We have demonstrated that a constitutively active form of the Neu/ErbB-2 receptor tyrosine kinase cooperates with TGF-β to promote the motility and invasion of breast cancer cells and that the removal of five autophosphorylation sites in the C terminus of Neu/ErbB-2 completely abrogates the synergistic interaction of these two pathways. Furthermore, through the use of a panel of Neu/ErbB-2 add-back mutants, we have demonstrated that specific signals emanating from tyrosines 1226/1227 (YD) and 1253 (YE) of Neu/ErbB-2 are critical for TGF-β-induced motility and invasion. This enhanced motility of TGF-β-treated Neu-NT and Neu-YD breast tumor explants is associated with the formation of smaller, less mature focal adhesions. The ShcA adaptor protein is known to bind the Neu/ErbB-2 receptor through tyrosines 1226/1227 (YD) and potentially through tyrosines 1201 (YC) and 1253 (YE) (14
). We demonstrate that the reduction of ShcA levels or the expression of a dominant-negative ShcA mutant (ShcA-3F) blocked TGF-β-induced motility and the invasion of Neu/ErbB-2-expressing breast cancer cells. Collectively, these observations demonstrate a central role for the ShcA adaptor protein in mediating the synergy between the ErbB-2 and TGF-β pathways.
The Neu/ErbB-2-expressing NMuMG tumor explant models that we have generated very closely resemble the phenotypes induced by these add-back receptors when they are expressed in the mammary epithelium of transgenic mice. In our system, Neu-NT-expressing explants displayed the most aggressive tumor outgrowth phenotype, and the longest latency was observed with Neu-NYPD cells. While intermediate effects on mammary tumor outgrowth were observed with the add-back mutants, Neu-YA, Neu-YB, and Neu-NYPD explants were less aggressive than Neu-YC-, Neu-YD-, and Neu-YE-transformed NMuMG cells. This is reflective of the results observed with the MMTV-based transgenic models, in which the onsets of mammary tumor formation were quite similar among Neu-YD-, Neu-YE-, and Neu-YC-expressing mice, followed by later onset in MMTV/Neu-YB transgenics and, finally, the longest latency in MMTV/Neu-NYPD animals (15
). We did observe that NMuMG cells expressing Neu-YA receptors were capable of forming mammary tumors, even though this tyrosine has been characterized as a negative regulatory site that suppressed the focus-forming ability of fibroblasts (16
). Transgenic mice expressing an MMTV/Neu-YA transgene have not been derived; thus, it is unknown whether this mutant receptor is indeed capable of transforming normal mammary epithelial cells in vivo. It is conceivable that the negative regulator of transformation that binds to tyrosine 1028 (YA) of Neu/ErbB-2 in fibroblast cells is not expressed in NMuMG epithelial cells.
