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.
Highlights
SMAD4 is a gastrointestinal malignancy-specific tumor suppressor gene found mutated in one third of colorectal cancer specimens and half of pancreatic tumors
To verify the restoration of SMAD4 in SMAD4-null AsPC-1 and CFPAC-1 cells, we first performed RT-qPCR analysis to examine the SMAD4 mRNA expression levels in those stable SMAD4 reconstituted pancreatic ductal adenocarcinoma (PDAC) cells; our results showed that the SMAD4 mRNA levels increased about 10-fold in comparison with puro control cells
We confirmed the reduced TGF-β1 signaling by phospho-SMAD2 western blot analysis and SBE4-luciferase activity assay in PANC-1 shSMAD4 cells when compared with control cells. (Figure 1B and C)
Summary
SMAD4 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). Excessive levels of TGF-β1 are associated with malignant tumor progression in many cancers, suggesting that inactivation of the SMAD proteins could be an important event in this process [11]. Several independent studies indicate that deletions or intragenic mutations of the SMAD4 gene are present in more than 50% of human PDACs, but are rare in other malignancies such as lung or breast cancer [12,13,14,15,16]. Many lines of evidence indicate that SMAD4 status in PDAC is associated with specific histopathological phenotypes, the detailed molecular basis of SMAD4dependent phenotypic changes in cancer biology has yet to be determined
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