Published OnlineFirst March 13, 2017; DOI: 10.1158/1541-7786.MCR-16-0482 Signal Transduction Molecular Cancer Research Distinct Afatinib Resistance Mechanisms Identified in Lung Adenocarcinoma Harboring an EGFR Mutation Toshimitsu Yamaoka1, Tohru Ohmori1, Motoi Ohba1, Satoru Arata1,2, Yasunori Murata3, Sojiro Kusumoto3, Koichi Ando3, Hiroo Ishida4, Tsukasa Ohnishi3, and Yasutsuna Sasaki4 Abstract EGFR tyrosine kinase inhibitors (TKI) are associated with and overexpression; however, these cells showed a progressive significant responses in non–smallcelllungcancer(NSCLC) decrease and eventual loss of the acquired KRAS dependence, as patients harboring EGFR-activating mutations. However, well as resensitization to afatinib, following a drug holiday. acquired resistance to reversible EGFR-TKIs remains a major Meanwhile, AFR2 cells exhibited increased expression of insu- obstacle. In particular, although the second-generation irrevers- lin-like growth factor-binding protein 3 (IGFBP3), which pro- ible EGFR-TKI afatinib is currently used for treating NSCLC moted insulin-like growth factor 1 receptor (IGF1R) activity patients, the mechanisms underlying acquired afatinib resis- and subsequent AKT phosphorylation, thereby indicating a tance remain poorly understood. Here, heterogeneous mechan- potential bypass signaling pathway associated with IGFR1. isms of acquired resistance were identified following long-term Finally, AFR3 cells harbored the secondary EGFR mutation exposure to increasing doses of afatinib in EGFR-mutant lung T790M. Our findings constitute the firstreportshowing adenocarcinoma PC-9 cells. Notably, three resistant cell lines, acquired wild-type KRAS overexpression and attenuation of PC-9AFR1, PC-9AFR2, and PC-9AFR3 (AFR1, AFR2, and AFR3, afatinib resistance following a drug holiday. respectively) employed distinct mechanisms for avoiding EGFR inhibition, with increased EGFR expression being detected in Implications: The heterogeneous mechanisms of afatinib resis- all resistant cell lines. Moreover, an activating EGFR mutation tance should facilitate the development of more effective thera- was partially lost in AFR1 and AFR2 cells. AFR1 cells exhibited peutic strategies for NSCLC patients. Mol Cancer Res; 15(7); 915–28. afatinib resistance as a result of wild-type KRAS amplification Ó2017 AACR. Introduction resistance to EGFR-targeted inhibitors (4, 5). The most com- mon mechanism related to acquired resistance to first-genera- Administration of the EGFR tyrosine kinase inhibitors (TKI) tion EGFR-TKIs (i.e., the reversible ATP-competitive inhibitors gefitinib and erlotinib has been showntoresultindramatic gefitinib and erlotinib) involves the presence of the secondary tumorregressioninnon–smallcelllungcancers(NSCLC) mutation EGFR T790M, which was detected in >50% of tumors involving EGFR-activating mutations, including exon-19 dele- (6, 7). Other resistance mechanisms, including bypass signals tions (dels) and the L858R point mutation (1). Notably, to MET [also known as hepatocyte growth factor receptor however, the tumors of many patients develop resistance to (HGFR)], insulin-like growth factor 1 receptor (IGF1R), and EGFR-TKIs within 9 to 15 months of treatment initiation (2, 3), HER2, transformation to SCLC, and induction of epithelial-to- and such acquired resistance constitutes a major difficulty in mesenchymal transition (EMT), were reported in preclinical improving clinical outcomes. Intensive research has therefore and clinical settings (8). focused on clarifying the mechanisms associated with acquired The second-generation EGFR-TKI afatinib is an irreversible drug that covalently binds to EGFR at Cys797 and was shown in 1Institute of Molecular Oncology, Showa University, Tokyo, Japan. 2Center for preclinical studies to be more potent than first-generation Biotechnology, Showa University, Tokyo, Japan. 3Division of Allergology and EGFR-TKIs against all EGFR variants, including wild-type (WT), Respiratory Medicine, Department of Medicine, Showa University School of L858R, T790M, and those harboring exon-19 dels. In clinical 4 Medicine, Tokyo, Japan. Division of Clinical Oncology, Department of Medicine, studies on patients harboring EGFR-activating mutations (exon- Showa University School of Medicine, Tokyo, Japan. 