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The Ron receptor tyrosine kinase positively regulates angiogenic chemokine production in prostate cancer cells.

Thobe MN, Gurusamy D, Pathrose P, Waltz SE - Oncogene (2009)

Bottom Line: The knockdown of Ron in PC-3 or DU145 cells results in a significant decrease in angiogenic chemokine production and is associated with a decreased activation of the transcription factor nuclear factor-kappaB (NF-kappaB).Moreover, exogenous overexpression of Ron in LNCaP cells is sufficient to induce a significant increase in angiogenic chemokines that can be abrogated by inhibition of NF-kappaB signaling.Our data show that knockdown of Ron in prostate cancer cells results in significantly less endothelial cell chemotaxis when compared with Ron-expressing cells in vitro as well as in reduced tumor growth and decreased microvessel density after orthotopic transplantation into the prostate in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA.

ABSTRACT
Overexpression of the Ron receptor tyrosine kinase has recently been shown in a wide variety of human cancers. However, no studies have examined Ron receptor expression or function during prostate tumorigenesis. In this study we report that Ron is highly expressed in human prostate adenocarcinoma and metastatic lymph nodes when compared with normal prostate or benign prostate hyperplasia. Furthermore, we show that Ron is overexpressed in PC-3 and DU145 prostate cancer cell lines, and that the levels of angiogenic chemokines produced by prostate cancer cells positively correlate with Ron expression. The knockdown of Ron in PC-3 or DU145 cells results in a significant decrease in angiogenic chemokine production and is associated with a decreased activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Moreover, exogenous overexpression of Ron in LNCaP cells is sufficient to induce a significant increase in angiogenic chemokines that can be abrogated by inhibition of NF-kappaB signaling. Given that the function of angiogenic chemokines is important in the development of new blood vessels, we also examined the ability of Ron to modulate endothelial cell migration. Our data show that knockdown of Ron in prostate cancer cells results in significantly less endothelial cell chemotaxis when compared with Ron-expressing cells in vitro as well as in reduced tumor growth and decreased microvessel density after orthotopic transplantation into the prostate in vivo. In total, our data suggest that the Ron receptor is important in modulating prostate tumor growth by modulating angiogenic chemokine production and subsequent endothelial cell recruitment.

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Ron knockdown in DU145 cells leads to decreased chemokine production, NF-κB activity, and endothelial cell chemoattractionA, DU145 cells were stably infected with either control shRNA (shNon) or a Ron-specific shRNA (shRon) and examined for Ron and IκBα expression (Figure 5A). B, DU145 cells with a loss of Ron produce significantly less CXCL8 compared to control cells. C, DU145 cells with a Ron knockdown have similar growth rates as control DU145 cells as determined by crystal violet analyses. D, DU145 Ron knockdown cells have decreased NF-κB activity compared to control cells. E, Supernatants from Ron knockdown DU145 cells exhibit less endothelial cell chemotaxis compared to supernatants from control cells. Data are expressed as means ± SE. Experiments were performed in triplicate and a representative experiment is shown. *p<0.05 compared to shNon group.
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Figure 5: Ron knockdown in DU145 cells leads to decreased chemokine production, NF-κB activity, and endothelial cell chemoattractionA, DU145 cells were stably infected with either control shRNA (shNon) or a Ron-specific shRNA (shRon) and examined for Ron and IκBα expression (Figure 5A). B, DU145 cells with a loss of Ron produce significantly less CXCL8 compared to control cells. C, DU145 cells with a Ron knockdown have similar growth rates as control DU145 cells as determined by crystal violet analyses. D, DU145 Ron knockdown cells have decreased NF-κB activity compared to control cells. E, Supernatants from Ron knockdown DU145 cells exhibit less endothelial cell chemotaxis compared to supernatants from control cells. Data are expressed as means ± SE. Experiments were performed in triplicate and a representative experiment is shown. *p<0.05 compared to shNon group.

Mentions: We have shown that Ron expression correlates with angiogenic chemokine production in prostate cancer cells, and next sought to determine the impact of Ron inhibition on chemokine production. PC-3 cells were transfected with either Ron-specific siRNA (Ron siRNA) or non-specific scrambled siRNA (Non siRNA). A significant loss of Ron mRNA was observed by 48 hours post-transfection (Figure 4A). To examine if loss of Ron expression impacted chemokine production in these cells, 30 hours following transfection, the cells were placed into serum free media and supernatant was collected at specific intervals. During this time frame, there was not significant change in cell number between control PC-3 cells and Ron knockdown PC-3 (Figure 4E). There are significant decreases in the angiogenic chemokines CXCL8 (Figure 4B), CXCL5 (Figure 4C), and CXCL1 (Figure 4D) secreted by Ron-knockdown PC-3 cells compared to control cells. Production of vascular endothelial growth factor (VEGF) and the angiostatic chemokine CXCL10 were also examined. PC-3 cells secrete relatively low levels of these factors (VEGF, Figure 3G) and there was no change in production in either CXCL10 or VEGF with Ron loss (data not shown). To complement our studies and to determine the specificity of Ron inhibition in another cell line, Ron expression was also knocked down in DU145 cells by infection with either a nonsense shRNA construct (shNon) as a control, or a Ron shRNA construct (shRon). As shown in Figure 5A, Ron expression was efficiently depleted in the Ron shRNA infected cells, which was associated with a significant decrease in CXCL8 production compared to control infected cells (Figure 5B). No change in cell proliferation was observed between DU145 control cells or DU145 Ron knockdown cells (Figure 5C).


