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. Author manuscript; available in PMC: 2014 Apr 1.
Published in final edited form as: Curr Opin Cell Biol. 2013 Feb 8;25(2):177–183. doi: 10.1016/j.ceb.2013.01.007

Epigenetic regulation of colon cancer and intestinal stem cells

Saher Sue Hammoud 1,2,3, Bradley R Cairns 1,2,3, David A Jones 1,3,4
PMCID: PMC3615052  NIHMSID: NIHMS440318  PMID: 23402869

Summary

The importance and role of the cellular epigenome in cell fating and development has been studied for decades. The epigenome encompasses a range of attributes including DNA methylation, histone modifications, and chromatin remodelers; together these components define the cellular transcriptome, identity, and function. The cellular epigenome is dynamic in response to environmental signals, modifiable during normal cell differentiation and is heritable in daughter cells. This plasticity, however, poses a risk for misregulation and may underlie a number of hereditary disorders, development defects, and cancer. Although the first epigenetic change described in cancer was gene hypomethylation [1,2], we know that cancers display global hypomethylation, as well as, site-specific gene hypermethylation in addition to changes in chromatin modifications. Mechanisms explaining the sometimes paradoxical epigenetic changes observed in cancer, their contributions to tumor initiation and progression and how epigenetics relate to genetic events are poorly understood. In this review we will briefly discuss recent findings on the epigenomic states observed in colon cancer, in particular, how perturbations to the genome and epigenome together may contribute to initiation and progression of colon cancer.

Introduction

Intestinal Epithelial Regeneration poses a risk for colon cancer development

Epithelia is a continuous sheet of tightly linked cells that line the digestive tract, urogenital, and respiratory tract. These epithelial layers protect from the external environment and aid in nutrient/water absorption and glandular secretions. Most epithelial layers are constantly regenerated in order to maintain normal adult organ function. Within the intestine a cyclical regeneration process [3-6] is maintained by adult stem cell populations that reside within the intestinal crypt [7-12]. The stem cells from the crypt bottoms give rise to a rapidly dividing transit-amplifying (TA) population. Near the mouth of the crypt, TA cells exit mitosis and differentiate into all mature cell types of the intestinal epithelium including absorptive enterocytes and three secretary cells types; goblet, enteroendocrine and Paneth cells [13]. Eventually, differentiated epithelial cells undergo apoptosis and are shed into the intestinal lumen. The average life span of a cell in the intestinal epithelium is just 3-5 days [14], so the mechanisms that regulate stem cell maintenance, proliferation, differentiation, and apoptosis must be precisely tuned to ensure proper organ maintenance. An imbalance in the proliferation, differentiation, and apoptosis patterns within the intestinal crypts can lead to aberrant crypt foci [3-6], which are thought to later progress to an adenoma. The progression from an adenoma to carcinoma in colon cancer may take decades, supporting the notion that accumulated genetic and epigenetic changes underlie the multistep developmental process of colorectal cancer (Figure 1).

Figure 1.

Figure 1

Model Figure

Colon cancer development is a multistep process with known perturbations to the genome and/or epigenome of colonic epithelial or progenitor cells. These perturbations together contribute to initiation and progression of colon cancer. Although, this may be an over simplification of the process, the loss of APC represents a common starting point. Upon loss of APC, the attenuated retionic acid levels and the concomitant upregulation in the DNA demethylases may perturb the differentiated cell fate or may prevent proper colonic progenitor cell differentiation. The upregulation in the DNA demethylases may contribute to the global hypomethylation or facilitate the second hit mutation in a tumor suppressor gene, which provides cancer cells a growth advantage and metastatic potential.

