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The RON receptor tyrosine kinase promotes MSP-independent cell spreading and survival in breast epithelial cells.

Feres KJ, Ischenko I, Hayman MJ - Oncogene (2008)

Bottom Line: In the absence of MSP, RON signaling promotes cell survival, increased cell spreading and enhanced migration in response to other growth factors.However, both RON-mediated proliferation and migration require the addition of MSP in MCF-10A cells.Both MSP-dependent and MSP-independent signaling by RON are mediated in part by Src family kinases.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics & Microbiology, Stony Brook University, Stony Brook, NY, USA.

ABSTRACT
The recepteur d'origine nantais (RON) is a receptor tyrosine kinase (RTK) in the scatter factor family, which includes the c-Met receptor. RON exhibits increased expression in a significant number of human breast cancer tissues as well as in many established breast cancer cell lines. Recent studies have indicated that in addition to ligand-dependent signaling events, RON also promotes signals in the absence of its only known ligand, MSP, when expressed in epithelial cells. In this study, we found that when expressed in MCF-10A breast epithelial cells, RON exhibits both MSP-dependent and MSP-independent signaling, which lead to distinct biological outcomes. In the absence of MSP, RON signaling promotes cell survival, increased cell spreading and enhanced migration in response to other growth factors. However, both RON-mediated proliferation and migration require the addition of MSP in MCF-10A cells. Both MSP-dependent and MSP-independent signaling by RON are mediated in part by Src family kinases. These data suggest that RON has two alternative modes of signaling that can contribute to oncogenic behavior in normal breast epithelial cells.

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MSP-independent activation of RON promotes cell survival(a)After 72 hours in serum-free media, floating and attached cells were pooled together, stained for Propidium Iodide (PI) and the percentage of PI-positive cells was determined by FACS. * indicates a P<0.002. Error bars represent + SEM, n=4. (b)Cells were cultured in regular MCF-10A media that lacked EGF. Phase contrast images were taken on Day 1 and Day 4 to demonstrate cell morphology
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Figure 6: MSP-independent activation of RON promotes cell survival(a)After 72 hours in serum-free media, floating and attached cells were pooled together, stained for Propidium Iodide (PI) and the percentage of PI-positive cells was determined by FACS. * indicates a P<0.002. Error bars represent + SEM, n=4. (b)Cells were cultured in regular MCF-10A media that lacked EGF. Phase contrast images were taken on Day 1 and Day 4 to demonstrate cell morphology

Mentions: MSP stimulation of RON has been associated with anti-apoptotic activity in epithelial cells (Danilkovitch et al., 2000). To examine whether RON can protect cells from cell death in the absence of MSP, we used a cell death assay in which we incubated cells in DMEM without added growth factors or serum for 72 hours. Floating cells were collected from the media and pooled with the remaining adherent cells after they were removed from the plate by trypsinization. Dead cells were identified by Propidium Iodide labeling and the percentage of cell survival was assessed by FACS analysis. As seen in Figure 6a, only 7% of the 10A/Vector cells survived under these conditions in contrast to 31% of the 10A/RON cells. MSP minimally increased the survival rate to 34%. These data imply that MSP-independent activation of RON improves 10A/RON cell survival in the absence of growth factors, and that MSP does not significantly contribute to this effect. Figure 6b presents the phenotypic difference between 10A/Vector and 10A/RON cells when cultured without EGF. After 4 days, most of the 10A/Vector cells were rounded-up, floating in the media, and stained positive for Propidium Iodide whereas the 10A/RON cells remained strongly adherent and began to proliferate when growth factors were added to the media (data not shown). The MCF-10A cells undergoing cell death in our survival assay appear to be dying by apoptosis, as Poly (ADP-ribose) polymerase (PARP) is cleaved in these cells (Supplementary Figure 2b). It was difficult to collect enough 10A/Vector cells for analysis, which is why in it is not possible to distinguish the cleaved form of PARP in the 10A/Vector cells, but it is clear that PARP is cleaved in dying 10A/RON cells from this data.


