Protein Expression Signatures for Inhibition of Epidermal Growth Factor Receptor-mediated 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. We asked whether changes in protein expression, which are more stable and easily analyzed, could reflect network stimulation and inhibition. We employed this approach 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 analysis verified differential expression of 12 of these proteins, which comprise a candidate EGFR inhibition signature. We then tested these 12 proteins by multiple reaction monitoring analysis in three other models: 1) a comparison of DiFi (EGFR inhibitor-sensitive) and HCT116 (EGFR-insensitive) cell lines, 2) in formalin-fixed, paraffin-embedded 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 that clinically used EGFR inhibitors generate characteristic protein expression changes. This work further outlines a prototypical approach to derive and test protein expression signatures for drug action on signaling networks.

Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content

“…We suggest that post-translational modifications, especially phosphorylation, may contribute to these processes, including with respect to EGFR (and beyond) (15,49,50,51). Indeed, recent findings suggest that HER2 may provoke widespread changes in the EV phosphoproteome (52), which is similar to the effects we observed in the case of EGFRvIII.…”

Quantitative Profiling of Protein Tyrosine Kinases in Human Cancer Cell Lines by Multiplexed Parallel Reaction Monitoring Assays

“…We compared PRM-based measurements to spectral count-based estimates and immunoblotting analyses. For the A431 cell model, the SW480 cell model and the 10 CRC cell lines, we used the previously published shotgun data sets (21,30,31). For the lung cancer cell lines study, we performed shotgun analyses for this work, as described in supplemental Methods.…”

“…Peptides containing cysteine or methionine residues were not excluded. Although priority was given to peptides that were previously identified in the shotgun data set with high MS/MS spectral quality (21,30,31), additional predicted peptides were selected in silico. Each PTK protein was monitored by 3-4 proteotypic peptides.…”

Oncogenic KRAS and BRAF Drive Metabolic Reprogramming in Colorectal Cancer

“…Multiplexed protein quantitation by multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) are powerful tools for systems characterization (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39). These multiplexed assays can interrogate coordinated expression of proteins in functional protein networks, such ␤-catenin signaling (35), nuclear factor-B signaling (36), protein expression changes because of EGFR signaling (37,38), and phosphotyrosine quantitation in EGFR signaling (39).…”

“…These multiplexed assays can interrogate coordinated expression of proteins in functional protein networks, such ␤-catenin signaling (35), nuclear factor-B signaling (36), protein expression changes because of EGFR signaling (37,38), and phosphotyrosine quantitation in EGFR signaling (39). Drabovich et al utilized a single MRM assay to quantify 134 proteotypic peptides from 76 proteins involved in glycolysis, the TCA cycle, the PPP and related reactions in a single MRM assay to analyze metabolic protein expression changes during hypoxia (40).…”