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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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Biochemical and biological characterization of EpH4 cells expressing Ras effector-specific mutants lacking signaling along the MAPK or PI3K pathways. (A) Clones of EpH4 cells expressing the Ras effector mutants S35-Ras, C40-Ras, or V12-Ras were tested for overexpression of Ras proteins in Western blots using V12-Ras–specific antibodies. Clones highly overexpressing the exogenous Ras proteins (top) were then tested for levels of phospho-Erk (middle) or phospho-Akt (bottom) by respective phospho-specific antibodies (as described in Materials and methods). (B) Mass cultures (pools of multiple clones) expressing lower but still clearly elevated levels of H-V12–Ras or Ras effector mutant proteins S35 or C40 (unpublished data) were analyzed for phospho-Erk and phospho-Akt expression as in A. (C) Representative clones expressing V12-Ras (left), S35-Ras (middle), and C40-Ras (right) were cultivated on porous supports for 7 d in the presence (panels) or absence of TGFβ (insets). Cells were immunostained for E-cadherin (green) and vimentin (red) plus DAPI counterstaining for DNA (blue; as described in Materials and methods) and analyzed by confocal immunofluorescence microscopy. Bars, 20 μm.
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fig4: Biochemical and biological characterization of EpH4 cells expressing Ras effector-specific mutants lacking signaling along the MAPK or PI3K pathways. (A) Clones of EpH4 cells expressing the Ras effector mutants S35-Ras, C40-Ras, or V12-Ras were tested for overexpression of Ras proteins in Western blots using V12-Ras–specific antibodies. Clones highly overexpressing the exogenous Ras proteins (top) were then tested for levels of phospho-Erk (middle) or phospho-Akt (bottom) by respective phospho-specific antibodies (as described in Materials and methods). (B) Mass cultures (pools of multiple clones) expressing lower but still clearly elevated levels of H-V12–Ras or Ras effector mutant proteins S35 or C40 (unpublished data) were analyzed for phospho-Erk and phospho-Akt expression as in A. (C) Representative clones expressing V12-Ras (left), S35-Ras (middle), and C40-Ras (right) were cultivated on porous supports for 7 d in the presence (panels) or absence of TGFβ (insets). Cells were immunostained for E-cadherin (green) and vimentin (red) plus DAPI counterstaining for DNA (blue; as described in Materials and methods) and analyzed by confocal immunofluorescence microscopy. Bars, 20 μm.

Mentions: To verify these results by an independent approach, two well-characterized Ras mutants were used, which selectively signal along the MAPK pathway, S35-V12–Ras (S35-Ras), or the PI3K pathway, C40-V12–Ras (C40-Ras), due to specific amino acid changes in the effector loop of the Ras protein (Rodriguez-Viciana et al., 1997; Downward, 1998). These mutant proteins and the parental oncogenic Ras protein (V12-Ras) were overexpressed in EpH4 cells using respective retroviral constructs (see Materials and methods). Since V12-Ras did not reach similar expression levels in mass cultures as seen for v-H-Ras in EpRas cells (unpublished data), clones were selected expressing particularly high levels of V12-Ras, S35-Ras, and C40-Ras. In Western blots, S35-Ras–expressing clones showed elevated phosphorylation of Erk1 but only basal levels of PKB/Akt phosphorylation compared with empty vector controls. In contrast, the C40-Ras clones showed only basal level phosphorylation of Erks but elevated levels of PKB/Akt phosphorylation (Fig. 4 A). As expected, the V12-Ras control cells showed elevated levels of both phospho-Erk and phospho-PKB/Akt. Similar results were obtained with pools of >10 clones also selected for high level expression of V12-Ras, S35-Ras, and C40-Ras proteins (Fig. 4 B; unpublished data; see Materials and methods).


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)

Biochemical and biological characterization of EpH4 cells expressing Ras effector-specific mutants lacking signaling along the MAPK or PI3K pathways. (A) Clones of EpH4 cells expressing the Ras effector mutants S35-Ras, C40-Ras, or V12-Ras were tested for overexpression of Ras proteins in Western blots using V12-Ras–specific antibodies. Clones highly overexpressing the exogenous Ras proteins (top) were then tested for levels of phospho-Erk (middle) or phospho-Akt (bottom) by respective phospho-specific antibodies (as described in Materials and methods). (B) Mass cultures (pools of multiple clones) expressing lower but still clearly elevated levels of H-V12–Ras or Ras effector mutant proteins S35 or C40 (unpublished data) were analyzed for phospho-Erk and phospho-Akt expression as in A. (C) Representative clones expressing V12-Ras (left), S35-Ras (middle), and C40-Ras (right) were cultivated on porous supports for 7 d in the presence (panels) or absence of TGFβ (insets). Cells were immunostained for E-cadherin (green) and vimentin (red) plus DAPI counterstaining for DNA (blue; as described in Materials and methods) and analyzed by confocal immunofluorescence microscopy. Bars, 20 μm.
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Related In: Results  -  Collection

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fig4: Biochemical and biological characterization of EpH4 cells expressing Ras effector-specific mutants lacking signaling along the MAPK or PI3K pathways. (A) Clones of EpH4 cells expressing the Ras effector mutants S35-Ras, C40-Ras, or V12-Ras were tested for overexpression of Ras proteins in Western blots using V12-Ras–specific antibodies. Clones highly overexpressing the exogenous Ras proteins (top) were then tested for levels of phospho-Erk (middle) or phospho-Akt (bottom) by respective phospho-specific antibodies (as described in Materials and methods). (B) Mass cultures (pools of multiple clones) expressing lower but still clearly elevated levels of H-V12–Ras or Ras effector mutant proteins S35 or C40 (unpublished data) were analyzed for phospho-Erk and phospho-Akt expression as in A. (C) Representative clones expressing V12-Ras (left), S35-Ras (middle), and C40-Ras (right) were cultivated on porous supports for 7 d in the presence (panels) or absence of TGFβ (insets). Cells were immunostained for E-cadherin (green) and vimentin (red) plus DAPI counterstaining for DNA (blue; as described in Materials and methods) and analyzed by confocal immunofluorescence microscopy. Bars, 20 μm.
Mentions: To verify these results by an independent approach, two well-characterized Ras mutants were used, which selectively signal along the MAPK pathway, S35-V12–Ras (S35-Ras), or the PI3K pathway, C40-V12–Ras (C40-Ras), due to specific amino acid changes in the effector loop of the Ras protein (Rodriguez-Viciana et al., 1997; Downward, 1998). These mutant proteins and the parental oncogenic Ras protein (V12-Ras) were overexpressed in EpH4 cells using respective retroviral constructs (see Materials and methods). Since V12-Ras did not reach similar expression levels in mass cultures as seen for v-H-Ras in EpRas cells (unpublished data), clones were selected expressing particularly high levels of V12-Ras, S35-Ras, and C40-Ras. In Western blots, S35-Ras–expressing clones showed elevated phosphorylation of Erk1 but only basal levels of PKB/Akt phosphorylation compared with empty vector controls. In contrast, the C40-Ras clones showed only basal level phosphorylation of Erks but elevated levels of PKB/Akt phosphorylation (Fig. 4 A). As expected, the V12-Ras control cells showed elevated levels of both phospho-Erk and phospho-PKB/Akt. Similar results were obtained with pools of >10 clones also selected for high level expression of V12-Ras, S35-Ras, and C40-Ras proteins (Fig. 4 B; unpublished data; see Materials and methods).

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