Research Article Effects of Vandetanib on Lung Adenocarcinoma Cells Harboring Epidermal Growth Factor Receptor T790M Mutation In vivo Eiki Ichihara, 1 Kadoaki Ohashi, 1 Nagio Takigawa, 2 Masahiro Osawa, 1 Atsuko Ogino, 1 Mitsune Tanimoto, 1 and Katsuyuki Kiura 1 1 Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences; 2 Department of Respiratory Medicine (Oncology), Okayama University Hospital, Okayama, Japan Abstract Vandetanib is a novel multitarget tyrosine kinase inhibitor (TKI) that inhibits vascular endothelial growth factor recep- tor-2 (VEGFR-2), with additional inhibition of epidermal growth factor receptor (EGFR) and rearranged during trans- fection receptor signaling, which has shown promising results in clinical trials for advanced non–small cell lung cancer. However, the mechanisms of acquired resistance to vandeta- nib remain unclear. Therefore, we established in vitro vande- tanib-resistant PC-9/VanR cells from PC-9, a vandetanib- sensitive lung adenocarcinoma cell line, by chronic exposure to this agent. PC-9/VanR cells were 50-fold more resistant to vandetanib than PC-9 cells in vitro. Compared with PC-9 cells, PC-9/VanR cells showed emergence of an EGFR T790M muta- tion, moderately elevated MET amplification, and similar VEGFR-2 inhibition by vandetanib. Note that phospho-MET in PC-9/VanR was suppressed following EGFR inhibition by an irreversible EGFR-TKI, indicating that MET signaling of PC-9/VanR was dependent on EGFR signaling and that MET amplification was not the primary mechanism of resistance to vandetanib. In contrast to the in vitro experiment, vande- tanib effectively inhibited the growth of PC-9/VanR tumors in an in vivo xenograft model through the antiangiogenesis ef- fects of VEGFR-2 inhibition. In conclusion, the multitarget TKI vandetanib induced or selected for the EGFR T790M mu- tation as observed previously with highly selective EGFR-TKIs. However, vandetanib retained significant efficacy in vivo against xenografts harboring the T790M mutation, providing a strong scientific rationale for investigating vandetanib in clinical settings where acquired resistance through emer- gence of EGFR T790M mutations limits the effectiveness of highly selective EGFR-TKIs. [Cancer Res 2009;69(12):5091–8] Introduction Dramatic clinical responses to the selective epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKI) gefitinib and erlotinib have been observed in patients with advanced non–small cell lung cancer (NSCLC), especially in those with tu- mors harboring activating EGFR mutations (1–3). However, selec- tive EGFR-TKIs have shown only limited survival benefits in NSCLC in some clinical trials (4, 5) and the majority of NSCLCs initially sensitive to gefitinib or erlotinib become resistant to these agents within 1 year of therapy (6). In patients with NSCLC who acquire resistance to gefitinib or erlotinib, ∼50% have tumors with a secondary T790M mutation in exon 20 of EGFR (7–10) and ∼20% have tumors with amplification of MET gene (11–13). Recently, multitarget treatment strategies have been developed for various solid cancers, and a variety of multitarget TKIs [e.g., lapatinib (14), sunitinib (15), and sorafenib (16)] have been intro- duced for clinical use. In particular, vascular endothelial growth factor receptor (VEGFR) and EGFR seem to be key molecular tar- gets in the treatment of NSCLC (5, 17). VEGFR-2 signaling pro- motes angiogenesis of the tumor microenvironment and is indirectly related to tumor growth. Bevacizumab, an anti-VEGF monoclonal antibody, has received Food and Drug Administration approval for treatment of nonsquamous NSCLC as well as for co- lorectal cancer and breast cancer (17, 18). A combination of beva- cizumab with erlotinib seemed to have clinical benefit in the treatment of nonsquamous NSCLC (19). Vandetanib (ZACTIMA) is an orally available multitarget TKI inhibiting VEGFR-2, with ad- ditional inhibition of EGFR and rearranged during transfection (RET) receptor signaling (20–22). Vandetanib has shown promising results in phase II clinical trials in patients with pretreated NSCLC (23–25). Currently, phase III trials of vandetanib versus erlotinib (ZEST) and vandetanib in combination with docetaxel versus doc- etaxel alone (ZODIAC) are under way in patients with advanced NSCLC who have received prior therapy. However, it remains un- known whether prolonged treatment with multitarget TKIs, such as vandetanib, results in the emergence of the T790M EGFR muta- tion or MET gene amplification as reported previously for acquired resistance to the highly selective EGFR-TKIs gefitinib and erlotinib (7–13). In this study, we established an in vitro vandetanib-resistant cell line from vandetanib-sensitive cells by chronic exposure to vande- tanib to elucidate the mechanism of acquired resistance to this agent. We also examined the sensitivity of the resistant cells to vandetanib in an in vivo xenograft model. Materials and Methods Establishmentofavandetanib-resistantcellline. PC-9 cells were cul- tured at 37°C with 5% CO 2 in RPMI 1640 supplemented with 10% heat-in- activated fetal bovine serum. To establish a vandetanib-resistant subline, the cells were treated with gradually increasing concentrations of vandeta- nib, starting at 0.01 μmol/L (lower than the IC 50 of PC-9 cells). After 3 mo, the cells grew rapidly in the presence of 4 μmol/L vandetanib, and then we performed a single-cell cloning by soft agar and established the vandetanib- resistant cell line (PC-9/VanR). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Growth inhibition was determined using a modified 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using Cell Counting Kit-8 (Dojindo; ref. 26). Briefly, the cells were placed on Note: Current address for A. Ogino: NHO Shikoku Cancer Center Hospital, Matsuyama 790-0280, Japan. Requestsforreprints: Katsuyuki Kiura, Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikatacho, Okayama 700-8558, Japan. Phone: 81-86-235-7225; Fax: 81-86-232-8226; E-mail: kkiura@md.okayama-u.ac.jp. ©2009 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-4204 5091 Cancer Res 2009; 69: (12). June 15, 2009 www.aacrjournals.org Research. on June 1, 2020. © 2009 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst June 2, 2009; DOI: 10.1158/0008-5472.CAN-08-4204