Abstract 2987: VHL dependent and independent regulation of HDAC expression and activity in renal cell carcinomas.

Abstract

Abstract Background: Histone modifications include addition and/or deletion of acetyl and methyl groups to lysine, serine and arginine residues of the histone tails leading to transcriptional inactivation or activation. The enzymes governing these modifications are histone acetyl transferases (HATs), histone deacetylases (HDACs), histone methylases (HMTs) and histone demethylases (HDMTs). HDAC inhibitors exert their inhibitory effects on tumor cell population through cell cycle inhibition, apoptosis, decreased cell proliferation as well as their induction of tumor suppressor genes (for eg: p53). Clear cell renal cell carcinoma (ccRCC) represents the dominant major subtype of RCC malignancy with frequent mutations, deletions or methylation in the tumor suppressor gene Von-Hippel Lindau (VHL). Class I HDACs have been reported to be overexpressed in ccRCCs and VHL protein is present in the same complex containing class I HDACs. The present study was aimed at defining the role of VHL in the regulation of HDAC expression and activity. Methods: Approximately 15 ccRCC tumors and matched normal kidney samples from nephrectomies were collected. Western blot analysis of UMRC-2 (VHL−/− RCC cell line) and UMRC2 (wt-VHL RCC cell line) were carried out under conditions of normoxia, hypoxia and in the presence of proteasomal inhibitor MG-132. Tumor cells (1 x 107) were injected subcutaneously in SCID mice to establish tumor xenografts. Results: Our preliminary studies in matched clinical tumor samples show overexpression of specific Class I (2 fold increase; p<0.05) and II HDACs (5 fold increase for HDAC 4). Conversely, HDAC 6 expression (2 fold decrease, p<0.01) and activity was down regulated in tumor samples. To investigate the role of VHL in RCCs, UMRC2 and UMRC2-VHL were utilized for HDAC expression and activity. Western blot analysis indicated HDAC 1 down regulation (1.5 fold) and HDAC 6 overexpression (1.5 fold) in the UMRC2 cells as compared to the UMRC2-VHL cells. In contrast, HDAC 6 activity, measured by expression of its target protein acetylated α-tubulin, was lower in the UMRC2 cells. Under hypoxic conditions both HDAC 1 and HDAC 6 were induced in the UMRC2-VHL cells suggesting VHL dependent hypoxic regulation of these HDACs. MG-132 induced HDAC 1 and 6 in UMRC2-VHL; whereas proteasome inhibitor down regulated HDAC 1 and 6 in UMRC2 cells suggesting a VHL independent mechanism of HDAC regulation. In vivo studies of tumor xenografts generated from the cell lines demonstrated hypoxic induction of HDAC 1 (5 fold increase) and HDAC 6 in UMRC2 when compared to the UMRC2-VHL tumors. Conclusion: Our results suggest that specific HDACs are differentially regulated by VHL dependent (via hypoxia regulation) and independent mechanisms, providing a rationale for testing selective HDAC inhibitors in RCCs. Citation Format: Swathi Ramakrishnan, Paula Sotomayor, Leigh Ellis, Sreenivasulu Chintala, Roberto Pili. VHL dependent and independent regulation of HDAC expression and activity in renal cell carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2987. doi:10.1158/1538-7445.AM2013-2987