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Updated: Mar 28, 2026
Deciphering the Structural Effects of Activating EGFR Somatic Mutations with Molecular Dynamics Simulation
Published on: May 20, 2020
Effects of Activating Mutations on EGFR Cellular Protein Turnover and Amino Acid Recycling Determined Using SILAC
Michael J Greig1, Sherry Niessen1, Scott L Weinrich2
1Worldwide Medicinal Chemistry, Pfizer, Inc., La Jolla Laboratories, 10770 Science Center Drive, San Diego, CA 92121, USA.
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.
Insights
Drug resistance in cancer therapy can arise from mutations in targeted proteins like epidermal growth factor receptor (EGFR). This study measured EGFR turnover rates, revealing variations influenced by mutations and amino acid recycling.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Protein mutations driving cancer therapy resistance can alter drug binding and protein stability.
- Epidermal growth factor receptor (EGFR) is a key target in non-small cell lung cancer (NSCLC).
- Understanding protein turnover is crucial for developing effective cancer treatments.
Purpose of the Study:
- To measure the cellular turnover rates of wild-type (WT) and mutant epidermal growth factor receptor (EGFR).
- To investigate the impact of specific EGFR mutations on protein turnover.
- To assess the influence of essential amino acid recycling on EGFR turnover rate calculations.
Main Methods:
- Stable Isotope Labeling by Amino acids in Cell culture (SILAC) coupled with Mass Spectrometry (MS) was used to measure protein turnover.
- Turnover rates were analyzed for WT EGFR, single mutants (Del 746-750, L858R), and a double mutant (L858R/T790M).
- Essential amino acid (Lysine and Arginine) recycling was quantified across different cell lines.
Main Results:
- EGFR turnover rates varied significantly across non-small cell lung cancer cell lines, ranging from 28 hours (WT EGFR in A431 cells) to 7.5 hours (Del 746-750 mutant EGFR in PC-9 cells).
- Measurement of EGFR turnover in cells treated with irreversible inhibitors presented challenges due to effects on cell viability.
- Amino acid recycling rates differed between cell lines, leading to a 10-20% reduction in calculated EGFR turnover rates when accounted for.
Conclusions:
- EGFR mutation status directly impacts its cellular turnover rate, a factor potentially contributing to drug resistance.
- Amino acid recycling significantly affects the accurate determination of protein turnover rates.
- These findings provide insights into the complex dynamics of EGFR in cancer and may inform future therapeutic strategies.
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