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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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Related in: MedlinePlus

Inhibitory effect of CP-1 and PD98059 on MSP or MSP plus TGF-β1-induced RSK2 phosphorylation: M-RON cells (3 × 106 cells per dish) in DMEM with 1% of FBS were first treated with CP-1 or PD98059 for 10 min followed by stimulation with MSP (2 nM) or MSP plus TGF-β1 (5 ng/ml). Cells were collected 30 min after stimulation. Phosphorylated RON was determined by Western blot analysis after Zt/g4 immunoprecipitation of cell lysates (250 μg proteins/sample). Phosphorylated Erk1/2 and RSK2 were directly determined by Western blot analysis using specific antibodies, respectively. Membranes were also reprobed with individual antibodies to detect non-phosphorylated proteins as the loading controls. Data shown here are from one of two experiments with similar results.
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Figure 2: Inhibitory effect of CP-1 and PD98059 on MSP or MSP plus TGF-β1-induced RSK2 phosphorylation: M-RON cells (3 × 106 cells per dish) in DMEM with 1% of FBS were first treated with CP-1 or PD98059 for 10 min followed by stimulation with MSP (2 nM) or MSP plus TGF-β1 (5 ng/ml). Cells were collected 30 min after stimulation. Phosphorylated RON was determined by Western blot analysis after Zt/g4 immunoprecipitation of cell lysates (250 μg proteins/sample). Phosphorylated Erk1/2 and RSK2 were directly determined by Western blot analysis using specific antibodies, respectively. Membranes were also reprobed with individual antibodies to detect non-phosphorylated proteins as the loading controls. Data shown here are from one of two experiments with similar results.

Mentions: To determine if MSP-induced RSK2 phosphorylation is indeed mediated by RON and Erk1/2 signaling, M-RON cells were stimulated in the presence or absence of specific RON inhibitor CP-1 and Erk1/2 inhibitor PD98059. RSK2 phosphorylation was determined by Western blot analysis. CP-1 inhibited MSP-induced RON phosphorylation in a dose-dependent manner (Figure 2A). CP-1 treatment also led to diminished Erk1/2 phosphorylation. Significantly, CP-1 inhibited MSP-induced RSK2 phosphorylation in a dose-dependent manner. We also observed the inhibitory effect of CP-1 in cells stimulated with MSP plus TGF-β1. However, levels of inhibition, as shown by the phosphorylation levels of Erk1/2 and RSK2, were not as strong as those shown in cells stimulated with MSP alone. Dramatic inhibition was only seen when high concentrations of CP-1 (up to 300 μg/ml) were used. Results from PD98059 experiments confirmed that inhibition of Erk1/2 had no effect on MSP-induced RON phosphorylation. However, levels of Erk1/2 phosphorylation were diminished by PD98059 in a dose-dependent manner (Figure 2B). Moreover, PD98059 inhibited MSP or MSP plus TGF-β1-induced RSK2 phosphorylation in a dose-dependent manner. Thus, the results in Figure 2 demonstrated that by inhibiting RON or Erk1/2 activation, both CP-1 and PD98059 are able to prevent MSP or MSP plus TGF-β1-induced RSK2 phosphorylation, suggesting that activated RON and Erk1/2 signaling is required for MSP-induced RSK2 phosphorylation.


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)

Inhibitory effect of CP-1 and PD98059 on MSP or MSP plus TGF-β1-induced RSK2 phosphorylation: M-RON cells (3 × 106 cells per dish) in DMEM with 1% of FBS were first treated with CP-1 or PD98059 for 10 min followed by stimulation with MSP (2 nM) or MSP plus TGF-β1 (5 ng/ml). Cells were collected 30 min after stimulation. Phosphorylated RON was determined by Western blot analysis after Zt/g4 immunoprecipitation of cell lysates (250 μg proteins/sample). Phosphorylated Erk1/2 and RSK2 were directly determined by Western blot analysis using specific antibodies, respectively. Membranes were also reprobed with individual antibodies to detect non-phosphorylated proteins as the loading controls. Data shown here are from one of two experiments with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Inhibitory effect of CP-1 and PD98059 on MSP or MSP plus TGF-β1-induced RSK2 phosphorylation: M-RON cells (3 × 106 cells per dish) in DMEM with 1% of FBS were first treated with CP-1 or PD98059 for 10 min followed by stimulation with MSP (2 nM) or MSP plus TGF-β1 (5 ng/ml). Cells were collected 30 min after stimulation. Phosphorylated RON was determined by Western blot analysis after Zt/g4 immunoprecipitation of cell lysates (250 μg proteins/sample). Phosphorylated Erk1/2 and RSK2 were directly determined by Western blot analysis using specific antibodies, respectively. Membranes were also reprobed with individual antibodies to detect non-phosphorylated proteins as the loading controls. Data shown here are from one of two experiments with similar results.
Mentions: To determine if MSP-induced RSK2 phosphorylation is indeed mediated by RON and Erk1/2 signaling, M-RON cells were stimulated in the presence or absence of specific RON inhibitor CP-1 and Erk1/2 inhibitor PD98059. RSK2 phosphorylation was determined by Western blot analysis. CP-1 inhibited MSP-induced RON phosphorylation in a dose-dependent manner (Figure 2A). CP-1 treatment also led to diminished Erk1/2 phosphorylation. Significantly, CP-1 inhibited MSP-induced RSK2 phosphorylation in a dose-dependent manner. We also observed the inhibitory effect of CP-1 in cells stimulated with MSP plus TGF-β1. However, levels of inhibition, as shown by the phosphorylation levels of Erk1/2 and RSK2, were not as strong as those shown in cells stimulated with MSP alone. Dramatic inhibition was only seen when high concentrations of CP-1 (up to 300 μg/ml) were used. Results from PD98059 experiments confirmed that inhibition of Erk1/2 had no effect on MSP-induced RON phosphorylation. However, levels of Erk1/2 phosphorylation were diminished by PD98059 in a dose-dependent manner (Figure 2B). Moreover, PD98059 inhibited MSP or MSP plus TGF-β1-induced RSK2 phosphorylation in a dose-dependent manner. Thus, the results in Figure 2 demonstrated that by inhibiting RON or Erk1/2 activation, both CP-1 and PD98059 are able to prevent MSP or MSP plus TGF-β1-induced RSK2 phosphorylation, suggesting that activated RON and Erk1/2 signaling is required for MSP-induced RSK2 phosphorylation.

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