Abstract 4487: Effect of everolimus on lung tumorigenesis in transgenic mice carrying activating EGFR mutation
Authors: Yasugi Masayuki , Takigawa Nagio , Ohashi Kadoaki , Takeda Hiromasa , Honda Yoshihiro , Murakami Toshi , Ichihara Eiki , Hotta Katsuyuki , Tanimoto Mitsune , Kiura Katsuyuki 
Published: June 2012

Abstract
The mammalian target of rapamycin (mTOR) plays a major role in the regulation of protein translation, cell growth, and metabolism. Alterations of the mTOR signaling pathway are common in cancer, and thus mTOR is being actively pursued as a therapeutic target. Everolimus is an orally available rapamycin analogue showing anticancer effects on renal cell carcinoma and used in clinical. Furthermore, in preclinical studies, everolimus shows anticancer effects in various cancer cell lines and xenograft models, including lung cancer. However, the effect of mTOR inhibitors on non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutation remains unclear. Our present study was carried out to investigate the preclinical efficacy of everolimus in NSCLC harboring EGFR mutation.
Growth inhibition was measured by MTT assays. RPC-9 cells that acquired T790M mutation of EGFR gene were established from parental PC-9 cells harboring exon 19 del mutation (delE746-A750) in our laboratory (Cancer Res 67, 2007). The drug concentration required to inhibit the growth of tumor cells by 50% (IC50) for 96 h exposure was used to evaluate the effectiveness of everolimus. Protein expression was determined by Western blotting. The IC50s were 2 μM for PC-9 cells and 4 μM for RPC-9 cells. RPC-9 cells that showed a 400-fold resistance to gefitinib compared with parental PC-9 cells remained the sensitivity to everolimus. Phosphorylated mTOR and phosphorylated S6 were suppressed by treatment with everolimus (2 μM), while phosphorylated AKT was increased presumably due to negative feedback of mTOR inhibition.
We generated transgenic mice expressing the L858R mutation of EGFR driven by the SP-C promoter (Cancer Res 69, 2009). In the absence of treatment, the transgenic mice developed atypical adenomatous hyperplasia at age 4 weeks and adenocarcinoma at 6 to 7 weeks of age, and died due to tumor progression at 25 to 40 weeks. To investigate the effect of everolimus on lung tumors induced by an activating EGFR mutation in vivo, the transgenic mice were treated with oral everolimus (10 mg/kg/day, n = 13) or vehicle alone (n = 14) from 5 to 20 weeks of age. The number of lung tumors (long axis exceeding 1 mm) in the everolimus-treated group and control group was 1.9 ± 0.9 and 9.4 ± 3.2 (log-rank test, p&lt;0.001), respectively. Phosphorylated S6 was suppressed during everolimus treatment. To evaluate the efficacy of everolimus on survival in EGFR transgenic mice, everolimus (10 mg/kg/day, n=12) or vehicle (n=12) was administered orally from 5 weeks of age. The median survival time in the everolimus-treated group (58 weeks) was significantly longer than that in the control group (33 weeks) (log-rank test, p&lt;0.001). In conclusion, these preclinical data suggest that everolimus may be effective for chemoprevention and treatment of lung tumors induced by an activating EGFR gene mutation.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4487. doi:10.1158/1538-7445.AM2011-4487