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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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Scattering induced by scatter factor (HGF/SF) and TGFβ can be distinguished from EMT by reversibility and lack of marker changes. (A–D) EpRas collagen gel structures were treated with SF/HGF and TGFβ. (A) EpRas cells seeded in collagen gels for 3 d were treated with HGF/SF or TGFβ (insets) for an additional 5 d and photographed (B). After factor removal, (as described in Materials and methods) cells were maintained for an additional 4 d and photographed again. White arrows indicate lumina of tubular structures; black arrows indicate unordered strands of spindle-like invasive cells. (C and D). Structures treated as in A and B were fixed, stained with antibodies to the epithelial markers β4-integrin, E-cadherin, and ZO-1 (C, insets) and the mesenchymal marker vimentin, and analyzed by confocal immunofluorescence microscopy (as described in Materials and methods). (C) Structures transiently treated with HGF/SF. (D) Structures transiently treated with TGFβ. Note that HGF/SF-treated cells retain nonpolar distributed β4-integrin (C, left), whereas cells completely repolarize after HGF/SF removal (basolateral localized E-cadherin, and ZO-1) (C, left and inset). In contrast, the TGFβ-treated control structures undergo EMT (loss of β4-integrin, and vimentin expression) (D, left), persisting after TGFβ withdrawal (D, right). (E–H) Bcl-2–expressing EpH4 cells (Ep–Bcl-2) and control EpH4 cells were treated with TGFβ, which induced apoptosis (revealed by in situ TUNEL staining) in the EpH4 cells (E, inset) but a spindle cell-like morphology (G) and no apoptosis in the Ep–Bcl-2 cells (G, inset). Untreated cells of both types form normal hollow tubules (E and F). After withdrawal of TGFβ, the EpH4–Bcl-2 cells revert to tubular structures (H). Bars: (A, B, and E–H) 50 μm; (C and D) 10 μm.
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fig1: Scattering induced by scatter factor (HGF/SF) and TGFβ can be distinguished from EMT by reversibility and lack of marker changes. (A–D) EpRas collagen gel structures were treated with SF/HGF and TGFβ. (A) EpRas cells seeded in collagen gels for 3 d were treated with HGF/SF or TGFβ (insets) for an additional 5 d and photographed (B). After factor removal, (as described in Materials and methods) cells were maintained for an additional 4 d and photographed again. White arrows indicate lumina of tubular structures; black arrows indicate unordered strands of spindle-like invasive cells. (C and D). Structures treated as in A and B were fixed, stained with antibodies to the epithelial markers β4-integrin, E-cadherin, and ZO-1 (C, insets) and the mesenchymal marker vimentin, and analyzed by confocal immunofluorescence microscopy (as described in Materials and methods). (C) Structures transiently treated with HGF/SF. (D) Structures transiently treated with TGFβ. Note that HGF/SF-treated cells retain nonpolar distributed β4-integrin (C, left), whereas cells completely repolarize after HGF/SF removal (basolateral localized E-cadherin, and ZO-1) (C, left and inset). In contrast, the TGFβ-treated control structures undergo EMT (loss of β4-integrin, and vimentin expression) (D, left), persisting after TGFβ withdrawal (D, right). (E–H) Bcl-2–expressing EpH4 cells (Ep–Bcl-2) and control EpH4 cells were treated with TGFβ, which induced apoptosis (revealed by in situ TUNEL staining) in the EpH4 cells (E, inset) but a spindle cell-like morphology (G) and no apoptosis in the Ep–Bcl-2 cells (G, inset). Untreated cells of both types form normal hollow tubules (E and F). After withdrawal of TGFβ, the EpH4–Bcl-2 cells revert to tubular structures (H). Bars: (A, B, and E–H) 50 μm; (C and D) 10 μm.

Mentions: In addition to oncogenic Ras (Oft et al., 1996), hepatocyte growth factor (HGF)/scatter factor (SF), fibroblast growth factor (FGF), and TGFβ alone induce mesenchymal features in diverse epithelial cell systems (Brinkmann et al., 1995; Piek et al., 1999; Thiery and Chopin, 1999). We investigated whether these different signals would induce similar or different types of mesenchymal phenotypes in EpRas cells. These represent Ha-Ras–transformed fully polarized EpH4 cells (Oft et al., 1996), spontaneously immortalized from mammary glands of midpregnant mice (Reichmann et al., 1992). EpRas cells were seeded in serum-free collagen gels and subjected to different factor treatments. HGF/SF induced EpRas cells to invade collagen gels and grow as disordered chords of nonpolarized cells (Fig. 1 A; true for >95% of >100 structures counted in two to three separate collagen gels; see Materials and methods). Upon HGF/SF removal, >90% of these factor-induced disordered structures reverted to hollow tubular structures after an additional 5 d (Fig. 1 B). The disordered collagen gel structures formed by the HGF/SF-treated cells continued to express β4-integrin, E-cadherin, and ZO-1 at the entire cell surface (indicating loss of polarity) and failed to express vimentin (Fig. 1 C, left; unpublished data). Upon factor withdrawal, β4-integrin regained polarized basal expression in the hollow tubules formed (Fig. 1 C, right). Likewise, E-cadherin and ZO-1 were again expressed basolaterally (Fig. 1 C, inset; unpublished data). Similar results were obtained using FGF (unpublished data).


