SMAD4 Loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells

Abstract

BackgroundSMAD4 is a gastrointestinal malignancy-specific tumor suppressor gene found mutated in one third of colorectal cancer specimens and half of pancreatic tumors. SMAD4 inactivation by allelic deletion or intragenic mutation mainly occurs in the late stage of human pancreatic ductal adenocarcinoma (PDAC). Various studies have proposed potential SMAD4-mediated anti-tumor effects in human malignancy; however, the relevance of SMAD4 in the PDAC molecular phenotype has not yet been fully characterized.MethodsThe AsPC-1, CFPAC-1 and PANC-1 human PDAC cell lines were used. The restoration or knockdown of SMAD4 expression in PDAC cells were confirmed by western blotting, luciferase reporter and immunofluorescence assays. In vitro cell proliferation, xenograft, wound healing, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry analysis were conducted using PDAC cells in which SMAD4 was either overexpressed or knocked down.ResultsHere, we report that re-expression of SMAD4 in SMAD4-null PDAC cells does not affect tumor cell growth in vitro or in vivo, but significantly enhances cells migration in vitro. SMAD4 restoration transcriptionally activates the TGF-β1/Nestin pathway and induces expression of several transcriptional factors. In contrast, SMAD4 loss in PDAC leads to increased expression of E-cadherin, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and CD133. Furthermore, SMAD4 loss causes alterations to multiple kinase pathways (particularly the phosphorylated ERK/p38/Akt pathways), and increases chemoresistance in vitro. Finally, PDAC cells with intact SMAD4 are more sensitive to TGF-β1 inhibitor treatment to reduced cell migration; PDAC cells lacking SMAD4 showed decreased cell motility in response to EGFR inhibitor treatment.ConclusionsThis study revealed the molecular basis for SMAD4-dependent differences in PDAC with the aim of identifying the subset of patients likely to respond to therapies targeting the TGF-β or EGFR signaling pathways and of identifying potential therapeutic interventions for PDAC patients with SMAD4 defects.

Epithelial-mesenchymal transition as a therapeutic target for overcoming chemoresistance in pancreatic cancer

“…Since Snail and Zeb family of transcriptional factors mediate E-cadherin translocation, loss of function or downregulation, they can potentially be targeted to avert EMT at its initial steps. Several studies have reported disruption in TGFβ signalling in pancreatic cancer[30-32]. Therefore, the mesenchymal transcriptional factors may be better druggable targets compared to TGF-β receptors to reduce EMT-derived chemoresistance in pancreatic cancer.…”

Smad4 inhibits cell migration via suppression of JNK activity in human pancreatic carcinoma PANC-1 cells

“…Furthermore, in isolated murine T cells, JNK phosphorylation was inversely associated with Smad4 protein expression (data not shown). Previous studies have indicated that constitutive JNK activity may promote the malignancy of B and T cells (21,31), while mice with a Smad4 deletion were observed to spontaneously develop gastrointestinal cancer (32,33). The possibility that a Smad4 deletion may initiate pancreatic cancer in humans is unclear.…”

Biomarkers for personalized medicine in GI cancers

“…In the same context, there is an ongoing phase II study of SMAD4 expression as a biomarker in unresectable pancreatic cancer to guide therapy in terms of high dose radiochemotherapy versus systemic therapy based on the likelihood of local or metastatic progression of disease (RTOG 1201). Targeting the TGF-β pathway in the setting of SMAD4 inactivation would be a rational strategy since loss of Smad4 function causes TGF-β to change from a tumor-suppressive to tumorigenic pathway (Chen et al, 2014b;Zhao et al, 2008). In line with these findings, it has been demonstrated that re-activation of SMAD4 restores TGF-β mediated growth inhibition and apoptosis in PDAC cell lines (Dai et al, 1999;Lagna et al, 1996).…”