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 found to form a stable complex with each other in several cell types. Further, a variety of studies have implicated both BCAR3 and p130Cas in signaling pathways that positively regulate Rac activation and cell motility. Not surprisingly, therefore, such earlier studies had suggested that BCAR3 was likely to act by facilitating p130Cas-mediated signaling in a manner that was dependent upon formation of a complex between the two proteins. Consistent with this model, elimination of the carboxy-terminal GEF-like domain of BCAR3 that binds to p130Cas resulted in loss of both BCAR3-induced anti-estrogen resistance and Rac activation [16
]. The current studies have addressed the role of BCAR3-p130Cas association more specifically by taking advantage of a recent structural study that identified a BCAR3 point mutation, R743A, that abrogates complex formation [19
]. In contrast to prior studies with carboxy-terminal BCAR3 deletion mutants, experiments with this point mutant now demonstrate that BCAR3 association with p130Cas is dispensable for BCAR3-induced growth of MCF-7 cells in the presence of fulvestrant (ICI 182,780), a clinically efficacious estrogen antagonist that induces proteosomal degradation of the estrogen receptor. BCAR3’s amino-terminal SH2 domain has previously been found to be required for anti-estrogen resistance [16
]. In aggregate, these studies suggest that fulvestrant resistance requires binding of the BCAR3 SH2 domain to a currently unknown tyrosine phosphorylated ligand and that formation of a BCAR3-p130Cas complex is not obligatory for the BCAR3-induced signaling that leads to such anti-estrogen resistance.
Despite the fact that p130Cas signaling has been linked to activation of the atypical Rac GDP exchange factor DOCK180, BCAR3-p130Cas complex formation was also found to be unnecessary for BCAR3-induced Rac activation and cellular discohesion in breast cancer epithelial cells. This result is in marked contrast to results obtained previously with the carboxy-terminal deleted form of BCAR3 which failed to induce Rac activation, suggesting that this region of BCAR3 plays a role in BCAR3-induced Rac activation independent of its ability to allow complex formation with CAS family members. Notably, immunofluorescent studies indicate that the localization of the R743A mutant form of BCAR3 to the leading edge membrane was indistinguishable from that of wildtype BCAR3 in both epithelial breast cancer and fibroblast cell lines. This finding is in keeping with a prior report by Schrecengost et al
indicating that both wildtype and carboxy-terminal-deleted BCAR3 localized to the membrane of breast cancer cell lines [18
]. BCAR3-induced lamellipodia formation in fibroblasts, a process characteristic of Rac-mediated signaling, also occurred equivalently with wildtype or R743A BCAR3 over-expression. Dail et al
have reported that when modified to constitutively localize to the plasma membrane, over-expression of NSP3 induces ruffling in NIH-3T3 fibroblasts in a manner that is dependent on p130Cas association [23
]. In the current study, BCAR3-induced lamellipodia formation in fibroblasts was not mimicked by over-expression of either wildtype non-hematopoietic NSP3, which does not constitutively localize to the membrane, or by a chimeric form of NSP3 in which the carboxy-terminal sequences responsible for binding to p130Cas were replaced with the analogous portion of BCAR3. These studies demonstrate that a recently identified complex-independent BCAR3-mediated signaling pathway that results in serine phosphorylation of p130Cas also does not correlate with either BCAR3-induced anti-estrogen resistance or lamellipodia formation.
Consistent with the concept of p130Cas-independent BCAR3-mediated signaling, it is notable that the morphologic effects of transduction of p130Cas knockout MEFs with a lentiviral BCAR3 expression construct are quite distinct from those observed following reconstitution with p130Cas. Upon reconstitution of p130Cas-/- MEFs with p130Cas, cells acquired a more rounded and branched phenotype but failed to alter their basal level of lamellipodia. BCAR3-transduced MEFs, in contrast, had augmented levels of lamellipodia, but failed to recapitulate the branched phenotype characteristic of p130Cas reconstitution. In cells transduced with both lentiviral constructs, fibroblasts exhibited cell rounding, branching and an increase in lamellipodia. These studies support the hypothesis that BCAR3 does not exert its morphologic effects in fibroblasts by way of a linear signaling pathway in which BCAR3 augments or facilitates p130Cas-mediated signaling.
