title: Toll-like receptor 4 variant D299G induces features of neoplastic progression in Caco-2 intestinal cells and is associated with advanced human colon cancer.
abstract: OBJECTIVE
  The Toll-like receptor (TLR) 4 mediates homeostasis of the intestinal epithelial cell (IEC) barrier. We investigated the effects of TLR4-D299G on IEC functions.
  
  
  METHODS
  We engineered IECs (Caco-2) to stably overexpress hemagglutinin-tagged wild-type TLR4, TLR4-D299G, or TLR4-T399I. We performed gene expression profiling using DNA microarray analysis. Findings were confirmed by real-time, quantitative, reverse-transcriptase polymerase chain reaction, immunoblot, enzyme-linked immunosorbent assay, confocal immunofluorescence, and functional analyses. Tumorigenicity was tested using the CD1 nu/nu mice xenograft model. Human colon cancer specimens (N = 214) were genotyped and assessed for disease stage.
  
  
  RESULTS
  Caco-2 cells that expressed TLR4-D299G underwent the epithelial-mesenchymal transition and morphologic changes associated with tumor progression, whereas cells that expressed wild-type TLR4 or TLR4-T399I did not. Caco-2 cells that expressed TLR4-D299G had significant increases in expression levels of genes and proteins associated with inflammation and/or tumorigenesis compared with cells that expressed other forms of TLR4. The invasive activity of TLR4-D299G Caco-2 cells required Wnt-dependent activation of STAT3. In mice, intestinal xenograft tumors grew from Caco-2 cells that expressed TLR4-D299G, but not cells that expressed other forms of TLR4; tumor growth was blocked by a specific inhibitor of STAT3. Human colon adenocarcinomas from patients with TLR4-D299G were more frequently of an advanced stage (International Union Against Cancer [UICC] ≥III, 70% vs 46%; P = .0142) with metastasis (UICC IV, 42% vs 19%; P = .0065) than those with wild-type TLR4. Expression of STAT3 messenger RNA was higher among colonic adenocarcinomas with TLR4-D299G than those with wild-type TLR4.
  
  
  CONCLUSIONS
  TLR4-D299G induces features of neoplastic progression in intestinal epithelial Caco-2 cells and associates with aggressive colon cancer in humans, implying a novel link between aberrant innate immunity and colonic cancerogenesis.
authors.affiliation: Division of Gastroenterology & Hepatology, University Hospital of Essen, Medical School, University of Duisburg-Essen, Essen, Germany.
authors.forename: Annette
authors.initials: A
authors.lastname: Eyking
authors.forename: Birgit
authors.initials: B
authors.lastname: Ey
authors.forename: Michael
authors.initials: M
authors.lastname: Rünzi
authors.forename: Andres I
authors.initials: AI
authors.lastname: Roig
authors.forename: Henning
authors.initials: H
authors.lastname: Reis
authors.forename: Kurt W
authors.initials: KW
authors.lastname: Schmid
authors.forename: Guido
authors.initials: G
authors.lastname: Gerken
authors.forename: Daniel K
authors.initials: DK
authors.lastname: Podolsky
authors.forename: Elke
authors.initials: E
authors.lastname: Cario
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D018938:Caco-2 Cells; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D018450:Disease Progression; D004797:Enzyme-Linked Immunosorbent Assay; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015151:Immunoblotting; D007249:Inflammation / Q000276:immunology; D007413:Intestinal Mucosa / Q000187:drug effects*; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050796:STAT3 Transcription Factor / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000502:physiology*; D060449:Wnt Signaling Pathway
chemical_list: D012333:RNA, Messenger; D050796:STAT3 Transcription Factor; D051197:Toll-Like Receptor 4