Inducible expression of TGFβ, Snail and Zeb1 recapitulates EMT in vitro and in vivo in a NSCLC model

  • Gretchen M. ArgastEmail author
  • Joseph S. Krueger
  • Stuart Thomson
  • Isabela Sujka-Kwok
  • Krista Carey
  • Stacia Silva
  • Matthew O’Connor
  • Peter Mercado
  • Iain J. Mulford
  • G. David Young
  • Regina Sennello
  • Robert Wild
  • Jonathan A. Pachter
  • Julie L. C. Kan
  • John Haley
  • Maryland Rosenfeld-Franklin
  • David M. Epstein
Research Paper


The progression of cancer from non-metastatic to metastatic is the critical transition in the course of the disease. The epithelial to mesenchymal transition (EMT) is a mechanism by which tumor cells acquire characteristics that improve metastatic efficiency. Targeting EMT processes in patients is therefore a potential strategy to block the transition to metastatic cancer and improve patient outcome. To develop models of EMT applicable to in vitro and in vivo settings, we engineered NCI-H358 non-small cell lung carcinoma cells to inducibly express three well-established drivers of EMT: activated transforming growth factor β (aTGFβ), Snail or Zeb1. We characterized the morphological, molecular and phenotypic changes induced by each of the drivers and compared the different end-states of EMT between the models. Both in vitro and in vivo, induction of the transgenes Snail and Zeb1 resulted in downregulation of epithelial markers and upregulation of mesenchymal markers, and reduced the ability of the cells to proliferate. Induced autocrine expression of aTGFβ caused marker and phenotypic changes consistent with EMT, a modest effect on growth rate, and a shift to a more invasive phenotype. In vivo, this manifested as tumor cell infiltration of the surrounding mouse stromal tissue. Overall, Snail and Zeb1 were sufficient to induce EMT in the cells, but aTGFβ induced a more complex EMT, in which changes in extracellular matrix remodeling components were pronounced.


Epithelial to mesenchymal transition In vivo model Non-small cell lung cancer Signaling networks Snail TGFβ Zeb1 



Epithelial to mesenchymal transition


Mesenchymal to epithelial transition


Non-small cell lung carcinoma


Quantitative polymerase chain reaction


Transforming growth factor β


Extracellular-signal regulated kinase


Mitogen-activated protein kinase


Radio-immunoprecipitation assay


Glyceraldehyde 3-phosphate dehydrogenase


Ingenuity pathway analysis


Platelet derived growth factor BB


Human chorionic gonadotropin


Nuclear factor kB


Vascular endothelial growth factor




Jun kinase


Fibronectin 1


Matrix metalloproteinase 2

Supplementary material

10585_2011_9394_MOESM1_ESM.ppt (6 mb)
Supplementary material 1. Supplemental Figure 1: Pathway analysis of differentially regulated genes in aTGFβ, Snail and Zeb1 models in vivo. Lists of differentially expressed human genes for each model in vivo were compared using IPA software. The highest ranking networks composed of genes regulated in vivo in each of the models (red = upregulated, green = downregulated), and genes unregulated but inferred as operating in the network (unfilled) are shown for aTGFβ, Snail and Zeb1. Signaling nodes (genes with 5 or more connections) are indicated by circles (yellow = experimental, blue = implied) (PPT 6186 kb)
10585_2011_9394_MOESM2_ESM.xls (16 kb)
Supplementary material 2 (XLS 17 kb)
10585_2011_9394_MOESM3_ESM.xls (15 kb)
Supplementary material 3 (XLS 15 kb)


