Methylation of CpG islands (CGIs) in promoter regions is an important mechanism inactivating tumor-suppressor genes in various human cancers. To identify novel tumor-suppressor genes in malignant melanomas, oligonucleotide microarray analysis was performed before and after treatment of three human melanoma cell lines, HMV-1, MeWo and WM-115, with a demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC). With a threshold of 16-fold, 79 known genes with CGIs were found to be up-regulated. Among them, seven genes were methylated in one or more of eight melanoma cell lines, expressed in cultured normal human epidermal melanocyte (HEM), and not expressed in melanoma cell lines with methylation. Among the seven genes, detailed analysis was performed on the tissue factor pathway inhibitor-2 gene (TFPI-2) because its growth-suppressive function in malignant melanomas was known. In one of 12 melanoma cell lines, the TFPI-2 promoter CGI was shown to be completely methylated by bisulfite sequencing, and its expression was completely lost by quantitative RT-PCR. In 5 of 37 (13.5 %) surgical melanoma specimens, the TFPI-2 promoter CGI was methylated by methylation-specific PCR. The five specimens with the methylation lacked immunoreactivity for TFPI-2. Importantly, methylation of the TFPI-2 promoter CGI was observed exclusively in metastatic specimens, not primary specimens (P = 0.009). It was demonstrated for the first time in this study that TFPI-2 is silenced by methylation of its promoter CGI in malignant melanomas, and it was indicated that TFPI-2 silencing is involved in their metastasis.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA