DNA methylation and histone modifications cause silencing of Wnt antagonist gene in human renal cell carcinoma cell lines.

Secreted frizzled-related protein 2 (sFRP2) is a negative modulator of the Wingless-type (Wnt) signaling pathway, and shown to be inactivated in renal cell carcinoma (RCC). However, the molecular mechanism of silencing of sFRP2 is not fully understood. Our study was designed to elucidate the silencing mechanism of sFRP2 in RCC. Expression of sFRP2 was examined in 20 pairs of primary cancers by immunohistochemistry. Kidney cell lines (HK-2, Caki-1, Caki-2, A-498 and ACHN) were analyzed for sFRP2 expression using real-time RT-PCR and Western blotting. The methylation status at 46 CpG sites of the 2 CpG islands in the sFRP2 promoter was characterized by bisulfite DNA sequencing. Histone modifications were assessed by chromatin immunoprecipitation (ChIP) assay using antibodies against AcH3, AcH4, H3K4 and H3K9. sFRP2 was frequently repressed in primary cancers and in RCC cells. The majority of sFRP2 negative cells had a methylated promoter. Meanwhile, sFRP2 expression was repressed by a hypomethylated promoter in Caki-1 cells, and these cells had a repressive histone modification at the promoter. In Caki-1 cells, sFRP2 was reactivated by trichostatin A (TSA). Repressive histone modifications were also observed in RCC cells with hypermethylated promoters, but sFRP2 was reactivated only by 5-aza-2'-deoxycytidine (DAC) and not by TSA. However, the activation of the silenced sFRP2 gene could be achieved in all cells using a combination of DAC and TSA. This is the first report indicating that aberrant DNA methylation and histone modifications work together to silence the sFRP2 gene in RCC cells.