Inhibition of STAT3 Phosphorylation by Smad4 Suppresses Transforming Growth Factor B–Mediated Invasion and Metastasis in Pancreatic Cancer Cells

The role of Smad4 in transforming growth factor B (TGFB)– mediated epithelial-mesenchymal transition (EMT), invasion, and metastasis was investigated using isogenically matched pancreatic cancer cell lines that differed only in expression of Smad4. Cells expressing Smad4 showed an enhanced TGFBmediated EMT as determined by increased expression of vimentin and decreased expression of B-catenin and E-cadherin. TGFB-mediated invasion was suppressed in Smad4-intact cells as determined by in vitro assays, and these cells showed a reduced metastasis in an orthotopic model of pancreatic cancer. Interestingly, TGFB inhibited STAT3 phosphorylation in Smad4-intact cells. The decrease in STAT3 phosphorylation was linked to a TGFB/Smad4dependent and enhanced activation of extracellular signalregulated kinases, which caused an increase in serine phosphorylation of STAT3. Down-regulating signal transducer and activator of transcription 3 (STAT3) expression by short hairpin RNA in Smad4-deficient cells prevented TGFBinduced invasion. Conversely, expressing a constitutively activated form of STAT3 (STAT3-C) in Smad4-intact cells enhanced invasion. This study indicates the requirement of STAT3 activity for TGFB-induced invasion in pancreatic cancer cells and implicates Smad4-dependent signaling in regulating STAT3 activity. These findings further suggest that loss of Smad4, leading to aberrant activation of STAT3, contributes to the switch of TGFB from a tumor-suppressive to a tumor-promoting pathway in pancreatic cancer. [Cancer Res 2008;68(11):4221–8]