Selective inhibition of tumor cell associated Vacuolar-ATPase ‘a2’ isoform overcomes cisplatin resistance in ovarian cancer cells

Abstract

Development of resistance to platinum compounds significantly hinders successful ovarian cancer (OVCA) treatment. In tumor cells, dysregulated pH gradient across cell membranes is a key physiological mechanism of metastasis/chemo-resistance. These pH alterations are mediated by aberrant activation of key multi-subunit proton pumps, Vacuolar-ATPases (V-ATPases). In tumor cells, its ‘a2’ isoform (V-ATPase-V0a2) is a component of functional plasma–membrane complex and promotes tumor invasion through tumor-acidification and immuno-modulation. Its involvement in chemo-resistance has not been studied. Here, we show that V-ATPase-V0a2 is over-expressed in acquired-cisplatin resistant OVCA cells (cis-A2780/cis-TOV112D). Of all the ‘a’ subunit isoforms, V-ATPase-V0a2 exhibited an elevated expression on plasma membrane of cisplatin-resistant cells compared to sensitive counterparts. Immuno-histochemistry revealed V-ATPase-V0a2 expression in both low grade (highly drug-resistant) and high grade (highly recurrent) human OVCA tissues indicating its role in a centralized mechanism of tumor resistance. In cisplatin resistant cells, shRNA mediated inhibition of V-ATPase-V0a2 enhanced sensitivity towards both cisplatin and carboplatin. This improved cytotoxicity was mediated by enhanced cisplatin-DNA-adduct formation and suppressed DNA-repair pathway, leading to enhanced apoptosis. Suppression of V0a2 activity strongly reduced cytosolic pH in resistant tumor cells, which is known to enhance platinum-associated DNA-damage. As an indicator of reduced metastasis and chemo-resistance, in contrast to plasma membrane localization, a diffused cytoplasmic localization of acidic vacuoles was observed in V0a2-knockdown resistant cells. Interestingly, pre-treatment with monoclonal V0a2-inhibitory antibody enhanced cisplatin cytotoxicity in resistant cells. Taken together, our findings suggest that the isoform specific inhibition of V-ATPase-V0a2 could serve as a therapeutic strategy for chemo-resistant ovarian carcinoma and improve efficacy of platinum drugs.