Abstract P5-04-09: Copper chaperons as novel targets for therapy in triple-negative breast cancer (TNBC)
Authors: Karginova O , Song A , Wang J , Luo C , Jiang H , He C , Olopade OI 
Published: May 2016

Abstract
Background: Copper metabolism is frequently dysregulated in cancer and promotes tumorigenesis. Copper chelation was shown to delay tumor development, attenuate tumor growth, block angiogenesis and inhibit metastases in preclinical breast cancer models. Copper depletion with tetrathiomolybdate (TM) in on-going phase II study for breast cancer patients at high risk for relapse resulted in significant improvement in progression-free survival, especially in patients with TNBC. We hypothesized that targeting ATOX-1 and CCS, copper chaperons that are major regulators of copper trafficking, with novel selective inhibitor may disrupt cellular copper transport and suppress TNBC cell growth, block angiogenic activity, and enhance cytotoxicity of available chemotherapy.
Methods:We measured ATOX-1 and CCS protein expression using western blot in a panel of breast cancer cell lines including TNBC cell lines with basal-like (BL) and claudin-low (CL) subtypes. We compared potency and efficacy of ATOX-1/CCS inhibitor to induce cytotoxicity in MDA-MB231, MDA-MB436, MDA-MB468 and primary normal mammary HMECs. We evaluated ability of the inhibitor to disrupt tubulogenesis of endothelial cells. To determine if blocking copper transport can enhance sensitivity of TNBC to chemotherapy we used novel ATOX-1/CCS inhibitor in combination with Cisplatin to treat TNBC in a schedule-dependent manner.
Results: ATOX-1 protein expression was elevated in all tested TNBC cell lines compared to normal HMEC (1.7±0.2 and 2.1± 0.3 folds higher in BL and CL cells, respectively). Upregulated CCS protein expression was also observed in majority of tested cell lines compared to HMEC (2.8±0.6 and 1.2±0.1 times higher in BL and CL cells, respectively). Treatment of MDA-MB231, MDA-MB436, MDA-MB468 with the inhibitor resulted in reduced cell proliferation. IC50 doses for 72h treatment with single agent were: 0.23±0.02uM (MDA-MB468), 0.29±0.03uM (MDA-MB231) and 0.35±0.02uM (MDA-MB436). Additional cytotoxicity was observed in TNBC when ATOX-1/CCS inhibitor was applied in combination with Cisplatin. Interestingly, sequential treatment resulted in synergistic effect (CI&lt; 1). Treatment with the inhibitor reduced growth of HMECs and HuVECs in vitro, and inhibited angiogenesis in tube formation assay with HuVECs.
Conclusions: Targeting copper trafficking by selective inhibition of chaperons ATOX-1 and CCS is promising and could potentially serve as a therapeutic approach to overcome resistance to chemotherapy in TNBC. In vivo studies investigating efficacy and biological activity of the novel compound in a xenograft model are ongoing and will help to elucidate molecular mechanisms of action, and further estimate potential clinical relevance of this approach.
Citation Format: Karginova O, Song A, Wang J, Luo C, Jiang H, He C, Olopade OI. Copper chaperons as novel targets for therapy in triple-negative breast cancer (TNBC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-09.