Skip to main content
NCBI home page
Carcinogenesis logoLink to Carcinogenesis
. 2011 May 11;32(8):1151–1156. doi: 10.1093/carcin/bgr086

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

Dawn V Jaquish 1, Peter T Yu 1, David J Shields 1, Randall P French 1, Karly P Maruyama 1, Sherry Niessen 2, Heather Hoover 2, David ACheresh 1, Ben Cravatt 2, Andrew M Lowy 1,*
PMCID: PMC3149203  PMID: 21565828

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.

Introduction

The need for more effective therapies to treat pancreatic cancer patients is indisputable. Based on work from several laboratories, the RON receptor tyrosine kinase (RTK) has emerged as a potential therapeutic target as it has been shown to mediate chemoresistance in pancreatic cancer cells and is overexpressed at high frequency in human pancreatic cancer (1–3). Despite this, we have only a rudimentary understanding of RON function and its participation in RTK-signaling networks in pancreatic cancer cells. To better understand RON function, we sought to identify RON protein interactants in pancreatic cancer cells using mass spectrometry. These experiments identified the insulin-like growth factor receptor 1 (IGF1-R) as a novel RON interactant and additional studies demonstrated receptor cross talk (4,5). RON appears to mediate IGF1-R signaling, a finding that was recently reported in childhood sarcoma as well (6). The downstream-signaling pathways in sarcoma and pancreatic cancer cells appear to differ, however, and thus demonstrate the need for independent study of RTK-signaling networks in specific tissue contexts.

Materials and methods

Cell lines

BxPC3, MiaPaCa-2 and AsPC-1 cells were obtained from the American Type Culture Collection. FG cell lines were provided by Dr David Cheresh (University of California, San Diego, La Jolla, CA). RON silenced cell lines were created as described previously (7).

Immunoblotting, immunoprecipitation

Cells were serum-starved overnight then stimulated for 5, 15, 30 min or 1 h with 100 ng/ml of either macrophage-stimulating protein (MSP) (R&D Systems, Minneapolis, MN), IGF-1, epidermal growth factor (EGF), hepatocyte growth factor (HGF) (Millipore, Temecula, CA) or a combination of MSP and IGF-1 at 100 ng/ml each. Studies using the RON kinase inhibitor BMS-777607 (Bristol-Myers Squibb, New York, NY) included a 1 h incubation of the inhibitor at a concentration of 100 nM after serum starvation followed by ligand treatment (8). IP and immunoblotting (IB) was performed as described previously, with the exception of the Immunoprecipitation (IP) for IGF-1R which used 2 μg of antibody on 1 mg of lysate (7). Antibodies are detailed in supplementary Table 1, available at Carcinogenesis Online.

Liquid chromatography—multidimensional protein identification

BxPC3 cells were serum-starved overnight, treated with 200 ng/ml of MSP or vehicle for 15 min and protein lysates collected. Fifteen micrograms of RON C-20 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was conjugated to 75 μl of A/G Ultralink Beads (Pierce) using the Seize X IP Kit (Pierce). Five milligrams of each sample was immunoprecipitated to the conjugated beads. Precipitated proteins were loaded onto a biphasic capillary column for multidimensional protein identification (MudPIT) then separated and analyzed by 2D-LC separation in combination with tandem MS as described (9).

Generation of mass spectrometry data

RAW files were generated from mass spectra using XCalibur version 1.4, and MS2 spectra data extracted using RAW Xtractor (version 1.9.1-available at http://fields.scripps.edu/?q=content/download). MS2 spectral data were searched using the SEQUEST algorithm (Version 3.0) against the human IPI database (v3.65) database containing 86 382 sequences concatenated to a decoy database in which the sequences for each entry in the original database was reversed. The resulting MS2 spectra matches were assembled and filtered using DTASelect (version 1.9). Peptides with cross correlation scores greater than 2.0 (+1), 2.5 (+2), 4.0 (+3), delta CN scores >0.09 and percent ion match >49% were included in the final data set. This resulted in a false-positive rate of 1.3% at the peptide level. To eliminate decoy database hits, a minimum peptide length of seven amino acids was imposed and protein identification required the matching of at least two peptides per protein.

