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

Molecular Cancer, May 2011

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.

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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

Molecular Cancer Differential roles of Smad2 and Smad3 in the regulation of TGF-b1-mediated growth inhibition and cell migration in pancreatic ductal adenocarcinoma cells: control by Rac1 Hendrik Ungefroren 0 1 Stephanie Groth 0 3 Susanne Sebens 2 Hendrik Lehnert 1 Frank Gieseler 1 Fred Fndrich 0 0 Clinic for Applied Cellular Medicine, University Hospital Schleswig-Holstein (UKSH) Campus Kiel , 24105 Kiel , Germany 1 First Department of Medicine, University Hospital Schleswig-Holstein (UKSH) , Campus Lubeck, 23538 Lubeck , Germany 2 Institute of Experimental Medicine c/o Laboratory of Molecular Gastroenterology and Hepatology, Department of Internal Medicine I, University Hospital Schleswig-Holstein (UKSH) Campus Kiel , 24105 Kiel , Germany 3 Current address: Department of Dermatology , UKSH, Campus Lubeck, 23538 Lubeck , Germany Background: Progression of pancreatic ductal adenocarcinoma (PDAC) is largely the result of genetic and/or epigenetic alterations in the transforming growth factor-beta (TGF-b)/Smad signalling pathway, eventually resulting in loss of TGF-b-mediated growth arrest and an increase in cellular migration, invasion, and metastasis. These cellular responses to TGF-b 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-b 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-b1-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-b1-mediated growth inhibition and cell migration (chemokinesis). Results: SiRNA-mediated silencing of Smad3 in the TGF-b responsive PDAC cell line PANC-1 reduced TGF-b1induced 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-b1induced growth inhibition, via upregulation of the cdk inhibitor p21WAF1, and cell migration. Inhibition of Rac1 activation reduced both TGF-b1-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-b 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-b growth inhibition. Conclusions: In malignant cells with a functional TGF-b signalling pathway Rac1 antagonizes the TGF-b1 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-b signalling at the R-Smad level from a tumoursuppressive towards a tumour-promoting outcome. Hence, Rac1 might represent a viable target for therapeutic intervention to inhibit PDAC progression. - Background TGF-b and its signalling effectors regulate many aspects of tumour cell biology, such as growth arrest, and cell motility the latter of which is important for the metastatic dissemination of tumour cells from their primary location to lymph or blood vessels [1,2]. TGF-bs cellular activities are mediated by specific receptor complexes that are assembled upon ligand binding and comprise the TGF-b type II receptor (TbRII) and TGF-b type I receptor (TbRI/ALK5). The activated ligand-receptor complex typically activates the Smad signalling pathway. The canonical Smad signalling cascade is initiated by Cterminal phosphorylation of receptor-regulated Smad transcription factors (R-Smads) Smad2 and/or Smad3 by activated ALK5 [3]. This allows R-Smad binding to Smad4 and translocation of the complex to the nucleus where it can recruit transcriptional coactivators or corepressors to Smad binding elements (SBEs) in the promoters of TGF-b target genes [1,2]. The TGF-b signalling effectors are also key players of tumour cell behaviour and are often deregulated in cancer cells [2,4]. For instance, human pancreatic ductal adenocarcinoma (PDAC) is characterized besides the common KRas mutations (representing an early event in PDAC tumourigenesis) by both TGF-boverexpression and mutational inactivation of the tumour suppressor Smad4/DPC4, the latter being a relatively late event. Recent studies in mice have shown that blockade of TGF-b/Smad signalling and activated Ras signalling cooperate to promote PDAC progression [5,6]. T (...truncated)


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Hendrik Ungefroren, Stephanie Groth, Susanne Sebens, Hendrik Lehnert, Frank Gieseler, Fred Fändrich. 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, Molecular Cancer, 2011, pp. 67, 10, DOI: 10.1186/1476-4598-10-67