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. 2014 Mar 14:14:181.
doi: 10.1186/1471-2407-14-181.

SMAD4 loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells

Yu-Wen Chen, Pi-Jung Hsiao, Ching-Chieh Weng, Kung-Kai Kuo, Tzu-Lei Kuo, Deng-Chyang Wu, Wen-Chun Hung, Kuang-Hung Cheng  1
Affiliations

SMAD4 loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells

Yu-Wen Chen et al. BMC Cancer. .

Abstract

Background: 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). 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.

Methods: The 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.

Results: Here, 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.

Conclusions: This 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.

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Figures

Figure 1
Figure 1
Generated stable SMAD4 rerexpressing or knockdown of SMAD4 in human PDAC cells. (A) Western blot analysis indicated that SMAD4 was successfully restored or knocked down in human PDAC cells as compared to control cells. β-actin was used as an internal control. (B) AsPC-1, CFPAC-1 and PANC-1 SMAD4 proficient and deficient cells were transiently transfected with SBE4 luciferase reporter and Renilla luciferase constructs. Cells were treated under the indicated conditions for 24 hours. Luciferase reporter assays were conducted using a dual luciferase assay, and Renilla luciferase activity was used as an internal control. Mean + SE (n = 3) *P <0.01. (C) Western blot detection of total and phosphorylated SMAD2 in SMAD4 proficient or deficient AsPC-1 and PANC-1 cells with or without TGF-β (10 ng/μl). Result confirmed TGF-β increased phosphorylation of SMAD2 in the SMAD4 restoration cell lines. β-actin was used as an internal control. (D) Immuno- fluorescence analysis confirmed that SMAD4 expression in AsPC-1 SMAD4 cells and mock control cells, and the nuclear localization of SMAD4 was observed in response to TGFβ1 treatment (magnification ×100).
Figure 2
Figure 2
SMAD4 does not significantly affect PDAC cell viability or proliferation, but increase PDAC cell motility in vitro. (A) SMAD4 does not significantly affect growth of PDAC cells in vitro. Cells were seeded (5X103 cells per well) in 96-well plates and cell proliferation rates were determined by MTT assay at indicated time points. (B) SMAD4 does not affect PDAC tumor growth in SCID mice. Xenograft tumors were established using SMAD4 proficient or deficient AsPC-1 and PANC-1 cells implanted by s.c injection (1 × 106 cells) and analyzed after 8 weeks. Tumor weights were measured at autopsy. Mean + SE (n = 6). (C) Wound healing assays indicated that SMAD4 restoration reduces PDAC cells migration in vitro. The closure rates of cell free gap were recorded by phase contrast microscope after overnight incubation. Similar results were reproduced in three independent experiments. (magnification x40). (D) SMAD4 promotes the invasive ability of PDAC cells in vitro. Invaded cells were fixed and stained with crystal violet, and quantitative results were normalized against vector controls. Mean + SEM (n > 3). *p < 0.05.
Figure 3
Figure 3
SMAD4 reduces E-cadherin, VEGF, EGFR and CD133 expression, but increases TGFβ1/Nestin and CD44 protein levels in PDAC. (A) SMAD4 modulates mRNA levels of EMT and CSC markers in PDAC cells. RT qPCR analyses were performed in AsPC-1 and PANC-1 SMAD4 deficient or proficient cells. Compared to the control, restoration of SMAD4 reduces mRNA level of EGFR, VEGF, CD133 and E-cadherin, but increased Vimentin, SMA, Nestin and CD44 mRNA expression in PDAC. Data were means ± SD of triplicates. *P < 0.05 (B) Western blot analysis of three PDAC cell lines, which had overexpression or knockdown for SMAD4 revealed that the expression levels of CD133, Nestin, EGFR, VEGF and EMT markers in indicated cell lines. β-actin was included as a loading control. (C) Luiferase activity assays for the analysis of CD133 and Nestin transcriptional activities in PDAC cells. Reproter assays were performed using CD133-luc (a) and Nestin-luc(b) reporter constructs in AsPC-1, CFPAC-1 and PANC-1 Smad4 proficient and deficient cells. Bars represent means derived from at least 3 independent experiments. *P < 0.01.
Figure 4
Figure 4
SMAD4 modulates multiple kinase pathways and influences transcriptional factors expression. (A) Restoration of SMAD4 results in a marked increase of p38 MAPK activation, but significantly attenuated the phosphorylation (activation) of EGFR and its downstream signals p44/42 (MEK) and Akt pathways in PDAC cells. (B) SMAD4 affects the expression transcriptional factors in PDAC cells. Total protein extracted from indicated cells were lysed for Western blot analysis. Western blots were then performed with the indicated antibodies. β-actin was served as an internal control.
Figure 5
Figure 5
SMAD4 loss contributes to chemoresistance of PDAC cells. AsPC-1, CFPAC-1 and PANC-1, SMAD4 deficient and proficient cells were treated with different concentration of cisplatin (Cis; 5 μM), paciltaxol (Pac; 1 μM) and gemcitabine, (Gem; 2 μM) for 3 days. Chemosensitivity testing of PDAC cells using the MTT colorimetric assay. The data indicates SMAD4 increases drug sensitivity to three different chemo drugs in PDAC cells. Data were means ± SD of triplicates. *P < 0.01.
Figure 6
Figure 6
SMAD4 proficient PDAC cells were more likely to respond to TGF-β1 inhibitor treatment, but SMAD4 deficient PDAC cells were more sensitive towards EGFR inhibitor treatment to block cell migration in vitro. AsPC-1 and PANC-1 SMAD4 deficient and proficient cells were treated with TGF-β1 inhibitor SB231542 (0.5 μM) or EGFR inhibitor gefitinib (0.5 μM) and subjected to in vitro wound-healing assay. Each monolayer was scratched and incubated for overnight. The closure rate was photographed to compare their migratory ability between SMAD4 deficient and proficient PDAC cells with or without TGF-β1 or EGFR inhibitors treatment. Images are representative of three independent experiments (magnification 40×). P < 0.05.
Figure 7
Figure 7
A Model of phenotypic alteration involving SMAD4 loss in PDAC cells. SMAD4 wild type PDAC cells exhibits more fibroblast-like morphology, which high express Nestin, SMA, CD44 gene, with the increase of activation of p38 MAPK and TGFβ1 signaling, and acquisition of a more high expression levels for the c-Jun, c-Fos, Hes1 NF-κb transcription factor genes. In contrast, inactivation of SMAD4 in PDAC cells exhibits more well differentiate epithelial like (cobblestone) morphology and leads to an overall increase in E-cadherin, CD133, VEGF, EGFR and Sp-1 expressions with high levels of activation p-44/42 and PI3K/Akt signaling pathways.
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