The Molecular Genetics of Colon Adenoma Formation

Familial Adenomatous Polyposis (FAP) results from mutations in a single gene known as adenomatous polyposis coli, APC [15,16]. This syndrome is defined by the appearance of hundreds to thousands of adenomatous polyps in affected individuals. The APC gene was discovered by genetic linkage analysis in FAP families [17-19]. Mutations in the APC gene appear in aberrant crypt foci and early adenomas, suggesting inactivation of APC very early in adenoma formation [20-22]. Furthermore, mutations in APC have been observed in 70-80% of sporadic colon cancers [23,24]. In support of APC loss as an initiating event in adenoma, mice lacking functional APC develop numerous intestinal adenomas[25] [26]. Together the data from human and mice place APC as a gatekeeper of colonic epithelial cell proliferation and differentiation; whose loss may lead to an imbalance in cell turnover. Supporting this gatekeeper role, a number of studies have investigated whether germline or de novo somatic mutations in genes such as p53 and RAS, commonly mutated genes in colon cancer [21,27-29], can efficiently initiate neoplastic processes. These studies show that the loss of these genes alone does not appear to lead to colorectal neoplasia, instead they assist in progression from adenoma to carcinoma. Therefore, these studies suggest that the sequence of mutations and the accumulation of mutations will determine the propensity of neoplasia [30,31]. A perplexing question in cancer biology is how a single gene mutation can lead to polyp formation or a marked predisposition to colorectal cancer? A possible answer to this question originates from the recent data suggesting that the loss of APC in a cell may affect both intestinal cell fating and cell proliferation, therefore, possibly explaining the enhanced disease penetrance in FAP patients. These findings suggest a model wherein genetic lesions coordinate with epigenetic changes that cause improper cell fating and may help dictate the response to subsequent transforming events.

The role DNA methylation in cancer and cell fating

Although genetic mutations have been implicated in the initiation of many cancers, epigenetic and genetic alterations are likely to act synergistically in cancer development. One of the first epigenetic abnormalities discovered in a number of cancers was the loss of DNA methylation at CpG dinucleotides [2,32]. This loss of methylation was observed in very early stages of premalignant adenomas with no significant bulk changes in methylation from adenoma to carcinoma [33,34]. This hypomethylation was thought to have significant implications on gene activation, loss of heterozygosity, and global chromosomal stability [35-37]. At the time, however, there were no obvious mechanisms explaining DNA demethylation or its biological importance. In parallel, however, the field made rapid progress in understanding DNA hypermethylation and the enzymes that facilitate this process. Evidence for targeted and predictable hypermethylation changes came from analysis of patient samples that resulted in a characteristic pattern of methylation referred to as a CpG island methylator phenotype or CIMP+ [38]. Clear examples of gene hypermethylation leading to inactivation of important tumor suppressor genes was first observed at the mismatch repair enzyme MLH1 in colon cancers [39]. This list of hypermethylated gene promoters in colorectal cancer (CRC) has grown extensively and now includes key tumor suppressor such as retinoblastoma (RB) [40], P16 [41], RARB, and SFRP [42,43]. It is important to note that epigenetic alterations commonly observed in colon cancers such as candidate gene hypermethylation and genome-wide DNA hypomethylation are also evident in normal aged colonic tissue [44-46]. These observations raise the question whether epigenetic marks acquired during aging or in response to oncogene activation might play important roles in priming tumorigenesis and cancer progression. Reversing the methylation state at these hypermethylated promoters/loci, such as MLH1, can be achieved using drugs that inhibit DNA methyltransferase and remain an interesting approach for reprogramming tumor cell with aberrant methylation. [47,48].

Today genome-wide analysis of DNA methylation using expanded promoter arrays have expanded our view of how and where methylation changes happen. Furthermore these genome-wide array studies have revealed that most of the methylation changes observed in a variety of cancers occur in CpG island shores rather than promoters [49]. In addition, this analysis showed that cancer specific differentially methylated loci varied across normal and colon, lung, breast, thyroid and wilms tumor subtypes [50]. Interestingly separate, parallel studies have revealed that these variable cancer loci are often differentially methylated or misregulated when comparing embryonic stem cells (ES) and induced pluripotent stem cells (iPSCs) [51]. Thus suggesting that the mechanisms of differentiation and reprogramming that are employed in normal development maybe shared in reprogramming toward cancer.

