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Ribosomal protein S6 kinase (RSK)-2 as a central effector molecule in RON receptor tyrosine kinase mediated epithelial to mesenchymal transition induced by macrophage-stimulating protein.

Ma Q, Guin S, Padhye SS, Zhou YQ, Zhang RW, Wang MH - Mol. Cancer (2011)

Bottom Line: These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF)-β1, an EMT-inducing cytokine.Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT.Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cancer Biology at State Key Laboratory for Diagnosis & Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University College of Medicine, Hangzhou 310003, China.

ABSTRACT

Background: Epithelial to mesenchymal transition (EMT) occurs during cancer cell invasion and malignant metastasis. Features of EMT include spindle-like cell morphology, loss of epithelial cellular markers and gain of mesenchymal phenotype. Activation of the RON receptor tyrosine kinase by macrophage-stimulating protein (MSP) has been implicated in cellular EMT program; however, the major signaling determinant(s) responsible for MSP-induced EMT is unknown.

Results: The study presented here demonstrates that RSK2, a downstream signaling protein of the Ras-Erk1/2 pathway, is the principal molecule that links MSP-activated RON signaling to complete EMT. Using MDCK cells expressing RON as a model, a spindle-shape based screen was conducted, which identifies RSK2 among various intracellular proteins as a potential signaling molecule responsible for MSP-induced EMT. MSP stimulation dissociated RSK2 with Erk1/2 and promoted RSK2 nuclear translocation. MSP strongly induced RSK2 phosphorylation in a dose-dependent manner. These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF)-β1, an EMT-inducing cytokine. Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT. In HT-29 cancer cells that barely express RSK2, forced RSK2 expression results in EMT-like phenotype upon MSP stimulation. Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration.

Conclusions: MSP-induced RSK2 activation is a critical determinant linking RON signaling to cellular EMT program. Inhibition of RSK2 activity may provide a therapeutic opportunity for blocking RON-mediated cancer cell migration and subsequent invasion.

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Effect of SL0101 on MSP or MSP plus TGF-β1-induced cell migration. The monolayer of M-RON cells in DMEM with 1% FBS) were wounded with a plastic tip and then stimulated with MSP (2 nM), TGF-β1 (5 ng/ml), or both in the presence or absence of CP-1 (300 μg/ml), PD98059 (100 μM), and SL0101 (50 μM). After incubation for 16 h, cells that migrated into the open spaces were observed under microscope and photographed. Wounded areas that covered by migrated cells were calculated as previously described [35]. Data shown here are from one of two experiments with similar results. Scale bars represent 50 μm.
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Figure 5: Effect of SL0101 on MSP or MSP plus TGF-β1-induced cell migration. The monolayer of M-RON cells in DMEM with 1% FBS) were wounded with a plastic tip and then stimulated with MSP (2 nM), TGF-β1 (5 ng/ml), or both in the presence or absence of CP-1 (300 μg/ml), PD98059 (100 μM), and SL0101 (50 μM). After incubation for 16 h, cells that migrated into the open spaces were observed under microscope and photographed. Wounded areas that covered by migrated cells were calculated as previously described [35]. Data shown here are from one of two experiments with similar results. Scale bars represent 50 μm.

Mentions: We performed the wound-healing assay to determine if SL0101 can prevent MSP-induced migration of M-RON cells. Increased migration is a function associated with EMT. Results in Figure 5 showed that M-RON cells had spontaneous migration (35.0%) and MSP stimulation further enhanced cell motility (86.0%). Treatment of cells with SL0101 alone had no effect on cell migration; however, SL0101 significantly prevented MSP or MSP-plus TGF-β1-induced cell migration. The percentages of cell migration induced by MSP and MSP plus TGF-β1 (86.0% and 89.3%, respectively) were dramatically reduced after SL0101 treatment (38.4% and 45.2%, respectively). We observed inhibition levels that were comparable to those treated with CP-1 and PD98059. Thus, results in Figure 4 and 5 demonstrated that SL0101 inhibition of RSK prevented MSP and TGF-β1-induced spindle-like morphology accompanied with redistribution of β-catenin and F-actin. E-cadherin and claudin-1 expression reappeared and vimentin expression was blocked. These activities were associated with the inhibition of transcription repressor Snail expression. Moreover, SL0101 significantly impairs MSP and TGF-β1 induced cell migration, which is a function associated with EMT.