Despite the negative results reported above, complex formation between BCAR3 and p130Cas is nonetheless likely to have important consequences in the physiology of cells that express both of these proteins. Bouton and colleagues have previously shown that over-expression of BCAR3 induces cell motility. In an initial study, while transient transfection of a plasmid encoding BCAR3 alone did not augment motility in C3H10T1/2-5H murine fibroblasts, the same BCAR3 construct enhanced migration induced by transient transfection of a plasmid encoding p130Cas [22
]. Deletion of BCAR3’s carboxy-terminal GEF domain abrogated the ability of BCAR3 to enhance such p130Cas over-expression-induced migration. The authors suggested that BCAR3 may have enhanced motility by recruiting p130Cas from focal adhesions to lamellipodial membranes. In a second study in epithelial breast cancer cell lines, BCAR3 expression induced motility even when used alone, although the role of BCAR3 association with p130Cas in this process was not specifically addressed [18
In some contrast to the prior work of Bouton and colleagues in fibroblast cell lines, we find that stable polyclonal lentiviral expression of BCAR3 in BALB/c-3T3 cells is sufficient to induce motility. In contrast to the hypothesis that BCAR3 induces motility solely as a result of its ability to associate with and recruit p130Cas to lamellipodial membranes, we observed BCAR3-induced migration in the absence of BCAR3-p130Cas complex formation in BALB/c-3T3 cells. Further, BCAR3-induced migration is observed in MEFs completely lacking p130Cas expression. However, our studies with the R743A mutant form of BCAR3 suggest that BCAR3 complex formation with CAS family members contributes to BCAR3-induced cell motility, as while R743A BCAR3 does induce motility in the BALB/c-3T3 cells relative to a vector-only control, the degree of motility induced was less than that observed with wildtype BCAR3. Consistent with this finding, in p130Cas-/-
MEFs, the R743A BCAR3 mutant’s loss of ability to bind to other CAS family members such as HEF1 led to a complete abrogation of induced motility. These observations parallel a report in which complex formation with p130Cas was shown to be required for NSP3’s ability to augment COS-7 cell migration to EGF [23
]. Similarly, Chat-H, a hematopoietic-restricted NSP3 isoform, has been reported to induce T lymphocyte migration in a manner that requires complex formation with HEF1 [24
Prior studies have demonstrated that BCAR3 binds endogenous HEF1 in murine B lymphocytes [13
]. In the current study, over-expression of BCAR3 induced a reduction in PAGE migration in endogenous HEF1 in both BALB/c-3T3 cells and p130Cas-/-
MEFs. This BCAR3-induced mobility shift was identified to be λ phosphatase-sensitive, confirming that as previously documented for p130Cas, BCAR3 over-expression induces phosphorylation of HEF1. However under the conditions utilized in these experiments, we failed to observe a coincident BCAR3-mediated reduction in HEF1 stability, suggesting that BCAR3-induced HEF1 phosphorylation is distinct from the previously reported adhesion-mediated phosphorylation of HEF1 that leads to proteasomal degradation [26
]. Whereas the non-hematopoietic isoform of NSP3 utilized in our study does not induce HEF1 phosphorylation, Chat-H, which constitutively localizes to the plasma membrane, has been reported to induce HEF1 phosphorylation [24
]. Our studies do however suggest that BCAR3 binding to p130Cas, or in the absence of p130Cas, HEF1, plays an important role in BCAR3’s ability to augment cell motility.
In conclusion, the studies reported here suggest that an important subset of the functional effects observed following over-expression of BCAR3 are independent of binding to CAS family members or, in certain instances, the presence of p130Cas. Should BCAR3 ultimately be found to play a meaningful role in clinical breast cancer resistance to tamoxifen or fulvestrant, these findings are of particular importance in that they suggest that small molecules that disrupt the BCAR3-p130Cas complex would be unlikely to reverse this process. Instead, the focus of future efforts in this field should center on identifying the signaling pathway activated by binding of the BCAR3 SH2 domain to its relevant ligand.