Proximity ligation assay

BxPC3 cells were grown to ∼50% confluence on eight chamber slides (Nunc Lab Tek, Rochester, NY). Cells were serum-starved overnight, then treated with 100 nM of BMS-777607 for 1 h followed by treatment with 100 nM of BMS-777607 + 100 ng/ml of MSP, IGF-1, a combination of MSP plus IGF-1 each at 100 ng/ml or ligands alone for 15 min. Cells were fixed with paraformaldehyde for 10 min, then permeablized with phosphate-buffered saline (PBS) + 0.1% Triton X-100 (PBS-T). Primary antibody labeling was performed using RON C-20 (Santa Cruz Biotechnology) at 1:1000 dilution and mouse anti IGF-1R (Abcam, Cambridge, MA) at 1 μg/ml in PBS-T overnight at 4°C. The proximity ligation assay was then performed as described previously (10). Images were obtained at the UCSD School of Medicine Light Microscope Facility using a Deltavision Deconvolution microscope (Applied Precision, Issaquah, WA) at ×20 magnification using Softworx version 4.0.0 software. To determine the mean ratio of red signals/blue nuclei for a specific image, quantification of the red signals was done by selecting four separate regions from an image and obtaining the number of red signals using the Softworx software 2D polygon setting at a threshold of 400 in the 607 channel. The number of blue nuclei in the designated region was hand counted in the 440 channel. Fold change was determined for each treatment compared with the serum-starved sample.

Scratch wound migration assay

BxPC3, FG and ASPC-1 cells were grown in six- well dishes to confluence in complete media. Cells were washed, placed in media containing 0.5% fetal bovine serum and incubated overnight. The next day four scratches were made in a plus shape with the end of a p200 barrier pipette tip. Fresh media containing 0.5% fetal bovine serum was then added to each plate plus either PBS, 100 ng/ml of MSP, 100 ng/ml IGF-1 or 100 ng/ml of MSP and IGF-1. Studies using BMS-777607 included a 1 h incubation of the inhibitor at a concentration of 100 nM followed by ligand treatment. Dishes were photographed under ×10 magnification using a Nikon inverted microscope at t = 0 and 18 h for BxPC3, 16 h for FG and 42 h for AsPC-1 cells. The wound area was calculated using the region setting in SPOT imaging software (SPOT Imaging Solutions, Sterling Heights, MI). Percent wound coverage was calculated as follows: [1−(area (μm2) at t = final hour/area (μm2) at t = 0)] × 100.

Generation of pancreatic tumor xenografts

Orthotopic tumor xenografts were developed by injecting 1 × 106 FG-mCherry cells in 20 μl of Dulbecco's modified Eagle's medium + growth factor reduced Matrigel (BD Biosciences, San Diego, CA) into the pancreata of 8-week-old nu/nu mice. Mice were killed 4 weeks postimplantation and the tumors processed for immunoblotting as described (7).

