Abstract LB-205: The calcium activated chloride channel ANO1 promotes breast cancer progression by activating EGFR- and CAMK-signaling.

Genomic alterations are the underlining cause of many human cancers: Amplified and overexpressed genes can drive neoplastic transformation and become essential survival factors for cancer cells. Thus, they represent promising targets for anti-cancer therapies and their identification and validation is of paramount importance. In a search for novel survival factors contributing to breast cancer oncogenesis, we performed genomic fine mapping of the 11q13 amplicon, one of the most frequently amplified chromosomal regions in human neoplasia, in a large dataset of breast cancer patients. Remarkably, we found ANO1, a calcium-activated chloride channel, to be located within the summit of the most frequently and highly amplified genomic region. Furthermore, amplification of ANO1 correlates with its overexpression and elevated protein levels and is a negative predictor for overall survival in breast cancer patients. Next, we examined how ANO1 contributes to cancer cell survival and proliferation. We found that inhibition of ANO1 expression or function reduced cancer cell viability and colony formation, and triggered apoptosis in 11q13 amplified breast cancer, HNSCC and ESCC. Furthermore, expression of ANO1 in non-transformed mammary cells increased cell viability and established a novel addiction to ANO1 biochemical activity. We next applied inducible shRNAs against ANO1 in vivo to assess the effect of ANO1 inhibition on the maintenance of established tumors. We found in four different 11q13-amplified tumor models that knockdown of ANO1 reduced tumor growth, indicating an important role for ANO1 not only in oncogenesis, but also in tumor maintenance of 11q13 amplified cancers. To explore the mechanism by which inhibition of ANO1 contributes to cancer cell viability and tumor growth, we performed antibody arrays to measure the activity of major oncogenic signaling pathways after knockdown of ANO1. Upon depletion of ANO1, activation of EGFR and several survival signaling pathways (AKT-, SRC- and ERK1/2 pathways) were reduced in different models of human cancer. Subsequent experiments showed that ANO1 modulates both EGFR- and Ca2+/calmodulin-dependent protein kinase (CAMK) signaling in breast cancer and HNSCC cells. Consistently, activation of EGFR- and CAMK correlated with the expression of ANO1 in human primary breast tumor samples. Lastly, we found that only the simultaneous stimulation of EGFR- and CAMK-signaling pathways rescued the effect of the ANO1-inhibition. In summary, our study establishes ANO1 as a key tumor-promoting factor in 11q13-amplified breast and other malignancies. Our results highlight the importance of chloride channels in cancer and provide the first detailed mechanistic insight into the activity of ANO1 in tumorigenesis. Most importantly, our findings open up new opportunities for therapeutic intervention in several prevalent cancers. Citation Format: Adrian Britschgi, Anke Bill, Heike Brinkhaus, Christopher Rothwell, Ieuan Clay, Stephan Duss, Michael Rebhan, Pichai Raman, Chantale Guy, Kristie Wetzel, Elizabeth George, M. Oana Popa, Sarah Lilley, Hedaythul Choudhury, Martin Gosling, Louis Wang, Stephanie Fitzgerald, Jason Borawski, Jonathan Baffoe, Mark Labow, L. Alex Gaither, Mohamed Bentires-Alj. The calcium activated chloride channel ANO1 promotes breast cancer progression by activating EGFR- and CAMK-signaling. [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 LB-205. doi:10.1158/1538-7445.AM2013-LB-205