Genome-wide DNA methylation profiling reveals cancer-associated changes within early colonic neoplasia

Abstract

Colorectal cancer (CRC) is characterized by genome-wide alterations to DNA methylation that influence gene expression and genomic stability. Less is known about the extent to which methylation is disrupted in the earliest stages of CRC development. In this study, we have combined laser-capture microdissection with reduced representation bisulfite sequencing to identify cancer-associated DNA methylation changes in human aberrant crypt foci (ACF), the earliest putative precursor to CRC. Using this approach, methylation profiles have been generated for 10 KRAS-mutant ACF and 10 CRCs harboring a KRAS mutation, as well as matched samples of normal mucosa. Of 811 differentially methylated regions (DMRs) identified in ACF, 537 (66%) were hypermethylated and 274 (34%) were hypomethylated. DMRs located within intergenic regions were heavily enriched for AP-1 transcription factor binding sites and were frequently hypomethylated. Furthermore, gene ontology analysis demonstrated that DMRs associated with promoters were enriched for genes involved in intestinal development, including homeobox genes and targets of the Polycomb repressive complex 2. Consistent with their role in the earliest stages of colonic neoplasia, 75% of the loci harboring methylation changes in ACF were also altered in CRC samples, though the magnitude of change at these sites was lesser in ACF. Although aberrant promoter methylation was associated with altered gene expression in CRC, this was not the case in ACF, suggesting the insufficiency of methylation changes to modulate gene expression in early colonic neoplasia. Altogether, these data demonstrate that DNA methylation changes, including significant hypermethylation, occur more frequently in early colonic neoplasia than previously believed, and identify epigenomic features of ACF that may provide new targets for cancer chemoprevention or lead to the development of new biomarkers for CRC risk.

Figure 1. DNA methylation patterns in Stage III–IV CRCs and ACF

(A) Gross appearance of a human ACF during HD-chromoendoscopy (top) and H&E-stained section of an ACF biopsy with serrated morphology (bottom). Serrated crypts, characterized by their star-shaped lumen in cross section, are indicated with black arrows. (B) Heat-map depicting differentially methylated regions (DMRs), defined as genomic regions with a change in methylation relative to matched normal >15% and a FDR-adjusted P-value<0.05, in CRCs. Tumor samples are generally segregated from their matched normal mucosa by unsupervised clustering. Of the 23,745 DMRs detected in cancer samples, 5,995 (25%) were hypermethylated, while 17,750 (75%) were hypomethylated. (C) Heat-map depicting DMRs in ACF. ACF are segregated from normal mucosal samples by unsupervised clustering. 811 DMRs were identified, 537 (66%) of which were hypermethylated and 274 of which (34%) were hypomethylated.

Figure 2. Direct comparison of DNA methylation changes in Stage III–IV CRCs and ACF

(A) Principal component analysis (PCA) bi-plot of DNA methylation changes demonstrates a clear separation of ACF (red) and tumor samples (black). CRCs are less tightly clustered than ACF, indicating a greater variability of DNA methylation. (B) Heat-map depicting overlapping DMRs detected in both CRCs and ACF. Of 608 shared DMRs, 66% were hypermethylated and 32% were hypomethylated; a small subset of DMRs (2%) were hypermethylated in ACF, but hypomethylated in cancers. The magnitude of these overlapping methylation changes was typically greater in CRCs than in ACF.

Figure 3. Functional enrichment analysis of overlapping DMRs

(A) 592 DMRs exhibited a methylation change in the same direction in both stage III–IV cancers and ACF; these DMRs are referred to as “overlapping” DMRs. (B) Gene ontology (GO) analysis for overlapping DMRs. These DMRs were significantly enriched (FDR-adjusted P-value <0.05) for homeobox genes, as well as genes involved in the regulation of transcription and cell fate commitment. (C) Representative homeobox genes that show increased methylation in both CRCs and ACF. All of the genes identified in this panel exhibit significantly increased methylation in both CRCs and ACF compared to matched normal tissues. Error bars represent means +/− SEM.

Figure 4. Volcano plots depicting DMRs in colon cancers and ACF grouped by genomic region

DMRs identified in CRCs and ACF were segregated according to their genomic location, and the methylation changes in gene bodies, intergenic and promoter regions were examined; DMRs are depicted as blue dots. (A) DMRs located within gene bodies. In cancer tissue, 12,258 DMRs (74%) were hypomethylated and 6,591 (26%) were hypermethylated. In contrast, in ACF, 136 gene body DMRs (39%) were hypomethylated and 213 (61%) were hypermethylated. (B) Intergenic DMRs in colon cancers and ACF. In cancers, 10,401 (85%) intergenic DMRs were hypomethylated while only 1,769 (15%) were hypermethylated. Of the 297 intergenic DMRs identified in ACF, 129 (43%) were hypomethylated, while 168 (57%) were hypermethylated. (C) Promoter-associated DMRs in colon cancers and ACF. In cancers, 758 DMRs (28%) were hypomethylated while 1,968 (72%) were hypermethylated. The same pattern was observed in promoter DMRs identified in ACF; only 9 promoter DMRs (5%) were hypomethylated, while 156 (95%) were hypermethylated.

Figure 5. Enrichment of AP-1 binding motifs in intergenic DMRs

Intergenic DMRs from cancers and ACF were subjected to Hypergeometric Optimization of Motif EnRichment (HOMER) analysis to determine whether they were enriched for known regulatory elements. (A) 103 motifs were enriched in intergenic DMRs in cancers, including 6 motifs which were also enriched in intergenic DMRs in ACF. (B) Motifs enriched in ACF intergenic regions. Five of the 6 motifs enriched in ACF intergenic DMRs corresponded to the binding sequences of AP-1 transcription factor family members, indicated with an asterisk. (C) Change in methylation of DMRs enriched for AP-1 binding motifs in ACF and cancer. In both sample sets, the DMRs containing AP-1 binding motifs were significantly hypomethylated compared to their respective matched normal samples. This pattern was especially pronounced in CRCs, where 95% of AP-1 enriched DMRs were hypomethylated. Furthermore, a greater number of AP-1 sites were affected in CRCs than in ACF. (D) Average change in percent methylation of DMRs enriched for AP-1 family motifs. Intergenic DMRs containing AP-1 binding motifs exhibited a ~15% reduction in methylation in ACF and a ~25% reduction in cancer. Bars represent average change in methylation of all DMRs containing the indicated motif. Error bars represent means +/− SEM.

Figure 6. Promoter methylation is correlated with gene expression in CRCs but not ACF

(A) Scatter-plot depicting the correlation between changes in promoter methylation and gene expression in CRCs (Pearson’s correlation coefficient = −0.55), indicating increased promoter methylation associated with gene silencing. (B) Scatter-plot depicting the lack of correlation between changes in promoter methylation and gene expression in ACF (Pearson’s correlation coefficient = −0.07). (C) Selected genes with promoter DMRs identified in both CRCs and ACF. GSG1L, DPP6, NEFL, GRIN2A, SORCS1, NPTX1, SFRP1, ST8SIA5, ADCYAP1R1, SNAP25, and GABRB3 were significantly down-regulated in cancer samples (at least 3-fold reduction in expression), but not significantly altered in ACF. (D) These same genes exhibit promoter hypermethylation in both stages of neoplasia, but the extend of hypermethylation is greater in CRC. Bars represent the average percentage change in methylation of promoter DMRs for indicated genes. Error bars represent 95% confidence interval and SEM in (C) and (D), respectively.