Results

RON interacts with IGF1-R

To better understand RON function in pancreatic cancer cells, we sought to identify novel RON protein interactants. Initially, RON protein was immunoprecipitated from BxPc-3 human pancreatic cancer cells in the presence and absence of the RON ligand, MSP. The immunoprecipitates were then subjected to MudPIT analysis (data not shown), which revealed IGF1-R as a potential RON-binding partner. Sequence coverage for IGF1-R was 1.7% which compared favorably to the 3.7% observed for the RON receptor itself. The RON/IGF1-R interaction was subsequently verified by IP/IB in both the BxPC-3 and FG cell lines and not the MiaPaca's which are negative for RON. This suggested that the two proteins are complexed both in the presence and absence of ligand (Figure 1A). We then examined RON/IGF1-R-binding in vivo by injecting FG cells into the mouse pancreatic tail. The resultant tumors were examined by IP/IB and, as shown in Figure 1B, RON and IGF1-R interact in vivo. Next, we utilized a proximity ligation assay to demonstrate direct interaction between the two kinases, and that in keeping with the IP/IB finding, RON and IGF1-R interact both in the absence and presence of ligand (Figure 1C and supplementary Figures S1C--D is available at Carcinogenesis Online). We did note an increased amount of interaction in the presence of both MSP and IGF-1. Finally, when the cells were treated with a small molecule tyrosine kinase inhibitor of RON, BMS 777607, the interaction between the two proteins was reduced, suggesting that RON kinase activity promotes its association with IGF1-R (supplementary Figure S1D is available at Carcinogenesis Online).

Fig. 1.

Fig. 1.

Open in a new tab

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.

RON is activated by multiple RTKs

After confirming the interaction between RON and IGF-1R, we next sought to determine if the receptors were capable of transphosphorylation. Cultured BxPc-3 and FG cells were stimulated with MSP and IB for phospho-IGF1-R was performed. No evidence of IGF1-R phosphorylation was observed, suggesting that RON cannot activate IGF1-R signaling (Figure 2A). In contrast, however, following exposure to IGF-1, rapid phosphorylation of the RON receptor was observed in the BxPC-3 and FG cell lines. For comparison, we also examined RON cross talk with the c-Met and EGF receptors (supplementary Figure S2A is available at Carcinogenesis Online). These experiments revealed that in pancreatic cancer cells exposed to the ligands HGF and EGF, RON is phosphorylated and that RON in turn, phosphorylates both c-Met and epidermal growth factor receptor after exposure to MSP. 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. No significant changes in the levels of total RON were observed (Figure 2B). c-Met is known to be activated by IGF1-R (11). To verify that the RON activation in response to IGF-1 was not a result of signaling form c-Met, we determined the status of c-Met phosphorylation in BxPc-3 and FG cells after exposure to IGF-1. No increase in phosphorylation of c-Met was observed in FG cells and an increase was observed in the BxPC-3 only after 1 h of exposure to IGF-1 (Figure 2C). Thus, we conclude that whereas RON displays bidirectional cross talk with c-Met and epidermal growth factor receptor, IGF1-R activates RON, but RON cannot activate IGF-1R and therefore signaling between the two is strictly unidirectional and is not due to IGF-1R/c-Met cross talk.

Fig. 2.

Fig. 2.

Open in a new tab

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.

IGF-1-induced migration is dependent on RON signaling

Having demonstrated that RON signaling may be activated by IGF1-R, we next investigated the role of RON signaling in IGF-1 mediated cell migration via a scratch wound assay. We chose to investigate cell migration as IGF1-R has been shown to be a potent regulator of migration in pancreatic cancer cells and it is readily quantifiable. Using a scratch wound assay, we observed enhanced migration in response to both IGF-1 and MSP in BxPC-3, FG and AsPC-1 cells. When both ligands were added together, we observed more rapid wound closure than when either ligand was added alone (Figure 3A). The degree of acceleration is less than additive suggesting some overlap in the pathways being activated downstream of RON and IGF1-R.

Fig. 3.

Fig. 3.

Open in a new tab

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 tfinal/area at t0)] × 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.

In the next series of experiments, we assayed cell migration in response to IGF-1 in the presence and absence of RON signaling. To achieve this, we utilized BxPc-3, FG and AsPC-1 cells in which RON expression had been stably silenced using shRNA. As depicted in Figure 3B and as expected, these cells did not respond to MSP stimulation. In comparison with RON competent cells, however, these cells exhibited a significantly reduced rate of wound closure in response to the IGF1-R ligand, IGF-1 and to the combination of MSP and IGF-1 (52, 47 and 33% reduction, respectively, for Bx-Pc3, FG and AsPC-1, P < 0.05 for all comparisons). These findings suggest that IGF-1 induced pancreatic cancer cell migration is partially regulated by RON signaling.

