Regulation of p130Cas/BCAR1 Expression in Tamoxifen-Sensitive and Tamoxifen-Resistant Breast Cancer Cells by EGR1 and NAB2

Abstract

Elevated levels of p130Cas/BCAR1 (Crk-associated substrate/breast cancer antiestrogen resistance 1) are found in aggressive breast tumors and are associated with tamoxifen resistance of mammary cancers. p130Cas promotes the integration of protein complexes involved in multiple signaling pathways frequently deregulated in breast cancer. To elucidate mechanisms leading to p130Cas up-regulation in mammary carcinomas and during acquired tamoxifen resistance, the regulation of p130Cas/BCAR1 was studied. Because multiple putative binding motifs for the inducible transcription factor EGR1 were identified in the 5′ region of BCAR1, the p130Cas/BCAR1 regulation by EGR1 and its coregulator NAB2 was investigated. Overexpression or short interfering RNA (siRNA)-mediated down-regulation of EGR1 or NAB2, and chromatin immunoprecipitations indicated that EGR1 and NAB2 act in concert to positively regulate p130Cas/BCAR1 expression in breast cancer cells. p130Cas depletion using siRNA showed that, in tamoxifen-sensitive MCF-7 cells, p130Cas regulates EGR1 and NAB2 expression, whereas in the derivative tamoxifen-resistant TAM-R cells, only NAB2 levels were influenced. BCAR1 messenger RNA and p130Cas protein were upregulated by phorbol esters following the kinetics of late response genes in MCF-7 but not in TAM-R cells. Thus, in MCF-7 cells, we identified a positive feedback loop where p130Cas positively regulates EGR1 and NAB2, which in turn induce p130Cas expression. Importantly, compared with MCF-7, enhanced NAB2 expression and increased EGR1 binding to the BCAR1 5′ region observed in TAM-R may lead to the constitutively increased p130Cas/BCAR1 levels in TAM-R cells. The uncovered differences in this EGR1/NAB2/p130Cas network in MCF-7 versus TAM-R cells may also contribute to p130Cas up-regulation during acquired tamoxifen resistance.