Abstract 4061: Evidence for hdac6 and er-α association in a subset of clear cell renal cell carcinoma
Authors: Ramakrishnan Swathi , Ku Sheng-Yu , Swetzig Wendy , Conroy Dylan , Shen Li , Chintala Sreenivasulu , Sotomayor Paula , Miles Kiersten M. , Adelaiye Remi , Ciamporcero Eric , Orillion Ashley , Ellis Leigh , Das Gokul , Pili Roberto 
Published: May 2015

Abstract
Introduction: Histone deacetylases are overexpressed in several tumors including prostate, breast and clear cell renal cell carcinoma (ccRCC). Our group has previously reported that class II HDACs, HDAC4 and HDAC6 regulate HIF-α stability in ccRCC cell lines. Interestingly, HDAC6 overexpression in ER-α positive breast cancer has been shown to correlate with overall and cancer specific survival in response to tamoxifen treatment. In addition, HDAC6 increases cell motility by deacetylating α-tubulin, and HDAC6 interaction with ER-α on the cell membrane increases its deacetylating activity. The objective of our study was to assess whether HDAC6 associates with ER-α in a subset of ccRCC and whether this association can be targeted therapeutically. Methods: Radical nephrectomy tumor samples (n=14) with matched adjacent non tumor tissues were collected and analyzed for HDAC6 expression by Western blot analysis. HDAC6 expression was also assessed in C2, C2VHL and 786-O ccRCC cell lines by Western blot and immunofluorescence analysis. HDAC6 and ER-α colocalization was examined by immunoprecipitation and immunofluorescence. HDAC 6 was overexpressed in cell lines and investigated for cell motility by scratch assay. Cell lines were also treated with hydroxy tamoxifen in short term (4 hours) and long term (24, 48 and 72 hours) culture experiments for evaluation of effects on acetylated α-tubulin and cell proliferation, respectively. Results: Analysis of matched patient tumor samples revealed that a subset of ccRCC had higher HDAC6 expression as compared to the adjacent non tumor tissue. HDAC6 and ER-α examined in ccRCC tumors (n=44) by immunofluorescence showed overexpression in 10% of tumor samples. Immunoprecipitation of HDAC 6 in ccRCC cell lines showed that ER-α is present in the same complex as HDAC 6 as confirmed also by fluorescence microscopy. HDAC6 overexpression in ccRCC cell lines increased cell motility, although overexpression did not affect cell proliferation. Cells treated for short term experiments with hydroxy tamoxifen showed an increase in acetylated α-tubulin when examined by immunofluorescence. Upon long term hydroxy tamoxifen treatment in regular DMEM medium with serum, ccRCC cell proliferation was affected at high concentrations (10-20µM), similar to MCF 7 cells treated under similar conditions. Conclusions: HDAC 6 and ER-α are overexpressed in a subset of ccRCC. HDAC6 overexpression affects cell motility but not proliferation. HDAC6 and ER-α are present in the same immunocomplex and this association may be targeted with therapeutic interventions. Ongoing studies are testing concomitant, either genetic or pharmacological, inhibition of both HDAC6 and ER-α in ccRCC and will provide the rationale for novel targeted therapies for a selected group of patients with ccRCC.
Citation Format: Swathi Ramakrishnan, Sheng-Yu Ku, Wendy Swetzig, Dylan Conroy, Li Shen, Sreenivasulu Chintala, Paula Sotomayor, Kiersten M. Miles, Remi Adelaiye, Eric Ciamporcero, Ashley Orillion, Leigh Ellis, Gokul Das, Roberto Pili. Evidence for hdac6 and er-α association in a subset of clear cell renal cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4061. doi:10.1158/1538-7445.AM2014-4061