We next chose to investigate this finding using pharmacologic inhibition of RON signaling. To accomplish this, pancreatic cancer cells were exposed to BMS 777607 prior to exposure to MSP and/or IGF-1. BMS 777607 is an ATP mimetic that has activity against both the RON and c-met kinases, but no activity against IGF1-R (11). Similar to our findings in the previous experiments using RON-silenced cells, BMS 777607 markedly inhibited both MSP and IGF-1-dependent migration (Figure 3C). Inhibition was more potent than that seen in the RON-silenced cells, a finding that can be explained in at least two ways; (i) BMS 777607 has effects on the c-met kinase which is also known to be activated downstream of IGF1-R as well as in conjunction with RON activation and,(ii) shRNA RON silencing is not complete and therefore, there is a degree of persistent RON activation as compared with pharmacologic inhibition. In summary, both genomic and pharmacologic inhibition of RON signaling result in significantly reduced pancreatic cancer cell migration in response to IGF-1, thereby demonstrating that RON signaling mediates this IGF-1 induced phenotype.

IGF1-R signaling is modified by RON silencing

In order to investigate the downstream pathways mediated by IGF1-R through RON, we chose to examine known downstream targets of RON and IGF1-R. In their recent study, Portratz et al. demonstrated that ribosomal protein S6 (RPS6) was downstream of both IGF1-R and RON and that its phosphorylation appeared critical to mediating therapeutic resistance to IGF1-R-directed therapy (6). Using conditions described previously for BxPC-3 and FG cells, we assayed for p-AKT, p-STAT-3 and p-RPS6 in the presence and absence of RON signaling. Figure 4 reveals that in BxPc-3 cells, while p-RPS6 is present in abundance, it does not appear to be regulated by RON or IGF1-R signaling. In contrast, while in the absence of RON signaling, phosphorylation of AKT is significantly reduced, there remains a significant response to IGF-1, thus indicating that IGF1-R activates AKT independent of RON. A similar pattern of activation was observed for extracellular signal-regulated kinase (data not shown). However, when we examined the response of STAT-3 to IGF-1, we observed a clear reduction in p-STAT-3 levels in the absence of RON signaling. This demonstrates that IGF-1-induced STAT-3 activation is at least partially dependent on RON signaling. Thus, the IGF1-R/RON-signaling axis in pancreatic cancer cells involves activation of STAT-3, and not RPS6.

Fig. 4.

Fig. 4.

Open in a new tab

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.

Discussion

Both the RON and IGF1-R receptors represent attractive potential targets for pancreatic cancer therapy as they are commonly overexpressed and mediate oncogenic phenotypes in pancreatic cancer cells. It has become clear; however, that the success of tyrosine kinase-directed therapy is inextricably linked to identification of those signaling pathways that are critical for cancer cell survival and to the extent to which the cancer cell can utilize alternate signaling networks to activate key effectors. In this study, we sought to better understand RON function by identifying its interactants in pancreatic cancer cells. Our studies identified IGF1-R as a RON partner, which to our knowledge has not previously reported. Both RON and IGF1-R have been shown to form heterodimers with other RTK’s and thus, this finding was not completely surprising, particularly given the fact that both proteins are known to be overexpressed in pancreatic cancer cells. The RON-IGF1-R interaction is distinct from previous reports of RON-c-Met and RON-epidermal growth factor receptor interactions; however, in that receptor, cross talk in this instance is uni- rather than bidirectional (4,5). Thus, it appears that IGF1-R signals through RON and that this signaling is necessary for the full migratory phenotype induced by exposure to IGF-1. This finding is concordant with a previous study that revealed IGF1-R-mediated migration to dependent on the expression of c-Met (12). The latter study did not report on the status of a direct interaction or transphosphorylation between c-Met and IGF1-R, however. We have shown in two pancreatic cancer cell lines that IGF-1 results in rapid phosphorylation of RON but not c-Met, thus demonstrating that signaling from IGF1-R is not being conducted to RON via an IGF1-R-c-Met interaction. A report by Liu et al. (13) revealed an interaction between FAK and IGF1-R that was required to conduct survival signals in pancreatic cancer cells. RON was also recently noted to conduct survival signals from c-Met even in the setting of c-Met amplification (14). The importance of RON-IGF1-R signaling in other oncogenic phenotypes such as survival is a subject of ongoing studies in our laboratory.

