Abstract
BackgroundCancer-specific hypermethylation of (promoter) CpG islands is common during the tumorigenesis of colon cancer. Although associations between certain genetic aberrations, such as BRAF mutation and microsatellite instability, and the CpG island methylator phenotype (CIMP), have been found, the mechanisms by which these associations are established are still unclear. We studied genome-wide DNA methylation differences between colorectal tumors carrying a BRAF mutation and BRAF wildtype tumors.ResultsUsing differential methylation hybridization on oligonucleotide microarrays representing 32,171 CpG-rich regions, we identified 1,770 regions with differential methylation between colorectal tumor and paired normal colon. Next, we compared the tumor/normal methylation ratios between different groups of patients. Related to CIMP, we identified 749 differentially methylated regions, of which 86% had a higher tumor/normal methylation ratio in the CIMP-positive group. We identified 758 regions with a BRAF mutation-specific methylation change, of which 96% had a higher tumor/normal methylation ratio in the BRAF mutant group. Among the genes affected by BRAF mutation-specific methylation changes, we found enrichment of several cancer-related pathways, including the PI3 kinase and Wnt signaling pathways. To focus on genes that are silenced in a tumor-specific rather than a lineage-specific manner, we used information on the epigenetic silencing mark H3K27me3 in embryonic stem (ES) cells. Among the genes showing BRAF mutation-specific promoter methylation but no H3K27me3 mark in ES cells were forkhead box (FOX) transcription factors associated with the PI3 kinase pathway, as well as MLH1 and SMO. Repression of FOXD3 gene expression in tumors could be related to its promoter hypermethylation.ConclusionsWe identified new BRAF mutation-specific methylation changes in colorectal cancer. Epigenetic downregulation of these targets may contribute to mutationally active BRAF-driven tumorigenesis, explaining its association with aberrant DNA methylation.
Highlights
IntroductionCancer-specific hypermethylation of (promoter) CpG islands is common during the tumorigenesis of colon cancer
Cancer-specific hypermethylation of CpG islands is common during the tumorigenesis of colon cancer
In an attempt to identify biologically relevant BRAF mutationspecific promoter methylation, we excluded loci with H3K27me3 pre-marking in embryonic stem (ES) cells from the functional pathway analyses. By both extending the number of screened loci and filtering out pre-marked genes, we identified new targets of BRAF mutation-specific methylation that could either create a favorable setting for the acquisition of BRAF mutations or function as an addition to upregulation of the RAS-RAF-MEK pathway
Summary
Cancer-specific hypermethylation of (promoter) CpG islands is common during the tumorigenesis of colon cancer. Subsequent studies regarding CIMP in colon cancer described a strong association between this epigenetic phenotype, BRAF mutations, and microsatellite instability (MSI) [2,3,4,5,6,7,8]. The field of epigenetic research has progressed from a candidate-gene to a genome-wide approach, which provides a plethora of new candidate targets of cancerspecific DNA methylation and a better understanding of transcription regulation by DNA methylation [11] Using such genome-wide DNA methylation approaches could help to identify new targets of BRAF mutation-specific promoter methylation. We screened 32,171 CpG sites located at 10,537 genes in a selected cohort of 19 patients with right-sided colon cancer to obtain additional insight into the association between BRAF mutations and DNA methylation in colon cancer tumorigenesis. The frequency of BRAF mutations in the CIMPpositive patients was comparable to those previously described in larger cohort studies [2,8,14,15]
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