Abstract LB-331: Protein expression signatures of epidermal growth factor receptor inhibition

Abstract

Abstract We have applied standardized shotgun and multiple reaction monitoring (MRM) proteomic platforms to detect protein expression changes associated with epidermal growth factor receptor (EGFR) stimulation and inhibition in A431 carcinoma cells. These experiments compared proliferating, stimulated (30 nmol EGF, 4h) with cells co-treated with EGF and either cetuximab (10 μg/ml) or gefitinib (500 nmol). The analyses are done by shotgun proteomics with liquid chromatography-tandem mass spectrometry (LC-MS/MS), using isoelectric focusing of peptide mixtures, followed by reverse phase LC-MS/MS on a Thermo LTQ instrument. Protein inventories (≥2 peptides per protein at 5% peptide FDR) for each set were approximately 3800 proteins at < 3% protein FDR. Differential protein expression was estimated by comparison of spectral count data with a generalized linear modeling (GLM) approach. EGF stimulation induced expression changes in 148 proteins. Effects of the inhibitors on this “stimulation signature” were then determined by GLM comparisons of spectral count data for cetuximab- and gefitinib-treated cell proteomes. These analyses identified 13 proteins whose EGF-induced expression was reversed by both drugs, 15 proteins reversed by cetuximab and 57 proteins reversed by gefitinib. Targeted MRM analysis verified differential expression of the 13 proteins reversed by both drugs, which comprise an “EGF inhibition signature”. These include the transcription factors c-Jun and JunD and the cell cycle inhibitor Cip1, which were upregulated by EGF and the protein CCDC50, which was decreased by EGF. MRM analyses also identified EGF-induced protein expression changes that were selectively reversed by either cetuximab or gefitinib. Serum deprivation, rather than EGF stimulation may account for a subset of the 106 protein changes that were not reversed by inhibitors in EGF treated cells. Our analyses also identified 87 and 149 proteins that were not affected by EGF, yet were significantly altered by cetuximab and gefitinib, respectively. Activation of the EGFR axis is typically measured via the phosphorylation status of EGFR tyrosine kinase substrates and downstream targets, but these measurements are difficult to achieve in tissues due to the low abundance of phosphorylated protein forms and the challenge of preserving phosphorylation status during sample preparation. Protein expression changes directed by EGFR activation may provide more robust measures of EGFR activation and inhibition. Ongoing studies will test the hypothesis that an EGF inhibition signature provides an early index of therapeutic responses to EGFR-targeted cancer therapies. (Supported by NIH Grant U54CA126479) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-331. doi:10.1158/1538-7445.AM2011-LB-331