Abstract 513: Breast cancers are notched up with a proton pump at their leading edge.

Abstract

Abstract The H+ vacuolar ATPase is a multimeric complex, which acidifies intracellular vesicles and extracellular space. Notch signaling is a key signaling pathway regulating cell proliferation and differentiation during development and cancer. The expression of both V-ATPases and Notch signaling is aberrant in most cancers and hence, are potential targets for therapeutics. It is speculated that the vacuolar ATPases regulate Notch signaling directly by endosomal acidification and indirectly by endocytosis. Here, we studied the role of the a2 isoform of vacuolar ATPase (a2V) in regulating Notch Signaling in Breast cancer. Using confocal microscopy, we traced the location of a2V in MCF7, SKBR3 and MDA-MB231 breast cancer cell lines. We found that a2V is present on the plasma membrane and not on the early endosomes. The amount of a2V correlated with the invasiveness of the cancer. Most importantly, the expression of a2V was restricted to one side of the cells marking their leading edge. Also, no a2V expression was found when cells contacted each other on the inside. Next, we evaluated the effect of a2V knockdown in MDA-MB231 cells. Using siRNA, a2V was knocked down. We found that decrease of a2V enhances the expression of Notch2 and Notch 4 significantly (>1.75 fold). Notch ligands (DLL family) and Notch targets also increased, supporting the finding. When we looked at Wnt pathway genes, we found a significant increase in Wnt4 (>2 fold) and c-Myc(∼3 fold) expression although levels of β-catenin were unchanged. Since c-Myc is known to mediate apoptosis, we looked at apoptotic genes Bax and Bcl2 to after siRNA knockdown of a2V. Although Bax increased, there was no significant change in apoptosis, as seen in PI-annexin assay. Taken together, our data suggests that a2V is important for tumor growth and its presence on leading edge marks aggressiveness of tumors. Interestingly, a2V depletion results in the activation of Notch and several proliferative markers in breast cancer. We postulate that this is due to indirect effects of a2V in endocytosis or in regulating pH dependent enzymes responsible for Notch and Wnt signal transduction. Citation Format: Sahithi Pamarthy, Alice Gilman-Sachs, Kenneth Beaman. Breast cancers are notched up with a proton pump at their leading edge. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 513. doi:10.1158/1538-7445.AM2013-513