Abstract

Abstract Goal: The goal of this experiment is to understand how kinase inhibitor therapies, like Erlotinib targeting the epidermal growth factor receptor (EGFR), modulate the signaling network using phosphoproteomics; developing a straightforward process to enable physicians to gather information that may assist in establishing a molecular basis for selection of chemotherapy, particularly small tyrosine inhibitor therapies, and for tracking patients’ responses to treatment. Methods: Non-small cell lung cancer (NSCLC) cell lines were used as model systems for examining lung cancer signaling, particularly the HCC827 cell line that overexpresses EGFR. To examine the response to inhibitor, EGFR from HCC827 cells was enriched by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) fractionation. After cell lysis, protein assays were carried out so that an equivalent amount of total protein was loaded into each lane. After excision of the appropriate region of the gel, reduction, and alkylation, in-gel trypsin digestion was performed. The resulting peptides were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) on a hybrid linear ion trap-orbital ion trap mass spectrometer. The high resolution and accurate mass measurements of the peptides enable relative quantification that can be used to examine dose response for each phosphorylation site. After detection and relative quantification of peptides including phosphorylation sites, quantitative assays based on liquid chromatography coupled to multiple reaction monitoring (LC-MRM) can be developed to increase sensitivity for subsequent translation to patient samples. Results: Using high resolution mass spectrometry, more than ten phosphotyrosine sites could be detected and quantified from EGFR and several of these phosphorylations were shown to be dose dependent to Erlotinib, a kinase inhibitor specific for EGFR. The decreases in phosphorylation at sites including tyrosine 1197 can be used as surrogates to monitor the inhibition of EGFR activity for determination of patient response. LC-MRM assays have been developed for EGFR expression and modification and implemented in the HCC827 cell line. Conclusion: The ability to monitor the effectiveness of the drug treatment at a molecular level allows physicians to modulate drug treatment based on the responses observed for each patient. This knowledge can maximize treatment effectiveness or suggest when other treatments may be more effective. The phosphoproteomics techniques illustrated here for the well characterized EGFR molecule can also be utilized to examine the drug induced molecular changes in other protein targets of clinical significance. Because of the potential importance of this experiment in clinical applications, a manuscript is being prepared for a chemical education journal that describes the lab activities and provides a virtual learning environment to monitor drug response with mass spectrometry. ∗This presenting student is supported by an ARRA supplement to the NCI Cancer Center Support Grant awarded to Moffitt (3P30 CA076292-11S6 PI WS Dalton) to create a training program for underrepresented undergraduate students in clinical proteomics Project LINK (Leaders In New Knowledge-Emerging Technologies) that also emphasizes education in health disparities and community outreach. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A68.

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