WNT INHIBITORY FACTOR 1 IS INACTIVATED BY HYPERMETHYLATION AND FUNCTIONS AS A TUMOR SUPPRESSOR IN KIDNEY CANCER CELLS

INTRODUCTION AND OBJECTIVE: Aberrant activation of the Wingless-type (Wnt) pathway plays a significant role in the carcinogenesis of several human cancers. Wnt inhibitory factor-1 (WIF-1) has been identified as one of the secreted antagonists that binds Wnt protein. The molecular function of WIF-1 has never been examined in kidney cancer. We hypothesize that WIF-1 functions as a tumor suppressor in kidney cancer. METHODS: WIF-1 expression levels were examined in 20 normal and 42 cancer regions of clinical samples using immunohistochemistry. WIF-1 mRNA expression was also checked by TaqMan real time RT-PCR in kidney cancer cell lines (Caki2, A498 and ACHN). Bisulfite sequencing was used to check the methylation status of the WIF-1 gene promoter. We also established stable cell line (A498) transfected with either empty vector (pcDNA3.1+) or the full-length WIF-1 gene. Cell proliferation assay and colony formation assay were carried out on the transfected A498 cell line. Microarray analysis was done on empty vector and WIF-1 transfected cells to determine which genes were differentially regulated by WIF-1 transfection. RESULTS: Immunohistochemistry and real time RT-PCR revealed that WIF-1 was significantly down regulated in kidney cancer samples and cell lines, respectively. Bisulfite sequencing of the WIF1 promoter region showed it to be frequently methylated. Significant inhibition of cell growth (p<0.01) and of colony formation efficiencies (p<0.01) in WIF-1 transfected cells compared to empty vector transfected cells were observed in cell proliferation assay and colony formation assay, respectively. Twenty one genes were found to be upor downregulated by WIF-1 transfection in microarray analysis and some genes, including Tumor Necrosis Factor (TNF) and Metastasis Suppressor 1 (MTSS1), were validated to be up-regulated in WIF-1 transfected cells by real time RT-PCR. CONCLUSIONS: Our current studies have demonstrated for the first time that the WIF-1 gene is frequently down-regulated by promoter hypermethylation and suppresses tumor growth activity of kidney cancer cells. These results suggest that WIF-1 functions as a potential tumor suppressor gene in kidney cancer cells.