Elsevier

Experimental Cell Research

Volume 326, Issue 2, 15 August 2014, Pages 201-209
Experimental Cell Research

Research Article
Everolimus prolonged survival in transgenic mice with EGFR-driven lung tumors

https://doi.org/10.1016/j.yexcr.2014.04.012Get rights and content

Highlights

  • Everolimus was similarly efficacious, in vitro, on cells harboring various EGFR mutations.

  • Everolimus suppressed lung tumors in EGFR transgenic mice and prolonged the overall survival of these mice.

  • Everolimus might be effective for EGFR-mutated lung cancer by inhibiting tumor angiogenesis.

Abstract

Everolimus is an orally administered mTOR inhibitor. The effect, and mechanism of action, of everolimus on lung cancers with an epidermal growth factor receptor (EGFR) mutation remain unclear. Four gefitinib-sensitive and -resistant cell lines were used in the present work. Growth inhibition was determined using the MTT assay. Transgenic mice carrying the EGFR L858R mutation were treated with everolimus (10 mg/kg/day), or vehicle alone, from 5 to 20 weeks of age, and were then sacrificed. To evaluate the efficacy of everolimus in prolonging survival, everolimus (10 mg/kg/day) or vehicle was administered from 5 weeks of age. The four cell lines were similarly sensitive to everolimus. Expression of phosphorylated (p) mTOR and pS6 were suppressed upon treatment with everolimus in vitro, whereas the pAKT level increased. The numbers of lung tumors with a long axis exceeding 1 mm in the everolimus-treated and control groups were 1.9±0.9 and 9.4±3.2 (t-test, p<0.001), respectively. pS6 was suppressed during everolimus treatment. Although apoptosis and autophagy were not induced in everolimus-treated EGFR transgenic mice, angiogenesis was suppressed. The median survival time in the everolimus-treated group (58.0 weeks) was significantly longer than that in the control group (31.2 weeks) (logrank test, p<0.001). These findings suggest that everolimus had an indirect effect on tumor formation by inhibiting angiogenesis and might be effective to treat lung tumors induced by an activating EGFR gene mutation.

Introduction

Somatic mutants of the epidermal growth factor receptor (EGFR), HER2, KRAS, BRAF, PIK3CA, and EML4-ALK of non-small cell lung cancers (NSCLCs) are viewed as targets of anti-cancer agents [1]. Of tumors in 52 East Asian never-smokers with lung adenocarcinomas, 78.8% harbored EGFR mutations, 5.8% (three) EML4-ALK fusions, 3.8% (two) HER2 mutations, and 1.9% (one) a KRAS mutation. Only 9.6% (five of 52) tumors did not harbor any of these known oncogenic driver mutations [2]. Never-smokers with NSCLC could be divided into four distinct genotypic groups based on genetic profiling of three major oncogenes (EGFR, EML4-ALK, and KRAS), yielding unique and non-overlapping subsets of patients with lung cancer who exhibited different therapeutic responses and survival outcomes [3]. The “Biomarker-integrated approaches of targeted therapy for lung cancer elimination” (BATTLE) program of personalized medicine has been recently developed [4]. Adaptive randomization was successfully used to assign NSCLC patients to the treatment offering the greatest potential benefit, based on the markers of prospectively biopsied tumors. Moreover, EGFR mutations and EML4-ALK gene rearrangements in NSCLC have been established as real molecular targets from the clinical studies [5]. Thus, it is important to treat patients with reference to their genetic profiles to determine whether patients can benefit from specifically targeted therapies.

EGFR mutations are more frequently observed in females, non-smokers, and adenocarcinoma patients, especially in Asian populations, wherein the mutation rate is 30–50% [6]. The EGFR tyrosine kinase inhibitors (TKIs), erlotinib and gefitinib, improved progression-free survival in patients with NSCLC harboring EGFR mutations [7], [8], [9], [10], [11]. The majority of EGFR mutant lung cancers initially sensitive to EGFR-TKI become resistant to these agents within 1 year. Some possible mechanisms for acquisition of resistance have been identified, the most common being development of an EGFR T790M “gatekeeper” mutation in about 50% of cases [12], [13]. Although clinical work seeking to overcome resistance acquired via T790M has been conducted, no generally accepted therapy has been established [14].

The mammalian target of rapamycin (mTOR) is located downstream of EGFR and plays a major role in regulation of protein translation, cell growth, and metabolism [15], [16], [17], [18]. Alterations in the mTOR signaling pathway are common in cancer patients and mTOR is being actively explored as a therapeutic target [17]. Inhibition of mTOR by targeting agents causes G1 cell-cycle arrest mediated by inactivation of phospho-S6 ribosomal protein (pS6) and hypophosphorylation of 4E-BP1 [16]. Everolimus is an orally administered rapamycin analog showing anticancer effects on renal cell carcinoma and pancreatic neuroendocrine tumors [17]. Furthermore, in preclinical studies, everolimus showed anticancer effects in various cancer cell lines and xenograft models, including lung cancer [16]. However, the effect of mTOR inhibitors on NSCLC harboring EGFR mutations remains unclear. In our present study, we investigated the preclinical efficacy of everolimus in NSCLC harboring EGFR mutations. Our findings suggest that everolimus might be effective for NSCLC by the inhibiting tumor angiogenesis.

Section snippets

Cell lines and mouse model

PC-9 is a lung adenocarcinoma cell line that has an in-frame deletion mutation at exon 19 of EGFR and amplification of the EGFR gene. We used RPC-9 cells with the T790M mutation of EGFR that were 400-fold more resistant to gefitinib than parental PC-9 cells [19]. The RPC-9 cell line was established in our laboratory by continuously exposing the PC-9 cell line to gefitinib. H3255 is a lung adenocarcinoma cell line that has a L858R point mutation at exon 21 of EGFR and amplification of the EGFR

Everolimus was effective regardless EGFR mutation status

Sensitivity to everolimus of lung cancer cell lines was examined at first. Everolimus exhibited similar efficacy on A549 cells (EGFR wild-type), H3255 cells (with the L858R mutation), PC-9 cells (with an exon 19 deletion mutation) and RPC-9 cells (with both the exon 19 deletion mutation and the T790M mutation). The curves of concentration versus surviving fraction were similar and all IC50s were around 0.01 μM (Fig. 1A and B).

Everolimus suppressed pmTOR and pS6

Next, effect of everolimus on EGFR-related signals was examined using

Discussion

We found that everolimus was similarly efficacious, in vitro, on cells harboring various EGFR mutations. The EGFR-tyrosine kinase inhibitor resistance-related T790M secondary mutation seemed to be neutralized by everolimus (Fig. 1B). In addition, everolimus suppressed lung tumors in transgenic mice expressing the L858R mutation of EGFR and prolonged the overall survival of these mice.

Signaling pathways of AKT/mTOR that include S6, 4E-BP, or the eukaryotic translation initiation factor complex

Conflict of interest

N. Takigawa and K. Kiura have honoraria from speakers׳ bureau from AstraZeneca. Other authors have no conflict of interest.

Acknowledgments

This study was partly supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan [grants 24591182 (N. Takigawa) and 23390221 (K. Kiura)] and by a Research Project Grant 23B-30 (M. Yasugi) from Kawasaki Medical School. We thank Ms. Chie Hada, Department of General Internal Medicine 4, Kawasaki Medical School for expert technical support. We also thank Drs. T. Kudo, H. Takeda, H. Hayakawa, M. Fujii, K. Rai, D. Minami and K. Hotta, Department of

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