Yonsei Med J.  2009 Jun;50(3):309-321. 10.3349/ymj.2009.50.3.309.

Promoter Methylation in the Genesis of Gastrointestinal Cancer

• Boland CR
• Shin SK
• Goel A

• KMID: 1758556
• DOI: http://doi.org/10.3349/ymj.2009.50.3.309

Abstract

Colorectal cancers (CRC)-and probably all cancers-are caused by alterations in genes. This includes activation of oncogenes and inactivation of tumor suppressor genes (TSGs). There are many ways to achieve these alterations. Oncogenes are frequently activated by point mutation, gene amplification, or changes in the promoter (typically caused by chromosomal rearrangements). TSGs are typically inactivated by mutation, deletion, or promoter methylation, which silences gene expression. About 15% of CRC is associated with loss of the DNA mismatch repair system, and the resulting CRCs have a unique phenotype that is called microsatellite instability, or MSI. This paper reviews the types of genetic alterations that can be found in CRCs and hepatocellular carcinoma (HCC), and focuses upon the epigenetic alterations that result in promoter methylation and the CpG island methylator phenotype (CIMP). The challenge facing CRC research and clinical care at this time is to deal with the heterogeneity and complexity of these genetic and epigenetic alterations, and to use this information to direct rational prevention and treatment strategies.

Keyword

Colorectal cancer; promoter methylation; CIMP; Lynch syndrome; HNPCC; microsatellite instability; chromosomal instability; hepatocellular carcinoma

MeSH Terms

Colorectal Neoplasms/genetics
DNA Methylation/*genetics
Gastrointestinal Neoplasms/*etiology/*genetics
Humans
Microsatellite Instability
Promoter Regions, Genetic/*genetics

Figure

• 

  Fig. 1 The relationships among various forms of genomic instability in CRC. (A) The pie chart illustrates the characterization of 209 sporadic CRCs for MSI and CIN.2 As shown, 48% of tumors had evidence of LOH, while 14% of tumors had MSI, including a minority of tumors (3%) that overlapped with LOH. We were most interested in the observation that nearly one third of the tumors (38%) did not have either MSI or LOH. (B) The bars demonstrate the mean methylation index based upon the number of markers methylated in the three subsets of CRCs.80 Methylation alterations were analyzed at 12 markers including 6 canonical CIMP markers (MINT-1, -2, -31, p16, p14 and MLH1) and 6 additional tumors suppressor genes (PTEN, TIMP3, RUNX3, HIC1, APC, and RARβ2). The three vertical columns represent the mean methylation ratios in MSI (blue), MSI-/LOH-(red) and LOH (white) CRCs. The error bars denote the S.D. The filled circles (color matched with vertical columns) represent the 95% confidence interval (CI) of the mean methylation ratios. The rectangular boxes in the upper panels represent the pair-wise correlation between the mean methylation ratios in each subset of CRC; p-values were calculated by the Wilcoxon test. As shown in the three panels, analysis of the methylation ratios using only four CIMP-related markers (versus using 12 or 6 CIMP-related markers) demonstrates a significant positive association for MSI and MSI-/LOH-tumors, but an inverse correlation for LOH CRCs. (C) The relationship between methylation ratios calculated using all 12 methylation markers was compared with LOH ratios in the total cohort of 126 CRCs. As shown, an inverse correlation was observed (p = -0.3690; p < 0.0001) between the methylation ratio and LOH. CRC, colorectal cancers; MSI, microsatellite instability; LOH, loss of heterozygosity.

