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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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Ras activity is required for induction and maintenance of EMT. (A–D) EpXT cells were seeded into collagen gels in the absence (A) or presence (B) of 10 μM Ras farnesylation inhibitor (L739749), and resulting structures were photographed after 6 d (as described in Materials and methods). Note the complete reversal of EpXT cells to tubular structures with lumina (white arrows), which persisted after removal of the inhibitor for 4 d (C). The inhibitor alone did not affect tubular structures formed by EpRas cells (B, inset) or after reversal (D, inset, control), whereas treatment of the reverted structures with TGFβ plus L739749 caused cell disintegration. (E and F) Immunostaining of frozen ultrathin sections of EpXT collagen gel structures before (E) or after treatment (F) with L739749 for 5 d. Sections were stained with antibodies to TGFβ (red) (Oft et al., 1996) and vimentin (green) plus DAPI (top, DNA). Parallel sections (bottom) were stained with antibodies to ZO-1 (red) and fibronectin (green). L739749 causes loss of intracellular TGFβ and vimentin (top) and extracellular fibronectin (bottom) and reexpression of ZO-1 at apicolateral sites of tight junctions (blue arrows, bottom left). Bars: (A–D) 50 μm; (E and F) 20 μm.
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fig2: Ras activity is required for induction and maintenance of EMT. (A–D) EpXT cells were seeded into collagen gels in the absence (A) or presence (B) of 10 μM Ras farnesylation inhibitor (L739749), and resulting structures were photographed after 6 d (as described in Materials and methods). Note the complete reversal of EpXT cells to tubular structures with lumina (white arrows), which persisted after removal of the inhibitor for 4 d (C). The inhibitor alone did not affect tubular structures formed by EpRas cells (B, inset) or after reversal (D, inset, control), whereas treatment of the reverted structures with TGFβ plus L739749 caused cell disintegration. (E and F) Immunostaining of frozen ultrathin sections of EpXT collagen gel structures before (E) or after treatment (F) with L739749 for 5 d. Sections were stained with antibodies to TGFβ (red) (Oft et al., 1996) and vimentin (green) plus DAPI (top, DNA). Parallel sections (bottom) were stained with antibodies to ZO-1 (red) and fibronectin (green). L739749 causes loss of intracellular TGFβ and vimentin (top) and extracellular fibronectin (bottom) and reexpression of ZO-1 at apicolateral sites of tight junctions (blue arrows, bottom left). Bars: (A–D) 50 μm; (E and F) 20 μm.

Mentions: Using a kinase-dead dominant negative mutant of the TGFβ-RII, we showed previously that maintenance of TGFβ-R signaling is required for EMT and metastasis (Oft et al., 1998). To address if EMT also required maintenance of oncogenic Ras activity, we used a specific nontoxic inhibitor of Ras-farnesylation (L739749 [Kohl et al., 1994]). L739749 reversed EMT in mesenchymal EpXT cells, causing reformation of hollow tubular structures (Fig. 2 B) from the unordered cords and strands of EpXT cells (Fig. 2 A) but did not affect EpH4 (Fig. 2 B, inset). After inhibitor withdrawal, the reverted cells maintained their polarized phenotype as indicated by persistence of tubular structures (Fig. 2 C). However, these structures could be reinduced to undergo EMT when again treated with TGFβ (unpublished data). This indicates that Ras inhibition reversed EMT completely, converting EpXT cells into cells with EpH4 cell properties. In line with this, reactivation of Ras in the reverted cells was insufficient for EMT, again requiring TGFβ as in the original EpRas cells (Oft et al., 1996). Furthermore, cotreatment of the reverted cells with L739749 and TGFβ resulted in cell death (Fig. 2 D), whereas their treatment with Ras inhibitor alone caused no phenotypical changes (Fig. 2 D, inset).


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)

Ras activity is required for induction and maintenance of EMT. (A–D) EpXT cells were seeded into collagen gels in the absence (A) or presence (B) of 10 μM Ras farnesylation inhibitor (L739749), and resulting structures were photographed after 6 d (as described in Materials and methods). Note the complete reversal of EpXT cells to tubular structures with lumina (white arrows), which persisted after removal of the inhibitor for 4 d (C). The inhibitor alone did not affect tubular structures formed by EpRas cells (B, inset) or after reversal (D, inset, control), whereas treatment of the reverted structures with TGFβ plus L739749 caused cell disintegration. (E and F) Immunostaining of frozen ultrathin sections of EpXT collagen gel structures before (E) or after treatment (F) with L739749 for 5 d. Sections were stained with antibodies to TGFβ (red) (Oft et al., 1996) and vimentin (green) plus DAPI (top, DNA). Parallel sections (bottom) were stained with antibodies to ZO-1 (red) and fibronectin (green). L739749 causes loss of intracellular TGFβ and vimentin (top) and extracellular fibronectin (bottom) and reexpression of ZO-1 at apicolateral sites of tight junctions (blue arrows, bottom left). Bars: (A–D) 50 μm; (E and F) 20 μm.
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fig2: Ras activity is required for induction and maintenance of EMT. (A–D) EpXT cells were seeded into collagen gels in the absence (A) or presence (B) of 10 μM Ras farnesylation inhibitor (L739749), and resulting structures were photographed after 6 d (as described in Materials and methods). Note the complete reversal of EpXT cells to tubular structures with lumina (white arrows), which persisted after removal of the inhibitor for 4 d (C). The inhibitor alone did not affect tubular structures formed by EpRas cells (B, inset) or after reversal (D, inset, control), whereas treatment of the reverted structures with TGFβ plus L739749 caused cell disintegration. (E and F) Immunostaining of frozen ultrathin sections of EpXT collagen gel structures before (E) or after treatment (F) with L739749 for 5 d. Sections were stained with antibodies to TGFβ (red) (Oft et al., 1996) and vimentin (green) plus DAPI (top, DNA). Parallel sections (bottom) were stained with antibodies to ZO-1 (red) and fibronectin (green). L739749 causes loss of intracellular TGFβ and vimentin (top) and extracellular fibronectin (bottom) and reexpression of ZO-1 at apicolateral sites of tight junctions (blue arrows, bottom left). Bars: (A–D) 50 μm; (E and F) 20 μm.
Mentions: Using a kinase-dead dominant negative mutant of the TGFβ-RII, we showed previously that maintenance of TGFβ-R signaling is required for EMT and metastasis (Oft et al., 1998). To address if EMT also required maintenance of oncogenic Ras activity, we used a specific nontoxic inhibitor of Ras-farnesylation (L739749 [Kohl et al., 1994]). L739749 reversed EMT in mesenchymal EpXT cells, causing reformation of hollow tubular structures (Fig. 2 B) from the unordered cords and strands of EpXT cells (Fig. 2 A) but did not affect EpH4 (Fig. 2 B, inset). After inhibitor withdrawal, the reverted cells maintained their polarized phenotype as indicated by persistence of tubular structures (Fig. 2 C). However, these structures could be reinduced to undergo EMT when again treated with TGFβ (unpublished data). This indicates that Ras inhibition reversed EMT completely, converting EpXT cells into cells with EpH4 cell properties. In line with this, reactivation of Ras in the reverted cells was insufficient for EMT, again requiring TGFβ as in the original EpRas cells (Oft et al., 1996). Furthermore, cotreatment of the reverted cells with L739749 and TGFβ resulted in cell death (Fig. 2 D), whereas their treatment with Ras inhibitor alone caused no phenotypical changes (Fig. 2 D, inset).

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