AND‐34/BCAR3 differs from other NSP homologs in induction of anti‐estrogen resistance, cyclin D1 promoter activation and altered breast cancer cell morphology

Abstract

Over-expression of AND-34/BCAR3/NSP2 (BCAR3) or its binding-partner p130Cas/BCAR1 generates anti-estrogen resistance in human breast cancer lines. Here, we have compared BCAR3 to two related homologs, NSP1 and NSP3/CHAT/SHEP, with regards to expression, anti-estrogen resistance, and signaling. BCAR3 is expressed at higher levels in ERα-negative, mesenchymal, than in ERα-positive, epithelial, breast cancer cell lines. Characterization of "intermediate" epithelial-like cell lines with variable ER-α expression reveals that BCAR3 expression correlates with both mesenchymal and ERα-negative phenotypes. Levels of the BCAR3/p130Cas complex correlate more strongly with the ERα-negative, mesenchymal phenotype than levels of either protein alone. NSP1 and NSP3 are expressed at lower levels than BCAR3 and without correlation to ERα/mesenchymal status. Among NSP-transfectants, only BCAR3 transfectants induce anti-estrogen resistance and augment transcription of cyclin D1 promoter constructs. Over-expression of all homologs results in activation of Rac, Cdc42 and Akt, suggesting that these signals are insufficient to induce anti-estrogen resistance. BCAR3 but not NSP1 nor NSP3 transfectants show altered morphology, transitioning from polygonal cell groups to rounded, single cells with numerous blebs. Whereas stable overexpression of BCAR3 in MCF-7 cells does not lead to classic epithelial-to-mesenchymal transition, it does result in down-regulation of cadherin-mediated adhesion and augmentation of fibronectin expression. These studies suggest that BCAR3's ability to induce anti-estrogen resistance is greater than that of other NSP homologs and may result from altered interaction of breast cancer cells with each other and the extracellular matrix.

Association of the Breast Cancer Antiestrogen Resistance Protein 1 (BCAR1) and BCAR3 Scaffolding Proteins in Cell Signaling and Antiestrogen Resistance

“…This is in line with earlier suggestions that the level of the BCAR1-BCAR3 complex in breast cancer cell lines correlates with malignancy more closely than the levels of each individual protein (23). Therefore, disrupting the complex could sensitize breast cancer cells to chemotherapeutic drugs and favor reversion to a less malignant state.…”

“…The Physical Association of BCAR1 and BCAR3 Leads to the Stabilization of Both Proteins-The increase in phosphorylated BCAR1 is accompanied by protein stabilization resulting in higher overall BCAR1 levels, which has been reported to be induced by BCAR3 and the related NSP3 (23,40). Accordingly, we found that both BCAR3 wild-type and the R748E mutant similarly increase BCAR1 protein levels, whereas the L744E/ R748E double mutant has lost this ability (Fig.…”

“…Overexpression of BCAR3 in the epithelial-like, estrogen receptor-expressing MCF7 breast cancer cells is known to lead to a more mesenchymal and migratory phenotype characterized by the formation of peripheral membrane ruffles and lamellipodia that are rich in filamentous actin and also BCAR1 and BCAR3 (23,30,31,33,39). To investigate the role of the association of BCAR3 with BCAR1 in the actin cytoskeleton rearrangements induced by BCAR3 in MCF7 cells, we used the BCAR3 L744E/R748E double mutant.…”

“…DOCK180 activates the RHO family GTPase RAC1, which promotes the formation of membrane ruffles and protrusions as well as cell migration and invasiveness. Notably, among the family members only BCAR1 has been shown to confer antiestrogen resistance (11,12,17,23).…”

“…Another multidomain signaling protein that has been associated with antiestrogen resistance and a mesenchymal phenotype in breast cancer is breast cancer antiestrogen resistance 3 (BCAR3, also known as AND-34) (23)(24)(25)(26)(27). BCAR3 is a member of the novel SH2-containing protein (NSP) family and was identified together with BCAR1 in a screen for gene products whose increased expression promotes proliferation of estrogen receptor-positive breast cancer cells treated with antiestrogens (3,24).…”