• 

  Fig. 2 KRAS and BRAF signaling play a role in aberrant methylation, and may contribute to epigenetic alterations in Lynch Syndrome cancers. (A) These 3 boxes show the average number of methylated loci in various subgroups of CRCs categorized by their BRAF/KRAS mutation status as published previously.31 The average number of methylated loci in each subset was calculated using all fourteen markers ("All Loci"), seven canonical CIMP markers ("CIMP") and the seven additional markers ("Non-CIMP"). The p values are based on Kruskal-Wallis one way analysis of variance on ranks, and represent the statistical differences among all three subsets of CRCs (BRAF-mutated, KRAS-mutated or wild type for both). BRAF-mutated cancers showed the highest degree of methylation at CIMP-related loci, and as a consequence, the highest degree of methylation when data were analyzed from "All Loci". KRAS mutated tumors exhibited methylation not only at the CIMP-markers, but interestingly, when data were analyzed from the additional (Non-CIMP) markers, there was a minimal difference in the number of markers methylated between BRAF- and KRAS-mutated cancers. (B) These 3 bar charts illustrate the average number of methylated loci in sporadic MSI (MSI-H), Lynch syndrome and non-MSI (MSI-L and MSS) CRCs.31 As expected, sporadic MSI tumors were most frequently methylated at all markers (CIMP and Non-CIMP). Lynch syndrome CRCs also demonstrated frequent methylation at canonical-CIMP markers, which was surprisingly slightly higher compared to the non-MSI tumors. CRC, colorectal cancers; CIMP, CpG island methylator phenotype; MSI, microsatellite instability; MSS, microsatellite stable.

• 

  Fig. 3 Hepatocellular carcinomas (HCCs) are classified based upon methylation and mutational status. In the upper figure, all 81 HCCs are classified by hierarchical clustering analysis based upon the degree of methylation and β-catenin or p53 mutations as published previously.39 The heat map (green-black-red) shows the tumors with a lower methylation density as green, and higher methylation density as red. The Y-axis shows the loci analyzed. A solid triangle represents a tumor with a β-catenin mutation, and an open triangle indicates a HCC with a p53 mutation. As depicted, β-catenin mutations were significantly associated with the Group 2 HCCs on the right (p < 0.0001). The table in the lower half of the figure highlights the significant association between higher methylation in Group 2 HCCs and β-catenin mutations. FAL indicates the "fractional allelic loss", or proportion of analyzed loci with allelic imbalance, or LOH.

• 

  Fig. 4 Treatment with a demethylating agent restores sensitivity to 5-FU toxicity in a MMR-deficient CRC cell line. (A) The top schematic summarizes the treatment plan and results from the in-vitro experiments. Four CRC cell lines - HCT116 and HCT116 + 2 (both MMR-deficient due to mutation in MLH1), HCT116 + 3 (MMR-proficient following restoration of MLH1 gene with chromosome 3 transfer in HCT116 cells) and SW48 (MMR-deficient due to hypermethylation of MLH1) - were cultivated in growth medium or exposed to 5-AZA for 24 h.66 Culture medium was exchanged and cells were allowed to form colonies over a period of 10-12 days. During this time, the cells were continuously exposed to increasing concentrations of 5-FU. Cells were washed, fixed, stained and colonies counted. MLH1 methylation status, its expression (mRNA and protein), and sensitivity to 5-FU sensitivity were monitored in each experiment. (B) A representative graph (in the left panel) depicts the mean ± S.D. of three different experiments for the colony forming assay from CRC cell lines exposed to different doses of 5-FU (1 µM, 2.5 µM, and 5 µM). Cells were allowed to form colonies for 10-12 days. The means and S.D. of three independently performed experiments are shown. As indicated, only HCT116 + 3 cells (MMR proficient cells) are sensitive to 5-FU, while all MMR-deficient cells demonstrate more colonies, indicating resistance to the chemotherapeutic drug. However, when SW48 cells were pretreated with 5 µM 5-Aza for 24 h before 5-FU exposure, MMR activity is restored, and they behave similarly to the MMR-proficient HCT116 + 3 cells (right panel). MMR, mismatch repair; CRC, colorectal cancers.

• 

  Fig. 5 An integrated model for different forms of genetic and epigenetic instability in CRC. Tumors can develop via chromosomal instability (CIN), microsatellite instability (MSI), or CpG island methylator phenotype (CIMP) pathways. MSI may develop either from Lynch syndrome (the hereditary form), or via the CIMP pathway (a presumably acquired form). Ultimately, all pathways converge pathologically as cancer. Based upon our current understanding, CIN and CIMP represent the two primary processes of genetic and epigenetic instability respectively, in the colon. In essence, MSI in sporadic CRCs originates because of CIMP, and once MLH1 becomes methylated, the tumors acquire MSI. Thus far the molecular mechanisms for CIN and CIMP are not clear, however, there are data to suggest that JC virus (JCV) may be a putative unifying mechanism for genetic and epigenetic instability in CRC. CRC, colorectal cancers; MMR, mismatch repair.

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