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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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 4

Downregulation of PI3K/Akt signaling is necessary for gefitinib to effectively induce apoptosis.

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Downregulation of PI3K/Akt signaling is necessary for gefitinib to effec...
(A) Retroviral expression of HA-tagged oncogenic PI3K (p110α E545K) or ca-Akt into HCC827 cells conferred resistance to gefitinib. Expression constructs were introduced into HCC827 cells by retroviral infection, and the resulting cell lines were subjected to an MTS survival assay in increasing concentrations of gefitinib. (B) Expression of p110α E545K abrogated apoptosis induced by gefitinib. HCC827 cells expressing GFP or p110α E545K were treated with 10 nM gefitinib for 72 hours and assessed for apoptosis by analyzing the percent of cells in SubG1 by FACS analysis as previously described (30). (C) Cells were treated as in B, lysed, and assessed for PARP cleavage. Increasing gefitinib concentrations led to increased appearance of the cleaved 89-kDa fragment in HCC827 GFP cells compared with HCC827 p110α E545K cells. (D) p110α E545K led to persistent Akt activation. HCC827 cells expressing GFP or p110α E545K were exposed to 1 μM gefitinib for 12 hours prior to lysis. Note that expression of HA-tagged P110α E545K led to diminished but persistent p-Akt expression in the presence of gefitinib. (E) ca-Akt was not inhibited by gefitinib. HCC827 ca-Akt and HCC827 GFP cells were grown with or without 1 μM gefitinib for 12 hours and lysed for Western blot analysis. Arrow indicates ca-Akt that migrates at a lower molecular weight because it lacks the pleckstrin homology domain. Gefitinib inhibited the phosphorylation of the endogenous but not the myristoylated Akt.