  1. 1.
    Thiery JP et al (2009) Epithelial-mesenchymal transitions in development and disease. Cell 139(5):871–890PubMedCrossRefGoogle Scholar
  2. 2.
    Kalluri R, Weinberg RA (2009) The basics of epithelial-mesenchymal transition. J Clin Invest 119(6):1420–1428PubMedCrossRefGoogle Scholar
  3. 3.
    Gavert N, Ben-Ze’ev A (2008) Epithelial-mesenchymal transition and the invasive potential of tumors. Trends Mol Med 14(5):199–209PubMedCrossRefGoogle Scholar
  4. 4.
    Turley EA et al (2008) Mechanisms of disease: epithelial-mesenchymal transition—does cellular plasticity fuel neoplastic progression? Nat Clin Pract Oncol 5(5):280–290PubMedCrossRefGoogle Scholar
  5. 5.
    Hollier BG, Evans K, Mani SA (2009) The epithelial-to-mesenchymal transition and cancer stem cells: a coalition against cancer therapies. J Mammary Gland Biol Neoplasia 14(1):29–43PubMedCrossRefGoogle Scholar
  6. 6.
    Orlichenko LS, Radisky DC (2008) Matrix metalloproteinases stimulate epithelial-mesenchymal transition during tumor development. Clin Exp Metastasis 25(6):593–600PubMedCrossRefGoogle Scholar
  7. 7.
    Voulgari A, Pintzas A (2009) Epithelial-mesenchymal transition in cancer metastasis: mechanisms, markers and strategies to overcome drug resistance in the clinic. Biochim Biophys Acta 1796(2):75–90PubMedGoogle Scholar
  8. 8.
    Chai Q et al (2003) Localisation and phenotypical characterisation of collagen-producing cells in TGF-beta 1-induced renal interstitial fibrosis. Histochem Cell Biol 119(4):267–280PubMedGoogle Scholar
  9. 9.
    Kim KK et al (2006) Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci USA 103(35):13180–13185PubMedCrossRefGoogle Scholar
  10. 10.
    Dooley S et al (2008) Hepatocyte-specific Smad7 expression attenuates TGF-beta-mediated fibrogenesis and protects against liver damage. Gastroenterology 135(2):642–659PubMedCrossRefGoogle Scholar
  11. 11.
    Nishioka R et al (2010) SNAIL induces epithelial-to-mesenchymal transition in a human pancreatic cancer cell line (BxPC3) and promotes distant metastasis and invasiveness in vivo. Exp Mol Pathol 89(2):149–157PubMedCrossRefGoogle Scholar
  12. 12.
    Wu MY, Hill CS (2009) Tgf-beta superfamily signaling in embryonic development and homeostasis. Dev Cell 16(3):329–343PubMedCrossRefGoogle Scholar
  13. 13.
    Li MO, Flavell RA (2008) TGF-beta: a master of all T cell trades. Cell 134(3):392–404PubMedCrossRefGoogle Scholar
  14. 14.
    Taylor MA, Parvani JG, Schiemann WP (2010) The pathophysiology of epithelial-mesenchymal transition induced by transforming growth factor-beta in normal and malignant mammary epithelial cells. J Mammary Gland Biol Neoplasia 15(2):169–190PubMedCrossRefGoogle Scholar
  15. 15.
    Tian M, Schiemann WP (2009) The TGF-beta paradox in human cancer: an update. Future Oncol 5(2):259–271PubMedCrossRefGoogle Scholar
  16. 16.
    Rahimi RA, Leof EB (2007) TGF-beta signaling: a tale of two responses. J Cell Biochem 102(3):593–608PubMedCrossRefGoogle Scholar
  17. 17.
    Xu J, Lamouille S, Derynck R (2009) TGF-beta-induced epithelial to mesenchymal transition. Cell Res 19(2):156–172PubMedCrossRefGoogle Scholar
  18. 18.
    Bhowmick NA et al (2001) Integrin beta 1 signaling is necessary for transforming growth factor-beta activation of p38MAPK and epithelial plasticity. J Biol Chem 276(50):46707–46713PubMedCrossRefGoogle Scholar
  19. 19.
    Wendt MK, Smith JA, Schiemann WP (2009) p130Cas is required for mammary tumor growth and transforming growth factor-beta-mediated metastasis through regulation of Smad2/3 activity. J Biol Chem 284(49):34145–34156PubMedCrossRefGoogle Scholar
  20. 20.
