Studies by us and others suggest that acquired resistance to aromatase inhibitors (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 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) and CSCs have been implicated in drug resistance. Therefore, this current study aimed to 1) compare the CSC characteristics of letrozole-resistant (LTLTCa) and parental letrozole-sensitive (MCF-7Ca) breast cancer cells; 2) determine the roles of HER2, BCRP, and HIF-1 on CSC characteristics; and 3) compare the effects of HER2 inhibition and of differentiating agent all-trans retinoic acid (ATRA) on CSC population of the LTLTCa cells. LTLTCa cells were found to have higher expression of CSC characteristics compared to parental MCF-7Ca cells: 9.17%±2.0% vs. 0.02% ±0.01% (p <0.01) measured by side population, and 3.08% vs. 0.53% (p<0.05) by aldehyde dehydrogenase (ALDH) activity. Consistent with previous results, inhibition of HER2, HIF-1, or BCRP resulted in decreased CSC characteristics in LTLTCa cells. Inhibition of HER2 by treatment with 1 μM lapatinib (Lap) decreased side population (from 7.91%±2.10 to 0.76%±0.20) and mammosphere formation (from >200 mammospheres/10,000 cells to ∼50 mammospheres/10,000 cells). Flow cytometry also demonstrated that LTLTCa cells high in both ALDH activity and CD44 positivity, were also high in HER2. Inhibition of HIF-1 (by siRNA) or BCRP (by pharmacological inhibitors or siRNA) significantly decreased mammosphere formation from 406 mammospheres/80,000 cells to 93 and 81 mammospheres/80,000 cells, respectively (p<0.01). Pharmacological inhibition of BCRP also decreased side population. Comparing the effects of Lap and ATRA on LTLTCa cells, both drugs decreased mammosphere formation after 48 h and cell viability after 7 days of treatment (p<0.05 for each). Both drugs also increased ERα and decreased BCRP expression in LTLTCa cells. Lastly, Lap and ATRA each increased the expression ratio of epithelial markers versus mesenchymal markers, but they did this by affecting different proteins. Lap increased expression of epithelial marker E-cadherin, but had no effect on mesenchymal marker N-cadherin. ATRA decreased N-cadherin but had no effect on E-cadherin. Overall, the results from this study suggest that HER2-HIF-1-BCRP signaling in contributes to acquired breast cancer cell resistance to letrozole by 1) increasing the cancer stem cell population, and possibly by 2) promoting epithelial mesenchymal transition (EMT), an indicator of cancer cell invasive and metastatic potential.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 787. doi:1538-7445.AM2012-787