body of evidence suggests that DNA methylation involving the promoters of various genes commences in the normal-appearing mucosa prior to the onset of recognizable preneoplastic changes, which may have huge preventive implications. Studies of normal gut mucosa collected near, or immediately adjacent to colorectal tumors have provided us with ample methylated genes in normal mucosa. Dig Dis 2012;30:310–315
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Field Effect The term ‘field cancerization’ was coined initially for oral cancers by Slaughter et al. [7] in 1953 and it was proposed to explain the development of multiple primary tumors and locally recurrent cancer. Since then it has been described for a wide variety of cancers [8], including CRC [9]. In that study DNA repair gene O6-methylguanine DNA methyltransferase (MGMT) was observed to be methylated and silenced in colorectal tumors and also in the surrounding mucosa [9]. MGMT removes mutagenic alkyl adducts from the O6 position of guanine. Silencing of MGMT results in O6-methylguanine being read as adenine and causing G]A point mutations among others in the KRAS gene [10]. Later it was associated with the development of low level microsatellite instability (MSI-L) CRC [11]. Field effect also explains how chronic inflammation through a genetically and epigenetically altered field can predispose the mucosa to the development of multiple primary tumors. More recent studies suggest that certain field abnormalities of the colon extend much further, perhaps encompassing the entire mucosal field. Two major contributors to field effect are aging and chronic inflammation. DNA Methylation and Aging The risk of cancer increases with age and so DNA methylation alterations have been implicated in the etiology of aging. Initial studies reported age-related global hypomethylation in morphologically normal colorectal mucosa [12]. Later on, age-dependent hypermethylation of CpG islands located in promoters was reported for several genes (table 1) and termed type A (age-related) methylation, in contrast to type C (cancer-specific) methylation, that was found exclusively in CIMP. These genes usually regulate growth and differentiation in normal colonic epithelial cells and CpG island methylation of their promoter might predispose colonic epithelial cells to neoplastic transformation. The first gene well characterized for a relation between aging and promoter methylation in normal tissues was the estrogen receptor gene (ER). ER methylation in normal colonic mucosa continuously increases with age, and its frequency in cancer is close to 100% [13]. Re-expression of ER in cell culture suppresses the growth of cancer cells, indicating that impairment of ER signaling might be an important event in the early stages of colorectal carcinogenesis [13]. Another gene affected by age-related methylation is the imprinted gene insulin-like growth factor 2 (IGF2) [14]. In healthy young individuals, the P2–4 promoters of IGF2 are methylated exclusively on the silenced maternal allele. During aging, this promoter methylation becomes more extensive and involves the original312
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Table 1. A selected list of hypermethylated genes along colorectal carcinogenesis
Gene
Reference
Aging ER IGF2 N33 MYOD UC and associated cancer ER p16 MYOD MINT1 RUNX3 CDH1 GDNF HPP1 CIMP – classic panel p16 MINT1 MINT2 MINT31 MLH1 CIMP – alternative panel CACNA1G IGF2 NEUROG1 RUNX3 SOCS1
Issa et al. [13] 1994 Issa et al. [14] 1996 Ahuja et al. [15] 1998 Ahuja et al. [15] 1998 Fujii et al. [17] 2005 Hsieh et al. [18] 1998 Issa et al. [19] 2001 Garrity-Park et al. [20] 2010 Garrity-Park et al. [20] 2010 Saito et al. [21] 2011 Saito et al. [21] 2011 Saito et al. [21] 2011 Park et al. [28] 2003
Weisenberger et al. [29] 2006
ly unmethylated paternal allele [14]. Further studies demonstrated age-related CpG island methylation for other genes, including N33 and MYOD [15]. These changes seem to be segment-specific: type A methylation is usually more pronounced at the distal colon [16]. All these results are consistent with the hypothesis that age-related methylation may lead to a field defect that reflects acquired predisposition to colorectal neoplasia.
