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

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.