title: Silencing of transforming growth factor-beta1 in situ by RNA interference for breast cancer: implications for proliferation and migration in vitro and metastasis in vivo.
abstract: OBJECTIVE
  Overexpression of transforming growth factor (TGF)-beta has been implicated in promoting immune suppression, tumor angiogenesis, tumor cell migration, and invasion in many cancers, including carcinoma of the breast. Thus, targeted down-regulation of TGF-beta1 expression in breast cancer in situ and determination of its implications would provide new treatment approaches for disease management.
  
  
  METHODS
  Small interfering RNA constructs targeting TGF-beta1 were validated and used to develop clonal derivatives of the metastatic breast cancer cell line MDA-MB-435. The cells were used in several in vitro analyses, including migration, invasion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, apoptosis, and signaling assays. A wound-healing assay was used to determine migration of the cells in culture and a Boyden chamber transwell assay was used for invasion. Further, the clones were used in an in vivo mouse model for the kinetics of tumor growth and gene expression in the primary site and in lungs upon metastasis.
  
  
  RESULTS
  Inhibition of TGF-beta1 expression in MDA-MB-435 cells showed a 35% decrease in migration and a 55% decrease in invasion in vitro, with a 50% increase in proliferation and no effect on apoptosis. In vivo analysis indicated a 90% decrease in the number of mice bearing macroscopic lung metastases; however, the primary tumors did not show any difference in the growth kinetics when compared with the parental MDA-MB-435 cells. Analysis of TGF-beta signaling pathways in the clonal derivatives showed a decrease in Smad2 activation and an increase in AKT and extracellular signal-regulated kinase activation. Interestingly, analysis of TGF-beta receptor expression showed a decrease in both receptor I and II expression in TGF-beta1 silenced cells. These results suggest that inhibition of TGF-beta1 ligand may act as a negative feedback loop to disrupt the function of all TGF-beta isoforms.
  
  
  CONCLUSIONS
  Therapies targeting the TGF-beta signaling pathway may be more effective in late-stage disease to prevent organ metastasis but not primary tumor formation and may be combined with other tumor-targeted therapies normally limited by increased circulating TGF-beta levels.
authors.affiliation: Department of Pathology, The University of Alabama at Birmingham, Birmingham, Alabama 35294-0007, USA.
authors.forename: Lakisha D
authors.initials: LD
authors.lastname: Moore
authors.forename: Tatyana
authors.initials: T
authors.lastname: Isayeva
authors.forename: Gene P
authors.initials: GP
authors.lastname: Siegal
authors.forename: Selvarangan
authors.initials: S
authors.lastname: Ponnazhagan
mesh_terms: D000818:Animals; D017209:Apoptosis; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D005260:Female; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D009368:Neoplasm Transplantation; D034622:RNA Interference; D053773:Transforming Growth Factor beta1 / Q000235:genetics* / Q000378:metabolism*
chemical_list: D053773:Transforming Growth Factor beta1