The RON-receptor regulates pancreatic cancer cell migration through phosphorylation-dependent breakdown of the hemidesmosome

Abstract

The recepteur d'origine nantais (RON) receptor tyrosine kinase is overexpressed and stimulates invasive growth in pancreatic cancer cells, yet the mechanisms that underlie RON-mediated phenotypes remain poorly characterized. To better understand RON function in pancreatic cancer cells, we sought to identify novel RON interactants using multidimensional protein identification analysis. These studies revealed plectin, a large protein of the spectrin superfamily, to be a novel RON interactant. Plectin is a multifunctional protein that complexes with integrin-β4 (ITGB4) to form hemidesmosomes, serves as a scaffolding platform crucial to the function of numerous protein signaling pathways and was recently described as an overexpressed protein in pancreatic cancer (Bausch D et al., Clin Cancer Res 2010; Kelly et al., PLoS Med 2008;5:e85). In this study, we demonstrate that on exposure to its ligand, macrophage-stimulating protein, RON binds to plectin and ITGB4, which results in disruption of the plectin-ITGB4 interaction and enhanced cell migration, a phenotype that can be recapitulated by small hairpin ribosomal nucleic acid (shRNA)-mediated suppression of plectin expression. We demonstrate that disruption of plectin-ITGB4 is dependent on RON and phosphoinositide-3 (PI3) kinase, but not mitogen-activated protein kinase (MEK), activity. Thus, in pancreatic cancer cells, plectin and ITGB4 form hemidesmosomes which serve to anchor cells to the extracellular matrix (ECM) and restrain migration. The current study defines a novel interaction between RON and plectin, provides new insight into RON-mediated migration and further supports efforts to target RON kinase activity in pancreatic cancer.

Figure 1

RON interacts with plectin in the presence of MSP. (a) IP/IB of RON/Plectin in BxPC-3 and FG cells demonstrating RON-plectin interaction after exposure to the RON ligand MSP (100 ng/ml following serum starvation). (b) OLINK proximity ligation assay on FG cells demonstrating that the RON-plectin interaction is direct in the presence of MSP, indicated by the production of red fluorescent dots. (c) Immunofluorescent confocal microscopy on FG cells for RON (red) and plectin (green) showing that before MSP stimulation (100 ng/ml following serum starvation), RON resides primarily in the cytoplasm where there appears to be little direct RON-plectin interaction. After MSP stimulation, the RON receptor relocates to colocalize with plectin at lamellipodia (denoted by yellow color and arrows). (d) Immunofluorescence on BxPC-3 cells demonstrating that the cellular extensions are true lamellipodia. Cells are costained for RON (green) and phalloidin (red), which recognizes the F-actin protein component of the lamellipodia. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2

Loss of plectin expression does not affect RON signaling. Parental (P), GFP missense and plectin knockdown (KD) of FG and BxPC-3 cells were serum starved overnight followed by treatment of MSP (100 ng/ml) for 30 min in the FG cells and 15 min for the BxPC-3 cells. (a) Immunoprecipitation of RON followed by immunoblotting with phosphor-tyrosine (Millipore #4G10) to determine RON phosphorylation. (b) Phosphorylation of Akt and Erk was determined by immunoblotting using phospho-specific antibodies phospho-Akt (Cell Signaling #9271) and phospho-Erk (Cell Signaling #9109). Note that the loss of plectin expression in plectin knockdown cells does not affect RON phosphorylation or RON-induced activation of either Akt or Erk, thus demonstrating that plectin is not required for RON signaling.

