Combining RON And EGFR Kinase Directed Therapy Markedly Inhibits Orthotopic Pancreatic Cancer Growth

Abstract

Introduction: The RON receptor tyrosine kinase is overexpressed in pancreatic cancer and mediates apoptotic resistance. We have recently demonstrated that RON inhibition can sensitize pancreatic cancer cells to the effects of Gemcitabine and that resistance to RON inhibition may be mediated by activation of EGFR. A previous study demonstrated crosstalk between RON and EGFR in 3T3 cells. The goals of this study were: 1) To determine if RON and EGFR crosstalk occurs in pancreatic cancer cells and 2) To determine the effects of combined inhibition of RON and EGFR signaling on orthotopic pancreatic cancer growth. Methods: BxPC3 and FG pancreatic cancer cell lines were cultured in RPMI/DMEM media with 10% FBS, serum starved overnight, and stimulated with MSP, the ligand for RON, or EGF over a 5-60 minute time course and compared with null and EGF/MSP stimulated controls. Cell lysates were used for RON immunoprecipitation and/or subjected to western blot analysis for phospho-Tyr or phospho-EGFR. Orthotopic xenografts were developed by injecting 1×106 FG-mCherry cells in 20 μL of DMEM + Growth Factor Reduced Matrigel into the pancreas of 8 week old nu/nu mice using a 28-guage needle. Seven days post-implantation, treatment was started with either PBS, RON8 (a human monoclonal antibody that inhibits RON), RON8+Erlotinib, or RON8+Erlotinib+Gemcitabine. Mice were sacrificed after 27 days post-implantation and tumor volume and weight were measured. Results: Stimulation with MSP resulted in increased EGFR phosphorylation after 60 minutes in BxPC3 pancreatic cancer cells. Similarly, stimulation with EGF resulted in increased RON phosphorylation after 30-60 minutes in both FG and BxPC3 cell lines, thereby demonstrating bidirectional crosstalk between these two receptors. Combination therapy directed at inhibition of both RON and EGFR kinase activity in orthotopic xenografts resulted in significantly decreased tumor volume and weight when compared with untreated controls. Conclusions: There is interaction between RON and EGFR and there is bidirectional crosstalk between the two receptors in pancreatic cancer cells. Combined RON and EGFR inhibition demonstrated a significant decrease in tumor growth in this orthotopic model of pancreatic cancer. Therefore, combined therapy targeting both RON and EGFR is a strategy worthy of further investigation.