Abstract 95: Inhibiiton of non-hypoxic HIF-1 expression in letrozole-resistant breast cancer cells reduces their cancer stem cell characteristics.

Abstract

Abstract Although aromatase inhibitors (AIs) have been shown to be highly effective in treating estrogen receptor positive (ER+) breast cancer, a significant percentage of patients either do not respond to AIs or become resistant to them. Studies suggest that acquired resistance to AIs (i.e., letrozole) involves a switch from dependence on ER signaling to dependence on growth factor-mediated pathways, such as epidermal growth factor receptor (EGFR)/HER2. Recent work in our lab has also linked hypoxia inducible factor 1 (HIF-1) and its target gene breast cancer resistance protein (BCRP, a stem cell marker) to HER2 and AI resistance. Other studies have associated each of these factors with cancer stem cells (CSCs),, which have been implicated in drug resistance. Thus, the purpose of this study is to 1) determine whether HIF-1 expression is enriched in the CSC subpopulation of letrozole-resistant breast cancer cells; and 2) determine the effects of inhibiting HIF-1 on CSC characteristics. The in vitro model used for acquired letrozole resistant breast cancer is the LTLTCa cell line, which was obtained through long-term letrozole treatment of MCF-7Ca xenograft tumors. The CSC subpopulation in LTLTCa cells was isolated by flow cytometry based on their aldehyde dehydrogenase (ALDH) expression, and the CSC characteristics studied include mammosphere formation, side population percentage, and CD44+/CD24+ expression. Inhibition of HIF-1 expression was accomplished using either commercially available HIF-1α siRNA or EZN-2968, a specific RNA antagonist against HIF-1α currently in clinical trials. HIF-1α is the inducible subunit of HIF-1. RT-PCR indicates that mRNA expression of HIF-1α and stem cell markers, such as BCRP, BMI-1, and Nanog, were significantly higher (p<0.05) in ALDHhigh (CSC) vs. ALDHlow (non-CSC) cells. HIF-1 siRNA and EZN-2968 decreased HIF-1α mRNA and protein expression in LTLTCa cells within 48h (0.4 to 0.01-fold vs. negative control siRNA/RNA antagonist). This correlated with decreases in mRNA expression of stem cell markers BCRP and BMI-1, and TWIST, an EMT marker and regulator of BMI-1. HIF-1α inhibition also reduced mammosphere formation by 33% (p<0.05). Interestingly, preliminary results indicate that while EZN-2968 decreased the ratio of CD44+/24+ expression (28% vs. 52% in negative control RNA antagonist) and side population percentage (8% vs. 16% in negative control RNA antagonist) in LTLTCa cells, the commercially available HIF-1α siRNA had no effect. These results suggest that nonhypoxic HIF-1 is involved in regulating cancer stem cell characteristics in letrozole-resistant breast cancer cells, and that EZN-2968 should be further explored in the prevention and/or treatment of AI-resistant breast cancer. EZN-2968 kindly provided by Enzon Pharmaceuticals. This work is funded by DOD Breast Cancer Research Program Postdoctoral Award (BC1039031). Citation Format: Armina A. Kazi, Preeti Shah, Amanda Schech, Gauri Sabnis, Saranya Chumsri, Angela Brodie. Inhibiiton of non-hypoxic HIF-1 expression in letrozole-resistant breast cancer cells reduces their cancer stem cell characteristics. [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 95. doi:10.1158/1538-7445.AM2013-95