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

Colorectal cancer (CRC), the third most common cancer in the Western world, is frequently characterized by aberrant activation of the Wnt signaling pathway.1 In up to 90% of cases this activation is caused by mutations to adenomatous polyposis coli (APC), an important component of the β-catenin destruction complex, which regulates Wnt signaling.2,3  In addition to mutational events, epigenetic changes arise frequently in  CRC, including genome-wide DNA hypomethylation along with promoter hypermethylation to silence tumor suppressor genes.

Another pathway contributing to CRC is DNA mismatch repair, leading to microsatellite instability (MSI) when dysregulated. MSI is frequently caused by promoter hypermethylation of the mismatch repair gene mutL homolog 1 (MLH1), but may also be caused by mutations to MLH1 or other mismatch repair genes.(4) A number of epigenetic events distinguishing the MSI subtype from microsatellite stable (MSS) CRCs have been demonstrated. We have previously shown that methylation of secreted frizzled-related protein 1 (SFRP1), an extracellular Wnt antagonist, is associated with MSS CRC.5 Conversely, methylation of a different Wnt antagonist, dickkopf Wnt signaling pathway inhibitor 1 (DKK1), is associated with MSI-high (MSI-H) CRC.5 Methylation of Wnt5a, which activates non-canonical Wnt signaling, is also associated with MSI. (6)

In order to explore CRC subtype-specific methylation events occurring at Wnt signaling genes, we selected two genes for further analysis, APC and immunoglobulin transcription factor 2 (ITF2). DNA methylation of the tumour suppressor APC has been observed in CRC. However, to what extent APC methylation plays a role in colorectal carcinogenesis is unclear, as a wide range of methylation levels has been reported. (7-10) Conflicting evidence exists regarding APC methylation in MSI CRCs. Some small-scale studies (MSI tumours N ≤ 29) have suggested that APC methylation may be associated with the MSI subtype but others show no significant difference (MSI tumours N ≤ 44).8,9,11-15 The transcription factor ITF2, a Wnt signaling target gene that is overexpressed in some adenomas and CRCs, is shown to incur methylation in several cancer types.(16-19)

In this study we quantified promoter methylation of ITF2 and APC using the semi-quantitative real-time PCR-based MethyLight technique 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 tumour subtypes stratified by MSI status. We also compared methylation of both genes in matched non-neoplastic colorectal mucosa and tumour (n = 47). We established a methylation cutoff of 10% in order to dichotomize values into two groups: unmethylated or methylated. Using this cutoff we determined that ITF2 and APC methylation are significantly associated with tumor versus normal state (both P < 1.0×10-). (6)

In our population from Ontario 26.1% of MSS cases incurred ITF2 methylation compared to 42.4% of MSI-H cases (P = 0.002). Similarly in Newfoundland 29.4% of MSS cases and 56.9% of MSI-H cases were methylated (P = 0.005). Ontario and Newfoundland data was then pooled for further analysis. Among MSI-H cases, ITF2 methylation was associated with MLH1 promoter hypermethylation (P = 6.72×10-4). ITF2 methylation was not found to be associated with age, sex, stage, grade, tumour location, histological type, CIMP status, or survival. APC methylation, although tumor-specific, did not show a significant association with MSI status, age, sex, stage, or other clinicopathological variables, indicating that it is a general tumour-specific CRC biomarker.

Based on its high specificity for CRC, APC methylation may offer usefulness as a marker within a panel of other genes for CRC detection and ITF2 may be useful for detection of MSI-H tumours. Future studies to independently validate these findings are warranted, especially in non-invasive biological sources such as serum or stool. Overall, this study has investigated methylation of the Wnt genes APC and ITF2 in two large cohorts of MSI-H and matched MSS CRC tumours and determined that ITF2 methylation is significantly associated with MSI-H tumours while APC methylation is a tumour- specific event in CRC, which does not differ significantly between MSI-H and MSS subtypes or other clinicopathological variables.

Written by Andrea J. Savio1,2 and Bharati Bapat1,2,3

  1. Lunenfeld-Tanenbaum Research Institute, Sinai Health System
  2. Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto
  3. Department of Pathology, University Health Network 

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