Applications of Quantitative Proteomics and Phosphoproteomics to Study the Development of Resistance to Targeted Therapy in Cancer

Abstract

Targeted inhibition of protein kinases is a major approach to treat cancer. However, the effectiveness of kinase inhibitors is limited due to intrinsic and acquired resistance mechanisms that promote the progression and survival of cancer cells. The objective of this dissertation is to use liquid chromatography coupled to mass spectrometry (LC MS) based quantitative proteomics to identify potential biomarkers of resistance and response to molecularly targeted therapies in cutaneous melanoma and lung adenocarcinoma in vitro. For the first part of this thesis, I conducted a proteomic analysis of the acquired drug resistance to extracellular signal-regulated kinase (ERK1/2) pathway inhibitors in a melanoma cell line model. A combination of immunoblot assays, global label-free bottom-up proteomics, phosphoproteomics and pathway analysis was used to characterize the differential protein expression in drug resistant melanoma cells. Examination of the quantitative data pointed to an invasive and metastatic phenotypic signature in the resistant cells. We also identified and verified the overexpression of β-catenin and Caveolin-1 (CAV-1) in MEK1/2 and ERK1/2 inhibitor resistant cells. These findings suggest that these proteins have a role in the development of resistance and may represent novel targets for co-therapy. For the second part of this thesis, I have utilized a multiple reaction monitoring (MRM) based targeted proteomic technique to verify previously identified potential biomarkers of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) response in lung adenocarcinoma. Published global phosphoproteomic data were used to select a list of phosphotyrosine peptides (pY) and MRM based relative and absolute quantitative methods were developed to measure their expression in TKI sensitive and resistant lung adenocarcinoma cells. Modified immuno-MRM assays were optimized using heavy labelled synthetic peptide standards which identified the targets with good reproducibility and repeatability. The results indicated that of the 11 chosen sites, EGFR-pY1197 can be used as potential biomarker of EGFR TKI sensitivity, regardless of the EGFR TKI used. Overall these data advance our understanding of the mechanisms of targeted therapy resistance and highlight candidate biomarkers of resistance and sensitivity.