RON and c-Met facilitate metastasis through the ERK signaling pathway in prostate cancer cells

Abstract

Prostate cancer (PCa) is a metastatic malignant cancer driven by complex pathological mechanisms and characterized by poor long-term prognosis. Metastasis is the main cause of death of PCa patients, yet the molecular mechanisms of this process are poorly understood. In the present study, positive co-expression of RON and c-Met was observed in human clinical PCa tissues (biopsy material), as detected by immunohistochemical staining and quantitative real-time PCR. We investigated this further in PCa cells, demonstrating that the inhibition of RON and c-Met with foretinib (GSK1363089) suppressed metastasis and promoted the reversal of the epithelial-to-mesenchymal transition (EMT) in PCa cells. Furthermore, the invasion and migration of PCa cells were enhanced by the exogenous activation of RON with MSP and c-Met with HGF, whereas silencing of RON and c-Met attenuated the invasion and metastasis of the PCa cells. Our data also demonstrated that HGF/c-Met, but not the MSP-RON signaling pathway may be the dominant mechanism for PCa EMT. We further revealed that RON and c-Met facilitate metastasis via ERK1/2 signaling. These findings indicate that RON and c-Met facilitate metastasis through ERK1/2 signaling and that targeting RON and c-Met with foretinib may be an attractive therapeutic option for suppressing PCa metastasis.

Figure 1.

RON and c-Met are overexpressed in prostate cancer tissues and are associated with metastasis in PCa cell lines. (A) H&E staining and immunohistochemistry using antibodies against RON and c-Met in normal prostate tissues and their corresponding prostate cancerous tissues (magnification, ×100). (B) Correlation between RON and c-Met mRNA expression levels in clinical specimens. Each mRNA level is relative to that of GAPDH. The Pearson correlation test and linear regression were used to determine the relationship between the two genes tested. There was a significant correlation between RON and c-Met (P<0.0001, R=0.7317, N=20). (C and D) RON and c-Met expression were examined by western blot analysis, RT-PCR and immunofluorescent analysis. (E) The metastatic potential of PCa cells was analyzed by migration and invasion assays (magnification, ×200). ***P<0.001.

Figure 2.

Inhibition of RON and c-Met using foretinib (GSK) inhibits the migration and invasion of PCa cell lines. (A) PC3 and DU145 cells were treated with foretinib (20 nM) for 24 h. Whole-cell lysates from indicated cells were analyzed by western blot analysis for RON, c-Met, E-cadherin, N-cadherin and GAPDH. (B) Wound healing assay was performed to determine the effect of foretinib (20 nM) on the migration of PC3 and DU145 cells. (C and D) PC3 and DU145 cells were treated with foretinib (20 nM) for 24 h. Migratory and invasive behaviors were analyzed by migration and invasion assays (magnification, ×200). ***P<0.001.

Figure 3.

Functional role of RON in the migration and invasion of PCa cell lines. (A) Detection of siRNA-mediated knockdown of RON in PC3 and DU145 cells by western blotting and real-time PCR. (B) The expression of RON, E-cadherin and N-cadherin was determined by western blotting after RON knockdown and MSP (100 ng/ml) stimulation in PC3 and DU145 cells. (C and D) Transwell assays for the effects of RON silencing and MSP (100 ng/ml) on the migratory and invasive potentials of PC3 and DU145 cells. *P<0.05, **P<0.01, ***P<0.001; NS, not significant.

Figure 4.

Functional role of c-Met in the metastasis and invasion of PCa cell lines. (A) Detection of the siRNA-mediated knockdown of c-Met in PC3 and DU145 cells by real-time PCR and western blotting. (B) The expression of c-Met, E-cadherin and N-cadherin was determined by western blotting after c-Met knockdown and HGF (50 ng/ml) stimulation in the PC3 and DU145 cells. (C and D) Transwell assays of the effects of c-Met silencing and HGF (50 ng/ml) on the migratory and invasive potentials of the PC3 and DU145 cells. (E) Morphological changes of PC3 and DU145 cells stimulated by HGF (50 ng/ml) and foretinib. (F and G) HGF (50 ng/ml) induced cell scattering in the PC3 and DU145 cells. *P<0.05, **P<0.01, ***P<0.001; NS, not significant.

Figure 5.

Functional role of RON and c-Met in the metastasis and invasion of PCa cell lines. (A) Transwell assays for the effects of si-RON and MSP (100 ng/ml) on migratory and invasive potentials of PC3 and DU145 cells (magnification, ×200). (B) Transwell assays for the effects of si-c-Met and HGF (50 ng/ml) on the migratory and invasive potentials of PC3 and DU145 cells (magnification, ×200).

Figure 6.

RON and c-Met mediates PCa metastasis via the ERK1/2 pathway. (A) Effect of RON and c-Met on phosphorylation of p-ERK1/2 was analyzed by western blotting. (B) PC3 and DU145 cells were treated with MSP or HGF for 24 h. Level of p-ERK1/2 was detected by western blotting. (C and D) Migration and invasion assays were carried out in the PC3 and DU145 cells following treatment with MSP, HGF and U0126 for 24 h (magnification, ×200). Each experiment was performed in triplicate. **P<0.01, ***P<0.001.