Epithelial to Mesenchymal Transition by TGFβ-1 Induction Increases Stemness Characteristics in Primary Non Small Cell Lung Cancer Cell Line

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. Materials and 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.

[1]  H. Tai,et al.  Reciprocal regulation of cyclooxygenase-2 and 15-hydroxyprostaglandin dehydrogenase expression in A549 human lung adenocarcinoma cells. , 2006, Carcinogenesis.

[2]  E. Hay,et al.  The mesenchymal cell, its role in the embryo, and the remarkable signaling mechanisms that create it , 2005, Developmental dynamics : an official publication of the American Association of Anatomists.

[3]  M. Biffoni,et al.  Identification and expansion of the tumorigenic lung cancer stem cell population , 2008, Cell Death and Differentiation.

[4]  Keith R. Johnson,et al.  Cadherins as modulators of cellular phenotype. , 2003, Annual review of cell and developmental biology.

[5]  M. Carroll,et al.  Human acute myelogenous leukemia stem cells are rare and heterogeneous when assayed in NOD/SCID/IL2Rγc-deficient mice. , 2011, The Journal of clinical investigation.

[6]  A. Molven,et al.  Expression of the "stem cell marker" CD133 in pancreas and pancreatic ductal adenocarcinomas , 2008, BMC Cancer.

[7]  B. Martín-Castillo,et al.  Metformin against TGFβ-induced epithelial-to-mesenchymal transition (EMT): From cancer stem cells to aging-associated fibrosis , 2010, Cell cycle.

[8]  D. Steinbach,et al.  ABC transporters and drug resistance in leukemia: was P-gp nothing but the first head of the Hydra? , 2007, Leukemia.

[9]  G. Dontu,et al.  Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. , 2006, Cancer research.

[10]  J. McNamara Cancer Stem Cells , 2007, Methods in Molecular Biology.

[11]  Seong-Doo Hong,et al.  Inhibition of Akt activity induces the mesenchymal-to-epithelial reverting transition with restoring E-cadherin expression in KB and KOSCC-25B oral squamous cell carcinoma cells , 2009, Journal of experimental & clinical cancer research : CR.

[12]  S. Bidlingmaier,et al.  The utility and limitations of glycosylated human CD133 epitopes in defining cancer stem cells , 2008, Journal of Molecular Medicine.

[13]  I. Weissman,et al.  Stem cells, cancer, and cancer stem cells , 2001, Nature.

[14]  C. Jordan Cancer stem cell biology: from leukemia to solid tumors. , 2004, Current opinion in cell biology.

[15]  G. Viglietto,et al.  The role of CD133 in the identification and characterisation of tumour-initiating cells in non-small-cell lung cancer. , 2009, European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery.

[16]  I. Mackenzie,et al.  Retention of intrinsic stem cell hierarchies in carcinoma-derived cell lines. , 2005, Cancer research.

[17]  R. Ueda,et al.  Ectopic expression of c‐kit in small‐cell lung cancer , 1994, International journal of cancer. Supplement = Journal international du cancer. Supplement.

[18]  Seppo Parkkila,et al.  BMC Cancer BioMed Central Research article Carbonic anhydrase IX in oligodendroglial brain tumors , 2008 .

[19]  Jean Paul Thiery,et al.  Epithelial-mesenchymal transitions in development and pathologies. , 2003, Current opinion in cell biology.

[20]  R. Weinberg,et al.  A Perspective on Cancer Cell Metastasis , 2011, Science.

[21]  Yau-Hua Yu,et al.  The epithelial-mesenchymal transition mediator S100A4 maintains cancer-initiating cells in head and neck cancers. , 2011, Cancer research.

[22]  G. Cavet,et al.  Epithelial versus Mesenchymal Phenotype Determines In vitro Sensitivity and Predicts Clinical Activity of Erlotinib in Lung Cancer Patients , 2005, Clinical Cancer Research.

[23]  M. Kitajima,et al.  Cooperation of Cancer Stem Cell Properties and Epithelial-Mesenchymal Transition in the Establishment of Breast Cancer Metastasis , 2010, Journal of oncology.

[24]  Tony Hunter,et al.  Downregulation of caveolin-1 function by EGF leads to the loss of E-cadherin, increased transcriptional activity of beta-catenin, and enhanced tumor cell invasion. , 2003, Cancer cell.

[25]  Véronique Delmas,et al.  IGF-II induces rapid β-catenin relocation to the nucleus during epithelium to mesenchyme transition , 2001, Oncogene.

[26]  O. Eremin,et al.  Breast cancer chemoresistance: emerging importance of cancer stem cells. , 2010, Surgical oncology.

[27]  J. Thiery,et al.  La transition épithéliomésenchymateuse au cours du développement dans la fibrose et dans la progression tumorale , 2010 .

[28]  Anna E. Lokshin,et al.  Drug-Selected Human Lung Cancer Stem Cells: Cytokine Network, Tumorigenic and Metastatic Properties , 2008, PloS one.

[29]  Takashi Aoi,et al.  Generation of Pluripotent Stem Cells from Adult Mouse Liver and Stomach Cells , 2008, Science.

[30]  K. Chua,et al.  [Epithelial mesenchymal transition during development in fibrosis and in the progression of carcinoma]. , 2010, Bulletin du cancer.

[31]  Shadan Ali,et al.  Up-Regulation of Sonic Hedgehog Contributes to TGF-β1-Induced Epithelial to Mesenchymal Transition in NSCLC Cells , 2011, PloS one.

[32]  G. Scambia,et al.  Targeting CD133 antigen in cancer , 2009, Expert opinion on therapeutic targets.

[33]  Cynthia Hawkins,et al.  Identification of a cancer stem cell in human brain tumors. , 2003, Cancer research.

[34]  Danila Coradini,et al.  Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. , 2005, Cancer research.

[35]  R. Mason,et al.  TGF-β1 induces human alveolar epithelial to mesenchymal cell transition (EMT) , 2005, Respiratory research.

[36]  Shulan Tian,et al.  Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells , 2007, Science.

[37]  Mary Anne Wheeler,et al.  Stem , 1985 .

[38]  Wenjun Guo,et al.  The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells , 2008, Cell.

[39]  M. Wicha,et al.  Stem cells in normal development and cancer. , 2010, Progress in molecular biology and translational science.

[40]  Michael Dean,et al.  Tumour stem cells and drug resistance , 2005, Nature Reviews Cancer.

[41]  Irving L Weissman,et al.  Biology of hematopoietic stem cells and progenitors: implications for clinical application. , 2003, Annual review of immunology.

[42]  J. Visvader,et al.  Cancer stem cells in solid tumours: accumulating evidence and unresolved questions , 2008, Nature Reviews Cancer.

[43]  Alfonso Bellacosa,et al.  Epithelial–mesenchymal transition in development and cancer: role of phosphatidylinositol 3′ kinase/AKT pathways , 2005, Oncogene.

[44]  Lucila Ohno-Machado,et al.  Snail2 is an essential mediator of Twist1-induced epithelial mesenchymal transition and metastasis. , 2011, Cancer research.

[45]  D. Santini,et al.  p66Shc/Notch‐3 Interplay Controls Self‐Renewal and Hypoxia Survival in Human Stem/Progenitor Cells of the Mammary Gland Expanded In Vitro as Mammospheres , 2007, Stem cells.

[46]  M. Clarke,et al.  Stem Cells and Cancer: Two Faces of Eve , 2006, Cell.

[47]  A. Lokshin,et al.  Elimination of human lung cancer stem cells through targeting of the stem cell factor-c-kit autocrine signaling loop. , 2010, Cancer research.