DNA Methylation and Chronic Inflammation: Ulcerative Colitis (UC) and UC-Associated CRC
It is well established that patients with long-standing and extensive UC have an increased risk of developing colorectal neoplasia. Although ulcerative colitis-associated CRC (UC-CRC) only accounts for 1% of all CRCs, it is a substantial cause of death in patients with UC. In order to detect colorectal neoplasia at an early stage, surveillance colonoscopy is widely recommended. However, there is a great need for additive markers to identify individuals at increased risk of neoplasia. UC is sometimes referred as an accelerated aging of the colorectum; therePatai /Molnár/Kalmár /Schöller /Tóth / Tulassay
fore, it seems to be evident, that various type A methylation markers were studied in UC and UC-CRC (table 1). As we discussed above, the ER shows age-related methylation in the colorectal epithelium and is also methylated frequently in sporadic colorectal neoplasia, suggesting that ER methylation may predispose to colorectal neoplasia. It was shown that ER methylation in rectal mucosa may be useful for predicting cases at high risk of neoplasia: methylation of ER was significantly more frequent in non-neoplastic epithelium from UC with neoplasia than in chronic inflamed epithelium from UC without neoplasia [17]. Several reports have revealed similar findings for genes including p16 [18] and MYOD [19]. A study comparing UC-CRC cases with UC controls showed that hypermethylated RUNX3 and MINT1 together with unmethylated COX-2 independently increased the chance for UCCRC, suggesting that COX-2 overexpression may be an important driving factor in the UC-CRC carcinogenesis [20]. A recent study involving 28 patients who underwent total proctocolectomy tried to identify mucosal inflammation-specific DNA methylation and showed CDH1, GDNF, HPP1 and MYOD1 to be more highly methylated in the active inflamed mucosa than in the quiescent mucosa in each UC patient. In addition, DNMT1 and DNMT3B were highly expressed in colonic epithelial cells with active mucosal inflammation, suggesting their involvement in inflammation-dependent methylation [21]. In light of these findings, UC might be considered as a disorder of premature aging in the colon, consistent with the fact that the development of CRC occurs at an earlier age in UC than in sporadic CRC. All of these studies indicate that methylation seems to precede dysplasia and affect the entire inflamed mucosa, suggesting a diagnostic value in identifying UC patients who may have an increased likelihood to progress to CRC even if it is undetectable with colonoscopy or routine histology. Based on these results, it is now widely accepted that exposure to chronic inflammation leads to DNA methylation in noncancerous tissues, which can be detected and could be used as a risk factor for cancer [22]. These data suggest that some of the epigenetic alterations induced by inflammation remain and accumulate in noncancerous tissues, thus detection and monitoring of field effect may have profound implications for cancer prevention.
CRC: New Classification
Colonoscopy is the gold standard method for the early detection and prevention of CRC, and it was shown that DNA Methylation in Colorectal Carcinogenesis
colonoscopy can reduce the risk of CRC by 77%; however, within the proximal colon this risk reduction was not as pronounced as for the distal colon and rectum [23]. Most sporadic CRCs develop through the adenoma-carcinoma sequence described by Vogelstein et al. [24]. According to this widely accepted model, sequentially acquired genetic mutations are thought to be the main drivers of carcinogenesis; each step from the normal mucosa towards the carcinoma involves specific and well-defined genetic alterations in a strict order. However, a distinct group of CRCs, predominantly found in the proximal colon, are short in genetic alterations and lack multistep progression from adenoma to colon carcinoma, but abound in characteristic epigenetic changes. They tend to arise between routine screening colonoscopies, representing a frequent cause of these interval cancers [25] and presumably follow a distinct sequence, the so-called serrated pathway [26]. These CRCs arise from different precursor lesions (hyperplastic polyps and serrated adenomas characterized by a sawtoothed growth pattern with epithelial dysplasia), are more common in elderly females, have different phenotypic features (e.g. flatness, paleness) and are supposed to have a relatively higher rate of malignization than CRCs developing along the traditional pathway. All of these characteristics hamper the detection of these lesions and it remains a challenge despite the advances in endoscopy. In the past decade since CIMP was described [27], several markers have been used to define it. The two most widely used sets in CIMP (table 1) consist of the traditional ‘classic’ panel (p16, MINT1, MINT2, MINT31 and hMLH1) [28] and the alternative panel (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1) proposed by Weisenberger et al. [29]. Most of these markers were also shown in precursor lesions. Depending on the definition used, the occurrence of CIMP is estimated at 15–25% among sporadic CRCs. Later CIMP cancers were further stratified into CIMP-high (CIMP-H) and CIMP-low (CIMP-L) categories according the degree of methylation. CIMP-H cancers arise in the proximal colon from sessile serrated adenomas, are usually MSI (due to the methylation of MLH1, a main mismatch repair gene) and tightly associated with BRAF V600E mutation [29], whereas CIMP-L cancers develop in the distal colon from traditional serrated adenomas and frequently exhibit KRAS mutation. The exact mechanisms underlying CIMP development are still unknown, several hypotheses exist and some risk factors have been identified. Overexpression of DNMT3B has been shown to induce tumors in mice [30] and Dig Dis 2012;30:310–315
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DNMT3B expression level sequentially increases during tumor progression congruent with DNA methylation [31]. A significant relationship has been reported between cigarette smoking and CIMP [32]. It has also been suggested that smoking is associated with hyperplastic polyps rather than traditional adenomatous polyps. In a recent study, severe caloric restriction during adolescence and early adulthood was associated with decreased risk of developing CIMP [33]. This study suggests that exposure to a transient environmental condition during this early period of life may result in persistent epigenetic changes that later influence CRC development.
