Differences in DNA methylation signatures reveal multiple pathways of progression from adenoma to colorectal cancer

Abstract

Background & aims: Genetic and epigenetic alterations contribute to the pathogenesis of colorectal cancer (CRC). There is considerable molecular heterogeneity among colorectal tumors, which appears to arise as polyps progress to cancer. This heterogeneity results in different pathways to tumorigenesis. Although epigenetic and genetic alterations have been detected in conventional tubular adenomas, little is known about how these affect progression to CRC. We compared methylomes of normal colon mucosa, tubular adenomas, and colorectal cancers to determine how epigenetic alterations might contribute to cancer formation.

Methods: We conducted genome-wide array-based studies and comprehensive data analyses of aberrantly methylated loci in 41 normal colon tissue, 42 colon adenomas, and 64 cancers using HumanMethylation450 arrays.

Results: We found genome-wide alterations in DNA methylation in the nontumor colon mucosa and cancers. Three classes of cancers and 2 classes of adenomas were identified based on their DNA methylation patterns. The adenomas separated into classes of high-frequency methylation and low-frequency methylation. Within the high-frequency methylation adenoma class a subset of adenomas had mutant KRAS. Additionally, the high-frequency methylation adenoma class had DNA methylation signatures similar to those of cancers with low or intermediate levels of methylation, and the low-frequency methylation adenoma class had methylation signatures similar to that of nontumor colon tissue. The CpG sites that were differentially methylated in these signatures are located in intragenic and intergenic regions.

Conclusions: Genome-wide alterations in DNA methylation occur during early stages of progression of tubular adenomas to cancer. These findings reveal heterogeneity in the pathogenesis of colorectal cancer, even at the adenoma step of the process.

Keywords: Colon Cancer; Epigenetic Modifications; Gene Regulation; Progression.

Conflict of interest statement

Conflicts of Interest: No conflicts of interest exist for any of the authors.

Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1. Differentially methylated CpG probes in the normal colon mucosa between people with no history of CRC and people with concurrent CRC

The strip-plot shows the top 65 probes that distinguish these 2 groups of tissue samples with a q-value < 1E-4. The Y-axis represents the methylation level (β value) of each case ranging from 0 (completely unmethylated) to 1 (completely methylated). The corresponding CpG probe ID on the HumanMethylation450 array is indicated on the X-axis. Each dot represents the β value of a single case for each targeted CpG probe. (Normal-Cancer (Normal-C) = normal colon mucosa samples from patients with concurrent CRC; Normal-Healthy (Normal-H) = samples from patients without a history of CRC).

Figure 2. Identification and validation of clustering of colorectal adenomas and heatmap representation of DNA methylation array data

DNA methylation status was assessed using HumanMethylation450 arrays. Each column represents one sample and each row represents one of the top 5,000 most variable probes. The probes are arranged based on the order of unsupervised hierarchical cluster analysis using a correlation distance metric and average linkage method. The DNA methylation M-values are represented by using a color scale from green (low DNA methylation) to red (high DNA methylation). The presence of TP53, PIK3CA,KRAS, BRAFV600E,CTNNB1 or APC mutations is indicated by a colored block (no color = wild-type). Two subgroups (adenoma-L and adenoma-H) were identified using the clustering analysis on the discovery set of 18 adenoma samples (middle panel). These results were confirmed in an independently collected validation set of 24 adenomas using the same probes identified in the discovery set of adenomas (right panel). Normal colon samples (left panel, N=41) were used for reference.

Figure 3. Cluster analysis of CRCs and heatmap representation of DNA methylation array data

DNA methylation status was assessed using HumanMethylation450 arrays. Each column represents one sample and each row represents one of the top 5,000 most variable probes. The DNA methylation M-values are represented by using a color scale from green (low DNA methylation) to red (high DNA methylation). Three subgroups were identified by clustering and are indicated above the heatmap (Methyl-Low, Methyl-Intermediate, and Methyl-High). The presence ofTP53, SRC PIK3CA, KRAS,FBXW7, CTNNB1,BRAFV600E or APC mutations and CIMP status, which was determined using the Weisenberger panel of CIMP CpGs with MethyLight assays, are indicated by a colored block (no color = wild-type). Normal colon samples (left panel, N=41) were used for reference.

Figure 4. Venn diagram of hypermethylated genes that are PcG-marked

A) PcG-marked hypermethylated genes in CRC samples (419 genes); B) PcG-marked hypermethylated genes in adenoma samples (545 genes); C) PcG-marked hypermethylated genes in adenoma and CRC samples (384 genes). The number in each area indicates the number of genes in that area.

Figure 5. Increased inter-sample methylation variability in colon adenomas and CRCs

A) The standard deviation of 1,000 randomly selected probes in normal colon samples and adenomas is shown; B) The standard deviation of 1,000 randomly selected probes in normal colon samples and CRCs is shown; C) The standard deviation of 1,000 randomly selected probes in adenomas and CRCs is shown; D) The standard deviation of 1,000 randomly selected probes in adenoma-L polyps and adenoma-H polyps is shown. The gray solid line in each image (from left low corner to the up right corner) is the identity line, which indicates the location of a1:1 ratio between the comparison sets, whereas the dashed red line indicates the best-fit linear regression of the CpG probes. The red line above the gray line means the group on the Y-axis has higher variability than the one on the X-axis.

Figure 6. Cluster analysis of M-H CRCs, M-I/M-L CRCs and adenoma-H adenomas using differentially methylated probes in intragenic/intergenic regions

Differentially methylated intragenic (A) and intergenic (B) CpG probes between M-H and M-I/M-L CRCs were used in unsupervised clustering analysis of the adenoma-H polyps and CRCs. The results show that the methylation pattern in adenoma-H polyps is similar to that in M-I/M-L CRCs but not to that in M-H CRCs. (C) Schematic of concept that there are multiple adenoma to cancer pathways, which can be identified by DNA methylation signatures in the normal colon, adenomas, and cancers (See Discussion).