Discovery of the methylation of the metastasis suppressor gene KiSS-1 in bladder cancer

Abstract

5023 Background: In an attempt to uncover the mechanisms by which the metastasis suppressor gene, KiSS1, is lost in bladder progression, we tested the hypothesis of epigenetic silencing. An enriched 5’-CpG islands was identified around the transcription start site of KiSS1, supporting its susceptibility to be epigenetically modified by hypermethylation. To the best of our knowledge, KiSS1 had not been reported to be epigenetically altered in bladder cancer or any other tumor type. In this report, the impact and clinical relevance of KiSS1 methylation along bladder cancer progression was evaluated using in vitro strategies as well as on tissue specimens. Methods: The methylation status of KiSS1 was analyzed by two polymerase chain reaction (PCR) analysis strategies of bisulfite-modified genomic DNA. First, by genomic sequencing of both strands of KiSS1 promoter and second, by methylated specific PCR (MS-PCR). The epigenetic silencing of KiSS1 by hypermethylation was tested in bladder cancer cells (n = 12) before and after azacytidine treatment. The methylation status of KiSS1 promoter was then evaluated by MS-PCR in a large series of 524 bladder tumors. KiSS1 transcript expression were analyzed by RTPCR on bladder tumors (n = 177) for which KiSS1 methylation was assessed by MS-PCR. Results: KiSS1 hypermethylation was frequent in the bladder cancer cells analyzed and associated with low gene expression by RT-PCR. KiSS-1 expression was increased in vitro by the demethylating agent azacytidine in methylated cells. KiSS1 was found to be frequently methylated in a large series of 524 bladder tumors (68.1%). KiSS1 methylation was significantly associated with tumor stage (p < 0.001) and tumor grade (p < 0.001). Interestingly, a significant association was found between transcript levels by quantitative RT-PCR in bladder tumors and increasing tumor stage (p < 0.001). Moreover, the presence of KiSS1 methylation was associated with low transcript expression (p < 0.001). Conclusions: KiSS1 was identified to be epigenetically modified in bladder cancer. The association of KiSS1 methylation with cancer progression suggests the utility of incorporating KiSS1 methylation assessment in the clinical management of patients affected by uroepithelial neoplasias. No significant financial relationships to disclose.