β(3) Integrin and Src facilitate transforming growth factor-β mediated induction of epithelial-mesenchymal transition in mammary epithelial cells

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