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Ras and TGF[beta] cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways.

Janda E, Lehmann K, Killisch I, Jechlinger M, Herzig M, Downward J, Beug H, Grünert S - J. Cell Biol. (2002)

Bottom Line: EMT requires continuous TGFbeta receptor (TGFbeta-R) and oncogenic Ras signaling and is stabilized by autocrine TGFbeta production.In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFbeta alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes.Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFbeta-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Pathology, A-1030 Vienna, Austria.

ABSTRACT
Multistep carcinogenesis involves more than six discrete events also important in normal development and cell behavior. Of these, local invasion and metastasis cause most cancer deaths but are the least well understood molecularly. We employed a combined in vitro/in vivo carcinogenesis model, that is, polarized Ha-Ras-transformed mammary epithelial cells (EpRas), to dissect the role of Ras downstream signaling pathways in epithelial cell plasticity, tumorigenesis, and metastasis. Ha-Ras cooperates with transforming growth factor beta (TGFbeta) to cause epithelial mesenchymal transition (EMT) characterized by spindle-like cell morphology, loss of epithelial markers, and induction of mesenchymal markers. EMT requires continuous TGFbeta receptor (TGFbeta-R) and oncogenic Ras signaling and is stabilized by autocrine TGFbeta production. In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFbeta alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes. Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFbeta-induced apoptosis. Hyperactivation of the PI3K pathway or the Raf/MAPK pathway are sufficient for tumorigenesis, whereas EMT in vivo and metastasis required a hyperactive Raf/MAPK pathway. Thus, EMT seems to be a close in vitro correlate of metastasis, both requiring synergism between TGFbeta-R and Raf/MAPK signaling.

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EpH4 cells expressing V12-Ras or S35-Ras undergo EMT upon TGFβ treatment; C40-Ras cells undergo scattering. (A) EpH4 cell clones overexpressing V12-Ras (left) and the Ras effector mutants S35-Ras (middle) and C40-Ras (right; Fig. 4) were seeded in collagen gels and either left untreated (top) or treated (middle) with 5 ng/ml TGFβ for 5 d followed by removal of TGFβ and further cultivation for 5 d (bottom). Photographs of representative tubular structures with lumina (white arrows) or distended chords and strands of invasive cells with mesenchymal morphology (black arrows) are shown. (B) Similar collagen gel structures as in A were stained in situ with antibodies to epithelial markers (green, β4-integrin and E-cadherin) and mesenchymal markers (red, vimentin and CD68; as described in the text) and analyzed by confocal immunofluorescence microscopy (as described in Materials and methods). (Top left, insets) tubular structures formed by untreated S35-Ras cells. (Top) “Invasive” structures formed by S35-Ras and C40-Ras cells after TGFβ treatment (note the persistent nonpolarized expression of epithelial markers in the C40-Ras structures). (Bottom) Structures formed by S35-Ras and C40-Ras after removal of TGFβ (mesenchymal shape and mesenchymal markers persist in the S35-Ras cells; C40-Ras cells revert to tubular structures with lumina [white dotted lines] basolaterally expressed epithelial markers). Bars: (A) 50 μm; (B) 20 μm.
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fig5: EpH4 cells expressing V12-Ras or S35-Ras undergo EMT upon TGFβ treatment; C40-Ras cells undergo scattering. (A) EpH4 cell clones overexpressing V12-Ras (left) and the Ras effector mutants S35-Ras (middle) and C40-Ras (right; Fig. 4) were seeded in collagen gels and either left untreated (top) or treated (middle) with 5 ng/ml TGFβ for 5 d followed by removal of TGFβ and further cultivation for 5 d (bottom). Photographs of representative tubular structures with lumina (white arrows) or distended chords and strands of invasive cells with mesenchymal morphology (black arrows) are shown. (B) Similar collagen gel structures as in A were stained in situ with antibodies to epithelial markers (green, β4-integrin and E-cadherin) and mesenchymal markers (red, vimentin and CD68; as described in the text) and analyzed by confocal immunofluorescence microscopy (as described in Materials and methods). (Top left, insets) tubular structures formed by untreated S35-Ras cells. (Top) “Invasive” structures formed by S35-Ras and C40-Ras cells after TGFβ treatment (note the persistent nonpolarized expression of epithelial markers in the C40-Ras structures). (Bottom) Structures formed by S35-Ras and C40-Ras after removal of TGFβ (mesenchymal shape and mesenchymal markers persist in the S35-Ras cells; C40-Ras cells revert to tubular structures with lumina [white dotted lines] basolaterally expressed epithelial markers). Bars: (A) 50 μm; (B) 20 μm.

