title: CITED4 gene silencing in colorectal cancer cells modulates adherens/tight junction gene expression and reduces cell proliferation.
abstract: OBJECTIVE
  CITED4 is one member of a family of transcriptional cofactors, several of which are deregulated in a variety of tumors, including colorectal cancer (CRC). We modulated CITED4 expression, in vitro, and analyzed the associated phenotypic and gene expression changes.
  
  
  METHODS
  CITED4-overexpressing and shRNA-mediated knockdown cell lines and control cell lines were established in the CRC cell line SW480. The cells were analyzed for changes in proliferation, apoptosis/cell cycle, migration, invasion, colony formation and adhesion. mRNA expression changes were determined by microarray and pathway analysis, and several deregulated genes were validated by qRT-PCR and Western blotting. Based on results obtained from these studies, the status of the actin cytoskeleton was evaluated by phalloidin/vinculin staining.
  
  
  RESULTS
  Phenotypically, the CITED4-overexpressing cell line showed only moderate changes in adhesion. Microarray analysis identified several deregulated genes, including several G protein-coupled receptors. Phenotypic analysis of the CITED4 shRNA knockdown cell line demonstrated decreased cell proliferation and G2 cell cycle blockage. Microarray analysis identified many deregulated genes, and pathway analysis discovered genes linked to actin-associated adherens junctions/tight junctions (claudin-4, claudin-7, ezrin, MET, ß-catenin). Phenotypically, no morphological changes of the actin cytoskeleton were seen.
  
  
  CONCLUSIONS
  Upregulation of CITED4 in SW480 resulted in no obvious phenotype. CITED4 shRNA-mediated knockdown led to decreased cellular proliferation and modulation of a large number of genes, including the c-MET tyrosine kinase and several actin-associated adherens junctions/tight junction genes.
authors.affiliation: Division of Molecular Genetics (B060), German Cancer Research Center, Im Neuenheimer Feld 580, 69120, Heidelberg, Germany.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Rogers
authors.affiliation: Division of Molecular Genetics (B060), German Cancer Research Center, Im Neuenheimer Feld 580, 69120, Heidelberg, Germany.
authors.forename: Verena
authors.initials: V
authors.lastname: Kalter
authors.affiliation: Division of Molecular Genetics (B060), German Cancer Research Center, Im Neuenheimer Feld 580, 69120, Heidelberg, Germany.
authors.forename: Gemma
authors.initials: G
authors.lastname: Marcias
authors.affiliation: Division of Molecular Genetics (B060), German Cancer Research Center, Im Neuenheimer Feld 580, 69120, Heidelberg, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Zapatka
authors.affiliation: Division of Molecular Genetics (B060), German Cancer Research Center, Im Neuenheimer Feld 580, 69120, Heidelberg, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Barbus
authors.affiliation: Division of Molecular Genetics (B060), German Cancer Research Center, Im Neuenheimer Feld 580, 69120, Heidelberg, Germany. peter.lichter@dkfz.de.
authors.forename: Peter
authors.initials: P
authors.lastname: Lichter
mesh_terms: D000199:Actins / Q000378:metabolism; D022005:Adherens Junctions / Q000235:genetics*; D017209:Apoptosis; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D020869:Gene Expression Profiling; D020868:Gene Silencing; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019108:Tight Junctions / Q000235:genetics*; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D000199:Actins; D014408:Biomarkers, Tumor; C451405:CITED4 protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors
keywords: Actin; Adherens junctions; CITED4; Claudins; Colorectal cancer; Ezrin; Gene expression; Microarray; Tight junctions; c-MET