Abstract 358: Identification of signaling pathways activated by BCAR4 expression in human breast cancer

Abstract

Abstract Background: The development of resistance to the antiestrogen tamoxifen remains a major challenge in the management of estrogen receptor alpha (ER)-positive breast cancer. Understanding the mechanisms leading to resistance is essential to develop more efficient therapies. In a functional screening for genes causing tamoxifen resistance, we have identified the breast cancer antiestrogen resistance 4 (BCAR4) gene. BCAR4 mRNA is expressed in 27% of primary breast tumors. High BCAR4 mRNA levels predict resistance to tamoxifen treatment and poor outcome, and are associated with a shorter metastasis-free survival and overall survival. Forced expression of BCAR4 in the estrogen-dependent breast cancer cell line ZR-75-1 induced antiestrogen resistance and phosphorylation of ERBB2, ERBB3, and their down-stream mediators ERK1/2 and AKT. The knockdown of ERBB2 or ERBB3 with specific siRNA inhibited cell proliferation, confirming the role of these receptors in BCAR4-induced tamoxifen resistance in vitro. These findings suggest that tumors expressing BCAR4 may rely on ERBB2/ERBB3 signaling and patients with such tumors may be eligible to receive ERBB-targeted therapy. Although ER is functional in ZR/BCAR4 cells, the knockdown of the receptor had no effect on the proliferation rate of BCAR4-expressing cells, showing that this mechanism of resistance is independent of ER signaling. Objective: To identify signaling pathways activated by BCAR4 expression and investigate the consequences of perturbations of the pathways. Experimental design: Reverse-phase protein microarray (RPPA) using phospho-specific antibodies was done to examine the activation status of several key signaling molecules in a cellular model for endocrine resistance of breast cancer. A panel of ZR-75-1-derived cell lines, each containing a different transgene conferring anti-estrogen resistance, was analyzed by RPPA. Results: By measuring the activity of a large number of phospho-protein isoforms in cell lysates from vector-containing control and BCAR4-expressing cells (ZR/BCAR4), it was confirmed that ERBB2 and ERBB3 were strongly phosphorylated in ZR/BCAR4 cells. This was observed in estradiol-containing medium and in medium supplemented with tamoxifen. Furthermore, many established down-stream signaling molecules of ERBB2/3 were found to be activated. Conclusions: ERBB2/ERBB3signaling is strongly activated in cells with forced expression of BCAR4 and completely independent of estrogen signaling. Both ERBB2 and ERBB3 are required for BCAR4-mediated tamoxifen resistance. Our results suggest that patients with ER-positive breast tumors expressing BCAR4 may benefit from ERBB-targeted therapies combined with tamoxifen treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 358. doi:10.1158/1538-7445.AM2011-358