by the FDA and are used clinically in low doses to treat myelodysplastic syndrome, providing proof of principal for epigenetic therapy. These agents mimic cytosines; during the S-phase of the cell cycle, cytidine analogues replace real cytosines in growing DNA strands and trap DNA methyltransferases to interfere with the ability of these enzymes to reproduce existing methylation in new cells [38]. Clinical trials are being extended to test DNMT inhibitors in solid tumors including CRC, in combination with histone deacetylase inhibitors which provide synergistic benefits in cell culture studies [38].
Conclusions and Future Directions Hypermethylated Genes in Surrogate Tissues as Prescreening Biomarkers for Colonoscopy
Much of the recent work indicates that hundreds of epigenetically silenced genes possibly exist even in individual tumors. Which epigenetic biomarkers could be used for detection of early-stage diseases is still an open question. Owing to space limitations we cannot go into details and discuss all the methylated genes that have been described in CRC. To our knowledge, there is currently only one commercially available test that measures the methylation status of the SEPT9 (sepin 9) promoter in plasma, and it seems to be able to select patients for colonoscopy (prescreening) with a higher sensitivity and specificity than currently used methods (e.g. FOBT) [34]. Furthermore, in combination with another DNA methylation marker, ALX4, this panel of two (SEPT9 and ALX4) was able to detect even advanced precancerous colorectal lesions [35]. However, the low sensitivity of many single methylation markers suggests that the assay of multiple DNA markers rather than a single marker is necessary to increase the sensitivity of DNA methylation in plasma. Such an approach was described by Lee et al. [36] using methylation status analysis of the APC, MGMT, RASSF2A and Wif-1 genes in plasma, and a panel of APC, MGMT, RARB2 and p16 in stool was also tested [37]. These approaches may provide new strategies to detect CRC at the earliest stage.
Many questions regarding DNA methylation and its role in CRC still remain open. These include, ‘Which environmental factors and which genes are responsible for enhanced disease susceptibility when epigenetically deregulated or do epimutations accumulate randomly with aging?’, ‘What are the consequences of this accumulation?’ and ‘Are these alterations selectively reversible?’. Answers to these might be provided by an emerging multidisciplinary field, ‘molecular pathological epidemiology’, proposed by Ogino et al. [39], which aims to elucidate the interaction of genetic factors and lifestyle exposures and find causal relationship with specific molecular (epi) genetic signatures of cancer. These studies can pave the way for the identification of early diagnostic biomarkers whose use could improve the efficacy of current tumor surveillance strategies and also allow for the development of novel epigenetic-based therapeutic strategies.
Acknowledgements We would like to thank Orsolya Bakonyi for the assistance in the graphic design. This study was supported by the National Office for Research and Technology, Hungary (TECH_08A1/2-2008-0114 grant). We apologize to all investigators whose research could not be cited owing to space limitations.
Disclosure Statement Therapeutic Potentials
Unlike genetic mutations, epigenetic alterations such as DNA methylation are potentially reversible by demethylating agents ‘reawakening’ silenced tumor suppressor genes. Two nucleoside DNA methylation inhibitors, azacytidine and deoxycytidine, have been approved 314
Dig Dis 2012;30:310–315
We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work. There is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, this paper.
Patai /Molnár/Kalmár /Schöller /Tóth / Tulassay
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