Insights Into SMAD4 Loss in Pancreatic Cancer From Inducible Restoration of TGF-β Signaling.
Pancreatic ductal adenocarcinoma (PDAC) is the fourth-leading cause of cancer death in the United States. The TGF-β signaling protein SMAD family member 4 is lost in 60% of PDAC, and this has been associated with poorer prognosis. However, the mechanisms by which SMAD4 loss promotes PDAC development are not fully understood. We expressed SMAD4 in human PDAC cell lines BxPC3 and CFPAC1 by selection of stable clones containing an inducible SMAD4 tetracycline inducible expression system construct. After 24 hours of SMAD4 expression, TGF-β signaling-dependent G1 arrest was observed in BxPC3 cells with an increase in the G1 phase fraction from 48.9% to 71.5%. Inhibition of cyclin-dependent kinase inhibitor 1A by small interfering RNA eliminated the antiproliferative effect, indicating that up-regulation of cyclin-dependent kinase inhibitor 1A/p21 by TGF-β signaling is necessary for the phenotype. SMAD4 expression had no impact on invasion in BxPC3 cells, but reduced migration. Microarray analysis of gene expression at 8, 24, and 48 hours after SMAD4 expression characterized the regulatory impact of SMAD4 expression in a SMAD4-null PDAC cell line and identified novel targets of TGF-β signaling. Among the novel TGF-β targets identified are anthrax toxin receptor 2 (3.58× at 8 h), tubulin, β-3 class III (7.35× at 8 h), cell migration inducing protein, hyaluronan binding (8.07× at 8 h), IL-1 receptor-like 1 (0.403× at 8 h), regulator of G protein signaling 4 (0.293× at 8 h), and THAP domain containing 11 (0.262× at 8 h). The gene expression changes we observed upon restoration of TGF-β signaling provide numerous new targets for future investigations into PDAC biology and progression.
Departments of Molecular and Human Genetics (P.T.F., M.M.M.), Pathology and Immunology (P.T.F., M.M.M.), Molecular and Cellular Biology (M.M.M.), Pharmacology (M.M.M.), and Medicine (C.J.C.); the Center for Drug Discovery (P.T.F., M.M.M.); and the Dan L. Duncan Cancer Center (P.T.F., C.J.C., M.M.M.), Baylor College of Medicine, Houston, Texas 77030.
Paul T
PT
Fullerton
Departments of Molecular and Human Genetics (P.T.F., M.M.M.), Pathology and Immunology (P.T.F., M.M.M.), Molecular and Cellular Biology (M.M.M.), Pharmacology (M.M.M.), and Medicine (C.J.C.); the Center for Drug Discovery (P.T.F., M.M.M.); and the Dan L. Duncan Cancer Center (P.T.F., C.J.C., M.M.M.), Baylor College of Medicine, Houston, Texas 77030.
Chad J
CJ
Creighton
Departments of Molecular and Human Genetics (P.T.F., M.M.M.), Pathology and Immunology (P.T.F., M.M.M.), Molecular and Cellular Biology (M.M.M.), Pharmacology (M.M.M.), and Medicine (C.J.C.); the Center for Drug Discovery (P.T.F., M.M.M.); and the Dan L. Duncan Cancer Center (P.T.F., C.J.C., M.M.M.), Baylor College of Medicine, Houston, Texas 77030.
Martin M
MM
Matzuk
D045744:Cell Line, Tumor; D049109:Cell Proliferation; D016922:Cellular Senescence; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics; D059585:G1 Phase Cell Cycle Checkpoints; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D005838:Genotype; D006801:Humans; D009361:Neoplasm Invasiveness; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D051901:Smad4 Protein / Q000172:deficiency* / Q000378:metabolism*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D015854:Up-Regulation / Q000235:genetics
D050759:Cyclin-Dependent Kinase Inhibitor p21; D034741:RNA, Small Interfering; D051901:Smad4 Protein; D016212:Transforming Growth Factor beta