IGF1-R signals through the RON receptor to mediate pancreatic cancer cell migration - PubMed (original) (raw)

IGF1-R signals through the RON receptor to mediate pancreatic cancer cell migration

Dawn V Jaquish et al. Carcinogenesis. 2011 Aug.

Abstract

The RON receptor tyrosine kinase (RTK) is overexpressed in the majority of pancreatic cancers, yet its role in pancreatic cancer cell biology remains to be clarified. Recent work in childhood sarcoma identified RON as a mediator of resistance to insulin-like growth factor receptor (IGF1-R)-directed therapy. To better understand RON function in pancreatic cancer cells, we sought to identify novel RON interactants. Using multidimensional protein identification analysis, IGF-1R was identified and confirmed to interact with RON in pancreatic cancer cell lines. IGF-1 induces rapid phosphorylation of RON, but RON signaling did not activate IGF-1R indicating unidirectional signaling between these RTKs. We next demonstrate that IGF-1 induces pancreatic cancer cell migration that is RON dependent, as inhibition of RON signaling by either shRNA-mediated RON knockdown or by a RON kinase inhibitor abrogated IGF-1 induced wound closure in a scratch assay. In pancreatic cancer cells, unlike childhood sarcoma, STAT-3, rather than RPS6, is activated in response to IGF-1, in a RON-dependent manner. The current study defines a novel interaction between RON and IGF-1R and taken together, these two studies demonstrate that RON is an important mediator of IGF1-R signaling and that this finding is consistent in both human epithelial and mesenchymal cancers. These findings demand additional investigation to determine if IGF-1R independent RON activation is associated with resistance to IGF-1R-directed therapies in vivo and to identify suitable biomarkers of activated RON signaling.

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Figures

Fig. 1.

RON binds IGF1-R. (A) IP/IB for IGF-1R/RON was performed in MiaPaca-2, BxPC-3 and FG cells in the presence and absence of MSP or IGF-1 (100 ng/ml following serum starvation) In both BxPC-3 and FG cells, the two proteins interact in the presence and absence of ligand. (B) IP/IB for RON/IGF1-R was performed on lysates prepared from orthotopic FG tumor xenografts. Lanes 1,2 and 3 represent tumors arising in three different mice and demonstrate that RON and IGF1-R interact in vivo. (C) Proximity ligation assay demonstrating that RON and IGF1-R (interaction indicated by red dots) directly interact in BxPc-3 cells, both in the absence and presence of ligand. Note that the interaction is increased in the presence of both MSP and IGF-1 and that the interaction is reduced when RON kinase activity is blocked by BMS 777607.

Fig. 2.

RON is activated by multiple RTKs. (A) BxPC3 and FG cells were serum-starved overnight followed by treatment of MSP at 100 ng/ml for 5, 15, 30 or 60 min. No treatment served as the negative control and IGF-1 treatment at 100 ng/ml for 5 or 15 min, as indicated, served as the positive control. Phosphorylation of IGF-1R was determined by immunoblotting using a phospho-specific antibody to IGF1-R (Cell Signaling #3024). (B) To determine the presence of p-RON following treatment with HGF, IGF-1 and EGF, an immunoprecipitation for RON C-20 was performed on lysates from BxPC3 and FG cells. Cells were serum-starved overnight followed by treatment with HGF, IGF-1 or EGF at 100 ng/ml for 5, 15, 30 or 60 min. An immunoblot looking for phospho-tyrosine using MAb p-Tyr (4G10) was then performed on the immunoprecipitates. No treatment served as the negative control while 15 or 30 min treatment with MSP (BxPC-3 or FG, respectively) acted as the positive control. To check for potential changes with total RON under the various treatments, IB on the lysates using RON C-20 Ab followed by actin as a loading control was performed. (C) To determine if IGF-1 induced RON phosphorylation was mediated by IGF-1R and c-Met cross talk, IGF-1 was used to stimulate BxPC-3 and FG cells for 5, 15, 30 or 60 min followed by IB for phospho-Met. No treatment served as the negative control while 15 and 30 min treatment with HGF acted as the positive control. No significant increase in phosphorylation of c-Met was observed in both cell lines up to 30 min, with the BxPC-3 cells only showing a slight increase after 60 min of IGF-1 treatment.

Fig. 3.

IGF-1 motility is dependent on RON signaling. (A) Confluent cell monolayers that were generated in six well dishes using BxPC3, FG and AsPC1 parental and RON-silenced cells. Scratches were made in the monolayer using a p200 tip after which treatment with either PBS, MSP, IGF or MSP + IGF at 100 ng/ml was added to its respective well. For studies using BMS 777607, cells were treated with BMS 777607 at 100 nM for 1 h prior to the addition of PBS, MSP, IGF or MSP + IGF at 100 ng/ml with the BMS compound being present at 100 nM throughout the assay. Images were taken at t = 0 h and t = 18 h at ×10 magnification on a Nikon inverted microscope. For the FG and AsPC-1 cells, the final time point was at t = 16 h and t = 42 h, respectively (scratch wound photos not shown). (B) Wound coverage data from scratch assays in BxPc-3, FG and AsPC1 cells is displayed. Scratch wound assays were measured by determining the area of the scratch at t = 0 h and at t = final hour (Final hour for BxPC3 cells = 16 h, for FG cells = 18 h and for ASPC-1 cells = 42 h) using the region setting in SPOT imaging software. To determine the percent Wound Coverage, the following equation was used: [1−(area at _t_final/area at _t_0)] × 100. The mean value was determined and graphed + SE. Two-tailed Student’s _t_-tests were performed for statistical analysis. Comparisons between PBS versus Treatment were done in parental (white*), RON-silenced cells (black*) and in BMS-treated cells (grey*). Comparison within a treatment group is shown with in black (*). Significant values were as follows: P < 0.05(*), P < 0.005(**), P < 0.0005 (***). Note that IGF-1-induced wound coverage is markedly reduced in the absence of RON signaling effected by either RON silencing or RON kinase inhibition by BMS 777607.

Fig. 4.

IGF1-R signaling is modified by RON silencing. Parental or RON-silenced BxPC-3 cells were serum-starved overnight and then treated with either MSP, IGF-1 or MSP + IGF-1 for 5, 15 or 30 min. For BMS 777607 lysates, the cells were treated with 100 nM compound after serum starvation and for 1 h prior to ligand treatment. No treatment acted as the negative control. Immunoblotting was performed to evaluate the expression of p-Akt, p-RPS6 and p-STAT-3 in the presence and absence of RON signaling. Total protein and actin loading controls are included. Note that p-STAT-3 is no longer induced by IGF-1 in the absence of RON signaling effected by either RON silencing or RON kinase inhibition with BMS 777607. In contrast, p-RPS6 levels are unaffected and p-AKT is affected in response to MSP, but not IGF-1.

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IGF1-R signals through the RON receptor to mediate pancreatic cancer cell migration - PubMed (original) (raw)