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)

Scattering induced by scatter factor (HGF/SF) and TGFβ can be distinguished from EMT by reversibility and lack of marker changes. (A–D) EpRas collagen gel structures were treated with SF/HGF and TGFβ. (A) EpRas cells seeded in collagen gels for 3 d were treated with HGF/SF or TGFβ (insets) for an additional 5 d and photographed (B). After factor removal, (as described in Materials and methods) cells were maintained for an additional 4 d and photographed again. White arrows indicate lumina of tubular structures; black arrows indicate unordered strands of spindle-like invasive cells. (C and D). Structures treated as in A and B were fixed, stained with antibodies to the epithelial markers β4-integrin, E-cadherin, and ZO-1 (C, insets) and the mesenchymal marker vimentin, and analyzed by confocal immunofluorescence microscopy (as described in Materials and methods). (C) Structures transiently treated with HGF/SF. (D) Structures transiently treated with TGFβ. Note that HGF/SF-treated cells retain nonpolar distributed β4-integrin (C, left), whereas cells completely repolarize after HGF/SF removal (basolateral localized E-cadherin, and ZO-1) (C, left and inset). In contrast, the TGFβ-treated control structures undergo EMT (loss of β4-integrin, and vimentin expression) (D, left), persisting after TGFβ withdrawal (D, right). (E–H) Bcl-2–expressing EpH4 cells (Ep–Bcl-2) and control EpH4 cells were treated with TGFβ, which induced apoptosis (revealed by in situ TUNEL staining) in the EpH4 cells (E, inset) but a spindle cell-like morphology (G) and no apoptosis in the Ep–Bcl-2 cells (G, inset). Untreated cells of both types form normal hollow tubules (E and F). After withdrawal of TGFβ, the EpH4–Bcl-2 cells revert to tubular structures (H). Bars: (A, B, and E–H) 50 μm; (C and D) 10 μm.
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Related In: Results  -  Collection

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fig1: Scattering induced by scatter factor (HGF/SF) and TGFβ can be distinguished from EMT by reversibility and lack of marker changes. (A–D) EpRas collagen gel structures were treated with SF/HGF and TGFβ. (A) EpRas cells seeded in collagen gels for 3 d were treated with HGF/SF or TGFβ (insets) for an additional 5 d and photographed (B). After factor removal, (as described in Materials and methods) cells were maintained for an additional 4 d and photographed again. White arrows indicate lumina of tubular structures; black arrows indicate unordered strands of spindle-like invasive cells. (C and D). Structures treated as in A and B were fixed, stained with antibodies to the epithelial markers β4-integrin, E-cadherin, and ZO-1 (C, insets) and the mesenchymal marker vimentin, and analyzed by confocal immunofluorescence microscopy (as described in Materials and methods). (C) Structures transiently treated with HGF/SF. (D) Structures transiently treated with TGFβ. Note that HGF/SF-treated cells retain nonpolar distributed β4-integrin (C, left), whereas cells completely repolarize after HGF/SF removal (basolateral localized E-cadherin, and ZO-1) (C, left and inset). In contrast, the TGFβ-treated control structures undergo EMT (loss of β4-integrin, and vimentin expression) (D, left), persisting after TGFβ withdrawal (D, right). (E–H) Bcl-2–expressing EpH4 cells (Ep–Bcl-2) and control EpH4 cells were treated with TGFβ, which induced apoptosis (revealed by in situ TUNEL staining) in the EpH4 cells (E, inset) but a spindle cell-like morphology (G) and no apoptosis in the Ep–Bcl-2 cells (G, inset). Untreated cells of both types form normal hollow tubules (E and F). After withdrawal of TGFβ, the EpH4–Bcl-2 cells revert to tubular structures (H). Bars: (A, B, and E–H) 50 μm; (C and D) 10 μm.
Mentions: In addition to oncogenic Ras (Oft et al., 1996), hepatocyte growth factor (HGF)/scatter factor (SF), fibroblast growth factor (FGF), and TGFβ alone induce mesenchymal features in diverse epithelial cell systems (Brinkmann et al., 1995; Piek et al., 1999; Thiery and Chopin, 1999). We investigated whether these different signals would induce similar or different types of mesenchymal phenotypes in EpRas cells. These represent Ha-Ras–transformed fully polarized EpH4 cells (Oft et al., 1996), spontaneously immortalized from mammary glands of midpregnant mice (Reichmann et al., 1992). EpRas cells were seeded in serum-free collagen gels and subjected to different factor treatments. HGF/SF induced EpRas cells to invade collagen gels and grow as disordered chords of nonpolarized cells (Fig. 1 A; true for >95% of >100 structures counted in two to three separate collagen gels; see Materials and methods). Upon HGF/SF removal, >90% of these factor-induced disordered structures reverted to hollow tubular structures after an additional 5 d (Fig. 1 B). The disordered collagen gel structures formed by the HGF/SF-treated cells continued to express β4-integrin, E-cadherin, and ZO-1 at the entire cell surface (indicating loss of polarity) and failed to express vimentin (Fig. 1 C, left; unpublished data). Upon factor withdrawal, β4-integrin regained polarized basal expression in the hollow tubules formed (Fig. 1 C, right). Likewise, E-cadherin and ZO-1 were again expressed basolaterally (Fig. 1 C, inset; unpublished data). Similar results were obtained using FGF (unpublished data).

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