The Ron receptor tyrosine kinase positively regulates angiogenic chemokine production in prostate cancer cells.

Thobe MN, Gurusamy D, Pathrose P, Waltz SE - Oncogene (2009)

Ron knockdown in DU145 cells leads to decreased chemokine production, NF-κB activity, and endothelial cell chemoattractionA, DU145 cells were stably infected with either control shRNA (shNon) or a Ron-specific shRNA (shRon) and examined for Ron and IκBα expression (Figure 5A). B, DU145 cells with a loss of Ron produce significantly less CXCL8 compared to control cells. C, DU145 cells with a Ron knockdown have similar growth rates as control DU145 cells as determined by crystal violet analyses. D, DU145 Ron knockdown cells have decreased NF-κB activity compared to control cells. E, Supernatants from Ron knockdown DU145 cells exhibit less endothelial cell chemotaxis compared to supernatants from control cells. Data are expressed as means ± SE. Experiments were performed in triplicate and a representative experiment is shown. *p<0.05 compared to shNon group.
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Figure 5: Ron knockdown in DU145 cells leads to decreased chemokine production, NF-κB activity, and endothelial cell chemoattractionA, DU145 cells were stably infected with either control shRNA (shNon) or a Ron-specific shRNA (shRon) and examined for Ron and IκBα expression (Figure 5A). B, DU145 cells with a loss of Ron produce significantly less CXCL8 compared to control cells. C, DU145 cells with a Ron knockdown have similar growth rates as control DU145 cells as determined by crystal violet analyses. D, DU145 Ron knockdown cells have decreased NF-κB activity compared to control cells. E, Supernatants from Ron knockdown DU145 cells exhibit less endothelial cell chemotaxis compared to supernatants from control cells. Data are expressed as means ± SE. Experiments were performed in triplicate and a representative experiment is shown. *p<0.05 compared to shNon group.
Mentions: We have shown that Ron expression correlates with angiogenic chemokine production in prostate cancer cells, and next sought to determine the impact of Ron inhibition on chemokine production. PC-3 cells were transfected with either Ron-specific siRNA (Ron siRNA) or non-specific scrambled siRNA (Non siRNA). A significant loss of Ron mRNA was observed by 48 hours post-transfection (Figure 4A). To examine if loss of Ron expression impacted chemokine production in these cells, 30 hours following transfection, the cells were placed into serum free media and supernatant was collected at specific intervals. During this time frame, there was not significant change in cell number between control PC-3 cells and Ron knockdown PC-3 (Figure 4E). There are significant decreases in the angiogenic chemokines CXCL8 (Figure 4B), CXCL5 (Figure 4C), and CXCL1 (Figure 4D) secreted by Ron-knockdown PC-3 cells compared to control cells. Production of vascular endothelial growth factor (VEGF) and the angiostatic chemokine CXCL10 were also examined. PC-3 cells secrete relatively low levels of these factors (VEGF, Figure 3G) and there was no change in production in either CXCL10 or VEGF with Ron loss (data not shown). To complement our studies and to determine the specificity of Ron inhibition in another cell line, Ron expression was also knocked down in DU145 cells by infection with either a nonsense shRNA construct (shNon) as a control, or a Ron shRNA construct (shRon). As shown in Figure 5A, Ron expression was efficiently depleted in the Ron shRNA infected cells, which was associated with a significant decrease in CXCL8 production compared to control infected cells (Figure 5B). No change in cell proliferation was observed between DU145 control cells or DU145 Ron knockdown cells (Figure 5C).

Bottom Line: The knockdown of Ron in PC-3 or DU145 cells results in a significant decrease in angiogenic chemokine production and is associated with a decreased activation of the transcription factor nuclear factor-kappaB (NF-kappaB).Moreover, exogenous overexpression of Ron in LNCaP cells is sufficient to induce a significant increase in angiogenic chemokines that can be abrogated by inhibition of NF-kappaB signaling.Our data show that knockdown of Ron in prostate cancer cells results in significantly less endothelial cell chemotaxis when compared with Ron-expressing cells in vitro as well as in reduced tumor growth and decreased microvessel density after orthotopic transplantation into the prostate in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA.

ABSTRACT
Overexpression of the Ron receptor tyrosine kinase has recently been shown in a wide variety of human cancers. However, no studies have examined Ron receptor expression or function during prostate tumorigenesis. In this study we report that Ron is highly expressed in human prostate adenocarcinoma and metastatic lymph nodes when compared with normal prostate or benign prostate hyperplasia. Furthermore, we show that Ron is overexpressed in PC-3 and DU145 prostate cancer cell lines, and that the levels of angiogenic chemokines produced by prostate cancer cells positively correlate with Ron expression. The knockdown of Ron in PC-3 or DU145 cells results in a significant decrease in angiogenic chemokine production and is associated with a decreased activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Moreover, exogenous overexpression of Ron in LNCaP cells is sufficient to induce a significant increase in angiogenic chemokines that can be abrogated by inhibition of NF-kappaB signaling. Given that the function of angiogenic chemokines is important in the development of new blood vessels, we also examined the ability of Ron to modulate endothelial cell migration. Our data show that knockdown of Ron in prostate cancer cells results in significantly less endothelial cell chemotaxis when compared with Ron-expressing cells in vitro as well as in reduced tumor growth and decreased microvessel density after orthotopic transplantation into the prostate in vivo. In total, our data suggest that the Ron receptor is important in modulating prostate tumor growth by modulating angiogenic chemokine production and subsequent endothelial cell recruitment.

Show MeSH
Related in: MedlinePlus