MP39-12 CARBONIC ANHYDRASE X, A NOVEL PUTATIVE TUMOR SUPPRESSOR SILENCED BY PROMOTER CPG METHYLATION, SUPPRESSES GROWTH AND INVASIVENESS OF RENAL CELL CARCINOMA

Abstract

INTRODUCTION AND OBJECTIVES: Metabolic alteration is closely associated with carcinogenesis. Carbonic anhydrase X (CA10) belongs to the carbonic anhydrase family of zinc metalloenzymes which involve in the hypoxic metabolism of tumors. It’s proposed to participate in the central nervous system development. However, the role of CA10 in tumorigenesis remains unclear. Here, our aim is to elucidate its epigenetic regulation and biological functions in renal cell carcinoma (RCC). METHODS: CA10 expression and methylation were detected in RCC cell lines and primary tumors by semi-quantitative RT-PCR, methylation-specific PCR and bisulfite genomic sequencing. Its tumor suppressive functions were further assessed in RCC cell lines 786-O and Caki-2. RESULTS: We reported that CA10 was expressed in immortalized normal epithelial cell line HEK293, but silenced or downregulated in 71% (5/7) of RCC cell lines, which is well correlated with its promoter status. The down-regulation of CA10 associated with the promoter hypermethylation was further evidenced by the re-expression by chemical demethylation treatment. Compared to adjacent noncancer tissues, paired RCC tumors expressed a lower level of CA10. We detected that CA10 was methylated in 100% (7/7) RCC cell lines and 59.7% (85/143) primary tumors, but not in adjacent normal renal tissues. High-resolution bisulite genomic sequencing (BGS) was performed to confirm the methylation. This methylation was associated with tumor diameters (p1⁄40.042). Ectopic expression of CA10 inhibited cell proliferation and migration abilities of RCC cells, through inducing apoptosis and regulating the protein expression of several key cell apoptotic regulators. Western blot analysis showed that CA10 could upregulate the expression pro-apoptosis protein: cleaved Caspase3, Bax and cleaved PARP, and inhibited the expression of pro-proliferation protein Bcl-2, and Bcl-xl. CONCLUSIONS: Thus, Epigenetic inactivation of CA10 is a frequent event in primary RCCs. CA10 appears to be a functional tumor suppressor involving in the RCC carcinogenesis.