Mentions: In collagen gels, untreated S35-Ras cells resembled V12-Ras cells (e.g., forming distended tubular structures with large lumina in collagen gels), whereas C40 cells more closely resembled EpH4 cells (thin tubules with tiny lumina; Fig. 5 A, top). Marker staining revealed basolateral E-cadherin staining and no expression of mesenchymal markers in all three cell types (Fig. 5 B, insets; unpublished data). Treatment of V12-Ras, S35-Ras, and C40-Ras cells with TGFβ resulted in unordered cell strands and cords with spindle-like cellular morphology (Fig. 5 A, middle). After withdrawal of TGFβ, these lumen-less disordered structures persisted in the V12-Ras and S35-Ras cells, whereas C40-Ras cells reverted to thin hollow structures (Fig. 5 A, bottom). Likewise, TGFβ-treated S35-Ras cells showed loss of E-cadherin/β4-integrin staining and induction of the mesenchymal markers vimentin and CD68 (Fig. 5 B, top), a marker distribution persisting after removal of TGFβ (Fig. 5 B, bottom). In contrast, C40-Ras–expressing cells maintained nonpolar E-cadherin staining in the disordered structures induced by TGFβ, whereas mesenchymal markers remained undetectable (Fig. 5 B, top). Upon withdrawal of TGFβ, the C40-Ras structures regained epithelial polarity as indicated by lumen formation and basolateral E-cadherin staining (Fig. 5 B, bottom).


Ras and TGF[beta] cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways.

Janda E, Lehmann K, Killisch I, Jechlinger M, Herzig M, Downward J, Beug H, Grünert S - J. Cell Biol. (2002)

