Smad7 abrogates transforming growth factor-beta1-mediated growth inhibition in COLO-357 cells through functional inactivation of the retinoblastoma protein.
Smad7 is overexpressed in 50% of human pancreatic cancers. COLO-357 pancreatic cancer cells engineered to overexpress Smad7 are resistant to the actions of transforming growth factor-beta1 (TGF-beta1) with respect to growth inhibition and cisplatin-induced apoptosis but not with respect to modulation of gene expression. To delineate the mechanisms underlying these divergent consequences of Smad7 overexpression, we studied the effects of Smad7 on TGF-beta1-dependent signaling pathways and cell cycle regulating proteins. TGF-beta1 induced the phosphorylation of MAPK, p38 MAPK, and AKT2 irrespective of the levels of Smad7, and inhibitors of these pathways did not alter TGF-beta1 actions on cell growth. By contrast, Smad7 overexpression interfered with TGF-beta1-mediated attenuation of cyclin A and B levels, inhibition of cdc2 dephosphorylation and CDK2 inactivation, up-regulation of p27, and the maintenance of the retinoblastoma protein (RB) in a hypophosphorylated state. Smad7 also suppressed TGF-beta1-mediated inhibition of E2F activity but did not alter TGF-beta1-mediated phosphorylation of Smad2, the nuclear translocation of Smad2/3/4, or DNA binding of the Smad2/3/4 complex. Although Smad7 did not associate with the type I TGF-beta receptor (TbetaRI), SB-431542, an inhibitor of the kinase activity of this receptor, blocked TGF-beta1-mediated effects on Smad-2 phosphorylation. These findings point toward a novel paradigm whereby Smad7 acts to functionally inactivate RB and de-repress E2F without blocking the activation of TbetaRI and the nuclear translocation of Smad2/3, thereby allowing for TGF-beta1 to exert effects in a cancer cell that is resistant to TGF-beta1-mediated growth inhibition.
Boyer Arnold
Nichole
N
Department of Medicine, Dartmouth-Hitchcock Medical Center and Dartmouth Medical School, Hanover, NH 03755, USA.
Korc
Murray
M
D021581:Active Transport, Cell Nucleus; D000818:Animals; D019556:COS Cells; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002469:Cell Separation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D050684:E2F Transcription Factors; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008156:Luciferases / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010766:Phosphorylation; D021381:Protein Transport; D016160:Retinoblastoma Protein / Q000378:metabolism*; D051906:Smad7 Protein; D013347:Subcellular Fractions / Q000378:metabolism; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D053773:Transforming Growth Factor beta1; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D016160:Retinoblastoma Protein; C497806:SMAD7 protein, human; D051906:Smad7 Protein; C508960:TGFB1 protein, human; D013778:Tetrazolium Salts; D013844:Thiazoles; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D008156:Luciferases; D048051:p38 Mitogen-Activated Protein Kinases; C022616:thiazolyl blue