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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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S35-Ras– and C40-Ras–overexpressing cells form tumors, but only S35-Ras cells undergo EMT in vivo. (A) Various clones (see also Fig. 4 A) overexpressing V12-Ras (black symbols) S35-Ras (red symbols) and C40-Ras (blue symbols) were injected into the mammary gland fat pads of mice (four mice with a total of 16 injection sites per clone). Growth of the multiple tumors developing in all mice was followed by measuring mean tumor sizes (as described in Materials and methods). (B) Ex-tumor cells recultivated from 4–5-wk-old tumors (as described in Materials and methods) induced by V12-Ras, S35-Ras, and C40-Ras clones (A) were seeded onto porous supports and analyzed by immunostaining for E-cadherin (green) and vimentin (red). Blue, DAPI counterstain for DNA. Bars, 20 μm.
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fig7: S35-Ras– and C40-Ras–overexpressing cells form tumors, but only S35-Ras cells undergo EMT in vivo. (A) Various clones (see also Fig. 4 A) overexpressing V12-Ras (black symbols) S35-Ras (red symbols) and C40-Ras (blue symbols) were injected into the mammary gland fat pads of mice (four mice with a total of 16 injection sites per clone). Growth of the multiple tumors developing in all mice was followed by measuring mean tumor sizes (as described in Materials and methods). (B) Ex-tumor cells recultivated from 4–5-wk-old tumors (as described in Materials and methods) induced by V12-Ras, S35-Ras, and C40-Ras clones (A) were seeded onto porous supports and analyzed by immunostaining for E-cadherin (green) and vimentin (red). Blue, DAPI counterstain for DNA. Bars, 20 μm.

Mentions: To analyze the importance of Ras downstream signaling pathways for tumor formation in vivo, clones strongly overexpressing V12-Ras, S35-Ras, and C40-Ras were injected into the mammary gland fat pads of nude mice. All cell types caused tumors with comparable efficiencies (16/16 injection sites; Fig. 7 A). Similar results were obtained with respective pools of clones overexpressing the different Ras signaling mutants except that tumor formation by cells from S35-Ras clone pools was significantly slower than from respective C40-Ras clone pools (unpublished data). Bcl-2–EpH4 control cells formed similarly small regressing nodules as EpH4 cells, which contained only well-polarized, E-cadherin–positive, vimentin-negative cells (unpublished data). Therefore, protection of EpH4 cells from TGFβ-dependent apoptosis is not sufficient for tumorigenesis.


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)

S35-Ras– and C40-Ras–overexpressing cells form tumors, but only S35-Ras cells undergo EMT in vivo. (A) Various clones (see also Fig. 4 A) overexpressing V12-Ras (black symbols) S35-Ras (red symbols) and C40-Ras (blue symbols) were injected into the mammary gland fat pads of mice (four mice with a total of 16 injection sites per clone). Growth of the multiple tumors developing in all mice was followed by measuring mean tumor sizes (as described in Materials and methods). (B) Ex-tumor cells recultivated from 4–5-wk-old tumors (as described in Materials and methods) induced by V12-Ras, S35-Ras, and C40-Ras clones (A) were seeded onto porous supports and analyzed by immunostaining for E-cadherin (green) and vimentin (red). Blue, DAPI counterstain for DNA. Bars, 20 μm.
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Related In: Results  -  Collection

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fig7: S35-Ras– and C40-Ras–overexpressing cells form tumors, but only S35-Ras cells undergo EMT in vivo. (A) Various clones (see also Fig. 4 A) overexpressing V12-Ras (black symbols) S35-Ras (red symbols) and C40-Ras (blue symbols) were injected into the mammary gland fat pads of mice (four mice with a total of 16 injection sites per clone). Growth of the multiple tumors developing in all mice was followed by measuring mean tumor sizes (as described in Materials and methods). (B) Ex-tumor cells recultivated from 4–5-wk-old tumors (as described in Materials and methods) induced by V12-Ras, S35-Ras, and C40-Ras clones (A) were seeded onto porous supports and analyzed by immunostaining for E-cadherin (green) and vimentin (red). Blue, DAPI counterstain for DNA. Bars, 20 μm.
Mentions: To analyze the importance of Ras downstream signaling pathways for tumor formation in vivo, clones strongly overexpressing V12-Ras, S35-Ras, and C40-Ras were injected into the mammary gland fat pads of nude mice. All cell types caused tumors with comparable efficiencies (16/16 injection sites; Fig. 7 A). Similar results were obtained with respective pools of clones overexpressing the different Ras signaling mutants except that tumor formation by cells from S35-Ras clone pools was significantly slower than from respective C40-Ras clone pools (unpublished data). Bcl-2–EpH4 control cells formed similarly small regressing nodules as EpH4 cells, which contained only well-polarized, E-cadherin–positive, vimentin-negative cells (unpublished data). Therefore, protection of EpH4 cells from TGFβ-dependent apoptosis is not sufficient for tumorigenesis.

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