Promoter hypermethylation leading to inactivation of MED1 DNA repair gene is present in human colorectal and ovarian cancer

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Introduction: The MED1 DNA mismatch repair gene acts as a tumor suppressor gene of the “caretaker” type. MED1 counteracts mutagenesis caused by spontaneous deamination of 5-methylcytosine and cytosine and functions as a guardian of genomic fidelity at CpG sites. The MED1 gene is frequently mutated in human colorectal and extracolonic carcinomas exhibiting microsatellite instability. Through analogy with other mismatch repair genes such as MLH1, we hypothesized that hypermethylation of the MED-1 gene promoter leads to decreased MED1 protein expression and thus an impaired mismatch repair system during colorectal and ovarian cancer tumorigenesis. Materials & Methods: Cell lines: Methylation status of the MED1 promoter was investigated in a panel of ovarian and colorectal cancer cell lines. The MED1 promoter region was sequenced following bisulfite treatment. Methylation frequency of the involved CpG islands was quantified by methylation-specific PCR of subclones. The effects of methylation on MED1 protein expression were evaluated by immunoblot following treatment of cancer cell lines with demethylating agents (5-azacytidine and trichostatin). Human cancer: The clinical relevance of MED1 promoter methylation was further investigated in colorectal cancer specimens from 21 patients (stages I-IV). Frequency of MED-1 methylation was analyzed by methylation specific PCR comparing matched normal mucosa, adenomas, and carcinoma. MED1 protein expression was evaluated by western blot. Results: Sequence analysis identified three CpG sites in the MED1 promoter that are frequently and preferentially methylated (≥ 50%) in ovarian cancer cell lines with low/reduced MED1 expression. Treatment of colorectal cancer cell lines with de-methylating agents reversed MED1 methylation and restored protein expression. The same three CpG islands identified in the ovarian cancer cell lines were found to be more frequently methylated in colorectal cancer specimens when compared to matched normal colon mucosa. Expression of MED1 protein was consistently decreased in colorectal polyps and cancers when compared to normal colon mucosa. Conclusions:Our findings indicate that the MED1 gene is inactivated by promoter hypermethylation in ovarian and colorectal cancer. Increased promoter methylation leads to a decrease in MED1 protein expression during the adenoma-carcinoma sequence. This suggests that MED1 promoter methylation is an early event in colorectal tumorigenesis. Inactivation of MED1 may endow cancer cells with a mutator phenotype at CpG sequences. MED1 promoter methylation may have prognostic and therapeutic implications for ovarian and colorectal cancer patients.