Epigenetic inactivation of the tissue inhibitor of metalloproteinase-2 (TIMP-2) gene in human prostate tumors

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Abstract
Gene silencing via CpG island methylation in the promoter region is one of the mechanisms by which tumor suppressor genes are inactivated in human cancers. Previous studies have shown that the tissue inhibitors of metalloproteinases (TIMP)-2 gene, which is an endogenous inhibitor of matrix metalloproteinases involved in cell invasion and tumorigenesis, is downregulated or silenced in a variety of human cancer cell lines. Here, we investigated the mechanism underlying TIMP-2 expression in prostate cancer cell lines and primary prostate tumor samples. We observed a strong correlation between promoter hypermethylation and lost expression of TIMP-2 gene, which was supported by other results demonstrating that promoter demethylation by 5-aza-2′-deoxycytidine and trichostatin A reactivated TIMP-2 and restored its expression in TIMP-2-silenced metastatic prostate cell lines. These results were further substantiated by a chromatin immunoprecipitation assay, showing the preferential binding of MeCP2 to methylated CpG island in TIMP-2-silenced metastatic prostate cell lines. In vitro Matrigel invasion assays showed that re-expression of TIMP-2 after a combined treatment with 5-aza and trichostatin-A in metastatic prostate cells resulted in a significant reduction of tumor cell invasion. Furthermore, CpG methylation of TIMP-2 promoter was also shown in primary prostate tumors that expressed decreased TIMP-2 protein levels. These results suggest that the downregulation of the TIMP-2 gene is associated with promoter methylation and that this may play an important role in prostate cancer progression during the invasive and metastatic stages of the disease.