Abstract 1466: Radiosensitization by the mTOR inhibitor RAD001 involves the induction of autophagy in two cancer cell lines

Abstract

Abstract Despite many recent technical advances, including the use of accelerated high linear energy transfer (LET) particles, cancer radiotherapy remains poorly effective in controlling certain types of tumors due to the frequent failure of malignant cells to undergo a cell death program after radiation exposure. Inactivating molecular components of signalling networks that are involved in tumor radioresistance thus represents an approach to improve cancer radiotherapy. Growing evidence suggests that one such component is the mammalian target of rapamycin (mTOR), a protein kinase that is commonly hyperactive in human tumors. We therefore evaluated the potent and selective mTOR inhibitor RAD001 (everolimus) for its capacity to enhance the effects of low-LET (X-rays and gamma rays) and high-LET (65MeV neutrons) radiation in two human cancer cell lines: SK-Hep1 (hepatocellular carcinoma) and U-87 (glioblastoma). Cellular growth (SRB assay) and the occurrence of DNA double-strand breaks (DSBs, determined by γH2AX foci) and cell death (apoptosis and autophagy, assessed by several methods) were monitored at various times post-irradiation. Clonogenic survival assays and western blotting of mTOR were also performed. RAD001 (20nM) was added to cell cultures 1 hr before the irradiation and maintained throughout the experiments. Irradiation was administered as a single fraction, at doses ranging from 2 to 16Gy. While highly resistant to low-LET radiation, both cell lines proved less resistant to fast neutrons, in agreement with previous studies. When associated with either type of radiation, RAD001 strongly reduced cell growth and also affected clonogenic survival, albeit marginally. Moreover, RAD001 treatment combined with neutron irradiation was found to induce the highest levels of γH2AX foci persistence and cytotoxicity in both cell lines. However, the type of cell death differed radically between the two cell lines. In the SK-Hep1 line, whereas only a low percent of cells underwent apoptosis after irradiation, the percent of autophagic cells was elevated and further enhanced by RAD001. By contrast, in the U-87 line, apoptosis contributed for a large part of cell death after irradiation, but its occurrence was decreased by the addition of RAD001 while the incidence of autophagic cells was concomitantly increased. Our results indicate that a correlation may exist between the persistence of DSBs and the induction of autophagy. Furthermore, autophagic cell death appears to account, almost completely in SK-Hep1 and partially in U-87, for the overall cytotoxicity induced by the combination of RAD001 and irradiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1466. doi:1538-7445.AM2012-1466