Phosphotyrosine Profiling of NSCLC Cells in Response to EGF and HGF Reveals Network Specific Mediators of Invasion

Abstract

Growth factor signaling is deregulated in cancer and often leads to invasion, yet receptor tyrosine kinase signaling pathways driving invasion under different growth factor conditions are not well understood. To identify specific signaling molecules regulating invasion of A549 non-small cell lung carcinoma (NSCLC) cells downstream of the epidermal growth factor receptor (EGFR) and Met, quantitative site-specific mass spectrometric analysis of tyrosine phosphorylation was performed following epidermal growth factor (EGF) or hepatocyte growth factor (HGF) stimulation, at three different growth factor concentrations and at two time points. Through this analysis, the temporal and concentration dependent phosphorylation profiles were obtained for 131 and 139 sites downstream of EGF and HGF stimulation, respectively. To characterize the effect of these signaling network alterations, we quantified 3D cell migration/invasion through Matrigel. Partial least-squares regression (PLSR) analysis was performed to identify the tyrosine phosphorylation sites most strongly correlated with EGF and/or HGF mediated invasion. Potential common and specific signaling events required for driving invasion downstream of EGFR and Met were identified using either a combined or two independent PLSR models, based on the quantitative EGF or HGF data. Our data highlight the integration and compartmentalization of signaling required for invasion in cancer.