In addition to the targeted changes at specific loci, factors such as nuclear organization, architecture, and genomic sequence may impart profound changes in genome wide methylation patterns. For example, a close examination of the genome using shotgun bisulfite sequencing in normal mucosa, three colorectal cancers, and two adenomatus polyps verified previous findings and revealed many new and interesting insights [52]. First, when investigating global DNA methylation changes by comparing the methylation of tumor to adjacent normal mucosa three distinct methylation profiles were found: (1) regions hypomethylated in both tumors and normals, (2) regions that are demethylated in tumors only (3) regions that acquired methylation in tumor. The methylation prone regions in tumors corresponded with CpG islands in and outside promoters, and were also highly enriched for marks of polycomb repressive complex 1 and 2 activity in hESCs and methylated in normal development [53-56]. Furthermore, the methylation prone loci were generally depleted of certain transcription factor sequences such as Sp1, YY1, and NRF1, which confer methylation protection in cancer [57,58]. Notably, the authors find that focal and large blocks of the genome are demethylated [50], and the regions of hypomethylation within tumors corresponded with an increase in gene expression in these tumors. Interestingly, these large blocks of hypomethylation overlapped with previously described partially methylated domains (PMDs) in IMR90 cells [59], demonstrating a shared attribute between immortalized cell lines and tumor cells. Furthermore, these large blocks of hypomethylation are defined by the nuclear lamina associated domains (LADs) [52]. These observations clearly indicate that changes in DNA methylation can occur throughout the genome and may require a variety of mechanisms all of which may serve to improperly fate cells. These changes could, therefore, be a major mechanism for initiating transformation or generating transformation competent cells.

Although the hypomethylation observed in adenomas and carcinomas [60-64] suggests altered cell plasticity and potential growth advantage, the mechanism through which this happens and the biological impacts are unclear. Many have speculated that this global change may be achieved passively (absence of maintenance methyltransferase activity) or actively (targeted enzymatic removal of mC mediated by the DNA demethylases) or a combination of both. Recent work, however, has shed light on this possibility by demonstrating that aberrant DNA methylation can occur soon after the loss of APC. For example, human FAP adenomas and apcmcr zebrafish have elevated transcript levels of the DNA demethylase machinery components (i.e. Mbd4, AID, Apobec2a, Gadd45a) [65-69]. The increase in DNA demethylase components corresponded with an increase in expression and reciprocal decrease in methylation at a number gene promoters implicated in intestinal cell fate specification and colorectal cancers such as aldh, and hoxd13 determined by MEDIP arrays. Knockdown of the DNA demethylase components restored DNA methylation in apcmor zebrafish. Furthermore, the upregulation of the DNA demethylase machinery was due to a previously described lack of retinoic acid [65-69]. Treatment of apcmcr zebrafish with retinoic acid, reduced the DNA demethylase components and restored intestinal cell fating; fating determined by an increase in IFABP expression and reduction in aldh1a2 levels. Furthermore, knockdown of the DNA demethylase components alone in apcmcr zebrafish also induced intestinal cell differentiation, suggesting that the upregulation of DNA demethylases maintained intestinal cells in a progenitor like state [65-69]. These data support a role for APC in controlling cell fate specification through its regulation of the DNA demethylases and place this demethylation as the initiating event that precedes disregulated cell proliferation. Consistent with this notion, Apc min mice carrying a genetic deletion for Apobec1 −/−, a cytidine deaminase, have reduced polyp formation [70].

