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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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Inhibitors of Mek-1 prevent and reverse EMT, and PI3K inhibitors abolish protection from TGFβ-induced apoptosis. (A) EpH4 cells (lanes marked E) or EpRas cells mock treated (lanes marked R) or treated with the inhibitors indicated (lanes marked iR) were lysed after 6 h of inhibitor treatment and analyzed for serine phosphorylation of Erk1/2 (MAPK) and PKB/Akt in Western blots (as described in Materials and methods). (B) EpRas cells seeded into collagen gels in the presence or absence (insets) of 5 ng/ml TGFβ. Both types of cultures were then left untreated (top, control) or treated with PD98059 (top right) and LY294002 (bottom) for another 5 d (as described in Materials and methods). Neither inhibitor was toxic to EpRas cells without TGFβ (insets; LY294002 slowed down structure growth). PD98059 (10 μM) completely reverted EMT (white arrows, top right), 5 μM LY294002 had no effect on the mesenchymal structures (black arrows, bottom left), whereas 30 μM caused cell death (bottom right, red circle). (C) Confocal analysis of in situ TUNEL staining (green) performed on collagen gel structures from EpRas cells treated for 4 d as indicated on the microphotographs (counterstaining for DNA in blue). (D) Quantitative analysis of cell death caused by inhibition of MEK-1 and PI3K in the absence or presence of TGFβ using trypan blue dye exclusion assay. The percentages of structures consisting of 25–60% (hatched bars, partially dead) or >60% trypan blue–positive cells (black bars, dead) are shown (as described in Materials and methods). Red asterisks indicate statistically significant induction of apoptosis by TGFβ. Bars: (B and C) 50 μm.
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fig3: Inhibitors of Mek-1 prevent and reverse EMT, and PI3K inhibitors abolish protection from TGFβ-induced apoptosis. (A) EpH4 cells (lanes marked E) or EpRas cells mock treated (lanes marked R) or treated with the inhibitors indicated (lanes marked iR) were lysed after 6 h of inhibitor treatment and analyzed for serine phosphorylation of Erk1/2 (MAPK) and PKB/Akt in Western blots (as described in Materials and methods). (B) EpRas cells seeded into collagen gels in the presence or absence (insets) of 5 ng/ml TGFβ. Both types of cultures were then left untreated (top, control) or treated with PD98059 (top right) and LY294002 (bottom) for another 5 d (as described in Materials and methods). Neither inhibitor was toxic to EpRas cells without TGFβ (insets; LY294002 slowed down structure growth). PD98059 (10 μM) completely reverted EMT (white arrows, top right), 5 μM LY294002 had no effect on the mesenchymal structures (black arrows, bottom left), whereas 30 μM caused cell death (bottom right, red circle). (C) Confocal analysis of in situ TUNEL staining (green) performed on collagen gel structures from EpRas cells treated for 4 d as indicated on the microphotographs (counterstaining for DNA in blue). (D) Quantitative analysis of cell death caused by inhibition of MEK-1 and PI3K in the absence or presence of TGFβ using trypan blue dye exclusion assay. The percentages of structures consisting of 25–60% (hatched bars, partially dead) or >60% trypan blue–positive cells (black bars, dead) are shown (as described in Materials and methods). Red asterisks indicate statistically significant induction of apoptosis by TGFβ. Bars: (B and C) 50 μm.

Mentions: The activity of the Mek1/MAPK- and PI3K-PKB/Akt pathways in EpRas and EpH4 cells and the ability of the two inhibitors to selectively suppress either pathway was analyzed by Western blots using phospho-specific antibodies to MAPK/extracellular signal–regulated kinase (Erk)1/2 and PKB/Akt. EpRas cells exhibited strongly elevated phospho-Erk/MAPK and phospho-PKB/Akt levels compared with EpH4 cells (Fig. 3 A, lanes E and R). These elevated levels were largely independent of cell density (unpublished data). Testing of the inhibitors in EpRas cells revealed that 10 μM PD98059 reduced MAPK phosphorylation to basal levels, whereas 40 μM completely abolished MAPK phosphorylation (unpublished data). Conversely, PKB/Akt phosphorylation levels were not affected by PD98059 (Fig. 3 A, left, lanes R and iR; unpublished data). Conversely, 5 μM LY294002 added at 12-h intervals stably reduced elevated PKB/Akt phosphorylation in EpRas cells to basal levels (Fig. 3 A, middle; unpublished data), whereas 30 μM LY294002 essentially abolished PKB/Akt phosphorylation (Fig. 3 A, right). In contrast, even high levels of LY294002 had no effect on MAPK phosphorylation in EpRas cells (Fig. 3 A).


