Page 137 - 2014 Printable Abstract Book
P. 137
(PS1-65) Radiation-induced endoplasmic reticulum stress contributes to vegf expression in glioblastoma
cell lines. David Dadey, BS; Arpine Khudanyan, BS; Vaishali Kapoor, PhD; Dinesh Thotala, PhD; and Dennis
Hallahan, MD, Washington University in St. Louis School of Medicine, St. Louis, MO
The advent of multimodal combination therapy for malignant gliomas has yielded limited
improvement in treatment outcomes, as radio-resistance and tumor recurrence remain significant
barriers to achieving efficacy. We found GRP78, a pro-survival stress protein and key regulator of the
endoplasmic reticulum (ER) stress response, to be induced by ionizing radiation (IR) in glioma tumor
models in vivo. This prompted us to investigate the biology of GRP78 and downstream elements of the ER
stress response, with the goal of identifying novel targets for improving the efficacy of radiation therapy
in the treatment of malignant gliomas. Recently, Pancreatic ER Kinase (PERK), a component of the ER-
stress response, has been implicated in the regulation of angiogenic factors and pro-survival signaling in
cancer. Here we show for the first time the impact of IR on ER stress signaling pathways, and the effect of
a small-molecule inhibitor of PERK on the expression of VEGF in irradiated glioma cell lines.
D54, LN827 and GL261 (two human and one mouse glioma cell line) were grown in culture and used to
study the effect of IR on ER homeostasis. By transducing cells with ER-localized red-ox and calcium sensors
(MERO-GFP and D1ER), we observed that radiation disrupted homeostasis by inducing an oxidative state
in the ER and increased calcium abundance. We subsequently found that IR induced ER stress chaperones,
GRP78 and GRP94, by western blot and immunofluorescent staining. The radiation-induced ER-stress
phenomenon was further substantiated by qRT-PCR analysis, which revealed increases in multiple
downstream mRNAs associated with the ER-stress response. Of the various up-regulated mRNAs, we
found PERK mRNA was induced 4 fold after IR. Concomitant increases in the mRNA of downstream targets
of PERK, such as ATF4 and VEGF, were also observed. Inhibition of PERK with GSK2606414 (1 µM)
abrogated PERK induction as well as phospho-eIF2a accumulation, and was sufficient to mitigate VEGF
mRNA induction. These results indicate that radiation-induced ER-stress contributes to adaptive
processes in glioblastoma. By regulating angiogenic factor production, PERK activation downstream of ER-
stress could be an important mediator of pro-survival signaling in irradiated gliomas, and may contribute
to radio-resistance.
135 | P a g e
cell lines. David Dadey, BS; Arpine Khudanyan, BS; Vaishali Kapoor, PhD; Dinesh Thotala, PhD; and Dennis
Hallahan, MD, Washington University in St. Louis School of Medicine, St. Louis, MO
The advent of multimodal combination therapy for malignant gliomas has yielded limited
improvement in treatment outcomes, as radio-resistance and tumor recurrence remain significant
barriers to achieving efficacy. We found GRP78, a pro-survival stress protein and key regulator of the
endoplasmic reticulum (ER) stress response, to be induced by ionizing radiation (IR) in glioma tumor
models in vivo. This prompted us to investigate the biology of GRP78 and downstream elements of the ER
stress response, with the goal of identifying novel targets for improving the efficacy of radiation therapy
in the treatment of malignant gliomas. Recently, Pancreatic ER Kinase (PERK), a component of the ER-
stress response, has been implicated in the regulation of angiogenic factors and pro-survival signaling in
cancer. Here we show for the first time the impact of IR on ER stress signaling pathways, and the effect of
a small-molecule inhibitor of PERK on the expression of VEGF in irradiated glioma cell lines.
D54, LN827 and GL261 (two human and one mouse glioma cell line) were grown in culture and used to
study the effect of IR on ER homeostasis. By transducing cells with ER-localized red-ox and calcium sensors
(MERO-GFP and D1ER), we observed that radiation disrupted homeostasis by inducing an oxidative state
in the ER and increased calcium abundance. We subsequently found that IR induced ER stress chaperones,
GRP78 and GRP94, by western blot and immunofluorescent staining. The radiation-induced ER-stress
phenomenon was further substantiated by qRT-PCR analysis, which revealed increases in multiple
downstream mRNAs associated with the ER-stress response. Of the various up-regulated mRNAs, we
found PERK mRNA was induced 4 fold after IR. Concomitant increases in the mRNA of downstream targets
of PERK, such as ATF4 and VEGF, were also observed. Inhibition of PERK with GSK2606414 (1 µM)
abrogated PERK induction as well as phospho-eIF2a accumulation, and was sufficient to mitigate VEGF
mRNA induction. These results indicate that radiation-induced ER-stress contributes to adaptive
processes in glioblastoma. By regulating angiogenic factor production, PERK activation downstream of ER-
stress could be an important mediator of pro-survival signaling in irradiated gliomas, and may contribute
to radio-resistance.
135 | P a g e