The RON receptor tyrosine kinase promotes MSP-independent cell spreading and survival in breast epithelial cells.

Feres KJ, Ischenko I, Hayman MJ - Oncogene (2008)

MSP-independent activation of RON promotes cell survival(a)After 72 hours in serum-free media, floating and attached cells were pooled together, stained for Propidium Iodide (PI) and the percentage of PI-positive cells was determined by FACS. * indicates a P<0.002. Error bars represent + SEM, n=4. (b)Cells were cultured in regular MCF-10A media that lacked EGF. Phase contrast images were taken on Day 1 and Day 4 to demonstrate cell morphology
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Related In: Results  -  Collection

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Figure 6: MSP-independent activation of RON promotes cell survival(a)After 72 hours in serum-free media, floating and attached cells were pooled together, stained for Propidium Iodide (PI) and the percentage of PI-positive cells was determined by FACS. * indicates a P<0.002. Error bars represent + SEM, n=4. (b)Cells were cultured in regular MCF-10A media that lacked EGF. Phase contrast images were taken on Day 1 and Day 4 to demonstrate cell morphology
Mentions: MSP stimulation of RON has been associated with anti-apoptotic activity in epithelial cells (Danilkovitch et al., 2000). To examine whether RON can protect cells from cell death in the absence of MSP, we used a cell death assay in which we incubated cells in DMEM without added growth factors or serum for 72 hours. Floating cells were collected from the media and pooled with the remaining adherent cells after they were removed from the plate by trypsinization. Dead cells were identified by Propidium Iodide labeling and the percentage of cell survival was assessed by FACS analysis. As seen in Figure 6a, only 7% of the 10A/Vector cells survived under these conditions in contrast to 31% of the 10A/RON cells. MSP minimally increased the survival rate to 34%. These data imply that MSP-independent activation of RON improves 10A/RON cell survival in the absence of growth factors, and that MSP does not significantly contribute to this effect. Figure 6b presents the phenotypic difference between 10A/Vector and 10A/RON cells when cultured without EGF. After 4 days, most of the 10A/Vector cells were rounded-up, floating in the media, and stained positive for Propidium Iodide whereas the 10A/RON cells remained strongly adherent and began to proliferate when growth factors were added to the media (data not shown). The MCF-10A cells undergoing cell death in our survival assay appear to be dying by apoptosis, as Poly (ADP-ribose) polymerase (PARP) is cleaved in these cells (Supplementary Figure 2b). It was difficult to collect enough 10A/Vector cells for analysis, which is why in it is not possible to distinguish the cleaved form of PARP in the 10A/Vector cells, but it is clear that PARP is cleaved in dying 10A/RON cells from this data.

Bottom Line: In the absence of MSP, RON signaling promotes cell survival, increased cell spreading and enhanced migration in response to other growth factors.However, both RON-mediated proliferation and migration require the addition of MSP in MCF-10A cells.Both MSP-dependent and MSP-independent signaling by RON are mediated in part by Src family kinases.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics & Microbiology, Stony Brook University, Stony Brook, NY, USA.

ABSTRACT
The recepteur d'origine nantais (RON) is a receptor tyrosine kinase (RTK) in the scatter factor family, which includes the c-Met receptor. RON exhibits increased expression in a significant number of human breast cancer tissues as well as in many established breast cancer cell lines. Recent studies have indicated that in addition to ligand-dependent signaling events, RON also promotes signals in the absence of its only known ligand, MSP, when expressed in epithelial cells. In this study, we found that when expressed in MCF-10A breast epithelial cells, RON exhibits both MSP-dependent and MSP-independent signaling, which lead to distinct biological outcomes. In the absence of MSP, RON signaling promotes cell survival, increased cell spreading and enhanced migration in response to other growth factors. However, both RON-mediated proliferation and migration require the addition of MSP in MCF-10A cells. Both MSP-dependent and MSP-independent signaling by RON are mediated in part by Src family kinases. These data suggest that RON has two alternative modes of signaling that can contribute to oncogenic behavior in normal breast epithelial cells.

Show MeSH
Related in: MedlinePlus