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Introduction: Non-small cell lung cancer (NSCLC) is the top cause of cancer-related mortality. We have identified a role for the tumor suppressor hepatocyte growth factor activator inhibitor type-1 (HAI-1) in human lung adenocarcinoma. HAI-1 loss results in unregulated downstream MET/RON tyrosine kinase receptor signaling. We hypothesized that HAI-1 is lost in human lung adenocarcinomas and could serve as a biomarker for therapy with crizotinib, a tyrosine kinase inhibitor (TKI) with activity against both MET and RON. We also hypothesized that unregulated RON signaling in the tumor microenvironment, via HAI-1 loss, may promote tumor metastasis by skewing tumor associated macrophages (TAMs) from an anti-tumor (M1) to a pro-tumor (M2) phenotype. Methods: Immunohistochemical staining with HAI-1 antibody (1N7) was performed on human tissue microarray containing 20 normal and 20 lung tumor specimens. Intensity grading (0, 1+, 2+, 3+) was performed, and percent cell expression was calculated for each group. For all in vitro assays, H358 lung adenocarcinoma cells, which express high levels of endogenous HAI-1, were stably transfected with mock shRNA or HAI-1 shRNA. Cell protein expression and phosphorylation was assessed via western blot. Cell viability after exposure to crizotinib was assessed by trypan blue staining. For flow cytometric analysis of macrophage phenotype, undifferentiated (M0) macrophages were exposed to conditioned medium from transfected H358 cells and macrophage phenotype was assessed by flow for surface markers CD68 (M0), CCR7 (M1), and CD206 (M2). Results: HAI-1 expression is significantly decreased in human lung adenocarcinoma compared to normal lung with overall HAI-1 IHC positivity ~20% compared to ~60% respectively. HAI-1 knockdown in H358 cells caused increased phosphorylation of RON and increased sensitivity to crizotinib. Culturing M0 macrophages in conditioned media from H358 shHAI-1 knockdown cells caused a decrease in the CCR7 positive M1 fraction from 8.4% to 1.9%, and an increase in the CD206 positive M2 fraction from 0.77% to 2.09%. Conclusion: We have demonstrated that loss of HAI-1 occurs in human lung adenocarcinoma consistent with previous studies in human NSCLC cell lines. Furthermore, we showed significantly increased RON signaling activity after HAI-1 knockdown. We have also demonstrated that HAI-1 loss can skew tumor associated macrophages (TAMs) from an anti-tumor (M1) to a pro-tumor (M2) phenotype. We have shown that loss of HAI-1 in vitro can increase sensitivity to crizotinib, suggesting that loss of HAI-1 may predict for tumor sensitivity to MET/RON inhibition. Our findings show that dysregulation of the HAI-1/MET/RON pathway exerts effects both on lung tumor cells as well as TAMs, making this pathway a potentially powerful therapeutic target. Citation Format: Austin J. McHenry, Gautam Sondarva, Vijayalakshmi Ananthanarayanan, Ashley Hess, Patricia E. Simms, Alhareth Alsayed, Hiroaki Kataoka, Jan Marusarz, Sandeep Kumar, Ravi Salgia, Arkadiusz Dudek, Stanley Borowicz, Ajay Rana. Loss of hepatocyte growth factor activator inhibitor type-1 (HAI-1) in human lung adenocarcinomas promotes RON receptor phosphorylation and increased sensitivity to crizotinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5514.

Rationale: Treatment of patients with triple-negative breast cancer (TNBC) remains challenging due to unpredictable disease progression and acquired resistance to chemotherapy. Finding novel targets in TNBC is important for the development of anticancer therapy. Cancer cells frequently adapt to cytotoxicity of existing pharmacological agents by exploiting metabolic pathways. Elevated copper metabolism and adaptations to oxidative stress have been linked to cancer progression. Copper chelation with tetrathiomolybdate (TM) was reported to control disease progression, especially in patients with TNBC. However, long-term consequences of global copper depletion are still under investigation. An alternative strategy is provided by a recently developed small molecule DCAC50 that blocks the copper transfer interface of two major copper chaperons, ATOX-1 and CCS. Protein levels of ATOX-1 and CCS are elevated in TNBC cells compared to normal cells. Thus, we hypothesized that disrupting copper transport by targeting ATOX-1 and CCS with DCAC50 may suppress TNBC progression. Results: We investigated efficacy and potency of novel small molecule DCAC50 to induce cytotoxicity in a panel of TNBC cells lines. DCAC50 reduced cell proliferation in dose-dependent manner, measured by MTS assay. IC50 doses ranged from 3-10uM. Most importantly, DCAC50 induced apoptosis in TNBC cells, detected by Caspase 3/7 activity and Annexin V/PI staining. As expected, DCAC50 elevated intracellular copper levels. Moreover, TNBC cells treated with DCAC50 had significantly higher levels of oxidized glutathione (GSSG) and caused increased oxidation of DCF-DA reagent, demonstrating elevated oxidative stress. Surprisingly, activity of CCS downstream target, SOD1, was not affected by selective inhibition of copper transport with DCAC50; suggesting that intracellular copper accumulation and oxidative stress is mediated by ATOX-1 inhibition. Interestingly, copper depletion with TM inhibited SOD1 activity but failed to significantly impact oxidative stress in TNBC cells. Recognizing DCAC50 generates oxidative stress triggering apoptosis in TNBC cells we evaluated benefits of combining DCAC50 treatment with paclitaxel. Multi-drug combination dose-response analysis revealed that co-treatment induced synergistic cytotoxicity and resulted in favorable dose reduction of both drugs. Conclusions: Selective inhibition of copper metabolism with novel small molecule DCAC50 elevates oxidative stress triggering apoptosis in TNBC cells. This approach may be valuable in combination with chemotherapy especially when tumor cells acquire resistance to first line therapy in TNBC. Ongoing studies comparing selective inhibition of copper transport to global copper chelation, and investigating efficacy and biological activity of DCAC50 in vivo will help further estimate benefits and clinical relevance of this approach for treatment of TNBC patients. Citation Format: Olga Karginova, Claire Weekley, Akila Raoul, Alhareth Alsayed, Tong Wu, Chuan He, Olufunmilayo I. Olopade. Selective inhibition of copper metabolism as a novel approach to treat triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3552. doi:10.1158/1538-7445.AM2017-3552