Increased MLH1, MGMT, and p16INK4a methylation levels in colon mucosa potentially useful as early risk marker of colon cancer

Abstract

The genes MutL Homolog 1 (MLH1), O6-methylguanine-DNA methyltransferase (MGMT), and cyclin-dependent kinase inhibitor p16INK4a are commonly downregulated by hypermethylation in colorectal cancer. Long interspersed nucleotide element 1 (LINE-1) can be used as marker for global hypomethylation. This study compared MLH1, MGMT, p16INK4a, and LINE-1 methylation with gene expression in colon tumors, matched non-cancerous mucosa, and control mucosa to identify signs of premalignancy. Tissues were obtained from 20 colon cancer patients and 40 controls. CpG site methylation was quantified by pyrosequencing, expression by qPCR, and MSI/KRAS status by fragment analysis and droplet digital PCR. MLH1, MGMT, and p16INK4a methylation was increasingly higher in control mucosa, non-cancerous mucosa, and tumors. MLH1 expression was lower in tumors compared to non-cancerous mucosa but higher compared to control mucosa. Tumoral LINE-1 methylation correlated negatively with MLH1 (r = −0.51, p =.021) and p16INK4a (r = −0.55, p =.012) methylation, but positively (r = 0.74, p =.0002) with MLH1 expression. A p16INK4a SNP (rs3814960 C>T) was associated with methylation, expression, and MSI/KRAS status. Aberrant methylation of tumor suppressor genes in colon mucosa could be an early cancer risk marker. Control mucosa is a more reliable reference than non-cancerous mucosa when identifying premalignant changes. Extended studies will evaluate the possible association between rs3814960 and cancer susceptibility. Trial registration: NCT03072641.