Abstract
BackgroundEpithelial 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.ResultsThe 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.ConclusionsMSP-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.
Highlights
Epithelial to mesenchymal transition (EMT) occurs during cancer cell invasion and malignant metastasis
By analyzing potential signaling proteins that are involved in macrophage-stimulating protein (MSP)-induced EMT-like activities, we discovered that RSK2 is a principle effector molecule responsible for MSP-induced EMT in Martin-Darby canine kidney (MDCK) and human cancer cells
Prevention of MSPinduced spindle-like morphology was not observed in MDCK cells stably expressing RON (M-RON) cells treated with wortmannin, SB203580, SP600125, Cay10512, and S31-201, suggesting that signaling from these pathways was not involved in MSPinduced EMT
Summary
Epithelial to mesenchymal transition (EMT) occurs during cancer cell invasion and malignant metastasis. Development of EMT in cancer cells is regulated and precisely controlled at different cellular levels [4,5] Various proteins such as receptor tyrosine kinases (RTK) [8,9,10], cytokine receptors [11,12], intracellular signaling molecules [13,14], and transcriptional factors [15,16] are involved in cellular EMT program. Convincing evidence indicates that signals coordinated among different pathways such as the RTKErk1/2 and TGF-b1-Smad pathways maximize transdifferentiation of epithelial tumor cells towards EMT [1,2] Such coordination raises the possibility that a converging signal for diverse pathways may exist, and may act as a central determinant controlling cellular EMT program
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