Oct4 plays a crucial role in the maintenance of gefitinib-resistant lung cancer stem cells Isao Kobayashi a, b, 1 , Fumiyuki Takahashi a, b, *, 1 , Fariz Nurwidya a, b , Takeshi Nara c , Muneaki Hashimoto c , Akiko Murakami a, b , Shigehiro Yagishita a, b , Ken Tajima a, b , Moulid Hidayat a, b , Naoko Shimada a, b, d , Kentaro Suina a, b , Yasuko Yoshioka a, b , Shinichi Sasaki a, b , Mariko Moriyama e , Hiroyuki Moriyama e , Kazuhisa Takahashi a, b, d a Department of Respiratory Medicine, Juntendo University, Graduate School of Medicine, Tokyo, Japan b Research Institute for Diseases of Old Ages, Juntendo University, Graduate School of Medicine, Tokyo, Japan c Department of Molecular and Cellular Parasitology, Juntendo University, Graduate School of Medicine, Tokyo, Japan d Leading Center for the Development and Research of Cancer Medicine, Juntendo University, Graduate School of Medicine, Tokyo, Japan e Pharmaceutical Research and Technology Institute, Kinki University, School of Medicine, Osaka, Japan article info Article history: Received 5 March 2016 Accepted 16 March 2016 Available online 17 March 2016 Keywords: Oct4 Gefitinib resistance Cancer stem cell NSCLC abstract Several recent studies have suggested that cancer stem cells (CSCs) are involved in resistance to gefitinib in non-small cell lung cancer (NSCLC). Oct4, a member of the POU-domain transcription factor family, has been shown to be involved in CSC properties of various cancers. We previously reported that Oct4 and the putative lung CSC marker CD133 were highly expressed in gefitinib-resistant persisters (GRPs) in NSCLC cells, and GRPs exhibited characteristic features of the CSCs phenotype. The aim of this study was to elucidate the role of Oct4 in the resistance to gefitinib in NSCLC cells with an activating epidermal growth factor receptor (EGFR) mutation. NSCLC cell lines, PC9, which express the EGFR exon 19 deletion mutation, were transplanted into NOG mice, and were treated with gefitinib in vivo. After 14e17 days of gefitinib treatment, the tumors still remained; these tumors were referred to as gefitinib-resistant tu- mors (GRTs). PC9-GRTs showed higher expression of Oct4 and CD133. To investigate the role of Oct4 in the maintenance of gefitinib-resistant lung CSCs, we introduced the Oct4 gene into PC9 and HCC827 cells carrying an activating EGFR mutation by lentiviral infection. Transfection of Oct4 significantly increased CD133-positive GRPs and the number of sphere formation, reflecting the self-renewal activity, of PC9 and HCC827 cells under the high concentration of gefitinib in vitro. Furthermore, Oct4-overexpressing PC9 cells (PC9-Oct4) significantly formed tumors at 1 Â 10 cells/injection in NOG mice as compared to control cells. In addition, PC9-Oct4 tumors were more resistant to gefitinib treatment as compared to control cells in vivo. Finally, immunohistochemical analysis revealed that Oct4 was highly expressed in tumor specimens of EGFR-mutant NSCLC patients with acquired resistance to gefitinib. Collectively, these findings suggest that Oct4 plays a pivotal role in the maintenance of lung CSCs resistant to gefitinib in EGFR mutation-positive NSCLC. © 2016 Elsevier Inc. All rights reserved. 1. Introduction Advanced non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide [1]. Somatic mutations in the epidermal growth factor receptor (EGFR) gene, such as an in- frame deletion mutation in exon 19, are associated with favorable response to the EGFR tyrosine kinase inhibitor (EGFR-TKI), gefitinib [2]. However, acquired resistance to gefitinib limits progression- free survival among NSCLC patients with activating EGFR muta- tions [3]. The most well-known mechanisms of acquired resistance are EGFR T790M secondary mutation, MET amplification, over- expression of HGF, mutation or amplification of HER2, and trans- formation to small cell lung cancer [4]. However, the mechanisms Abbreviations: GRP, gefitinib-resistant persister; GRT, gefitinib-resistant tumor; qPCR, quantitative real-time PCR. * Corresponding author. Department of Respiratory Medicine, Juntendo Univer- sity, Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-Ku, Tokyo,113-8421, Japan. E-mail address: fumiyuki@dol.hi-ho.ne.jp (F. Takahashi). 1 These authors contributed equally to this study. Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc http://dx.doi.org/10.1016/j.bbrc.2016.03.064 0006-291X/© 2016 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications 473 (2016) 125e132