Abstract 4939: The baffling effect of bafilomycin on Notch signaling in triple-negative breast cancer

Abstract

Abstract Triple Negative Breast Cancers (TNBC) constitute 15 to 20% of all breast cancers and lack expression of ER/PR and Her2 receptors. Owing to an aggressive phenotype and lack of targeted therapies, TNBC has poor prognosis. Notch signaling is an evolutionarily conserved signaling pathway known to be important for normal mammary development and breast cancer progression. Although there has been considerable research on the role of Notch in TNBC, little is known about its regulation. Identifying novel molecules that regulate Notch can lead to the development of effective combinatorial treatments. The Vacuolar ATPase (V-ATPase) is a proton pump responsible for maintaining pH. Expression of V-ATPase is upregulated in tumor cells, promoting cancer proliferation and metastasis. We have previously identified the role of V-ATPase a2 isoform (V0a2) in pregnancy and cancer. Here, we report that V-ATPase regulates Notch Signaling in TNBC. Upon treatment with Bafilomycin A1 (BafA1), a specific V-ATPase inhibitor, Notch and Wnt Signaling genes were significantly upregulated in TNBC cell lines MDA-MB-231 and MDA-MB-468 (p<0.005) as determined by PCR arrays. BafA1 induced dose dependent increase of Notch pathway genes as measured by Notch luciferase reporter assay. To investigate the hypothesis that V-ATPase inhibition activates Notch Signaling by autophagy, we tested the expression of LC3B. BafA1 treatment in MDA-MB-231 cells led to accumulation of LC3B, thereby inhibiting autophagy. V-ATPase inhibition also induced a cytoprotective response by increasing expression of Hif1α, a gene recently reported to activate Notch signaling. Given that both Notch pathway and V-ATPases are involved in tumor cell proliferation, we tested the effect of Baf A1 and Notch Signaling inhibitor - GSI on TNBC cell viability. MDA-MB-231 cells showed a significant decrease in proliferation upon treatment with Baf A1 (p<0.0001) or GSI (p<0.001) as measured by MTT assay. However, combinatorial treatment with BafA1 and GSI was not as efficient, an effect that can be attributed to enhanced Notch Signaling through V-ATPase inhibition. A similar trend was observed with apoptosis induction as measured by Caspase 3/7 activation assay. Effect of V-ATPase inhibition on cell proliferation was corroborated by Concanamycin A, another V-ATPase inhibitor. In summary, our results show that V-ATPase regulates cell survival pathways like Notch Signaling in TNBC. Through this study, we have deciphered a novel role of V-ATPase inhibitors in cell signaling regulation, a finding that may be useful for combinatorial therapy regimens. Citation Format: Sahithi Pamarthy, Mukesh K. Jaiswal, Arpita Kulshrestha, Gajendra Katara, Safaa Ibrahim, Alice Gilman-Sachs, Kenneth D. Beaman. The baffling effect of bafilomycin on Notch signaling in triple-negative breast 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 4939. doi:10.1158/1538-7445.AM2015-4939