Promoter methylation of ITF2, but not APC, is associated with microsatellite instability in two populations of colorectal cancer patients

Andrea J. Savio, Darshana Daftary, Elizabeth Dicks, Daniel D. Buchanan, Patrick S. Parfrey, Joanne P. Young, Daniel Weisenberger, Roger C. Green, Steven Gallinger, John R. McLaughlin, Julia A. Knight, Bharati Bapat

Aberrant Wnt signaling activation occurs commonly in colorectal carcinogenesis, leading to upregulation of many target genes. APC (adenomatous polyposis coli) is an important component of the β-catenin destruction complex, which regulates Wnt signaling, and is often mutated in colorectal cancer (CRC). In addition to mutational events, epigenetic changes arise frequently in CRC, specifically, promoter hypermethylation which silences tumor suppressor genes. APC and the Wnt signaling target gene ITF2 (immunoglobulin transcription factor 2) incur hypermethylation in various cancers, however, methylation-dependent regulation of these genes in CRC has not been studied in large, well-characterized patient cohorts. The microsatellite instability (MSI) subtype of CRC, featuring DNA mismatch repair deficiency and often promoter hypermethylation of MutL homolog 1 (MLH1), has a favorable outcome and is characterized by different chemotherapeutic responses than microsatellite stable (MSS) tumors. Other epigenetic events distinguishing these subtypes have not yet been fully elucidated. Here, we quantify promoter methylation of ITF2 and APC by MethyLight in two case-case studies nested in population-based CRC cohorts from the Ontario Familial Colorectal Cancer Registry (n = 330) and the Newfoundland Familial Colorectal Cancer Registry (n = 102) comparing MSI status groups. ITF2 and APC methylation are significantly associated with tumor versus normal state (both P < 1.0×10(-6)). ITF2 is methylated in 45.8 % of MSI cases and 26.9 % of MSS cases and is significantly associated with MSI in Ontario (P = 0.002) and Newfoundland (P = 0.005) as well as the MSI-associated feature of MLH1 promoter hypermethylation (P = 6.72×10(-4)). APC methylation, although tumor-specific, does not show a significant association with tumor subtype, age, gender, or stage, indicating it is a general tumor-specific CRC biomarker. This study demonstrates, for the first time, MSI-associated ITF2 methylation, and further reveals the subtype-specific epigenetic events modulating Wnt signaling in CRC.