Our NMuMG cell-based mammary tumor explants also recapitulate features of the metastatic phenotypes induced by specific Neu/ErbB-2 add-back receptors. For instance, MMTV/Neu-YB animals display a more aggressive lung metastatic phenotype than MMTV/Neu-YD transgenic mice (15
). In our NMuMG mammary tumor explants, we consistently observe higher baseline invasion of the Neu-YB explants compared to that of cells expressing the Neu-YD receptor. Interestingly, only Neu-YD and Neu-YE explant cultures displayed TGF-β-induced motility and invasion. The observation that Neu-YB-expressing NMuMG explants possess high baseline invasiveness that is not TGF-β inducible, whereas Neu-YD cells display lower baseline invasiveness that is enhanced by TGF-β stimulation, also is reminiscent of our earlier transgenic studies. Indeed, we demonstrated that the overexpression of TβRI(AAD) in the mammary gland increased the number of extravascular lung metastases in MMTV/Neu-YD mice but could not enhance the aggressive lung metastatic phenotype exhibited by MMTV/Neu-YB transgenic animals (53
To better understand the underlying basis for Neu/ErbB-2 and TGF-β cooperativity in enhancing cell motility, we examined focal adhesion formation in each of the NMuMG explant cultures. Several types of focal adhesions have been described, including focal complexes or contacts, focal adhesions, and fibrillar adhesions (72
). Focal complexes are small (<0.25 μm2
), short-lived adhesions that form close to the leading edge of cells, whereas focal adhesions are larger (1 to 10 μm2
) and persist for longer periods of time. It has been postulated that the small, nascent focal complexes apply strong propulsive traction that facilitates cell migration, whereas larger, more mature focal adhesions promote a passive anchorage function (7
). Other groups argue that small focal contacts (less than 1 μm2
) exert negligible traction forces, whereas the tractional forces generated by larger adhesions are proportional to their areas (4
). One possible resolution to this apparent contradiction is that small, closely spaced focal complexes at the leading edge of a cell are functionally interconnected, and the resulting integration of multiple small contacts produces greater tractional forces than individual, widely spaced large adhesions (29
). When we examined the nature of focal adhesion formation by FAK immunofluorescence (with adhesions greater than 1 μm2
in size), we observed a TGF-β-mediated increase in the formation of large, mature focal adhesions only in those explants that did not demonstrate a TGF-β-inducible increase in migration (empty vector cells and Neu-NYPD, Neu-YA, and Neu-YB explants). In contrast, cells with a high-baseline migratory phenotype (Neu-YC explants), or those that displayed enhanced motility in response to TGF-β stimulation (Neu-NT, Neu-YD, and Neu-YE explants), had smaller and less numerous mature focal adhesions.
The receptor-like protein tyrosine phosphatase kappa (PTPRK) recently has been implicated as an important TGF-β target, the expression of which is required for TGF-β-induced cell adhesion, motility, and focal adhesion formation in ErbB-2-expressing MCF-10A cells (66
). We did not observe TGF-β-induced PTPRK mRNA expression at 24 h after cytokine treatment (Northey and Siegel, unpublished), and PTPRK does not appear to be a TGF-β-inducible target in parental NMuMG cells (68
). Focal adhesion formation and cytoskeletal changes associated with cell motility are regulated by small GTPases, such as Rho, Rac, and Cdc42. The TGF-β-induced activations of RhoA and Rac1 have been shown to be important for EMT and cell migration in NMuMG cells (2
). Furthermore, Rac1 activity is elevated in MCF-10A cells transformed by ErbB-2, which is further induced by TGF-β stimulation (62
). However, in our NMuMG system, we did not observe a unique or more robust induction of RhoA or Rac1 activation in Neu-NT-expressing cells in response to TGF-β that would explain the observed synergy in motility and invasion.
Our results implicate the ShcA adaptor protein as an important mediator of TGF-β-inducible focal adhesion turnover. Our observation that ShcA null breast cancer cells possess numerous, large vinculin-positive focal adhesions and are impaired in TGF-β-induced motility correlates well with studies demonstrating that vinculin overexpression leads to the formation of large focal adhesions that possess enhanced adhesive strength in fibroblast cells (19
). Conversely, it has been shown that vinculin-deficient fibroblasts are less adherent, form fewer and smaller focal adhesions, and are more motile than wild-type cells (12
). Thus, it is possible that TGF-β signaling enhances focal adhesion turnover in Neu-NT, Neu-YD, and Neu-YE explants in a ShcA-dependent fashion and prevent them from becoming larger, more stable adhesions. In contrast, cells with a less motile phenotype form large, mature adhesions that anchor them and limit their movement. This hypothesis will require further investigation using live-imaging techniques to quantify focal adhesion turnover.
