Epigenetic regulation of the Wnt-signaling pathway in CIMP-H BRAFV600E mCRC.

Abstract

110 Background: BRAFV600E mutation identifies mCRC patients with poor prognosis with only little benefit from standard therapy. Analysis from TCGA revealed that 89% of BRAFV600E CRC tumors were associated with a high CpG island methylator phenotype (CIMP-H), which may result in epigenetic silencing of tumor suppressor genes, while only 29% of BRAF wild-type tumors are CIMP-H. In this study, we define key pathways regulated by global DNA hypermethylation in the context of BRAFV600E mutation. Methods: We analyzed the TCGA Illumina 450k array methylation datasets and RNA-sequencing datasets for 97 CIMP-H CRC tumors (27 BRAFV600E; 69 BRAF WT) identified by five universal CIMP annotations ( p14, p16, MLH1, MINT1, MINT2, MINT31). We defined differential methylation profile according to BRAF mutation status and calculated Spearman correlation between methylation and gene expression to identify CIMP-H BRAF-associated genes. Next, pathways enriched with CIMP-H BRAFV600E tumors were defined using PANTHER pathway analysis. Additionally, β-catenin IHC were conducted on 145 MD Anderson CRC patient samples and compared by CIMP and BRAF status. Results: BRAF mutation is associated with lower rates of APC mutation as has previously been shown (32%, 82%). We identified 6,097 differentially methylated probes by BRAF mutation status (FDR = 10-4), and as expected, our data suggests a higher methylation profile in BRAFV600E mutated tumors compared to BRAF WT. Intriguingly, CIMP-H BRAF-associated genes showed enrichment in the Wnt-signaling and cadherin signaling pathways ( p< 0.0001 (FDR < 0.0001)). Despite the epigenetic Wnt-signaling, nuclear β-catenin expression (as a measure of Wnt activity) in CIMP-H and BRAF tumors remains lower than for non-CIMP, and BRAF wild-type ( p= 0.0003 for comparison of CIMP). Conclusions: Genes under methylation regulation in the BRAF-mutant context showed enrichment in Wnt-signaling pathway. Since BRAFV600E CRC tumors have a low association with APC mutation, this data suggests role of epigenetic regulation of the Wnt-pathway activation. However, as measured by nuclear β-catenin, Wnt activation in these tumors is not as high as traditional APC-mutated CRC tumors. CIMP-H tumors with BRAFV600E mutation is a unique subset of CRC tumor that have Wnt-pathway activation regulated by epigenetic modifications more than a β-catenin activation.