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-beta 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-beta 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-beta 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-beta signaling. Among the novel TGF-beta targets identified are anthrax toxin receptor 2 (3.58x at 8 h), tubulin, beta-3 class III (7.35x at 8 h), cell migration inducing protein, hyaluronan binding (8.07x at 8 h), IL-1 receptor-like 1 (0.403x at 8 h), regulator of G protein signaling 4 (0.293x at 8 h), and THAP domain containing 11 (0.262x at 8 h). The gene expression changes we observed upon restoration of TGF-beta signaling provide numerous new targets for future investigations into PDAC biology and progression.