The transient knockdown of ShcA in Neu-NT, Neu-YD, and Neu-YE tumor explants clearly is associated with reduced cell motility and invasion following TGF-β stimulation. Likewise, the stable expression of a dominant-negative ShcA-3F molecule impaired TGF-β-induced motility and the invasion of Neu-NT-transformed cells, while it paradoxically rendered them more motile and invasive in the absence of TGF-β. This is unlikely to be a clonal effect, since the NeuNT/ShcA-3F cells are a pool of three independent ShcA-3F expressors. Unlike the transient but complete knockdown of ShcA expression, the ShcA-3F dominant mutant still retains intact PTB and SH2 domains and thus is capable of mediating phosphotyrosine-independent adaptor functions. Thus, it is conceivable that ShcA-3F expression in NMuMG cells sequesters a factor(s) that normally impairs basal, but not TGF-β-inducible, cell motility and invasion. In addition, stable ShcA-3F expression, but not acute ShcA loss, may allow for the selection of adaptive responses that favor enhanced motility and invasion in these cells.
The use of the Neu/ErbB-2 add-back mutants indicates that signaling pathways initiating from tyrosines 1226/1227 (YD) and 1253 (YE) are responsible for the observed synergy with TGF-β. The ShcA adaptor molecule has been shown to bind directly to site D (Y1227) (14
), while others have shown that ShcA also may bind to tyrosines 1201 (YC) and 1253 (YE) (27
). Our results indicate that the ShcA adaptor protein plays an essential role in the TGF-β-induced migration and invasion of Neu-NT-, Neu-YD-, and Neu-YE-expressing breast cancer cells. Numerous studies have indicated an important role for ShcA in promoting cancer cell motility and metastasis. While transgenic mice expressing the polyomavirus middle T oncogene (PyV mT) in the mammary gland develop aggressive mammary tumors that metastasize to the lung (21
), the mutation of the ShcA binding site on PyV mT diminished mammary tumor formation (67
). Interestingly, 7% of the mammary tumors and 36% of lung metastases displayed point mutations or in-frame deletions within PyV mT that re-created a functional ShcA binding site (67
). These observations argue strongly for an important role for the ShcA adaptor in promoting mammary tumorigenesis and metastasis. More recent studies have demonstrated that the expression of activated Neu-NT, Neu-YC, Neu-YD, or Neu-YE receptors promotes the scattering and enhanced motility of MDCK cells (28
). It is interesting that Neu-YC-expressing explants display a higher baseline motility than the other add-back receptor-expressing cells, which is not further induced by TGF-β stimulation. Moreover, the knockdown of ShcA has no effect on the motility of Neu-YC-expressing cells in either the absence or presence of TGF-β. Thus, the Neu-YC receptor signals enhanced cell motility through a distinct pathway that neither requires ShcA nor synergizes with TGF-β. Interestingly, it has been shown by phosphopeptide interactions that YC may be a docking site for Crk or CrkL (27
), two adaptor proteins that have been shown to be important for cell motility and invasion (41
ShcA can recruit Grb2 through interactions with tyrosine residues 239 and 313 (317 in human ShcA) (44
). While it is conceivable that Grb2 bound to ShcA is important for TGF-β-induced motility and invasion, it should be noted that tumor explants expressing Neu-YB, which directly binds Grb2, did not synergize with TGF-β to enhance breast cancer migration and invasion. This suggests that ShcA interacts with other proteins to promote these TGF-β responses. Indeed, ShcA has been shown to be important for heregulin-induced motility of ErbB-2-expressing T47D breast cancer cells via the recruitment of Memo to tyrosine residue 1227 of ErbB-2 (31
). In this study, Memo was implicated as an important mediator of ErbB-2-induced motility by virtue of its role in microtubule formation. Whether Memo plays a role in TGF-β-induced motility and invasion of Neu/ErbB-2-expressing cells awaits further investigation; however, antitubulin staining did not reveal any discernible differences in microtubule outgrowth between Neu-NT- and Neu-NYPD-expressing mammary tumor explants (Northey and Siegel, unpublished). Future studies will address potential mechanisms downstream of ShcA that are responsible for Neu/ErbB-2 and TGF-β synergy in breast cancer motility and invasion.