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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

Abstract

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.

Keywords

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

Abbreviations

EMT

Epithelial to mesenchymal transition

MET

Mesenchymal to epithelial transition

NSCLC

Non-small cell lung carcinoma

qPCR

Quantitative polymerase chain reaction

TGFβ

Transforming growth factor β

ERK

Extracellular-signal regulated kinase

MAPK

Mitogen-activated protein kinase

RIPA

Radio-immunoprecipitation assay

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

IPA

Ingenuity pathway analysis

PDGFBB

Platelet derived growth factor BB

hCG

Human chorionic gonadotropin

NFkB

Nuclear factor kB

VEGF

Vascular endothelial growth factor

IgG

Immunoglobulin

JNK

Jun kinase

FN1

Fibronectin 1

MMP2

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)

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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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