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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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V12-Ras and C40-Ras but not S35-Ras protect EpH4 cells from TGFβ-induced apoptosis. (A) For apoptosis determination in collagen gels, EpH4 cells expressing Ras or Ras effector–specific mutants were allowed to form structures in collagen gels for 3 d and were treated with various concentrations of TGFβ (0.5–40 ng/ml) for 4 d (dotted bar). Cells were either TUNEL stained in suspension (C, top) or in situ (B, and C, bottom panel; as described in Materials and methods). (B) Confocal immunofluorescence analysis of gel structures formed by clones overexpressing S35-Ras (left) and C40-Ras cells (right) treated with moderate (top, 2.5 ng/ml) or high levels of TGFβ (40 ng/ml; see A) and subjected to in situ TUNEL staining (green). DAPI staining (blue) indicates live cells. (C) Quantitation of TUNEL-positive cells from collagen gel structures either stained and counted in suspension after collagenase digestion (top; red asterisks indicate statistically significant increases in apoptosis induced by TGFβ) or stained and counted in situ (bottom; as described in Materials and methods). Mean percentages of apoptotic cells from multiple gel structures plus standard deviations (error bars) from three independent determinations are shown. Bars, 50 μm.
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fig6: V12-Ras and C40-Ras but not S35-Ras protect EpH4 cells from TGFβ-induced apoptosis. (A) For apoptosis determination in collagen gels, EpH4 cells expressing Ras or Ras effector–specific mutants were allowed to form structures in collagen gels for 3 d and were treated with various concentrations of TGFβ (0.5–40 ng/ml) for 4 d (dotted bar). Cells were either TUNEL stained in suspension (C, top) or in situ (B, and C, bottom panel; as described in Materials and methods). (B) Confocal immunofluorescence analysis of gel structures formed by clones overexpressing S35-Ras (left) and C40-Ras cells (right) treated with moderate (top, 2.5 ng/ml) or high levels of TGFβ (40 ng/ml; see A) and subjected to in situ TUNEL staining (green). DAPI staining (blue) indicates live cells. (C) Quantitation of TUNEL-positive cells from collagen gel structures either stained and counted in suspension after collagenase digestion (top; red asterisks indicate statistically significant increases in apoptosis induced by TGFβ) or stained and counted in situ (bottom; as described in Materials and methods). Mean percentages of apoptotic cells from multiple gel structures plus standard deviations (error bars) from three independent determinations are shown. Bars, 50 μm.

Mentions: Oncogenic Ras and Raf abolish TGFβ-induced apoptosis in several epithelial cell systems (Oft et al., 1996; Lehmann et al., 2000). V12-Ras, S35-Ras, and C40-Ras cells (from both clones and clone pools; Fig. 4 A) were therefore tested for their apoptotic response upon TGFβ treatment. This was done using cells pregrown in collagen gels for 3–4 d (Fig. 6 A), since detection of apoptosis was much easier in these cultures than on plastic (Lehmann et al., 2000). Collagen structures were either collagenase digested and the cells subjected to TUNEL staining in suspension (Fig. 6 C, top) or TUNEL stained and counted in situ, that is, within the intact collagen gels (Fig. 6, B and C, bottom; 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)

V12-Ras and C40-Ras but not S35-Ras protect EpH4 cells from TGFβ-induced apoptosis. (A) For apoptosis determination in collagen gels, EpH4 cells expressing Ras or Ras effector–specific mutants were allowed to form structures in collagen gels for 3 d and were treated with various concentrations of TGFβ (0.5–40 ng/ml) for 4 d (dotted bar). Cells were either TUNEL stained in suspension (C, top) or in situ (B, and C, bottom panel; as described in Materials and methods). (B) Confocal immunofluorescence analysis of gel structures formed by clones overexpressing S35-Ras (left) and C40-Ras cells (right) treated with moderate (top, 2.5 ng/ml) or high levels of TGFβ (40 ng/ml; see A) and subjected to in situ TUNEL staining (green). DAPI staining (blue) indicates live cells. (C) Quantitation of TUNEL-positive cells from collagen gel structures either stained and counted in suspension after collagenase digestion (top; red asterisks indicate statistically significant increases in apoptosis induced by TGFβ) or stained and counted in situ (bottom; as described in Materials and methods). Mean percentages of apoptotic cells from multiple gel structures plus standard deviations (error bars) from three independent determinations are shown. Bars, 50 μm.
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getmorefigures.php?uid=PMC2199233&req=5

fig6: V12-Ras and C40-Ras but not S35-Ras protect EpH4 cells from TGFβ-induced apoptosis. (A) For apoptosis determination in collagen gels, EpH4 cells expressing Ras or Ras effector–specific mutants were allowed to form structures in collagen gels for 3 d and were treated with various concentrations of TGFβ (0.5–40 ng/ml) for 4 d (dotted bar). Cells were either TUNEL stained in suspension (C, top) or in situ (B, and C, bottom panel; as described in Materials and methods). (B) Confocal immunofluorescence analysis of gel structures formed by clones overexpressing S35-Ras (left) and C40-Ras cells (right) treated with moderate (top, 2.5 ng/ml) or high levels of TGFβ (40 ng/ml; see A) and subjected to in situ TUNEL staining (green). DAPI staining (blue) indicates live cells. (C) Quantitation of TUNEL-positive cells from collagen gel structures either stained and counted in suspension after collagenase digestion (top; red asterisks indicate statistically significant increases in apoptosis induced by TGFβ) or stained and counted in situ (bottom; as described in Materials and methods). Mean percentages of apoptotic cells from multiple gel structures plus standard deviations (error bars) from three independent determinations are shown. Bars, 50 μm.
Mentions: Oncogenic Ras and Raf abolish TGFβ-induced apoptosis in several epithelial cell systems (Oft et al., 1996; Lehmann et al., 2000). V12-Ras, S35-Ras, and C40-Ras cells (from both clones and clone pools; Fig. 4 A) were therefore tested for their apoptotic response upon TGFβ treatment. This was done using cells pregrown in collagen gels for 3–4 d (Fig. 6 A), since detection of apoptosis was much easier in these cultures than on plastic (Lehmann et al., 2000). Collagen structures were either collagenase digested and the cells subjected to TUNEL staining in suspension (Fig. 6 C, top) or TUNEL stained and counted in situ, that is, within the intact collagen gels (Fig. 6, B and C, bottom; 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