Abstract PR08: HNF1β confers resistance to oxidative stress of ovarian clear cell carcinoma

Abstract

Abstract Ovarian clear cell carcinoma (OCCC) is a histologic subtype of epithelial ovarian cancer (EOC) with unique characteristics including chemoresistance and carcinogenic origins from endometriotic cysts. We previously reported that a high concentration of free iron in endometriotic cysts causes oxidative stress. (Yamaguchi K, Clin Can Res, 2008). We also reported that resistance to oxidative stress is an inherent characteristic of OCCC, and that HNF1β pathway genes are upregulated exclusively in OCCC relative to the other EOCs (Yamaguchi K, Oncogene, 2010). Recent findings indicate that cancer cell-specific metabolism (the Warburg effect) confers resistance to oxidative stress by suppressing the mitochondrial TCA cycle, thus leading to decreased production of intracellular reactive oxygen species (ROS). The aim of this study was to verify our hypothesis that HNF1β mediates resistance to oxidative stress in OCCC through the cancer-specific metabolic process. Expression of HNF1β in RMGII and JHOC5 OCCC cell lines was repressed using two different shRNAs, sh1-HNF1β and sh2-HNF1β. HNF1β knockdown was associated with increased sensitivity (reduced IC50) to ferric nitrilotriacetate (Fe-NTA; a Fe-mediated inducer of oxidative stress), and increased intracellular ROS (p<0.05 for both cell lines). Comprehensive metabolomic analyses using capillary electrophoresis time-of-flight mass spectrometry was used in RMGII cells to compare HNF1β knockdown to controls. Knockdown of HNF1β was associated with decreased intracellular lactic acid (p<0.05), increased pyruvic acid (p<0.01) and increased citric acid (p<0.01), indicating that HNF1β increases anaerobic glycolysis while suppressing the TCA cycle, consistent with the Warburg effect. Glutathione, another major antioxidant molecule, was significantly decreased in the HFN1β knockdown cells (p<0.0005). A quantitative intracellular glutathione assay confirmed that intracellular glutathione was decreased by HNF1β knockdown in both cell lines (p<0.05, respectively). We analyzed gene expression microarray data (Affymetrix U133 plus 2) for sh1-HNF1β-RMGII (n=5), sh2-HNF1β-RMGII (n=5) and sh-control-RMGII (n=10) cells. Of the genes encoding key molecules involved in glutathione biosynthesis such as cystine transporters (SLC3A1 and SLC7A9) and biosynthesis enzymes (GSS, GCLC and GCLM), only SLC3A1 transcription was significantly downregulated by HNF1β knockdown (p<0.0001). Western blots showed that SLC3A1 protein expression was also downregulated by knockdown of HNF1β in both RMGII and JHOC5 cells. Furthermore, from analysis of microarray dataset GSE39204, HNF1β and SLC3A1 expression are positively correlated in clinical ovarian cancer specimens (p<0.0001). In summary, we found HNF1β, exclusively expressed in OCCC, confers resistance to Fe-mediated oxidative stress which is abundant in endometriotic cysts, the precursor lesion of OCCC. This may be mechanistically driven by decreased TCA cycle activity combined with increased intracellular glutathione through increased expression of cystine transporter SLC3A1. Further investigation of this mechanism may lead to development of new therapeutic modalities against OCCC. This abstract is also presented as Poster B37. Citation Format: Yasuaki Amano, Ken Yamaguchi, Masaki Mandai, Noriomi Matsumura, Junzo Hamanishi, Tsukasa Baba, Koji Yamanoi, Susan K. Murphy, Ikuo Konishi. HNF1β confers resistance to oxidative stress of ovarian clear cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR08.