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International Journal of Cell Biology
Volume 2015, Article ID 798936, 8 pages
http://dx.doi.org/10.1155/2015/798936
Research Article

Effects of Activating Mutations on EGFR Cellular Protein Turnover and Amino Acid Recycling Determined Using SILAC Mass Spectrometry

1Worldwide Medicinal Chemistry, Pfizer, Inc., La Jolla Laboratories, 10770 Science Center Drive, San Diego, CA 92121, USA
2Oncology Research Unit, Pfizer, Inc., La Jolla Laboratories, 10770 Science Center Drive, San Diego, CA 92121, USA

Received 29 June 2015; Revised 2 October 2015; Accepted 20 October 2015

Academic Editor: Pavel Hozak

Copyright © 2015 Michael J. Greig et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Rapid mutations of proteins that are targeted in cancer therapy often lead to drug resistance. Often, the mutation directly affects a drug’s binding site, effectively blocking binding of the drug, but these mutations can have other effects such as changing the protein turnover half-life. Utilizing SILAC MS, we measured the cellular turnover rates of an important non-small cell lung cancer target, epidermal growth factor receptor (EGFR). Wild-type (WT) EGFR, EGFR with a single activating mutant (Del 746–750 or L858R), and the drug-resistant double mutant (L858R/T790M) EGFR were analyzed. In non-small cell lung cancer cell lines, EGFR turnover rates ranged from 28 hours in A431 cells (WT) to 7.5 hours in the PC-9 cells (Del 746–750 mutant). The measurement of EGFR turnover rate in PC-9 cells dosed with irreversible inhibitors has additional complexity due to inhibitor effects on cell viability and results were reported as a range. Finally, essential amino acid recycling (K and R) was measured in different cell lines. The recycling was different in each cell line, but the overall inclusion of the effect of amino acid recycling on calculating EGFR turnover rates resulted in a 10–20% reduction in rates.