Elsevier

Cellular Signalling

Volume 23, Issue 6, June 2011, Pages 1030-1040
Cellular Signalling

BCAR3/AND-34 can signal independent of complex formation with CAS family members or the presence of p130Cas

https://doi.org/10.1016/j.cellsig.2011.01.018Get rights and content

Abstract

BCAR3 binds to the carboxy-terminus of p130Cas, a focal adhesion adapter protein. Both BCAR3 and p130Cas have been linked to resistance to anti-estrogens in breast cancer, Rac activation and cell motility. Using R743A BCAR3, a point mutant that has lost the ability to bind p130Cas, we find that BCAR3–p130Cas complex formation is not required for BCAR3-mediated anti-estrogen resistance, Rac activation or discohesion of epithelial breast cancer cells. Complex formation was also not required for BCAR3-induced lamellipodia formation in BALB/c-3T3 fibroblasts but was required for optimal BCAR3-induced motility. Although both wildtype and R743A BCAR3 induced phosphorylation of p130Cas and the related adapter protein HEF1/NEDD9, chimeric NSP3:BCAR3 experiments demonstrate that such phosphorylation does not correlate with BCAR3-induced anti-estrogen resistance or lamellipodia formation. Wildtype but not R743A BCAR3 induced lamellipodia formation and augmented cell motility in p130Cas−/− murine embryonic fibroblasts (MEFs), suggesting that while p130Cas itself is not strictly required for these endpoints, complex formation with other CAS family members is, at least in cells lacking p130Cas. Overall, our work suggests that many, but not all, BCAR3-mediated signaling events in epithelial and mesenchymal cells are independent of p130Cas association. These studies also indicate that disruption of the BCAR3–p130Cas complex is unlikely to reverse BCAR3-mediated anti-estrogen resistance.

Introduction

The interaction of integrin receptors with extracellular matrix proteins regulates both the ability of breast cancer cells to grow in the presence of anti-estrogens and their invasive properties. Breast tumors that develop resistance to anti-estrogen therapy often simultaneously acquire a more aggressive and metastatic phenotype. In a cell line model of anti-estrogen resistance, Dorssers and colleagues identified BCAR1 (p130Cas) and BCAR3 as genes whose over-expression induce estrogen independent growth in normally estrogen-dependent cell lines [1], [2]. p130Cas is a focal adhesion adapter protein best known for its ability to convert integrin receptor signals to Src family kinase-mediated tyrosine phosphorylation of the p130Cas “substrate domain” [3], [4]. Such p130Cas phosphorylation in turn leads to recruitment of Crk, and the atypical Rac GDP exchange factor DOCK180, with resultant Rac activation and augmented cell motility [5], [6]. While elimination of p130Cas expression in mice by homologous recombination results in embryonic lethality due to a cardiac phenotype, p130Cas−/− murine embryonic fibroblasts (MEFs) are notable both for defects in cell motility and Src-mediated transformation [7]. In keeping with such cell line studies, Dorssers and colleagues have reported that high-level expression of p130Cas in primary breast tumors confers an increased likelihood of lack of response to hormonal therapy, early disease progression and shorter overall survival [8], [9], [10].

Although BCAR3 and p130Cas were identified quite independently as potential “anti-estrogen resistance” genes in the studies cited above, contemporaneous studies of the murine homologue of BCAR3, AND-34, demonstrated that these two proteins normally can be found in cells as part of a complex, with the carboxy-terminus of AND-34 bound to the carboxy-terminus of p130Cas [11], [12]. AND-34 (both human and murine proteins will henceforth be referred to as BCAR3) is a 95 kDa protein with an amino-terminal SH2 domain and a carboxy-terminal domain with sequence homology to the cdc25 Ras subfamily GDP exchange factor catalytic domain. Both BCAR3 and p130Cas are members of gene families. In humans, there are three BCAR3-like proteins: NSP1, NSP2/BCAR3 and NSP3/SHEP1/CHAT and four p130Cas-like proteins: p130Cas, HEF1/NEDD9/Cas-L, Efs/Sin and HEPL [4]. BCAR3 also forms a complex in lymphoid cells with endogenous HEF1 [13]. Among NSP family members, over-expression of only BCAR3 induces significant anti-estrogen resistance [14].

Consistent with the fact that BCAR3 binds to an adapter protein known to facilitate integrin-mediated signaling and motility, BCAR3 levels regulate breast cancer cell line morphology and cell motility. Despite the Ras subfamily GEF-like domain, initial studies in murine fibroblasts stably transfected with BCAR3 revealed lamellipodia and ruffling characteristic of Rac GTPase signaling [15]. Subsequent “pulldown” studies confirmed that BCAR3 expression resulted in robust activation of both Rac and Cdc42 GTPases, most likely as an indirect result of PI3K activation [13], [15], [16]. Over-expression of BCAR3 in ERα-positive epithelial breast cancer cell lines that normally have quite low basal levels of BCAR3 expression results in conversion of cells to a mesenchymal-like growth pattern, with a loss of cell cohesiveness and a loss of cadherin-mediated homotypic adhesion at cell borders [14]. In contrast, siRNA-mediated down-regulation of BCAR3 in mesenchymal ERα-negative breast cancer cell lines that normally express substantial levels of BCAR3 results in conversion to an epithelioid growth pattern, with cohesive apposition of cells to each other [17]. Concordantly, studies by Bouton and colleagues have shown that the over-expression of BCAR3 in epithelial breast cancer cell lines induces cell motility, while the down-regulation of BCAR3 in mesenchymal breast cancer cell lines attenuates migration and invasion [18].

