title: TGF-β-activated SMAD3/4 complex transcriptionally upregulates N-cadherin expression in non-small cell lung cancer.
abstract: OBJECTIVE
  Epithelial-mesenchymal transition (EMT) is a key process in early stage of cancer metastasis. TGF-β-mediated EMT is characterized by repression of E-cadherin and induction of N-cadherin (CDH2) in various cancers. Although many investigations have focused on the regulation of E-cadherin expression, the transcription-mediated events that directly induce N-cadherin expression in TGF-β-induced EMT are not fully clear. Here, we mainly focus on non-small cell lung cancer (NSCLC) cells, in which expression of CDH2 can be activated upon TGF-β stimulation, to investigate the underlying mechanisms of CDH2 expression regulation.
  
  
  METHODS
  Western blot analysis, real-time quantitative reverse transcriptase PCR, luciferase reporter gene assays, RNA interference and in vivo chromatin immunoprecipitation (ChIP) assay were performed on human NSCLC cell lines A549 and SPC-A1. Twenty-six paired NSCLC tissues and adjacent noncancerous lung tissues were collected.
  
  
  RESULTS
  Luciferase reporter assay revealed that a functional TGF-β-response element was located at position -1078 to -891 in the CDH2 promoter region. Furthermore, in vivo ChIP experiment indicated that TGF-β-activated SMAD3/4 complex was directly recruited to CDH2 promoter region (-1078 to -891). Upon TGF-β1 stimulation, knockdown of SMAD3 or/and SMAD4 led to a significant reduction in CDH2 promoter activity, and silencing of SMAD3 or SMAD4 significantly inhibited CDH2 mRNA and protein expression in A549 and SPC-A1 cells. In human NSCLC tissues, SMAD3 or SMAD4 mRNA level was positively correlated with CDH2 mRNA level, respectively.
  
  
  CONCLUSIONS
  We found that TGF-β-activated SMAD3/4 complex may upregulate CDH2 expression by directly interacting with a specific SMAD-binding element in CDH2 promoter. Our findings provide insights into mechanisms underlying the transcriptional regulation of CDH2 expression in TGF-β-induced EMT and SMADs-based therapeutic strategies for NSCLCs.
authors.affiliation: Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou 215123, China; Suzhou Key Laboratory for Molecular Cancer Genetics, Suzhou 215123, China.
authors.forename: Haiping
authors.initials: H
authors.lastname: Yang
authors.affiliation: Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou 215123, China; Suzhou Key Laboratory for Molecular Cancer Genetics, Suzhou 215123, China.
authors.forename: Longqiang
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Thoracic and Cardiovascular Surgery, The First Affiliated Hospital of Soochow University, Medical College of Soochow University, Suzhou 215006, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Department of Thoracic and Cardiovascular Surgery, The Second Affiliated Hospital of Soochow University, Medical College of Soochow University, Suzhou 215004, China.
authors.forename: Yongbing
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou 215123, China; Suzhou Key Laboratory for Molecular Cancer Genetics, Suzhou 215123, China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Lei
authors.affiliation: Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou 215123, China; Suzhou Key Laboratory for Molecular Cancer Genetics, Suzhou 215123, China.
authors.forename: Xia
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Pathology, The Second Affiliated Hospital of Soochow University, Medical College of Soochow University, Suzhou 215004, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Xia
authors.affiliation: Department of Pathology, The Second Affiliated Hospital of Soochow University, Medical College of Soochow University, Suzhou 215004, China.
authors.forename: Lingling
authors.initials: L
authors.lastname: Guo
authors.affiliation: Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou 215123, China; Suzhou Key Laboratory for Molecular Cancer Genetics, Suzhou 215123, China. Electronic address: htzhang@suda.edu.cn.
authors.forename: Hong-Tao
authors.initials: HT
authors.lastname: Zhang
mesh_terms: D015703:Antigens, CD / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D015820:Cadherins / Q000235:genetics*; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism*; D059372:Nucleotide Motifs; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051900:Smad3 Protein / Q000378:metabolism*; D051901:Smad4 Protein / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D015703:Antigens, CD; C050361:CDH2 protein, human; D015820:Cadherins; D051900:Smad3 Protein; D051901:Smad4 Protein; D016212:Transforming Growth Factor beta
keywords: CDH2; EMT; NSCLC; SMAD; TGF-β; Transcription regulation