Abstract 3323: Radiation-induced ER stress contributes to survival in glioblastoma cell lines

Abstract

Abstract Glioblastoma is the most common primary malignant brain tumor in adults in the United States. While the standard of care for afflicted patients has evolved to include surgery, ionizing radiation (IR) and temozolomide chemotherapy, the prognoses of these patients remain dismal. With the median survival of glioblastoma patients remaining in the range of 12-15 months, there is an unmet need for approaches that can overcome the inherent therapeutic resistance of these tumors. In recent years, several studies have demonstrated that IR can activate pro-survival signaling that may facilitate adaptation to therapy and the development of radio-resistance. The endoplasmic reticulum stress response (ERSR) is a conserved program known to be deregulated in cancer and can mediate pro-survival signaling in the context of therapy. The ATF6, IRE1 and PERK pathways of the ERSR are known to be important mediators in the initiation of this signaling. In this research, we show for the first time that IR, a key component in glioblastoma therapy, can induce genes downstream of ATF6, IRE1 and PERK in glioblastoma cell lines. Furthermore, we identify the ATF6 and PERK-eIF2a-ATF4 pathways as important contributors to the radiation-response in glioblastoma. Our study began with the observation of protein and mRNA induction of targets associated with the ERSR in irradiated glioblastoma cell lines. In addition to ATF6 target genes, we observed robust induction of PERK target genes 48h after 6 Gy IR. This induction of PERK target genes was accompanied with increased phosphorylation of eIF2a in a time and dose-dependent manner. To evaluate the requirement of PERK for radiation-induced eIF2a phosphorylation, we used a specific inhibitor of PERK - GSK2606414, and found that PERK inhibition was sufficient to prevent radiation-induced eIF2a phosphorylation. We also analyzed expression of several genes downstream of PERK after treatment with GSK2606414, and found that PERK inhibition lead to 70-80% reduction in radiation-induction of downstream genes. Since ATF4 is downstream of PERK-eIF2a, we examined ATF4 expression in irradiated glioblastoma and found that it was also induced by radiation. Using RNA-interference, we found that knockdown of ATF4 reduced proliferation and clonogenic survival by 50%, and resulted in 38% increase in PARP cleavage in irradiated glioblastoma. We observed similar trends when we knocked down ATF6, suggesting that multiple aspects of the ERSR can contribute to the radiation response in glioblastoma. These results indicate that IR-induced activation of ER-stress signaling through PERK and ATF6 contributes to adaptive survival mechanisms in glioblastoma. Further characterization of the mechanism by which ATF6 and ATF4 mediate cell survival after irradiation may reveal novel targets for the enhancement of radiotherapy for glioblastoma. Citation Format: David Dadey, Vaishali Kapoor, Arpine Khudanyan, Dinesh Thotala, Dennis Hallahan. Radiation-induced ER stress contributes to survival in glioblastoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3323. doi:10.1158/1538-7445.AM2015-3323