Given that BCAR3 binds to p130Cas and that p130Cas is an important integrator of adhesion-related signaling, it seems reasonable to hypothesize that BCAR3 expression regulates cell morphology and motility as a direct result of its ability to modulate p130Cas-mediated signaling. Consistent with such a hypothesis, prior experiments with BCAR3 deletion constructs lacking the GEF-like domain (BCAR3 ΔGEF) required for association with p130Cas have demonstrated both a loss of BCAR3-mediated Rac activation and BCAR3-induced cell motility [15], [18]. Interestingly, however, not all BCAR3-associated signaling requires formation of a BCAR3–p130Cas complex. Recent phosphoamino-acid analysis studies have shown that BCAR3 expression induces serine phosphorylation of p130Cas, characterized by a reduction in p130Cas migration on low bis-acrylamide PAGE gels [17]. Among three sites of p130Cas serine phosphorylation identified by mass spectrometry, mutation of serine 437 (rat p130Cas) to alanine reduced such a BCAR3-induced shift in p130Cas PAGE migration. Such BCAR3-mediated p130Cas phosphorylation is adhesion and actin microfilament-dependent and temporally distinct from previously well-characterized rapid FAK and Src-kinase-mediated p130Cas tyrosine phosphorylation. BCAR3-mediated p130Cas phosphorylation requires the amino-terminal BCAR3 SH2 domain but occurs in the absence of the GEF-like domain and thus is independent of BCAR3–p130Cas complex formation.

The association of both BCAR3 and p130Cas over-expression with breast cancer anti-estrogen resistance has suggested that disrupting the BCAR3–p130Cas complex might be an important novel approach to restoring sensitivity to anti-estrogens. Small-angle X-ray scattering studies of the complex of BCAR3 with the CAS family member HEF1 revealed that the GEF-like domain of BCAR3, which folds similarly to a Cdc25-like GEF domain, binds tightly to helix 2 of a four-helix bundle carboxy-terminal binding domain in HEF1 [19]. As predicted by these structural data, mutation of arginine 743 of BCAR3 to alanine eliminated BCAR3 association with HEF1 and, as shown in the current study, p130Cas. As deletion of BCAR3's GEF-like domain could have effects on BCAR3-associated signaling quite independent of BCAR3's ability to form a complex with p130Cas, we here have utilized the R743A BCAR3 mutant to more directly examine the importance of BCAR3–p130Cas complex formation in BCAR3-associated anti-estrogen resistance, cytoskeletal changes, Rac activation and cell motility. Finally, we have addressed the role of p130Cas itself in BCAR3-associated lamellipodia formation and motility using p130Cas−/− MEFs.

Section snippets

Cell lines

MCF-7 human breast cancer cell lines were obtained from ATCC. The HA-BCAR3 stably transfected MCF-7 cell line II-6 has been previously described [14]. BALB/c-3T3 murine fibroblasts were a gift of Dr. Douglas Faller (Cancer Center, BUSM). MCF-7 and BALB/c-3T3 lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Mediatech, Inc.) supplemented with 10% heat-inactivated fetal calf serum (Biomeda), 2.2 mM l-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. p130Cas−/− mouse embryonic

BCAR3-mediated anti-estrogen resistance is independent of p130Cas complex formation and BCAR3-mediated p130Cas phosphorylation

The ERα-positive human epithelial breast cancer cell line MCF-7 is known to express only low basal levels of BCAR3 and over-expression of BCAR3 in MCF-7 cells has been shown to induce both anti-estrogen resistance and a mesenchymal-like morphologic shift in growth pattern [14], [15]. To assess the role of BCAR3–p130Cas complex formation in these processes, we transduced MCF-7 cells with a control ZsGreen-expressing lentiviral vector or the same bicistronic vector also expressing either

Discussion

In the studies described in this report, we have shown that BCAR3 complex formation with p130Cas (or the related family member HEF1) is not required for BCAR3-induced ERα-positive breast cancer cell line anti-estrogen resistance, cell discohesion, Rac activation, or fibroblast lamellipodia formation. These results are surprising in that both BCAR3 and p130Cas (BCAR1) were identified in a screen for genes capable of inducing anti-estrogen resistance and the endogenous proteins were subsequently

Acknowledgements

The authors gratefully acknowledge excellent technical assistance from Alex Bloom, as well as advice and reagents from Dr.'s Douglas Faller (Cancer Center, BUSM), Kathrin Kirsch (Biochemistry, BUSM), and Maria Kukuruzinska (Molecular and Cell Biology, BU Goldman School of Dental Medicine). This work was supported by NIH RO1 CA114094, the Logica Foundation and the Breast Cancer Alliance.

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