Changes in Chromatin Packaging in Cancer Initiation and Progression

The histone code at or outside promoters affects DNA methylation dynamics in a stem/progenitor or differentiated cell. For example regions highly enriched for marks of the polycomb repressive complex 1 and 2 activity in hESCs commonly acquire DNA methylation in normal development/differentiation [53-56]. Interestingly, these same gene promoters acquire methylation in colon adenomas. Recently, the misregulation of post-translational histone modifications has become increasingly apparent in a number of human cancers, and is caused by the deregulation of factors that mediate the reading, writing, and removal. For example, in addition to global changes to DNA methylation and H3K27me, a generalized loss of H4K16 acetylation and H4K20 methylation is found in both lymphoma and colorectal cancer and correlated with transcriptional silencing [71]. Future studies are needed to explore the role of histone modifications, remodelers, and transcription factors in the facilitation in intestinal cell turnover and tumor initiation and progression. Achieving a comprehensive understanding of the roles of chromatin remodelers and modifiers in normal intestinal fating and how they are misregulated in cancer may be of therapeutic potential.

Uncontrolled cell proliferation and its role in Colon cancer initiation and progression

The current data now point to misregulation of epigenetics as a major factor in governing intestinal cell fating and colon tumor initiation. Changes in the intestinal cell epigenome may precede and/or enhance the activity of other oncogenes such as Wnt, RAS and p53, which are needed for neoplastic progression. Indeed, a number of studies suggest that loss of cell fating precedes disregulation of proliferation stimulated by signaling pathways such as Wnt/beta - catenin [23,75-82]. In support of this possibilitiy, a number of studies that have failed to correlate the loss of APC with activation of WNT signaling as determined by the presence of nuclear b-catenin, particularly in early adenomas [83-87]. An absence of nuclear b-catenin suggests a role for others factors, such as mehthylation changes as key to adenoma initiation upon loss of APC. Support for this model comes from work in human cells lines, human FAP adenomas, and APC morphant zebrafish [86] showing a need for loss of APC as well as RAS activation in promoting proliferation of undifferentiated intestinal cells. These findings are consistent with previous observations in mice, wherein, loss of APC and KRAS mutation causes an increase in adenoma size, number and invasiveness [88,89]. Further, this enhanced proliferation appears to expand the number of cells bearing putative stem cell markers within the tumor epithelium [90]. Taken together the data suggest that epigenetic changes work in concert with proliferative signals for initiation and progression of colon tumors. Future studies with help define whether specific epigenetic landscapes may be necessary and permit transformation by oncogenes such as RAS.

Concluding remarks

Emerging evidence suggests that both genetic alterations and epigenetic aberrations contribute to the initiation and progression of human cancers, including colon cancer. Loss of a major tumor suppressor, APC, appears to induces aberrant DNA methylation and that this misprogramming contributes to mis-fating of intestinal cells as a common mechanism to drive colon tumorigenesis. These changes in DNA methylation, along with changes in histone modifications, create a new landscape for the correct interpretation of cell signals that usually govern normal cell turnover. Mis-interpretation of these signals due an altered epigenetic state can lead to the misregulation of gene expression and selected growth advantage of tranformed cells. Therefore, further definition of mechanisms targeting epigenetic modifications in the normal intestinal epithelium and in precancerous, and cancerous lesions offers the promise of identifying opportunities for early cancer detection and intervention.

  1. APC modulates the intestinal epigenomic landscape and cell fate through its regulation of RA/DNA demethylases.

  2. Misregulation of the epithelial cell epigenome governs intestinal cell fate and colon tumor initiation.

  3. Global intestinal epigenomic changes may enhance oncogene activity and may facilitate neoplastic progression

Acknowledgements

This work is supported by grants from the National Cancer Institute (CA073992 and CA96934) awarded to D.A.J., the National Institute of Child Health and Development (HD058506 to B.R.C. and D.A.J), the Howard Hughes Medical Institute (to B.R.C), the Huntsman Cancer Foundation, The Helen Hay Whitney awarded to S. H., and by the Cancer Center Support Grant (CA042014).

Footnotes

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