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.
Highlights
From the ‡Jim Ayers Institute for Precancer Detection and Diagnosis, the Vanderbilt Institute of Imaging Sciences, the ‡‡Epithelial Biology Center, the §Vanderbilt-Ingram Cancer Center, and the Departments of ¶Biochemistry, **Radiology and Radiological Sciences, and §§Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
One of the most extensively studied signaling pathways is driven by the epidermal growth factor receptor (EGFR),1 a receptor tyrosine kinase that influences a broad range of signaling events and biological processes
EGFR protein expression inversely varied with activation, which reflects enhanced receptor internalization and down-regulation upon activation [43,44,45,46]. Increasing concentrations of both cetuximab and gefitinib lead to a decrease of EGFR phosphorylation at Tyr-1173 and Tyr-998 compared with EGF only stimulated cells
Summary
Materials and Reagents—Iodoacetamide, ammonium bicarbonate, sodium molybdate, -glycerophosphate, sodium molybdate, sodium orthovanadate, 4-(2-aminoethyl)benzenesulfonyl fluoride, aprotinin, leupeptin, betastatin, pepstatin A, dimethyl sulfoxoide, and sodium dodecyl sulfate (all Ͼ99% purity) were purchased from Sigma. Rehydrated mouse xenograft samples and Menetrier disease lysates were prepared for MRM analysis as described above for cultured cells, except that in the initial protein solubilization step, the tissue samples were sonicated three times at 30 watts and 20% output continuously for 20 s both before and after 60 min of incubation in 50 mM ammonium bicarbonate with 50% TFE. Sample solutions (2 l) containing 0.5 ng/l peptide mixture (based on protein concentration) were loaded for 15 min onto the column using 100% solvent A at 1 l/min followed by a gradient elution (400 nl/min) from 3 to 20% solvent B in 7 min, 20 to 60% solvent B over 35 min, 60 to 95% solvent B in 6 min, and held at 95% for 11 min before returning to 3% solvent B. Significance of measured differences for target proteins of all MRM analytes was determined with a two-tailed unpaired t test using Prism 5.0 (GraphPad Software, San Diego, CA)
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