The process of EMT recently has been subject to intense research investigation because of its importance in mediating cancer cell motility, invasion, and metastasis [11
]. TGF-β is a major regulator of both developmental and pathological EMT, particularly that in diseased MECs, which frequently exhibit decreased cytostasis and increased invasiveness in response to TGF-β [56
]. Unfortunately, the molecular mechanisms underlying the conversion of TGF-β from an inhibitor of MEC proliferation to an inducer of their EMT are poorly understood. Because cell microenvironmental changes regulate cancer development and progression, including the acquisition of EMT, and because integrins and TGF-β are major regulators of cell microenvironments, we hypothesized that differential integrin expression induced by TGF-β would contribute to its regulation of MEC proliferation and EMT.
To this end, we now show that β3 integrin expression alters the response of MECs to TGF-β, particularly its ability to regulate cell proliferation, invasion, and EMT. Indeed, β3 integrin expression appears essential for TGF-β stimulation of EMT (Figure ). Moreover, we demonstrate that β3 integrin directly couples to the TGF-β signaling system by interacting physically with TβR-II (Figure ), resulting in enhanced MEC proliferation, invasion, and EMT when stimulated with TGF-β (Figure ). More importantly, β3 integrin promotes Src mediated tyrosine phosphorylation of TβR-II (Figure ), which we show to be essential for the ability of TGF-β to activate MAPKs (Figure ) and, consequently, to induce EMT in MECs (Figure ). More importantly, we show for the first time that induction of β3 integrin expression during mammary tumorigenesis correlates with metastasis development, and that antagonizing β3 integrin signaling abolishes TGF-β stimulation of breast cancer cell invasion (Figure ). Collectively, we identified a potentially important mechanism whereby β3 integrin expression selectively facilitates TGF-β stimulation of MEC invasion and EMT.
The joint activation of integrin and RTK signaling systems is essential for the positional control of cells. More importantly, cell attachment to the ECM also determines the nature and context of how cells interpret and respond to cytokine and growth factor binding [52
]. For instance, the ability of EGF and platelet-derived growth factor receptors to stimulate fibroblast migration requires RTK receptor association with integrins and FAK [24
]. Similarly, Scaffidi and coworkers [42
] showed that αv
integrin bound TβR-II in lung fibroblasts, thereby enhancing fibroblast proliferation stimulated by TGF-β. Those authors further speculated that the formation of αv
integrin:TβR-II complexes may exacerbate TGF-β mediated wound healing and fibrotic reactions [42
], processes reminiscent of the ability of β3
integrin:TβR-II complexes to drive TGF-β stimulation of EMT in MECs. Most recently, the adapter protein Dab2, which mediates Smad2/3 activation by TβRs [57
], was shown to participate in TGF-β stimulated EMT by interacting with integrins and preventing MEC apoptosis [58
]. These findings, together with those presented herein, indicate an important and underappreciated role for integrins in regulating cellular response to TGF-β. Future studies in MECs must determine the relationship between TGF-β stimulated expression of β3
integrin and Dab2, as well as β3
integrin stimulated TβR-II expression in MECs; whether Dab2 links β3
integrin to TβR-II during EMT; and the ability of additional integrins to influence, either positively or negatively, MEC response to TGF-β.
A particularly novel finding of our study was the demonstration that β3
integrin induced TβR-II tyrosine phosphorylation via Src kinase. Indeed, we show for the first time that Src activity mediates tyrosine phosphorylation of TβR-II in MECs, leading to TGF-β mediated MAPK activation, and to MEC invasion and EMT. Previous studies established that TβR-II is phosphorylated predominantly on Ser and Thr residues [8
], which, depending on the site of phosphorylation, either augments or attenuates TβR-II protein kinase activity [60
]. However, tyrosine phosphorylation of TβR-II is not without precedent. Indeed, Lawler and coworkers [61
] showed that TβR-II is a dual-specificity protein kinase that autophosphorylates not only on Ser/Thr residues but also on tyrosine 259, 336, and 424. Moreover, although Phe substitution at these positions significantly reduced TβR-II protein kinase activity, these mutations had little affect on the ability of TGF-β to induce gene expression in lung epithelial cells [61
], and as such the role of tyrosines 259, 336, and 424 in mediating TGF-β action remain to be clarified. We too have converted tyrosines 259, 336, and 424 to phenylalanine in all possible combinations (i.e. single, double, and triple mutations), all of which failed to alter Src mediated tyrosine phosphorylation of GST-TβR-II (K277R) in vitro
(Galliher AJ, Schiemann WP, unpublished data). Sequence analysis of the cytoplasmic domain of TβR-II revealed two possible Src phosphorylation consensus motifs at tyrosines 284 and 470. These two sites were individually mutated to phenylalanines, and preliminary data suggest that Src phosphorylates TβR-II at tyrosine 284 (Galliher AJ, Schiemann WP, unpublished data). Further analysis of this site in mediating TGF-β stimulation of EMT in MECs is currently under investigation.
Activation of MAPKs, particularly that of ERK1/2 [5
] and p38 MAPK [2
], are necessary for TGF-β to stimulate EMT. We also found that TGF-β stimulation of ERK1/2 and p38 MAPK are necessary for induction of EMT in MECs. More importantly, we show for the first time that Src activity is essential for activation of MAPKs by TGF-β and for its stimulation of EMT. Indeed, our results indicate that β3
integrin expression and Src activity are sufficient in overcoming TGF-β mediated cytostasis in MECs. Src activity also has been associated with protecting hepatocytes from apoptosis induced by TGF-β [62
] and with the ability of TGF-β to stimulate ovarian cancer cell invasion [65
]. More recently, TGF-β treatment of MECs was shown to induce their expression of RPTPκ, a protein tyrosine phosphatase that activates Src and mediates the antiproliferative and the promigratory effects of TGF-β [66
]. These findings, together with those presented herein, implicate Src as an important player operant in dictating the MEC response to TGF-β; they also suggest that Src inhibition, similar to integrin interdiction, may one day be used to enhance the tumor suppressing activities of TGF-β in breast cancer cells.
Finally, based on the fact that phosphotyrosine residues often create binding motifs that couple receptors to various signaling pathways [67
], including the MAPK cascade, it is tempting to speculate that Src-mediated tyrosine phosphorylation of TβR-II functions similarly as a receptor docking site to recruit SH2 and/or PTB containing signaling molecules to β3
integrin:TβR-II complexes. If correct, then such a mechanism could account for the augmented ability of TGF-β to activate MAPKs in β3
integrin expressing MECs, and for the shift in MAPK and Smad2/3 signaling that favored EMT over cytostasis in response to TGF-β. Indeed, the ability of β3
integrin to increase Smad2/3 transcriptional activity without altering Smad2/3 phosphorylation suggests that other integrin or MAPK stimulated nuclear factors converge on TGF-β targeted promoters and synergize with Smad2/3 in coordinating gene expression regulated by TGF-β. This notion is wholly consistent with previous work demonstrating the ability of Smad2/3 to synergize with activated TβR complexes in mediating EMT [37