Abstract 4682: Reversal of cisplatin resistance in human ovarian cancer through autophagy inhibition and caspase 1-α activation: Role of tumor associated Vacuolar-ATPase

Abstract

Abstract The development of resistance to cisplatin drugs significantly hinders successful ovarian cancer treatment. An emerging centralized mechanism of chemo-resistance is a dysregulation in tumor cell pH (alkaline intracellular, acidic extracellular). Vacuolar-ATPase (V-ATPase) is a key proton pump that alters the pH and mediates metastasis/chemo-resistance in tumor cells. The major pH sensing unit of the V-ATPase complex is the ‘a2’ isoform(V0a2) and has been previously shown to promote metastasis in ovarian cancer cells. Here, we show that V0a2 is over-expressed in acquired cisplatin resistant ovarian tumor cells (cis-A2780 and cis-TOV112D) at both mRNA and protein levels. The shRNA mediated inhibition of V0a2 activity in cisplatin resistant cells (sh-V0a2-cis) sensitized the cells to cisplatin with a 3 fold reduction in inhibitory concentration 50% [IC50] (sh-V0a2-cis, IC50 = 11.47 ± 3.02 μM, p<0.05) compared to control resistant cells [IC50 = 37.75 ± 6.62 μM] as determined by Alamar blue cell cytotoxicity assay. Suppression of V0a2 activity strongly reduced the cytosolic pH in the resistant tumor cells (cis-A2780- pH = 7.19 ± 0.03; sh-V0a2-cis- pH = 6.87 ± 0.04; cisplatin sensitive cells- pH = 6.96± 0.01). This cytosolic acidification significantly enhanced DNA damage by cisplatin-DNA adduct formation as revealed by FACS and immuno-fluorescence analysis (p<0.05). To further define the cisplatin mediated multi-factorial responses in resistant cells upon V0a2 inhibition, we performed a PCR array for cell death pathway related genes. Several pro-apoptotic genes including caspase 3, caspase 9, Apaf-1, FASL, CD40,CD40L, TNF, TNFR1 were up-regulated (p<0.05). Notably, caspase-1α was significantly up-regulated upon cisplatin treatment in sh-V0a2-cis compared to control resistant cells (p<0.05). Caspase-1α is known to induce apoptosis of neurons and macrophages and is recently indicated to be pro-apoptotic in ovarian cancer cells. Additionally, several autophagy related genes such as LC3A, IGFR, ESR1, IRGM were also found up-regulated upon V0a2 inhibition. Impaired autophagy in sh-V0a2-cis was further confirmed at protein level by FACS analysis using autophagy detection kit. Taken together, our findings suggest that the isoform specific inhibition of V-ATPase-V0a2 inhibits autophagy and activates caspase-1α upon cisplatin treatment in resistant ovarian cancer cells. V0a2 inhibition can therefore serve as a therapeutic strategy to overcome cisplatin resistance and in improving the treatment efficacy in chemo-resistant ovarian carcinoma. Citation Format: Arpita Kulshrestha, Gajendra K. Katara, Sahithi Pamarthy, Alice Gilman-Sachs, Kenneth D. Beaman. Reversal of cisplatin resistance in human ovarian cancer through autophagy inhibition and caspase 1-α activation: Role of tumor associated Vacuolar-ATPase. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4682.