EpH4 cells expressing V12-Ras or S35-Ras undergo EMT upon TGFβ treatment; C40-Ras cells undergo scattering. (A) EpH4 cell clones overexpressing V12-Ras (left) and the Ras effector mutants S35-Ras (middle) and C40-Ras (right; Fig. 4) were seeded in collagen gels and either left untreated (top) or treated (middle) with 5 ng/ml TGFβ for 5 d followed by removal of TGFβ and further cultivation for 5 d (bottom). Photographs of representative tubular structures with lumina (white arrows) or distended chords and strands of invasive cells with mesenchymal morphology (black arrows) are shown. (B) Similar collagen gel structures as in A were stained in situ with antibodies to epithelial markers (green, β4-integrin and E-cadherin) and mesenchymal markers (red, vimentin and CD68; as described in the text) and analyzed by confocal immunofluorescence microscopy (as described in Materials and methods). (Top left, insets) tubular structures formed by untreated S35-Ras cells. (Top) “Invasive” structures formed by S35-Ras and C40-Ras cells after TGFβ treatment (note the persistent nonpolarized expression of epithelial markers in the C40-Ras structures). (Bottom) Structures formed by S35-Ras and C40-Ras after removal of TGFβ (mesenchymal shape and mesenchymal markers persist in the S35-Ras cells; C40-Ras cells revert to tubular structures with lumina [white dotted lines] basolaterally expressed epithelial markers). Bars: (A) 50 μm; (B) 20 μm.
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fig5: EpH4 cells expressing V12-Ras or S35-Ras undergo EMT upon TGFβ treatment; C40-Ras cells undergo scattering. (A) EpH4 cell clones overexpressing V12-Ras (left) and the Ras effector mutants S35-Ras (middle) and C40-Ras (right; Fig. 4) were seeded in collagen gels and either left untreated (top) or treated (middle) with 5 ng/ml TGFβ for 5 d followed by removal of TGFβ and further cultivation for 5 d (bottom). Photographs of representative tubular structures with lumina (white arrows) or distended chords and strands of invasive cells with mesenchymal morphology (black arrows) are shown. (B) Similar collagen gel structures as in A were stained in situ with antibodies to epithelial markers (green, β4-integrin and E-cadherin) and mesenchymal markers (red, vimentin and CD68; as described in the text) and analyzed by confocal immunofluorescence microscopy (as described in Materials and methods). (Top left, insets) tubular structures formed by untreated S35-Ras cells. (Top) “Invasive” structures formed by S35-Ras and C40-Ras cells after TGFβ treatment (note the persistent nonpolarized expression of epithelial markers in the C40-Ras structures). (Bottom) Structures formed by S35-Ras and C40-Ras after removal of TGFβ (mesenchymal shape and mesenchymal markers persist in the S35-Ras cells; C40-Ras cells revert to tubular structures with lumina [white dotted lines] basolaterally expressed epithelial markers). Bars: (A) 50 μm; (B) 20 μm.
Mentions: In collagen gels, untreated S35-Ras cells resembled V12-Ras cells (e.g., forming distended tubular structures with large lumina in collagen gels), whereas C40 cells more closely resembled EpH4 cells (thin tubules with tiny lumina; Fig. 5 A, top). Marker staining revealed basolateral E-cadherin staining and no expression of mesenchymal markers in all three cell types (Fig. 5 B, insets; unpublished data). Treatment of V12-Ras, S35-Ras, and C40-Ras cells with TGFβ resulted in unordered cell strands and cords with spindle-like cellular morphology (Fig. 5 A, middle). After withdrawal of TGFβ, these lumen-less disordered structures persisted in the V12-Ras and S35-Ras cells, whereas C40-Ras cells reverted to thin hollow structures (Fig. 5 A, bottom). Likewise, TGFβ-treated S35-Ras cells showed loss of E-cadherin/β4-integrin staining and induction of the mesenchymal markers vimentin and CD68 (Fig. 5 B, top), a marker distribution persisting after removal of TGFβ (Fig. 5 B, bottom). In contrast, C40-Ras–expressing cells maintained nonpolar E-cadherin staining in the disordered structures induced by TGFβ, whereas mesenchymal markers remained undetectable (Fig. 5 B, top). Upon withdrawal of TGFβ, the C40-Ras structures regained epithelial polarity as indicated by lumen formation and basolateral E-cadherin staining (Fig. 5 B, bottom).

Bottom Line: EMT requires continuous TGFbeta receptor (TGFbeta-R) and oncogenic Ras signaling and is stabilized by autocrine TGFbeta production.In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFbeta alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes.Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFbeta-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Pathology, A-1030 Vienna, Austria.

ABSTRACT
Multistep carcinogenesis involves more than six discrete events also important in normal development and cell behavior. Of these, local invasion and metastasis cause most cancer deaths but are the least well understood molecularly. We employed a combined in vitro/in vivo carcinogenesis model, that is, polarized Ha-Ras-transformed mammary epithelial cells (EpRas), to dissect the role of Ras downstream signaling pathways in epithelial cell plasticity, tumorigenesis, and metastasis. Ha-Ras cooperates with transforming growth factor beta (TGFbeta) to cause epithelial mesenchymal transition (EMT) characterized by spindle-like cell morphology, loss of epithelial markers, and induction of mesenchymal markers. EMT requires continuous TGFbeta receptor (TGFbeta-R) and oncogenic Ras signaling and is stabilized by autocrine TGFbeta production. In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFbeta alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes. Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFbeta-induced apoptosis. Hyperactivation of the PI3K pathway or the Raf/MAPK pathway are sufficient for tumorigenesis, whereas EMT in vivo and metastasis required a hyperactive Raf/MAPK pathway. Thus, EMT seems to be a close in vitro correlate of metastasis, both requiring synergism between TGFbeta-R and Raf/MAPK signaling.

Show MeSH
Related in: MedlinePlus