Figure 3

RON signaling regulates integrin beta-4 (ITGB4) interactions. (a) OLINK proximity ligation assay on BxPC-3 cells demonstrating a direct interaction between plectin and ITGB4 in the absence of RON activation, as evidenced by red fluorescent dots. After RON activation, this direct interaction is disrupted as indicated by loss of red signal. (b) IP/IB of ITGB4/Plectin was performed on BxPC-3 and FG cells showing the disruption of the plectin-ITGB4 interaction following exposure to MSP (100 ng/ml following serum starvation). (c) Proximity ligation assay on FG cells demonstrating that MSP (100 ng/ml following serum starvation) prompts the association of RON with ITGB4 as shown by red fluorescent dots. (d) IP/IB of RON/ITGB4 on BxPC-3 and FG cells again demonstrating that MSP (100 ng/ml following serum starvation) prompts the interaction of RON and ITGB4. (e) A time-course experiment was performed in which FG cells were exposed to 100 ng/ml MSP following serum starvation for 5, 10, 20, 30, 40, 50 min and 1 hr. No treatment served as the negative control. Cell lysates were collected and a RON immunoprecipitation was performed, followed by immunoblotting for either plectin (BD Biosciences #611348) or ITGB4 (Santa Cruz #sc-55514). An immunoprecipitation of ITGB4 (Santa Cruz #sc-9090) followed by immunoblotting with Plectin (BD Biosciences #6113348) was also performed on these same lysates looking out 20 min following MSP treatment. To check for equal pull down for the immunoprecipitations, immunoblotting for RON (Santa Cruz #sc-322) was done following the IP. After exposure to MSP, the interaction of RON and plectin/ITGB4 is characterized by repetitive association and disassociation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 4

RON and PI3 kinase activity are required for MSP-induced disruption of the plectin-ITGB4 interaction. (a) Proximity ligation assay on BxPC-3 cells showing that RON activity is required for MSP-induced disruption of the plectin-ITGB4 interaction. Serum starved image shows a direct plectin-ITGB4 interaction before 100 ng/ml MSP treatment, as evidenced by red fluorescent dots. For the RON-Met kinase inhibitor (BMS 777607, Bristol Myers Squib), serum starved cells were treated with 100 nM of the inhibitor for 1 hr before MSP treatment with BMS being present during MSP stimulation. As seen in the BMS777607 + MSP image, the presence of the inhibitor prevented MSP to disrupt plectin-ITGB4, demonstrating that RON kinase activity is required. The BMS 777607 image shows that exposure to this inhibitor alone does not affect the plectin-ITGB4 interaction. (b) Proximity ligation assay on BxPC-3 cells showing that PI3 kinase activity is required for MSP-induced disruption of the plectin-ITGB4 interaction. The PI3 kinase inhibitor (LY294002, Cell Signaling) was used on serum starved cells at 50 μM for 1 hr before MSP treatment with the LY294002 compound being present during MSP stimulation. As shown in the MSP + LY294002 image, we see that this inhibitor serves to preserve the plectin-ITGB4 interaction in the presence of MSP. (c) Proximity ligation assay on BxPC-3 cells showing that a MEK inhibitor (U0126, Cell Signaling) does not affect the plectin–ITGB4 interaction in the presence of MSP. The MEK inhibitor U0126 (25 μM) was added to serum starved cells for 1 hr before MSP stimulation (100 ng/ml) with U0126 being present during MSP stimulation. As shown in the MSP + U0126 image, we see that the presence of the inhibitor does not prevent MSP from disrupting the plectin–ITGB4 interaction. This finding suggests that PI3 kinase activity is required for plectin-ITGB4 disruption, and that RON may achieve this disruption via its activation of PI3K. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 5

Loss of plectin expression in pancreatic cancer cells enhances migration. (a) Confluent cell monolayers were generated in six-well dishes using BxPC3 and FG parental, GFP missense and plectin knockdown cells. Scratches were made in the monolayer using a p200 tip after which treatment with either PBS or 100 ng/ml of MSP added to its respective well. Images were taken at t = 0 and t = 16 hr for the FG cells and t = 18 hr for the BxPC-3 cells (scratch wound images not shown). Each condition was performed in triplicate, and each experiment was repeated three times. (b) Wound coverage data from scratch assays in FG and BxPC-3 cells is displayed. Scratch would assays were measured by determining the area of the scratch at t = 0 hr and at t = final hour (Final hour for FG cells = 16 hr and for BxPC3 cells = 18 hr) 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 t₀)} × 100. The mean value was determined and graphed + SE. Two-tailed Student’s t-test was performed for statistical analysis. Comparisons between PBS vs. treatment were done in parental (grey), GFP missense (white) and in knockdown (black). Significant values were as follows: p < 0.005(**), p < 0.0005(***). In both cell lines, plectin knockdown cells achieved a higher percentage of wound closure in the absence and presence of MSP, ***(p < 0.0005).