During the final preparation of this manuscript, Portratz et al. (6) reported the finding that RON could mediate resistance to IGF1-R-directed therapy in childhood sarcomas. This group identified RON via a siRNA screen for genes that could modulate such resistance. They subsequently identified the ribosomal protein RPS6 as the downstream target of IGF1-R signaling that appears to modulate the resistant phenotype. Their studies revealed that RON could activate RPS6 independently of IGF1-R, thereby accounting for RON’s place as a modifier of anti-IGF1-R therapeutic efficacy. In our study, while high levels of phospho-RPS6 were present in pancreatic cancer cells, we could not detect meaningful differences in the activation of RPS6 either in the presence or absence of activated RON or IGF1-R signaling. Rather, we found that in BxPc-3 cells, IGF-1-induced STAT-3 activation was associated with the presence of activated RON signaling, thus revealing differences in downstream signaling between pancreatic cancer and sarcoma. Though our results directly place RON downstream as demonstrated by its direct binding to, and transphosphorylation of IGF1-R, it is unclear that the extent to which this occurs in sarcoma. These studies strongly suggest that signaling networks while sharing common features also may display critical tissue context-dependent differences that could be highly relevant when considering nodes for therapeutic intervention.

Defining RTK-signaling networks in pancreatic cancer and other solid tumors will be critical to devising effective therapeutic strategies. Our findings reveal a physical and physiologic interaction between RON and IGF1-R that along with the findings of Portratz et al. suggest that RON activation may represent both an important mediator of IGF1-R phenotypes as well as a potential mechanism of resistance to IGF1-R therapy. Still, it will be critical to understand how RON activation may play a role in resistance to IGF1-R in vivo. Since RON activation probably occurs through multiple mechanisms, including the expression of active splice variants, receptor autophosphorylation and transactivation from other RTK’s, it will probably prove difficult to develop a single biomarker of activated RON signaling, but rather an ‘activated RON profile’ based on genomics or proteomics will be required. Our findings demand additional investigation to identify such suitable biomarkers of RON activation and to determine if IGF-1-R-independent RON activation is associated with resistance to IGF-1R-directed therapies in vivo. A current Southwest Oncology group trial is investigating the benefit of IGF1-R monoclonal antibody therapy added to Gemcitabine and Erlotinib for patients with Stage IV pancreatic cancer. While the results of this study are not yet known, studies such as these will provide the opportunity to determine the relevance of the IGF1-R/RON-signaling axis to the outcomes of pancreatic cancer therapy.

Supplementary material

Supplementary Table 1 and Figures S1–S2 can be found at http://carcin.oxfordjournals.org/

Funding

National Institutes of Health (CA 45726 and CA 137692 to A.M.L.).

Acknowledgments

Conflict of Interest Statement: None declared.