    Liu X et al (1997) Transforming growth factor beta-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells. Proc Natl Acad Sci USA 94(20):10669–10674PubMedCrossRefGoogle Scholar
  21. 21.
    Nieto MA (2002) The snail superfamily of zinc-finger transcription factors. Nat Rev Mol Cell Biol 3(3):155–166PubMedCrossRefGoogle Scholar
  22. 22.
    Peinado H, Olmeda D, Cano A (2007) Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer 7(6):415–428PubMedCrossRefGoogle Scholar
  23. 23.
    Browne G, Sayan AE, Tulchinsky E (2010) ZEB proteins link cell motility with cell cycle control and cell survival in cancer. Cell Cycle 9(5):886–891PubMedCrossRefGoogle Scholar
  24. 24.
    Moreno-Bueno G, Portillo F, Cano A (2008) Transcriptional regulation of cell polarity in EMT and cancer. Oncogene 27(55):6958–6969PubMedCrossRefGoogle Scholar
  25. 25.
    Savagner P, Yamada KM, Thiery JP (1997) The zinc-finger protein slug causes desmosome dissociation, an initial and necessary step for growth factor-induced epithelial-mesenchymal transition. J Cell Biol 137(6):1403–1419PubMedCrossRefGoogle Scholar
  26. 26.
    Cano A et al (2000) The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol 2(2):76–83PubMedCrossRefGoogle Scholar
  27. 27.
    Eger A et al (2005) DeltaEF1 is a transcriptional repressor of E-cadherin and regulates epithelial plasticity in breast cancer cells. Oncogene 24(14):2375–2385PubMedCrossRefGoogle Scholar
  28. 28.
    Yang J et al (2004) Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117(7):927–939PubMedCrossRefGoogle Scholar
  29. 29.
    Comijn J et al (2001) The two-handed E box binding zinc finger protein SIP1 downregulates E-cadherin and induces invasion. Mol Cell 7(6):1267–1278PubMedCrossRefGoogle Scholar
  30. 30.
    Barrallo-Gimeno A, Nieto MA (2005) The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development 132(14):3151–3161PubMedCrossRefGoogle Scholar
  31. 31.
    Takeichi M (1991) Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251(5000):1451–1455PubMedCrossRefGoogle Scholar
  32. 32.
    Yauch RL et al (2005) Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients. Clin Cancer Res 11(24 Pt 1):8686–8698PubMedCrossRefGoogle Scholar
  33. 33.
    Buck E et al (2007) Loss of homotypic cell adhesion by epithelial-mesenchymal transition or mutation limits sensitivity to epidermal growth factor receptor inhibition. Mol Cancer Ther 6(2):532–541PubMedCrossRefGoogle Scholar
  34. 34.
    Thomson S et al (2005) Epithelial to mesenchymal transition is a determinant of sensitivity of non-small-cell lung carcinoma cell lines and xenografts to epidermal growth factor receptor inhibition. Cancer Res 65(20):9455–9462PubMedCrossRefGoogle Scholar
  35. 35.
    Argast GM et al (2011) Cooperative signaling between oncostatin M, hepatocyte growth factor and transforming growth factor-beta enhances epithelial to mesenchymal transition in lung and pancreatic tumor models. Cells Tissues Organs 193(1–2):114–132PubMedCrossRefGoogle Scholar
  36. 36.
    Thomson S et al (2008) Kinase switching in mesenchymal-like non-small cell lung cancer lines contributes to EGFR inhibitor resistance through pathway redundancy. Clin Exp Metastasis 25(8):843–854PubMedCrossRefGoogle Scholar
  37. 37.
    Muraoka RS et al (2003) Increased malignancy of Neu-induced mammary tumors overexpressing active transforming growth factor beta1. Mol Cell Biol 23(23):8691–8703PubMedCrossRefGoogle Scholar
  38. 38.
    Guaita S et al (2002) Snail induction of epithelial to mesenchymal transition in tumor cells is accompanied by MUC1 repression and ZEB1 expression. J Biol Chem 277(42):39209–39216PubMedCrossRefGoogle Scholar
  39. 39.
    Thomson S et al (2011) A systems view of epithelial-mesenchymal transition signaling states. Clin Exp Metastasis 28(2):137–155PubMedCrossRefGoogle Scholar