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)

Inhibitors of Mek-1 prevent and reverse EMT, and PI3K inhibitors abolish protection from TGFβ-induced apoptosis. (A) EpH4 cells (lanes marked E) or EpRas cells mock treated (lanes marked R) or treated with the inhibitors indicated (lanes marked iR) were lysed after 6 h of inhibitor treatment and analyzed for serine phosphorylation of Erk1/2 (MAPK) and PKB/Akt in Western blots (as described in Materials and methods). (B) EpRas cells seeded into collagen gels in the presence or absence (insets) of 5 ng/ml TGFβ. Both types of cultures were then left untreated (top, control) or treated with PD98059 (top right) and LY294002 (bottom) for another 5 d (as described in Materials and methods). Neither inhibitor was toxic to EpRas cells without TGFβ (insets; LY294002 slowed down structure growth). PD98059 (10 μM) completely reverted EMT (white arrows, top right), 5 μM LY294002 had no effect on the mesenchymal structures (black arrows, bottom left), whereas 30 μM caused cell death (bottom right, red circle). (C) Confocal analysis of in situ TUNEL staining (green) performed on collagen gel structures from EpRas cells treated for 4 d as indicated on the microphotographs (counterstaining for DNA in blue). (D) Quantitative analysis of cell death caused by inhibition of MEK-1 and PI3K in the absence or presence of TGFβ using trypan blue dye exclusion assay. The percentages of structures consisting of 25–60% (hatched bars, partially dead) or >60% trypan blue–positive cells (black bars, dead) are shown (as described in Materials and methods). Red asterisks indicate statistically significant induction of apoptosis by TGFβ. Bars: (B and C) 50 μm.
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fig3: Inhibitors of Mek-1 prevent and reverse EMT, and PI3K inhibitors abolish protection from TGFβ-induced apoptosis. (A) EpH4 cells (lanes marked E) or EpRas cells mock treated (lanes marked R) or treated with the inhibitors indicated (lanes marked iR) were lysed after 6 h of inhibitor treatment and analyzed for serine phosphorylation of Erk1/2 (MAPK) and PKB/Akt in Western blots (as described in Materials and methods). (B) EpRas cells seeded into collagen gels in the presence or absence (insets) of 5 ng/ml TGFβ. Both types of cultures were then left untreated (top, control) or treated with PD98059 (top right) and LY294002 (bottom) for another 5 d (as described in Materials and methods). Neither inhibitor was toxic to EpRas cells without TGFβ (insets; LY294002 slowed down structure growth). PD98059 (10 μM) completely reverted EMT (white arrows, top right), 5 μM LY294002 had no effect on the mesenchymal structures (black arrows, bottom left), whereas 30 μM caused cell death (bottom right, red circle). (C) Confocal analysis of in situ TUNEL staining (green) performed on collagen gel structures from EpRas cells treated for 4 d as indicated on the microphotographs (counterstaining for DNA in blue). (D) Quantitative analysis of cell death caused by inhibition of MEK-1 and PI3K in the absence or presence of TGFβ using trypan blue dye exclusion assay. The percentages of structures consisting of 25–60% (hatched bars, partially dead) or >60% trypan blue–positive cells (black bars, dead) are shown (as described in Materials and methods). Red asterisks indicate statistically significant induction of apoptosis by TGFβ. Bars: (B and C) 50 μm.
Mentions: The activity of the Mek1/MAPK- and PI3K-PKB/Akt pathways in EpRas and EpH4 cells and the ability of the two inhibitors to selectively suppress either pathway was analyzed by Western blots using phospho-specific antibodies to MAPK/extracellular signal–regulated kinase (Erk)1/2 and PKB/Akt. EpRas cells exhibited strongly elevated phospho-Erk/MAPK and phospho-PKB/Akt levels compared with EpH4 cells (Fig. 3 A, lanes E and R). These elevated levels were largely independent of cell density (unpublished data). Testing of the inhibitors in EpRas cells revealed that 10 μM PD98059 reduced MAPK phosphorylation to basal levels, whereas 40 μM completely abolished MAPK phosphorylation (unpublished data). Conversely, PKB/Akt phosphorylation levels were not affected by PD98059 (Fig. 3 A, left, lanes R and iR; unpublished data). Conversely, 5 μM LY294002 added at 12-h intervals stably reduced elevated PKB/Akt phosphorylation in EpRas cells to basal levels (Fig. 3 A, middle; unpublished data), whereas 30 μM LY294002 essentially abolished PKB/Akt phosphorylation (Fig. 3 A, right). In contrast, even high levels of LY294002 had no effect on MAPK phosphorylation in EpRas cells (Fig. 3 A).

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