Ribosomal protein S6 kinase (RSK)-2 as a central effector molecule in RON receptor tyrosine kinase mediated epithelial to mesenchymal transition induced by macrophage-stimulating protein.

Ma Q, Guin S, Padhye SS, Zhou YQ, Zhang RW, Wang MH - Mol. Cancer (2011)

Effect of SL0101 on MSP or MSP plus TGF-β1-induced cell migration. The monolayer of M-RON cells in DMEM with 1% FBS) were wounded with a plastic tip and then stimulated with MSP (2 nM), TGF-β1 (5 ng/ml), or both in the presence or absence of CP-1 (300 μg/ml), PD98059 (100 μM), and SL0101 (50 μM). After incubation for 16 h, cells that migrated into the open spaces were observed under microscope and photographed. Wounded areas that covered by migrated cells were calculated as previously described [35]. Data shown here are from one of two experiments with similar results. Scale bars represent 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117816&req=5

Figure 5: Effect of SL0101 on MSP or MSP plus TGF-β1-induced cell migration. The monolayer of M-RON cells in DMEM with 1% FBS) were wounded with a plastic tip and then stimulated with MSP (2 nM), TGF-β1 (5 ng/ml), or both in the presence or absence of CP-1 (300 μg/ml), PD98059 (100 μM), and SL0101 (50 μM). After incubation for 16 h, cells that migrated into the open spaces were observed under microscope and photographed. Wounded areas that covered by migrated cells were calculated as previously described [35]. Data shown here are from one of two experiments with similar results. Scale bars represent 50 μm.
Mentions: We performed the wound-healing assay to determine if SL0101 can prevent MSP-induced migration of M-RON cells. Increased migration is a function associated with EMT. Results in Figure 5 showed that M-RON cells had spontaneous migration (35.0%) and MSP stimulation further enhanced cell motility (86.0%). Treatment of cells with SL0101 alone had no effect on cell migration; however, SL0101 significantly prevented MSP or MSP-plus TGF-β1-induced cell migration. The percentages of cell migration induced by MSP and MSP plus TGF-β1 (86.0% and 89.3%, respectively) were dramatically reduced after SL0101 treatment (38.4% and 45.2%, respectively). We observed inhibition levels that were comparable to those treated with CP-1 and PD98059. Thus, results in Figure 4 and 5 demonstrated that SL0101 inhibition of RSK prevented MSP and TGF-β1-induced spindle-like morphology accompanied with redistribution of β-catenin and F-actin. E-cadherin and claudin-1 expression reappeared and vimentin expression was blocked. These activities were associated with the inhibition of transcription repressor Snail expression. Moreover, SL0101 significantly impairs MSP and TGF-β1 induced cell migration, which is a function associated with EMT.

Bottom Line: These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF)-β1, an EMT-inducing cytokine.Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT.Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cancer Biology at State Key Laboratory for Diagnosis & Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University College of Medicine, Hangzhou 310003, China.

ABSTRACT

Background: Epithelial to mesenchymal transition (EMT) occurs during cancer cell invasion and malignant metastasis. Features of EMT include spindle-like cell morphology, loss of epithelial cellular markers and gain of mesenchymal phenotype. Activation of the RON receptor tyrosine kinase by macrophage-stimulating protein (MSP) has been implicated in cellular EMT program; however, the major signaling determinant(s) responsible for MSP-induced EMT is unknown.

Results: The study presented here demonstrates that RSK2, a downstream signaling protein of the Ras-Erk1/2 pathway, is the principal molecule that links MSP-activated RON signaling to complete EMT. Using MDCK cells expressing RON as a model, a spindle-shape based screen was conducted, which identifies RSK2 among various intracellular proteins as a potential signaling molecule responsible for MSP-induced EMT. MSP stimulation dissociated RSK2 with Erk1/2 and promoted RSK2 nuclear translocation. MSP strongly induced RSK2 phosphorylation in a dose-dependent manner. These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF)-β1, an EMT-inducing cytokine. Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT. In HT-29 cancer cells that barely express RSK2, forced RSK2 expression results in EMT-like phenotype upon MSP stimulation. Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration.

Conclusions: MSP-induced RSK2 activation is a critical determinant linking RON signaling to cellular EMT program. Inhibition of RSK2 activity may provide a therapeutic opportunity for blocking RON-mediated cancer cell migration and subsequent invasion.

Show MeSH
Related in: MedlinePlus