19 del or L858R), afatinib was an effective first-line treatment and Note: Supplementary data for this article are available at Molecular Cancer resulted in significantly prolonged survival rates compared with Research Online (http://mcr.aacrjournals.org/). gefitinib treatment (9, 10). However, for patients with EGFR- Corresponding Author: Toshimitsu Yamaoka, Institute of Molecular Oncology, mutant lung cancers that exhibit disease progression during Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. treatment with gefitinib or erlotinib, the efficacy of treatment Phone: 81-33784-8146; Fax: 81-33784-2299; E-mail: with afatinib was limited (11). Although the secondary EGFR [email protected] mutation T790M was frequently observed following the devel- doi: 10.1158/1541-7786.MCR-16-0482 opment of resistance to afatinib in clinical samples, other resis- Ó2017 American Association for Cancer Research. tance mechanisms have yet to be fully confirmed (12, 13). www.aacrjournals.org 915 Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst March 13, 2017; DOI: 10.1158/1541-7786.MCR-16-0482 Yamaoka et al. The emergence of acquired resistance remains a significant ogy. Gefitinib and afatinib were provided by AstraZeneca Phar- obstacle for afatinib-treated patients. In vitro, FGFR1 activation maceuticals and Boehringer-Ingelheim, respectively, and other via a ligand of the FGF2-autocrine loop was reported as a inhibitors were obtained from Selleck Chemicals. bypass signal in the human lung cancer cell line PC-9, indi- cating that treatment with a combination of afatinib and an Establishment of PC-9 cells with acquired resistance to afatinib FGFR inhibitor resulted in drug resensitization (14). In addi- To obtain cell lines with acquired resistance, PC-9 cells were tion, PC-9 cells that are resistant to dacomitinib (another exposed to increasing concentrations of afatinib in the growth irreversible EGFR-TKI) maintain PI3K/AKT signaling through medium. Starting with a dose that was nearly one tenth of the IC50, activation of insulin-like growth factor 1 receptor (IGF1R) the dosage was progressively increased over 6 to 9 months to signaling, as mediated by downregulation of insulin-like 1 mmol/L afatinib. The three resulting PC-9 afatinib-resistant cell growth factor-binding protein 3 (IGFBP3; ref. 15). Moreover, lines were designated AFR1, AFR2, and AFR3, and were main- Eberlein and colleagues observed that PC-9 cells exhibit ampli- tained continuously in culture medium containing 1 mmol/L fied KRAS or NRAS expression, resulting in afatinib resistance afatinib. (16). These PC-9AR_1 cells subsequently exhibited significant decreases in ERK1/2 phosphorylation and cell proliferation Cell proliferation assay following KRAS knockdown. However, the mechanisms asso- Cell proliferation was measured using the MTT assay (Pro- fl  2 ciated with the development of acquired resistance to irrevers- mega), as previously described (21). Brie y, cells (5 10 /well) ible EGFR-TKIs remain uncharacterized. were seeded in 96-well plates and incubated overnight, and assays Clinical reports suggest that retained sensitivity occurs upon were performed on days 0, 1, 2, 3, 5, and 7. To inhibit cell  3 gefitinib or erlotinib re-administration following disease progres- proliferation, 5 10 cells/well were seeded in 96-well plates sion (17, 18), and several studies have indicated that re-treatment and incubated overnight, followed by continuous exposure to the with the EGFR-TKIs gefitinib or erlotinib might be effective indicated concentrations of inhibitor for 72 hours. The optical following a drug holiday in certain patients (19, 20). Although density at 570 nm (OD570) was then measured with a Powerscan the definitive rationale for the re-challenge of EGFR-TKIs remains HT microplate reader (BioTek) and expressed as a percentage of unclear, it may constitute a promising therapeutic approach the value obtained from the control cells. We prepared 6 to 12 in NSCLC, particularly as subsequent lines of therapy remain replicates, and the experiments were repeated at least three times. undefined. Data were graphically displayed using GraphPad Prism version Here, we established three cell lines, PC-9AFR1, PC-9AFR2, and 5.0 software (GraphPad, Inc.). PC-9AFR3 (designated AFR1, AFR2, and AFR3, respectively), exhibiting resistance to long-term treatment with afatinib. Nota- Western blot analysis bly, although each of these cell lines displayed enhanced EGFR Treated cells were washed twice with ice-cold PBS and lysed expression, this effect was mediated via three distinct mechan- with modified RIPA buffer consisting of 50 mmol/L Tris (pH 7.4), isms. Together, these results provide insight into the pharmaco- 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Nonidet P-40, 0.25% logical basis underlying requirements for alternative treatment sodium deoxycholate, 0.1% SDS, and 1.0% protease- and phos- strategies. phatase-inhibitor cocktails (Sigma-Aldrich). Cell suspensions were centrifuged for 5 minutes at 1,200 rpm at 4C, and protein concentrations were determined using the bicinchoninic acid Materials and Methods assay (Sigma-Aldrich). Equal amounts of protein were mixed and Cell