Abstract 17: Tumor-suppressive function of SLC5A8 in renal cancer cells

Abstract

Abstract SLC5A8, a Na+-coupled high-affinity transporter for monocarboxylates, and the closely related SLC5A12, a Na+-coupled low-affinity transporter for monocarboxylates, are both highly expressed in kidney, where they mediate the reuptake of lactate and pyruvate from the glomerular filtrate. Recent studies have shown that SLC5A8 is a tumor-suppressor that is silenced in many cancers by promoter hypermethylation. We investigated if SLC5A8 serves as a tumor suppressor in renal cancer cells (RCC). Comparison of the expression of SLC5A8 at the mRNA and protein levels in two RCC (A498 & 786-0) and two normal kidney cell lines (HK-2 & HEK-293) revealed that the expression of SLC5A8 was significantly lower in RCC. Analysis of methylation status of the CpG island in the regulatory region of SLC5A8 showed that the extent of methylation was significantly higher in the two RCC compared to the two normal cells. Culturing A498 cells in the presence of 5-aza-deoxycytidine resulted in re-expression of SLC5A8, confirming that the silencing of the gene is due to DNA methylation. Ectopic expression of SLC5A8 in A498 cells caused apoptosis when cultured in the presence of HDAC inhibitors butyrate and pyruvate. These data show that SLC5A8-mediated concentrative entry of HDAC inhibitors causes cell death in RCC. Interestingly, normal proximal tubular cells, which express SLC5A8 robustly and are exposed to pyruvate, do not undergo apoptosis; HDAC inhibitor-mediated apoptosis is specific to cancer cells. To investigate if HDAC isoforms play a role in this differential sensitivity of normal and RCC to pyruvate, we compared the expression of various HDAC isoforms in HK-2 and A498 cells by RT-PCR. The results indicated that the expression of HDAC1 and HDAC3 was higher in RCC compared to normal cells. The total HDAC enzyme activity was ∼2-3-fold higher in RCC compared to normal cells. If intracellular pyruvate is detrimental to the survival of tumor cells and the tumor cells silence SLC5A8 to prevent entry of extracellular pyruvate into cells, the levels of pyruvate inside tumor cells should be lower in RCC compared to normal cells. To confirm this, we measured pyruvate and lactate levels in normal cells and RCC and found that pyruvate levels were ∼4-fold lower while lactate was ∼2-fold higher in RCC cells compared to normal cells. Finally, to conclusively demonstrate the tumor-suppressive role for SLC5A8 in RCC, we performed mouse xenograft studies using control A498 & 786-0 cells, which do not express SLC5A8 constitutively, and A498 & 786-0 cells, which were made to express SLC5A8 by lentiviral mediated transfection of SLC5A8 cDNA. The growth of the tumor was significantly slower with SLC5A8-expressing cells than with SLC5A8-negative parent cells. These studies demonstrate that SLC5A8 is indeed a tumor suppressor in kidney, suggesting that pharmacological induction of the transporter expression in renal cancer might be a novel strategy for treatment of renal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 17. doi:1538-7445.AM2012-17