Proteomic Signatures of Epidermal Growth Factor Receptor Signaling

Abstract

Analysis of cellular signaling networks typically involves targeted measurements of phosphorylated protein intermediates. However, phosphoproteomic analyses usually require affinity enrichment of phosphopeptides and can be complicated by artifactual changes in phosphorylation caused by uncontrolled preanalytical variables, particularly in the analysis of tissue specimens. I hypothesized that changes in protein expression, which are more stable and easily analyzed, could reflect network stimulation and inhibition. This approach was employed to analyze stimulation and inhibition of the epidermal growth factor receptor (EGFR) by EGF and selective EGFR inhibitors. Shotgun analysis of proteomes from proliferating A431 cells, EGF-stimulated cells and cells co-treated with the EGFR inhibitors cetuximab or gefitinib identified groups of differentially expressed proteins. Comparisons of these protein groups identified 13 proteins whose EGF-induced expression changes were reversed by both EGFR inhibitors. Targeted multiple- reaction-monitoring (MRM) analysis verified differential expression of 12 of these proteins, which comprise a candidate EGFR inhibition signature. I then tested these 12 proteins by MRM analysis in 3 other models: 1) a comparison of DiFi (EGFR inhibitor-sensitive) and HCT116 (EGFR-insensitive) cell lines, 2) in formalin-fixed, paraffin-embedded (FFPE) mouse xenograft DiFi and HCT116 tumors, and 3) in tissue biopsies from a patient with the gastric hyperproliferative disorder Ménétrier’s disease, who was treated with cetuximab. Of the proteins in the candidate signature, a core group, including c-Jun, jagged-1, and claudin 4 were decreased by EGFR inhibitors in all three models. Although the goal of these studies was not to validate a clinically-useful EGFR inhibition signature, the results confirm the hypothesis and outline a prototypical approach to derive and test protein expression signatures for drug action on signaling networks. A secondary goal of this research was to apply a new method to quantify protein modification changes to EGFR using internal reference peptides (IRP). The major focus of this work was to assess the performance of this newly developed MS-based quantitation method to detect phosphorylation changes on EGFR by comparing the performance characteristics to stable isotope dilution (SID) methods. Initial studies are presented along with suggestions for future studies using overall findings in this dissertation.