Oct4 plays a crucial role in the maintenance of gefitinib-resistant lung cancer stem cells

https://doi.org/10.1016/j.bbrc.2016.03.064Get rights and content

Highlights

  • Oct4 is highly expressed in gefitinib-resistant tumors of EGFR-mutant NSCLC in vivo.

  • Oct4 induces gefitinib resistance in vitro and in vivo.

  • Oct4 increases CD133-positive gefitinib-resistant persisters.

  • Oct4 promotes properties of cancer stem cells under the gefitinib treatment.

  • Oct4 is highly expressed in tumors of patients with acquired resistance to gefitinib.

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 14–17 days of gefitinib treatment, the tumors still remained; these tumors were referred to as gefitinib-resistant tumors (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 × 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.

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 mutations [3]. The most well-known mechanisms of acquired resistance are EGFR T790M secondary mutation, MET amplification, overexpression of HGF, mutation or amplification of HER2, and transformation to small cell lung cancer [4]. However, the mechanisms responsible for intrinsic resistance and other acquired forms of resistance to EGFR-TKI are not fully understood.

Cancer stem cells (CSCs), also known as tumor-initiating cells and stem-like cancer cells, exhibit self-renewal capacity and are responsible for tumor maintenance, metastases, and resistance to cancer therapeutics [5]. Several recent studies have demonstrated that CSCs are involved in resistance to gefitinib in NSCLC as non-mutational mechanisms [6], [7], [8]. However, molecular factors that regulate CSCs in gefitinib resistance have not been fully clarified.

Oct4, a member of the POU-domain family of transcription factors, is essential to maintain self-renewal and is expressed in pluripotent embryonic stem (ES) cells and germ cells [9], [10], [11]. A recent report has revealed that Oct4 expression plays a crucial role in maintaining CSC properties in lung cancer-derived CD133-positive cells [12]. However, the role of Oct4 in the maintenance of lung CSCs that results in acquired resistance to gefitinib has not been elucidated.

We previously reported that stem cell genes, including Oct4, are highly expressed in gefitinib-resistant persisters (GRPs) of NSCLC cell lines, PC9, which could survive and remain after gefitinib exposure [13]. PC9-GRPs exhibited a high potential for sphere formation in vitro and tumorigenicity in vivo, suggesting CSC properties of GRPs. These previous findings prompted us to investigate the role of Oct4 in the persistence of gefitinib-resistant lung CSCs.

In this study, we established an in vivo model to obtain gefitinib-resistant-tumors (GRTs) of EGFR-mutant NSCLC, PC9 cells. We found that Oct4 and CD133 were highly expressed in our GRT model. We also introduced the Oct4 gene into PC9 and HCC827 cells, and high expression of Oct4 significantly increased CD133-positive GRPs and promoted self-renewal ability under the high concentration of gefitinib in vitro. Furthermore, Oct4-overexpressing PC9 cells exhibited a high potential for tumorigenicity in vivo and significantly induced gefitinib resistance in vitro and in vivo. Oct4 expression was much higher in tumor specimens of EGFR-mutant NSCLC patients with acquired resistance to gefitinib as compared with that in tumor tissues obtained before treatment. The biological significance of Oct4 for the maintenance of gefitinib-resistant lung CSCs was investigated.

Section snippets

Cell culture and reagents

The NSCLC cell lines, PC9 and HCC827, which express EGFR exon 19 deletion mutations (ΔE746-A750), were used in this study. PC9 cells were established at the Tokyo Medical University (Tokyo, Japan) as previously described [14], and were kindly provided by Dr. Kazuto Nishio (Department of Genome Biology, School of Medicine; Kinki University, Osaka). Culture conditions and reagents are described in Supplementary Materials and Methods.

Quantitative real-time PCR

PCR conditions and primer sequences used for detection of

Oct4 and CD133 were highly expressed in gefitinib-resistant tumors (GRTs) in vivo

To investigate the gefitinib resistance of EGFR-mutant NSCLC cells in vivo, we transplanted 1 × 105 PC9 cells into NOG mice. When the tumor volumes reached approximately 75 mm3, as measured with digital calipers, we started to treat these tumor-bearing mice with gefitinib (20 mg/kg) or vehicle by intraperitoneal injection (6 times/week). As shown in Fig. 1A, tumor growth was inhibited by gefitinib. However, after 14–17 days of gefitinib treatment, the tumors still remained, and resumed growth

Discussion

In this study, we demonstrated that Oct4 was highly expressed in gefitinib-resistant tumors (GRTs) of EGFR-mutant NSCLC, PC9 cells, which remained after gefitinib treatment in vivo. We also found that transfection of the Oct4 gene promoted CSC properties including self-renewal ability in vitro and tumorigenicity in vivo, and significantly increased CD133-positive gefitinib-resistant persisters (GRPs). Furthermore, the overexpression of Oct4 significantly induced gefitinib resistance in vitro,

Conflict of interest

The authors declare no conflicts of interest for this work.

Acknowledgments

This study was supported by a Grant-in-Aid for Scientific Research (No. 24591176) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. This study was also supported by Research Grants from Takeda Science Foundation, Kobayashi Foundation, and Yasuda Medical Foundation. This work was supported by Promotion Plan for the Platform of Human Resource Development for Cancer in Juntendo University Graduate School of Medicine. We are grateful to Dr. Sonoko Habu for providing

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