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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

Requirement of RSK2 expression in MSP and TGF-β1 induced EMT-like activity in cancer cells. A) Expression of RON, RSK1, RSK2, and TGF-β receptors in human cancer L3.6pl and HT-29 cells. Cell lysates (50 μg/sample) were subjected to Western blot analysis using antibodies specific to individual proteins. B) MSP induces EMT-like activities in pancreatic cancer L3.6pl cells. L3.6pl cells (0.5 × 106 cells per dish) were cultured overnight and then stimulated at 37°C with MSP (2 nM), TGF-β1 (5 ng/ml), or both for 24 h. Cell morphological changes were observed by Olympus microscope and photographed with CCD camera. E-cadherin and vimentin expression was determined by Western blot analysis using cell lysates as described previously [35]. Actin was used as the loading control. Scale bars represent 20 μm. C) Forced RSK2 expression facilitates MSP and TGF-1-induced EMT-like activity in HT-29 cells. Cells (2 × 106 cells per dish) were transiently transfected with 3 μg of pRKS2 plasmid or control vector pcDNA3.1 for 48 h and then stimulated with MSP and TGF-β1 as described above. Morphological changes and expression of individual proteins were determined as described in A. Scale bars represent 20 μM. D) RSK2 expression diminishes E-cadherin expression and increases vimentin expression. HT-29 cells were transiently transfected with pRSK2 plasmid for 48 h followed by stimulation with MSP, TGF-β1 or both for 24 h. Cell lysates were subjected to Western blot analysis using antibodies specific to E-cadherin or vimentin. β-actin was used as the loading control. Data shown here are from one of two experiments with similar results.
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Figure 6: Requirement of RSK2 expression in MSP and TGF-β1 induced EMT-like activity in cancer cells. A) Expression of RON, RSK1, RSK2, and TGF-β receptors in human cancer L3.6pl and HT-29 cells. Cell lysates (50 μg/sample) were subjected to Western blot analysis using antibodies specific to individual proteins. B) MSP induces EMT-like activities in pancreatic cancer L3.6pl cells. L3.6pl cells (0.5 × 106 cells per dish) were cultured overnight and then stimulated at 37°C with MSP (2 nM), TGF-β1 (5 ng/ml), or both for 24 h. Cell morphological changes were observed by Olympus microscope and photographed with CCD camera. E-cadherin and vimentin expression was determined by Western blot analysis using cell lysates as described previously [35]. Actin was used as the loading control. Scale bars represent 20 μm. C) Forced RSK2 expression facilitates MSP and TGF-1-induced EMT-like activity in HT-29 cells. Cells (2 × 106 cells per dish) were transiently transfected with 3 μg of pRKS2 plasmid or control vector pcDNA3.1 for 48 h and then stimulated with MSP and TGF-β1 as described above. Morphological changes and expression of individual proteins were determined as described in A. Scale bars represent 20 μM. D) RSK2 expression diminishes E-cadherin expression and increases vimentin expression. HT-29 cells were transiently transfected with pRSK2 plasmid for 48 h followed by stimulation with MSP, TGF-β1 or both for 24 h. Cell lysates were subjected to Western blot analysis using antibodies specific to E-cadherin or vimentin. β-actin was used as the loading control. Data shown here are from one of two experiments with similar results.

Mentions: To study the effect of RSK2 on MSP-induced EMT in more detail, two human cancer cell lines L3.6pl and HT-29 were selected based on their differences in RSK1 and RSK2 levels and similarities in RON and TGF-β receptor expression (Figure 6A). Pancreatic cancer L3.6pL cells expressed regular levels of RSK1 and RSK2. MSP and TGF-β1 stimulation caused elongated cell morphology, reduced E-cadherin expression, and increased vimentin expression (Figure 6B). Combined MSP and TGF-β1 treatment further enhanced the modulating effect on E-cadherin and vimentin expression. These results indicated that L3.6pl cells show EMT-like phenotypic changes after MSP and TGF-β1 stimulation and a synergistic activity between RON and TGF-βRI/II signaling in induction of EMT-like phenotype.


