Insights Into SMAD4 Loss in Pancreatic Cancer From Inducible Restoration of TGF-β Signaling

Abstract

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.

SMAD4-independent activation of TGF-β signaling by MUC1 in a human pancreatic cancer cell line

“…When the role of TGF-β changes from tumor suppressor to tumor promoter, as reviewed in Lebrun 2012, the tumor promoting effects of TGF-β includes induction of EMT, resistance to apoptosis, migration, invasion, and tumor metastasis [ 58 ]. It has been shown that SMAD-4 deleted WT BxPC3 cells constitutively activates ERK, has an increased anti-apoptotic response but does not promote invasiveness [ 43 , 59 ]. Finally, it has also been shown that MUC1 expression can confer resistance of epithelial cancer cells to cell death via anoikis [ 60 ].…”

PDAC-derived exosomes enrich the microenvironment in MDSCs in a SMAD4-dependent manner through a new calcium related axis

“…colorectal adenocarcinoma), which occurs in about 50% of PDAC [ 2 ], appears to be of particular interest. PDAC patients carrying SMAD4 loss have a worse prognosis [ 6 , 7 ], and this event, in vitro, favors cell proliferation and migration, while antagonizing cell senescence [ 8 ]. It is widely believed that the molecular basis linking SMAD4 loss with increased tumor growth and aggressiveness depends on the absence of Smad4 protein, which mediates the effects of the transforming growth factor (TGF)-β and bone morphogenetic protein, which inhibit cell proliferation and migration, and trigger apoptosis [ 9 ].…”

Targeting reactive oxygen species in development and progression of pancreatic cancer

“…Sustained activation of signaling pathways downstream of mutant KRas then drive cancer cell survival, proliferation, migration and invasion [11]. Inactivating mutations in the tumor suppressor genes CDKN2A , TP53 and SMAD4 also promote pancreatic carcinogenesis by facilitating enhanced tumor growth and survival and are associated with poor prognosis [6,12–14]. In addition to above genetic components, development of PDA is also driven by environmental factors.…”