Genome-wide DNA methylation profiling of primary colorectal laterally spreading tumors identifies disease-specific epimutations on common pathways

Abstract

Colorectal laterally spreading tumors (LSTs) grow to extremely large size while rarely invade deeply. Also, there is a low tendency to become cancerous. We used the Illumina Human Methylation 450K array to query the main epigenetic difference of LSTs. We built a discovery cohort with 10 matched cases, and a validation cohort with 9 additional matched cases. Our results suggest that LST displays significant decrease in DNA methylation, highlighted by the discovery of 1,018 hypomethylated intergenic regions (IGRs). Comparing to classic differentially methylated probes and regions that overlap transcription starting site and CpG island, IGR-regions were associated more closely with genes involved in functional biological processes and correlated with specific histone modifications. Hypomethylated IGR regions were often annotated as tissue-specific regulatory elements for noncolon tissues and were typically epigenetically silenced in normal colon mucosa. By integration of public data, we defined the commonality and specific epigenetic signatures for adenomas, LSTs and colon adenocarcinomas. Only 435 hypermethylated differentially methylated probes (DMPs) and differentially methylated regions (DMRs) and 517 hypomethylated DMPs and DMRs were shared by the three diseases. However, our pathway-level analysis discovered that genes in four pathways were common target of epimutations in LSTs, adenomas and CRCs. More interestingly, different diseases seem to employ distinct epigenetic insult to disturb specific pathways. Between LST and adenoma, we found eight pathways including Ras signaling and Rap1 signaling pathway were commonly targeted but the epimutation patterns were opposite. Comparison between precancerous conditions and invasive states revealed the key pathways governing the progression to malignancy, including PI3K-Akt pathways.

Keywords: DNA methylation; DNA methylation array; epimutation; laterally spreading tumors.

Figure 1:

Differentially methylated positions (DMP) analysis in laterally spreading tumors (LST) cases and controls. (A) Manhattan plot of top differentially methylated positions (DMPs) in laterally spreading tumors (LST) versus control. (B) Volcano plot of top DMPs and position of methylation probes in relation to the gene (IGR, intergenic region; TSS, transcription start site; UTR, untranslated region). The percentages of hyper- and hypo-methylated DMPs are displayed on top. The proportions of different genomic features are shown on the right.

Figure 2:

Distribution of LST-associated DMPs across different categories of genomic feature. The top panel displays for the hypermethylated DMPs, and the bottom panel displays for the hypomethylated DMPs.

Figure 3:

Gene ontology (GO) analysis of different groups of hypomethylated DMPs. Different biological functions were enriched in IGR-hypomethylated DMPs (A) and TSS1500-hypomethylated DMPs (B). (C) Histone modification enrichment patterns across different categories of DMPs. Enrichment of six histone modifications (H3K27ac, H3K27me3, H3k36me3, H3K4me1, H3K4me3 and H3K9me3) at CGI-hypomethylated DMPs, TSS-hypomethylated DMPs and IGR-hypomethylated DMPs in normal colon mucosa. Each line represents histone modification ChIP-seq signal around DMPs extended 500bp window ±3 kb.

Figure 4:

Loss of DNA methylation in repressed regions in LSTs may non-colon cell type-specific regulatory elements. (A) Relative enrichment/depletion of LST-associated hypomethylated DMPs in different chromatin states (defined by the Roadmap project chromHMM-18 state model across 78 diverse cell and tissue types). Orange dot represents primary colon muscosa sample (Roadmap E075); gray dots represents the remaining 77 Roadmap samples; black line represents odds ratio of 1. (B) Regions that repressed in normal colon mucosa and lose DNA methylation in LSTs were enriched for regulatory elements specific for non-colon tissues, but not for digestive tissues. (C) Enrichment of transcription factor binding sites in these regions.

Figure 5:

Distinct DNA methylation abnormality pattern across three colon neoplasms. (A) Overlapping relationship between DMPs. (B) Overlapping relationships between pathways that underwent epigenetic lesion. (C) The disparities of deregulated pathways in adenomas and LSTs. (D) Specific genes in the Rap1 and Ras signaling pathway exhibited opposite DNA methylation abnormality in LSTs and in adenomas.

Figure 6:

The PI3K-Akt pathway was repeated epi-mutated in three neoplasms. The genes were grouped into seven subcategories: LST specific (Black Russian), Adenoma specific(Jacksons Purple), CRC specific(Pear), Adenoma & CRC (Yellow), LST & CRC(Carrot Orange), LST & Adenoma(Outrageous Orange) and LST & Adenoma & CRC(Red).