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)

Requirement of RSK2 expression in MSP and TGF-β1 induced EMT-like activity in cancer cells. A) Expression of RON, RSK1, RSK2, and TGF-β receptors in human cancer L3.6pl and HT-29 cells. Cell lysates (50 μg/sample) were subjected to Western blot analysis using antibodies specific to individual proteins. B) MSP induces EMT-like activities in pancreatic cancer L3.6pl cells. L3.6pl cells (0.5 × 106 cells per dish) were cultured overnight and then stimulated at 37°C with MSP (2 nM), TGF-β1 (5 ng/ml), or both for 24 h. Cell morphological changes were observed by Olympus microscope and photographed with CCD camera. E-cadherin and vimentin expression was determined by Western blot analysis using cell lysates as described previously [35]. Actin was used as the loading control. Scale bars represent 20 μm. C) Forced RSK2 expression facilitates MSP and TGF-1-induced EMT-like activity in HT-29 cells. Cells (2 × 106 cells per dish) were transiently transfected with 3 μg of pRKS2 plasmid or control vector pcDNA3.1 for 48 h and then stimulated with MSP and TGF-β1 as described above. Morphological changes and expression of individual proteins were determined as described in A. Scale bars represent 20 μM. D) RSK2 expression diminishes E-cadherin expression and increases vimentin expression. HT-29 cells were transiently transfected with pRSK2 plasmid for 48 h followed by stimulation with MSP, TGF-β1 or both for 24 h. Cell lysates were subjected to Western blot analysis using antibodies specific to E-cadherin or vimentin. β-actin was used as the loading control. Data shown here are from one of two experiments with similar results.
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Related In: Results  -  Collection

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Figure 6: Requirement of RSK2 expression in MSP and TGF-β1 induced EMT-like activity in cancer cells. A) Expression of RON, RSK1, RSK2, and TGF-β receptors in human cancer L3.6pl and HT-29 cells. Cell lysates (50 μg/sample) were subjected to Western blot analysis using antibodies specific to individual proteins. B) MSP induces EMT-like activities in pancreatic cancer L3.6pl cells. L3.6pl cells (0.5 × 106 cells per dish) were cultured overnight and then stimulated at 37°C with MSP (2 nM), TGF-β1 (5 ng/ml), or both for 24 h. Cell morphological changes were observed by Olympus microscope and photographed with CCD camera. E-cadherin and vimentin expression was determined by Western blot analysis using cell lysates as described previously [35]. Actin was used as the loading control. Scale bars represent 20 μm. C) Forced RSK2 expression facilitates MSP and TGF-1-induced EMT-like activity in HT-29 cells. Cells (2 × 106 cells per dish) were transiently transfected with 3 μg of pRKS2 plasmid or control vector pcDNA3.1 for 48 h and then stimulated with MSP and TGF-β1 as described above. Morphological changes and expression of individual proteins were determined as described in A. Scale bars represent 20 μM. D) RSK2 expression diminishes E-cadherin expression and increases vimentin expression. HT-29 cells were transiently transfected with pRSK2 plasmid for 48 h followed by stimulation with MSP, TGF-β1 or both for 24 h. Cell lysates were subjected to Western blot analysis using antibodies specific to E-cadherin or vimentin. β-actin was used as the loading control. Data shown here are from one of two experiments with similar results.
Mentions: To study the effect of RSK2 on MSP-induced EMT in more detail, two human cancer cell lines L3.6pl and HT-29 were selected based on their differences in RSK1 and RSK2 levels and similarities in RON and TGF-β receptor expression (Figure 6A). Pancreatic cancer L3.6pL cells expressed regular levels of RSK1 and RSK2. MSP and TGF-β1 stimulation caused elongated cell morphology, reduced E-cadherin expression, and increased vimentin expression (Figure 6B). Combined MSP and TGF-β1 treatment further enhanced the modulating effect on E-cadherin and vimentin expression. These results indicated that L3.6pl cells show EMT-like phenotypic changes after MSP and TGF-β1 stimulation and a synergistic activity between RON and TGF-βRI/II signaling in induction of EMT-like phenotype.

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