High Expression Of Ncx-1 In Triple Negative Breast Cancer Cell Lines Identifies A Potential Biomarker For Sensitivity To Cardiac Glycosides

Triple negative breast cancers (TNBCs) represent 15-20% of all breast cancers and are defined by lack of the estrogen and progesterone receptors and HER2 gene amplification. Therefore, TNBC patients do not benefit from available therapies targeting these receptors. The significant heterogeneity of TNBC precludes the expectation that a single molecular target will be identified for this disease. Our goal is to identify compounds with selective cytotoxic effects in subsets of TNBC cells to identify new therapeutic targets for TNBC subgroups. Our studies show that the TNBC cell lines BT-549 and Hs578T are distinct from other TNBC cells in that they are highly sensitive to 9 cardiac glycosides/cardenolides isolated from Calotropis gigantea as well as digoxin. BT-549 cells are 15-times more sensitive to the isolated cardiac glycoside calotropin and 9-times more sensitive to digoxin as compared to MDA-MB-231 TNBC cells. Cardiac glycosides bind to the Na+/K+ ATPase and inhibit its activity, leading to increased intracellular Na+, which results in higher intracellular Ca2+ through reversal of the Na+/Ca2+ exchanger (NCX). Notably, among the 10 cardiac glycosides/cardenolides, there was a significant correlation between potency for inhibition of purified Na+/K+ ATPase and cytotoxic potency and selectivity for BT-549 cells. These data suggest that inhibition of Na+/K+ ATPase is critical for the selective cytotoxic effects of these compounds. BT-549 and Hs578T cells express high levels of non-specific TRPC1/4 cation channels1 and low levels of SERCA2 Ca2+ pumps2 compared to other TNBC cells, suggesting that they have impaired ability to handle intracellular Ca2+. Consistent with this hypothesis, within 3 h, concentrations of calotropin and digoxin that are selectivity cytotoxic each initiated a significant increase in intracellular Ca2+ in BT-549 cells. To test whether sensitivity to cardiac glycosides was due to higher Na+/K+ ATPase expression, message and protein levels were evaluated in 10 TNBC cell lines. The results show that there is no correlation between levels of Na+/K+ ATPase and sensitivity to cardiac glycosides. In contrast, NCX-1 expression was found to be 120 and 60-fold higher in the sensitive BT-549 and Hs578T cells, respectively, as compared to the resistant TNBC cell lines. Ongoing studies are evaluating whether genetic manipulation of NCX-1 is sufficient to modulate sensitivity to cardiac glycosides. These findings demonstrate that NCX-1 expression is associated with the selective sensitivity of a subgroup of TNBC cell lines to the cytotoxic effects of cardiac glycosides. Our results suggest that NCX-1 could be a biomarker to identify TNBC patients that could ultimately benefit from use of a cardiac glycoside for anticancer indications. 1. Grant CV, et al. Breast Cancer Res Treat. 2019;177(2):345. 2. Varga K, et al. BMC Cancer. 2018;18(1):1029. Citation Format: Petra E. Pederson, Shengxin Cai, Chase M. Carver, Tanja Grkovic, Barry R. O9Keefe, April L. Risinger, Robert H. Cichewicz, Susan L. Mooberry. High expression of NCX-1 in triple negative breast cancer cell lines identifies a potential biomarker for sensitivity to cardiac glycosides [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1786.