title: Differential roles of Smad2 and Smad3 in the regulation of TGF-β1-mediated growth inhibition and cell migration in pancreatic ductal adenocarcinoma cells: control by Rac1.
abstract: BACKGROUND
  Progression of pancreatic ductal adenocarcinoma (PDAC) is largely the result of genetic and/or epigenetic alterations in the transforming growth factor-beta (TGF-β)/Smad signalling pathway, eventually resulting in loss of TGF-β-mediated growth arrest and an increase in cellular migration, invasion, and metastasis. These cellular responses to TGF-β are mediated solely or partially through the canonical Smad signalling pathway which commences with activation of receptor-regulated Smads (R-Smads) Smad2 and Smad3 by the TGF-β type I receptor. However, little is known on the relative contribution of each R-Smad, the possible existence of functional antagonism, or the crosstalk with other signalling pathways in the control of TGF-β1-induced growth inhibition and cell migration. Using genetic and pharmacologic approaches we have inhibited in PDAC cells endogenous Smad2 and Smad3, as well as a potential regulator, the small GTPase Rac1, and have analysed the consequences for TGF-β1-mediated growth inhibition and cell migration (chemokinesis).
  
  
  RESULTS
  SiRNA-mediated silencing of Smad3 in the TGF-β responsive PDAC cell line PANC-1 reduced TGF-β1-induced growth inhibition but increased the migratory response, while silencing of Smad2 enhanced growth inhibition but decreased chemokinesis. Interestingly, siRNA-mediated silencing of the small GTPase Rac1, or ectopic expression of a dominant-negative Rac1 mutant largely mimicked the effect of Smad2 silencing on both TGF-β1-induced growth inhibition, via upregulation of the cdk inhibitor p21WAF1, and cell migration. Inhibition of Rac1 activation reduced both TGF-β1-induction of a Smad2-specific transcriptional reporter and Smad2 C-terminal phosphorylation in PDAC cells while Smad3-specific transcriptional activity and Smad3 C-terminal phosphorylation appeared increased. Disruption of autocrine TGF-β signalling in PANC-1 cells rendered cells less susceptible to the growth-suppressive effect of Rac1 inhibition, suggesting that the decrease in "basal" proliferation upon Rac1 inhibition was caused by potentiation of autocrine TGF-β growth inhibition.
  
  
  CONCLUSIONS
  In malignant cells with a functional TGF-β signalling pathway Rac1 antagonizes the TGF-β1 growth inhibitory response and enhances cell migration by antagonistically regulating Smad2 and Smad3 activation. This study reveals that Rac1 is prooncogenic in that it can alter TGF-β signalling at the R-Smad level from a tumour-suppressive towards a tumour-promoting outcome. Hence, Rac1 might represent a viable target for therapeutic intervention to inhibit PDAC progression.
authors.affiliation: Clinic for Applied Cellular Medicine, University Hospital Schleswig-Holstein (UKSH) Campus Kiel, 24105 Kiel, Germany. hendrik.ungefroren@uk-sh.de
authors.forename: Hendrik
authors.initials: H
authors.lastname: Ungefroren
authors.forename: Stephanie
authors.initials: S
authors.lastname: Groth
authors.forename: Susanne
authors.initials: S
authors.lastname: Sebens
authors.forename: Hendrik
authors.initials: H
authors.lastname: Lehnert
authors.forename: Frank
authors.initials: F
authors.lastname: Gieseler
authors.forename: Fred
authors.initials: F
authors.lastname: Fändrich
mesh_terms: D019898:Autocrine Communication / Q000502:physiology; D021441:Carcinoma, Pancreatic Ductal / Q000503:physiopathology*; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D010190:Pancreatic Neoplasms / Q000503:physiopathology*; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051899:Smad2 Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051900:Smad3 Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; D051899:Smad2 Protein; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1; D020830:rac1 GTP-Binding Protein