title: Beta3 integrin and Src facilitate transforming growth factor-beta mediated induction of epithelial-mesenchymal transition in mammary epithelial cells.
abstract: BACKGROUND
  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
  beta3 Integrin expression or function in MECs was manipulated by retroviral transduction of active or inactive beta3 integrins, or by transient transfection of small interfering RNA (siRNA) against beta3 integrin. Altered proliferation, invasion, and epithelial-mesenchymal transition (EMT) stimulated by TGF-beta in control and beta3 integrin manipulated MECs was determined. Src involvement in beta3 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 alphavbeta3 integrin expression in a manner that coincided with EMT in MECs. Introduction of siRNA against beta3 integrin blocked its induction by TGF-beta and prevented TGF-beta stimulation of EMT in MECs. beta3 integrin interacted physically with the TGF-beta receptor (TbetaR) type II, thereby enhancing TGF-beta stimulation of mitogen-activated protein kinases (MAPKs), and of Smad2/3-mediated gene transcription in MECs. Formation of beta3 integrin:TbetaR-II complexes blocked TGF-beta mediated growth arrest and increased TGF-beta mediated invasion and EMT. Dual beta3 integrin:TbetaR-II activation induced tyrosine phosphorylation of TbetaR-II, a phosphotransferase reaction mediated by Src in vitro. Inhibiting Src activity in MECs prevented the ability of beta3 integrin to induce TbetaR-II tyrosine phosphorylation, MAPK activation, and EMT stimulated by TGF-beta. Lastly, wild-type and D119A beta3 integrin expression enhanced and abolished, respectively, TGF-beta stimulation of invasion in human breast cancer cells.
  
  
  CONCLUSIONS
  We show that beta3 integrin alters TGF-beta signaling in MECs via Src-mediated TbetaR-II tyrosine phosphorylation, which significantly enhanced the ability of TGF-beta to induce EMT and invasion. Our findings suggest that beta3 integrin interdiction strategies may represent an innovative approach to re-establishing TGF-beta mediated tumor suppression in progressing human breast cancers.
authors.affiliation: UCHSC, Fitzsimons Campus, Department of Pharmacology, Mail Stop 8303, RC1 South Tower, Rm L18-6110, PO Box 6511, Aurora, CO 80045, USA. Amy.Galliher@UCHSC.edu
authors.forename: Amy J
authors.initials: AJ
authors.lastname: Galliher
authors.forename: William P
authors.initials: WP
authors.lastname: Schiemann
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000503:physiopathology; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000502:physiology*; D005260:Female; D018014:Gene Transfer Techniques; D006801:Humans; D039661:Integrin beta3 / Q000096:biosynthesis*; D008321:Mammary Glands, Animal / Q000166:cytology / Q000503:physiopathology*; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D051379:Mice; D009361:Neoplasm Invasiveness / Q000503:physiopathology; D015398:Signal Transduction / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D019061:src-Family Kinases / Q000096:biosynthesis*
chemical_list: D039661:Integrin beta3; D016212:Transforming Growth Factor beta; D019061:src-Family Kinases