Abstract 686: Regulation of intracellular sequestration of sunitinib by cystine transporter xCT in renal cancer
Authors: Chintala Sreenivasulu , Nguyen Hillary , Ramakrishnan Swathi , Ku Sheng-Yu , Ciamporcero Eric , Miles Kiersten M. , Pili Roberto 
Published: May 2015

Abstract
Background: Acquired and/or intrinsic resistance to sunitinib, a multikinase inhibitor, is one of the major obstacles for the treatment of renal cell carcinoma (RCC). Recently, lysosomal sequestration of sunitinib has been shown as one of the potential mechanisms of resistance in renal cancer. The current study was initiated to understand the role of the Cystine Transporter xCT (encoded product of Solute Carrier family 7A member 11, SLC7A11 gene), a lysosome regulatory gene, on intracellular sequestration of sunitinib and to evaluate the cytotoxic effects of the xCT inhibitor sulfasalazine alone and in combination with sunitinib on RCC cells. Methods: Intracellular sequestration of sunitinib was evaluated using the fluorescent microscopy technique by determining the autofluorescence of sunitinib. Cytotoxic effects of sulfasalazine alone and in combination with sunitinib were assessed by sulforhodamine B assay. Quantitative RT-PCR analysis and Western blot analysis were utilized to determine the expression of xCT in normal renal tubular HK2 cells, human umbilical vein endothelial cells (HUVEC), and RCC 786-O cells, and to determine the association of sunitinib sequestration and xCT expression. Expression of xCT interacting partner CD44s and its variant forms CD44v6 and CD44v8, which influence the expression of xCT, was determined in 786-O and HK2 cells. Additionally, we utilized The Cancer Genome Atlas (TCGA) data to determine the expression of xCT in ccRCC patients and evaluated the significance of its expression and correlation of patient survival. Results: Our results revealed higher intracellular sequestration of sunitinib in RCC 786-O cells compared to HK2 and HUVEC cells. Furthermore, we found higher expression of xCT in 786-O cells compared to HK2 and HUVEC cells, which was associated with high intracellular sequestration of sunitinib. The pharmacological inhibition of xCT by sulfasalazine revealed the reversal of intracellular sequestration of sunitinib in 786-O cells. The SRB assay showed the enhanced cytotoxic effects of sunitinib in combination with sulfasalazine, which indicates that, the reversal of intracellular sequestration of sunitinib may led to the enhanced cytotoxic effects. Moreover, there was a significant decrease of xCT stabilizing isoform CD44v6 mRNA in 786-O cells compared to HK2 and no change was observed in CD44s and CD44v8. Bioinformatics analysis using cBioPortal Cancer Genomics from MSKCC data of TCGA revealed the significant up regulation of xCT in ccRCC patients correlated with poor prognosis. Conclusions: Our results indicate that lysosome regulatory protein xCT inhibition by sulfasalazine leads to the reversal of intracellular sequestration of sunitinib and enhances the efficacy of sunitinib in renal cancer. These results provide the rationale for the preclinical and clinical testing of sulfasalazine in combination with sunitinib in the treatment of ccRCC.
Citation Format: Sreenivasulu Chintala, Hillary Nguyen, Swathi Ramakrishnan, Sheng-Yu Ku, Eric Ciamporcero, Kiersten M. Miles, Roberto Pili. Regulation of intracellular sequestration of sunitinib by cystine transporter xCT in renal cancer. [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 686. doi:10.1158/1538-7445.AM2014-686