β3 Integrin and Src facilitate transforming growth factor-β mediated induction of epithelial-mesenchymal transition in mammary epithelial cells

Introduction Transforming growth factor (TGF)-beta suppresses breast cancer formation by preventing cell cycle progression in mammary epithelial cells (MECs). During the course of mammary tumorigenesis, genetic and epigenetic changes negate the cytostatic actions of TGF-beta, thus enabling TGF-beta to promote the acquisition and development of metastatic phenotypes. The molecular mechanisms underlying this conversion of TGF-beta function remain poorly understood but may involve signaling inputs from integrins. Methods beta(3) Integrin expression or function in MECs was manipulated by retroviral transduction of active or inactive beta(3) integrins, or by transient transfection of small interfering RNA ( siRNA) against beta(3) integrin. Altered proliferation, invasion, and epithelial-mesenchymal transition (EMT) stimulated by TGF-beta in control and beta(3) integrin manipulated MECs was determined. Src involvement in beta(3) integrin mediated alterations in TGF-beta signaling was assessed by performing Src protein kinase assays, and by interdicting Src function pharmacologically and genetically. Results TGF-beta stimulation induced alpha(v)beta(3) integrin expression in a manner that coincided with EMT in MECs. Introduction of siRNA against beta(3) integrin blocked its induction by TGF-beta and prevented TGF-beta stimulation of EMT in MECs. beta(3) integrin interacted physically with the TGF-beta receptor (T beta R) type II, thereby enhancing TGF-beta stimulation of mitogen-activated protein kinases (MAPKs), and of Smad2/3-mediated gene transcription in MECs. Formation of beta(3) integrin: T beta R-II complexes blocked TGF-beta mediated growth arrest and increased TGF-beta mediated invasion and EMT. Dual beta(3) integrin: T beta R-II activation induced tyrosine phosphorylation of T beta R-II, a phosphotransferase reaction mediated by Src in vitro. Inhibiting Src activity in MECs prevented the ability of beta(3) integrin to induce T beta R-II tyrosine phosphorylation, MAPK activation, and EMT stimulated by TGF-beta. Lastly, wild-type and D119A beta(3) integrin expression enhanced and abolished, respectively, TGF-beta stimulation of invasion in human breast cancer cells. Conclusion We show that beta(3) integrin alters TGF-beta signaling in MECs via Src-mediated T beta R-II tyrosine phosphorylation, which significantly enhanced the ability of TGF-beta to induce EMT and invasion. Our findings suggest that beta(3) integrin interdiction strategies may represent an innovative approach to reestablishing TGF-beta mediated tumor suppression in progressing human breast cancers.