Frequent hypermethylation of 5′ flanking region of TIMP‐2 gene in cervical cancer

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Abstract
Tissue inhibitor of metalloproteinase-2 (TIMP-2) is an endogenous inhibitor of matrix metalloproteinases (MMPs). This multifunctional protein regulates activities of MMPs and possesses growth promoting effect in cell culture, anti-tumoral, anti-apoptotic and anti-angiogenic effects in animal model systems in vivo. It has been shown that this gene is downregulated in cervical carcinomas. The mechanism of inhibition of TIMP-2 expression remains obscure. We have examined whether aberrant DNA methylation of the 5′CpG island of the TIMP-2 gene is involved in its inhibition during cervical carcinogenesis. Bisulfite-modified DNA sequencing and MSP assay showed aberrant methylation of TIMP-2 5′-CpG island in 17 of 36 (47%) invasive cervical carcinomas and in 2 of 3 cervical cancer cell lines. TIMP-2 gene was mostly unmethylated in the morphologically normal tissues adjacent to the tumors, whereas methylated alleles of this gene were found in 4 samples. Each tumor and each cell line DNA was characterized by unique methylation pattern, however a discrete region of TIMP-2 CpG island upstream to the transcription start site was densely methylated in all hypermethylated DNA samples examined. The expression of TIMP-2 mRNA can be restored in the cell lines, in which this discrete region of TIMP-2 CpG island is methylated, by treatment with demethylating agents, 5-azacytidine and 5-aza-2′-deoxycytidine. Our data suggest that the aberrant methylation of TIMP-2 favors the development of primary cervical tumors. We describe for the first time the aberrant hypermethylation of TIMP-2 gene in human cancer.