Abstract 5426: Tumor cell surface-associated vacuolar-ATPase ‘a2′ isoform promotes invasion by regulating matrix metalloproteinase activity in ovarian cancer
Authors: Kulshrestha Arpita , Katara Gajendra K. , Pamarthy Sahithi , Ibrahim Safaa A. , Sachs Alice Gilman , Beaman Kenneth D. 
Published: August 2015

In tumors, an acidic pH is an important feature of tumor microenvironment and is a major determinant of tumor metastasis. Efforts are required to identify novel molecular targets that alter this acidic microenvironment to control tumor spread. The Vacuolar-H + −ATPases (V-ATPases) are multi-subunit enzyme complexes that acidify the extracellular space, and activate matrix degrading proteases, thereby promoting tumor invasion. Identifying specific V-ATPase subunit isoforms that are selectively expressed on tumor cells will provide biomarkers and novel targets for V-ATPase inhibition. The ‘a’ subunit of V-ATPase V0 domain is the major pH sensing unit and exists in four different isoforms. The present study describes an elevated expression of V-ATPase ‘a’ subunit isoforms in ovarian cancer. Our findings indicate that among all the ‘a’ subunit isoforms, ‘a2′ is most prominently expressed on the ovarian tumor cell surface. To understand the clinical relevance, the immuno-histochemistry in human OVCA tissues revealed over-expression of V-ATPase-V0a2 compared to control tissues (normal ovary and benign tumors). V-ATPase-V0a2 was upto 4 fold up-regulated at the mRNA level in human OVCA cell lines (A2780, SKOV-3 and TOV-112D) compared to normal ovary epithelia using quantitative reverse transcription-PCR. This up-regulation was also observed at the protein level by western blot and flow cytometry analysis. Immunofluorescence analysis showed selective V0a2 distribution only on tumor cell surface and not on normal ovarian epithelia. Confocal microscopy analysis was performed for understanding the sub-cellular distribution of V-ATPase-V0a2, which confirmed its localization on plasma-membrane and early endosomes. Interestingly, the V0a2 isoform exhibited co-association with cortactin, an F-actin stabilizing protein at leading edges of cancer cells, that are the sites of focal protease release. To understand the possible effect of V0a2 on protease activation, an in vitro targeted inhibition of V-ATPase-V0a2 in OVCA cells using monoclonal anti-V0a2 antibody assay was used. The antibody reduced the matrix metalloproteinase (MMP-9 & MMP-2) activity by 2 fold. Measurement of invasion using an in vitro wound healing assay revealed that inhibition of V-ATPase-V0a2 reduced the invasion capacity and therefore wound healing, in SKOV-3 cells. In conclusion, V-ATPase-V0a2 isoform is highly expressed on ovarian tumor cell surface and is associated with invasion assembly related proteins, therefore playing a critical role in tumor invasion by modulating the activity of matrix-degrading proteases. Our data suggests that the ‘V-ATPase-V0a2 isoform is a potential biomarker in OVCA and isoform specific inhibition of V-ATPase-V0a2 may aid in better clinical outcome of OVCA.

Citation Format: Arpita Kulshrestha, Gajendra K. Katara, Sahithi Pamarthy, Safaa A. Ibrahim, Alice Gilman Sachs, Kenneth D. Beaman. Tumor cell surface-associated vacuolar-ATPase ‘a2′ isoform promotes invasion by regulating matrix metalloproteinase activity in ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5426. doi:10.1158/1538-7445.AM2015-5426