Glossary

Abbreviations

EGF, epidermal growth factor; HGF, hepatocyte growth factor; IB, immunoblotting; IP, Immunoprecipitation; MSP

macrophage-stimulating protein

MudPIT

multidimensional protein identification

PBS

phosphate-buffered saline

RTK

receptor tyrosine kinase

References

  • 1.Camp ER, et al. Tyrosine kinase receptor RON in human pancreatic cancer: expression, function, and validation as a target. Cancer. 2007;109:1030–1039. doi: 10.1002/cncr.22490. [DOI] [PubMed] [Google Scholar]
  • 2.O'Toole JM, et al. Therapeutic implications of a human neutralizing antibody to the macrophage-stimulating protein receptor tyrosine kinase (RON), a c-MET family member. Cancer Res. 2006;66:9162–9170. doi: 10.1158/0008-5472.CAN-06-0283. [DOI] [PubMed] [Google Scholar]
  • 3.Thomas RM, et al. The RON receptor tyrosine kinase mediates oncogenic phenotypes in pancreatic cancer cells and is increasingly expressed during pancreatic cancer progression. Cancer Res. 2007;67:6075–6082. doi: 10.1158/0008-5472.CAN-06-4128. [DOI] [PubMed] [Google Scholar]
  • 4.Follenzi A, et al. Cross-talk between the proto-oncogenes Met and Ron. Oncogene. 2000;19:3041–3049. doi: 10.1038/sj.onc.1203620. [DOI] [PubMed] [Google Scholar]
  • 5.Peace BE, et al. Cross-talk between the receptor tyrosine kinases Ron and epidermal growth factor receptor. Exp. Cell Res. 2003;289:317–325. doi: 10.1016/s0014-4827(03)00280-5. [DOI] [PubMed] [Google Scholar]
  • 6.Potratz JC, et al. Synthetic lethality screens reveal RPS6 and MST1R as modifiers of insulin-like growth factor-1 receptor inhibitor activity in childhood sarcomas. Cancer Res. 2010;70:8770–8781. doi: 10.1158/0008-5472.CAN-10-1093. [DOI] [PubMed] [Google Scholar]
  • 7.Logan-Collins J, et al. Silencing of RON receptor signaling promotes apoptosis and Gemcitabine sensitivity in pancreatic cancers. Cancer Res, 70, 1130--1140. 2010 doi: 10.1158/0008-5472.CAN-09-0761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Schroeder GM, et al. Discovery of N-(4-(2-amino-3-chloropyridin-4-yloxy)-3-fluorophenyl)-4-ethoxy-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (BMS-777607), a selective and orally efficacious inhibitor of the Met kinase superfamily. J. Med. Chem. 2009;52:1251–1254. doi: 10.1021/jm801586s. [DOI] [PubMed] [Google Scholar]
  • 9.Washburn MP, et al. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 2001;19:242–247. doi: 10.1038/85686. [DOI] [PubMed] [Google Scholar]
  • 10.Greenberg JI, et al. A role for VEGF as a negative regulator of pericyte function and vessel maturation. Nature. 2008;456:809–813. doi: 10.1038/nature07424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Dai Y, et al. BMS-777607, a small-molecule met kinase inhibitor, suppresses hepatocyte growth factor-stimulated prostate cancer metastatic phenotype in vitro. Mol. Cancer Ther. 2010;9:1554–1561. doi: 10.1158/1535-7163.MCT-10-0359. [DOI] [PubMed] [Google Scholar]
  • 12.Bauer TW, et al. Regulatory role of c-Met in insulin-like growth factor-I receptor-mediated migration and invasion of human pancreatic carcinoma cells. Mol. Cancer Ther. 2006;5:1676–1682. doi: 10.1158/1535-7163.MCT-05-0175. [DOI] [PubMed] [Google Scholar]
  • 13.Liu W, et al. FAK and IGF-IR interact to provide survival signals in human pancreatic adenocarcinoma cells. Carcinogenesis. 2008;29:1096–1107. doi: 10.1093/carcin/bgn026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Benvenuti S, et al. Ron kinase transphosphorylation sustains MET oncogene addiction. Cancer Res. 2011;71:1945–1955. doi: 10.1158/0008-5472.CAN-10-2100. [DOI] [PubMed] [Google Scholar]

Articles from Carcinogenesis are provided here courtesy of Oxford University Press

RESOURCES