  40. 40.
    Yang J, Weinberg RA (2008) Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis. Dev Cell 14(6):818–829PubMedCrossRefGoogle Scholar
  41. 41.
    Lei S et al (2004) The murine gastrin promoter is synergistically activated by transforming growth factor-beta/Smad and Wnt signaling pathways. J Biol Chem 279(41):42492–42502PubMedCrossRefGoogle Scholar
  42. 42.
    Neve RM et al (2006) A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 10(6):515–527PubMedCrossRefGoogle Scholar
  43. 43.
    Hennessy BT et al (2009) Characterization of a naturally occurring breast cancer subset enriched in epithelial-to-mesenchymal transition and stem cell characteristics. Cancer Res 69(10):4116–4124PubMedCrossRefGoogle Scholar
  44. 44.
    Taube JH et al (2010) Core epithelial-to-mesenchymal transition interactome gene-expression signature is associated with claudin-low and metaplastic breast cancer subtypes. Proc Natl Acad Sci USA 107(35):15449–15454PubMedCrossRefGoogle Scholar
  45. 45.
    Blick T et al (2010) Epithelial mesenchymal transition traits in human breast cancer cell lines parallel the CD44(hi/)CD24 (lo/−) stem cell phenotype in human breast cancer. J Mammary Gland Biol Neoplasia 15(2):235–252PubMedCrossRefGoogle Scholar
  46. 46.
    de Herreros AG et al (2010) Snail family regulation and epithelial mesenchymal transitions in breast cancer progression. J Mammary Gland Biol Neoplasia 15(2):135–147PubMedCrossRefGoogle Scholar
  47. 47.
    Halachmi S et al (2000) Genetic alterations in urinary bladder carcinosarcoma: evidence of a common clonal origin. Eur Urol 37(3):350–357PubMedCrossRefGoogle Scholar
  48. 48.
    Ceppi P et al (2010) Loss of miR-200c expression induces an aggressive, invasive, and chemoresistant phenotype in non-small cell lung cancer. Mol Cancer Res 8(9):1207–1216PubMedCrossRefGoogle Scholar
  49. 49.
    Oft M, Heider KH, Beug H (1998) TGFbeta signaling is necessary for carcinoma cell invasiveness and metastasis. Curr Biol 8(23):1243–1252PubMedCrossRefGoogle Scholar
  50. 50.
    Yang L et al (2008) Abrogation of TGF beta signaling in mammary carcinomas recruits Gr-1+ CD11b+ myeloid cells that promote metastasis. Cancer Cell 13(1):23–35PubMedCrossRefGoogle Scholar
  51. 51.
    van Zijl F et al (2009) Hepatic tumor-stroma crosstalk guides epithelial to mesenchymal transition at the tumor edge. Oncogene 28(45):4022–4033PubMedCrossRefGoogle Scholar
  52. 52.
    Dumont N, Arteaga CL (2000) Transforming growth factor-beta and breast cancer: tumor promoting effects of transforming growth factor-beta. Breast Cancer Res 2(2):125–132PubMedCrossRefGoogle Scholar
  53. 53.
    Massague J (2008) TGFbeta in cancer. Cell 134(2):215–230PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Gretchen M. Argast
    • 1
    Email author
  • Joseph S. Krueger
    • 1
    • 2
  • Stuart Thomson
    • 1
  • Isabela Sujka-Kwok
    • 1
  • Krista Carey
    • 1
  • Stacia Silva
    • 1
  • Matthew O’Connor
    • 1
  • Peter Mercado
    • 1
  • Iain J. Mulford
    • 1
  • G. David Young
    • 1
    • 2
  • Regina Sennello
    • 1
  • Robert Wild
    • 1
    • 3
  • Jonathan A. Pachter
    • 1
  • Julie L. C. Kan
    • 1
    • 4
  • John Haley
    • 1
  • Maryland Rosenfeld-Franklin
    • 1
  • David M. Epstein
    • 1
  1. 1.Departments of Translational Research, Biochemical and Cellular Pharmacology and In Vivo PharmacologyOSI Pharmaceuticals, Inc.FarmingdaleUSA
  2. 2.Flagship BioscienceFlagstaffUSA
  3. 3.Eli Lilly and Company, Lilly Corporate CenterIndianapolisUSA
  4. 4.Pfizer IncSan DiegoUSA

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