title: SMAD4 loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells.
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.
authors.forename: Yu-Wen
authors.initials: YW
authors.lastname: Chen
authors.forename: Pi-Jung
authors.initials: PJ
authors.lastname: Hsiao
authors.forename: Ching-Chieh
authors.initials: CC
authors.lastname: Weng
authors.forename: Kung-Kai
authors.initials: KK
authors.lastname: Kuo
authors.forename: Tzu-Lei
authors.initials: TL
authors.lastname: Kuo
authors.forename: Deng-Chyang
authors.initials: DC
authors.lastname: Wu
authors.forename: Wen-Chun
authors.initials: WC
authors.lastname: Hung
authors.affiliation: Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 80424, Taiwan. khcheng@faculty.nsysu.edu.tw.
authors.forename: Kuang-Hung
authors.initials: KH
authors.lastname: Cheng
mesh_terms: D000818:Animals; D021441:Carcinoma, Pancreatic Ductal / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D002464:Cell Migration Inhibition / Q000235:genetics; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D005260:Female; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D016513:Mice, SCID; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D010641:Phenotype; D051901:Smad4 Protein / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism
chemical_list: D051901:Smad4 Protein; C490810:Smad4 protein, mouse