The Src Family Kinase Inhibitors PP2 and PP1 Block TGF-Beta1-Mediated Cellular Responses by Direct and Differential Inhibition of Type I and Type II TGF-Beta Receptors

Both the nonreceptor tyrosine kinase Src and the receptors for transforming growth factor (TGF)-beta (T beta RI, T beta RII) play major roles during tumorigenesis by regulating cell growth, migration/invasion and metastasis. The common Src family kinase inhibitors PP2 and PP1 effectively block Src activity in vitro and in vivo, however, they may exert non-specific effects on other kinases. In this study, we have evaluated PP2 and PP1 for their ability to inhibit TGF beta 1-mediated responses in the TGF-beta-responsive pancreatic adenocarcinoma cell line Panc1. We show that PP2 and PP1 but not the more specific Src inhibitor SU6656 effectively relieved TGF-beta 1-induced growth arrest and p21(WAF1) induction, while basal growth was enhanced by PP2 and PP1, and suppressed by SU6656. PP2 and PP1 but not SU6656 also suppressed TGF-beta 1-induced epithelial-to-mesenchymal transition (EMT) as evidenced by their ability to inhibit downregulation of the epithelial marker E-cadherin, and upregulation of the EMT-associated transcription factor Slug. Likewise, PP2 and PP1 but not SU6656 effectively blocked TGF-beta 1-induced activation of Smad2 and p38 MAPK and partially suppressed Smad activation and transcriptional activity on TGF-beta/Smad-responsive reporters of a kinase-active T beta RI mutant ectopically expressed in Panc1 cells. Interestingly, PP2 and PP1 strongly inhibited recombinant T beta RI in an in vitro kinase assay, with PP1 being more potent and PP2 being nearly as potent as the established T beta RI inhibitor SB431542. PP2 but not PP1 also weakly inhibited the T beta RII kinase. Together, these data provide evidence that PP2 and PP1 are powerful inhibitors of T beta R function that can block TGF-beta/Smad signaling in a Src-unrelated fashion. Both agents may be useful as dual TGF-beta/Src inhibitors in experimental therapeutics of late stage metastatic disease.