title: Epithelial to mesenchymal transition by TGFβ-1 induction increases stemness characteristics in primary non small cell lung cancer cell line.
abstract: BACKGROUND
  Cancer Stem Cells (CSCs) hypothesis asserts that only a small subset of cells within a tumour is capable of both tumour initiation and sustainment. The Epithelial-Mesenchymal Transition (EMT) is an embryonic developmental program that is often activated during cancer invasion and metastasis. The aim of this study is to shed light on the relationship between EMT and CSCs by using LC31 lung cancer primary cell line.
  
  
  METHODS
  A549 and LC31 cell lines were treated with 2 ng/ml TGFβ-1 for 30 days, and 80 days, respectively. To evaluate EMT, morphological changes were assessed by light microscopy, immunofluorescence and cytometry for following markers: cytokeratins, e-cadherin, CD326 (epithelial markers) and CD90, and vimentin (mesenchymal markers). Moreover, RT-PCR for Slug, Twist and β-catenin genes were performed. On TGFβ-1 treated and untreated LC31 cell lines, we performed stemness tests such as pneumospheres growth and stem markers expression such as Oct4, Nanog, Sox2, c-kit and CD133. Western Blot for CD133 and tumorigenicity assays using NOD/SCID mice were performed.
  
  
  RESULTS
  TGFβ-1 treated LC31 cell line lost its epithelial morphology assuming a fibroblast-like appearance. The same results were obtained for the A549 cell line (as control). Immunofluorescence and cytometry showed up-regulation of vimentin and CD90 and down-regulation of cytocheratin, e-cadherin and CD326 in TGFβ-1 treated LC31 and A549 cell lines. Slug, Twist and β-catenin m-RNA transcripts were up-regulated in TGFβ-1 treated LC31 cell line confirming EMT. This cell line showed also over-expression of Oct4, Nanog, Sox2 and CD133, all genes of stemness. In addition, in TGFβ-1 treated LC31 cell line, an increased pneumosphere-forming capacity and tumours-forming ability in NOD/SCID mice were detectable.
  
  
  CONCLUSIONS
  The induction of EMT by TGFβ-1 exposure, in primary lung cancer cell line results in the acquisition of mesenchymal profile and in the expression of stem cell markers.
authors: Department of Experimental Oncology, National Cancer Institute, Naples, Italy. pinopiro@tin.it
authors: Giuseppe
authors: G
authors: Pirozzi
authors: Virginia
authors: V
authors: Tirino
authors: Rosa
authors: R
authors: Camerlingo
authors: Renato
authors: R
authors: Franco
authors: Aantonello
authors: A
authors: La Rocca
authors: Eleonora
authors: E
authors: Liguori
authors: Nicola
authors: N
authors: Martucci
authors: Francesca
authors: F
authors: Paino
authors: Nicola
authors: N
authors: Normanno
authors: Gaetano
authors: G
authors: Rocco
mesh_terms: D000071916:AC133 Antigen; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism*; D045744:Cell Line, Tumor; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D006023:Glycoproteins; D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C000606023:PROM1 protein, human; D010455:Peptides; C000606024:Prom1 protein, mouse; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D053773:Transforming Growth Factor beta1