Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer
Jeffrey A. Engelman, … , Lewis C. Cantley, Pasi A. Jänne
Jeffrey A. Engelman, … , Lewis C. Cantley, Pasi A. Jänne
Published October 2, 2006
Citation Information: J Clin Invest. 2006;116(10):2695-2706. https://doi.org/10.1172/JCI28656.
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Research Article Oncology

Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer

Abstract

EGFR is frequently mutated and amplified in lung adenocarcinomas sensitive to EGFR inhibitors gefitinib and erlotinib. A secondary mutation, T790M, has been associated with acquired resistance but has not been shown to be sufficient to render EGFR mutant/amplified lung cancers resistant to EGFR inhibitors. We created a model for studying acquired resistance to gefitinib by prolonged exposure of a gefitinib-sensitive lung carcinoma cell line (H3255; EGFR mutated and amplified) to gefitinib in vitro. The resulting resistant cell line acquired a T790M mutation in a small fraction of the amplified alleles that was undetected by direct sequencing and identified only by a highly sensitive HPLC-based technique. In gefitinib-sensitive lung cancer cells with EGFR mutations and amplifications, exogenous introduction of EGFR T790M effectively conferred resistance to gefitinib and continued ErbB-3/PI3K/Akt signaling when in cis to an activating mutation. Moreover, continued activation of PI3K signaling by the PIK3CA oncogenic mutant, p110α E545K, was sufficient to abrogate gefitinib-induced apoptosis. These findings suggest that allelic dilution of biologically significant resistance mutations may go undetected by direct sequencing in cancers with amplified oncogenes and that restoration of PI3K activation via either a T790M mutation or other mechanisms can provide resistance to gefitinib.

Authors

Jeffrey A. Engelman, Toru Mukohara, Kreshnik Zejnullahu, Eugene Lifshits, Ana M. Borrás, Christopher-Michael Gale, George N. Naumov, Beow Y. Yeap, Emily Jarrell, Jason Sun, Sean Tracy, Xiaojun Zhao, John V. Heymach, Bruce E. Johnson, Lewis C. Cantley, Pasi A. Jänne

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Figure 1

Generation of a cell line, H3255 GR, with acquired resistance to gefitinib.

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Generation of a cell line, H3255 GR, with acquired resistance to gefitin...
(A) H3255 and H3255 GR cell lines were subjected to MTS survival assays in increasing concentrations of gefitinib (see Methods). (B) ErbB-3 was immunoprecipitated from H3255 and H3255 GR cells grown with or without 1 μM gefitinib for 6 hours. Half of the immunoprecipitate was used for ErbB-3 quantification; the other half was used for PI3K assays (see Methods). PI3K activity normalized to ErbB-3 protein is shown as a percent of control (no gefitinib). (C) H3255 and H3255 GR cells were exposed to increasing amounts of gefitinib for 24 hours prior to lysis. Blots of H3255 and H3255 GR extracts for each antibody are the same exposure from a single gel and blot; an irrelevant lane between the H3255 and H3255 GR extracts was omitted. (D) H3255 and H3255 GR cells were serum-starved overnight followed by treatment with or without 1 μM gefitinib for 3 hours followed by stimulation with EGF. Immunofluorescence was performed using anti–p-EGFR (pY1068) antibodies. Left: p-EGFR membrane staining by immunofluorescence in untreated H3255 and H3255 GR cells and corresponding phase-contrast images (bottom panels). Right: Immunofluorescence and phase-contrast images of H3255 and H3255 GR cells treated with gefitinib. Most H3255 GR cells maintained detectable membrane EGFR staining in the presence of gefitinib (arrows). The gefitinib-treated panels were equally overexposed to highlight the residual membrane staining. Magnification, ×60.