Loss of Activating EGFR Mutant Gene Contributes to Acquired Resistance to EGFR Tyrosine Kinase Inhibitors in Lung Cancer Cells

Keisuke Tabara,Mayumi Ono,Masayoshi Kage,Kazuto Nishio,Kahori Sonoda,Aki Yoshinaga,Takuya Kubo,Kenshi Hayashi,Michihiko Kuwano,Rafael Rosell,Tokuzo Arao,Akihiko Kawahara,Yuichi Murakami,Rina Kanda,Tomoko Tahira,Koichi Azuma,Hideyuki Abe,Yiqun G Shellman

doi:10.1371/journal.pone.0041017

Abstract

Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11–18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11–18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11–18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.

Highlights

Non-small-cell lung cancer (NSCLC) is one of the most widespread malignant cancers and a leading cause of death worldwide
Our study indicates that loss of addiction to mutant epidermal growth factor receptor (EGFR) resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-tyrosine kinase inhibitors (TKIs) resistance
Anti-HER2 and anti-phospho-HER2 antibodies were purchased from Upstate Biotechnology, Anti-phosphoEGFR, anti-EGFR, anti-phospho-HER3, anti-phospho-c-Met, anti-phospho-Akt, anti-Akt, anti-PTEN, anti-phospho-ERK1/2, anti-ERK1/2, and mutation-specific (L858R in exon 21 and deletion E746-A750 in exon 19) antibodies were from Cell Signaling Technology, anti-HER3 and anti-c-Met antibodies were from Santa Cruz Biotechnology, anti-a-tubulin antibody was from Sigma-Aldrich, and anti-GAPDH antibody was from Trevigen

Summary

Introduction

Non-small-cell lung cancer (NSCLC) is one of the most widespread malignant cancers and a leading cause of death worldwide. Two representative EGFR-tyrosine kinase inhibitors (EGFR-TKIs), gefitinib and erlotinib, have a common quinazoline structure and have been approved for the treatment of progressive NSCLC. The most common activating EGFR mutations are in-frame deletion in exon 19 (delE746-A750) and the point mutation replacing leucine with arginine at codon 858 of exon (L858R) [5,6,7,8,9]. These two major mutations account for 85–90% of all mutations and enhance the therapeutic efficacy of EGFR-targeted drugs [10,11,12,13]. These activating mutations gained addiction to EGFR in lung cancer cells, resulting in enhanced susceptibility to EGFR-TKI such as gefitinib and erlotinib [6,14,15,16]

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Conclusion