Abstract 445: The unfolded protein response sensor PERK mediates extrinsic ER stress-induced inhibition of glioblastoma stem cell self-renewal

Abstract

Abstract Introduction: Glioblastoma (GBM) is the most prevalent and aggressive brain tumor in adults and has a very poor prognosis. GBM cancer stem cells (GSCs) rather than differentiated offspring are thought to be critical for tumor progression and therapy resistance. Hence, targeting GSCs is essential for effective therapy. In this study we aimed to investigate whether ER-stress aggravation is an effective approach for targeting GSCs. ER stress is known to impair protein production leading to activation of the unfolded protein response (UPR). This adaptive response attempts to restore protein homeostasis and cell survival, but induces cell death when damage is overwhelming. Methods: Patient-derived GBM neuropsphere cell lines enriched for GSCs were used. Differentiated counterparts were obtained by serum exposure. Thapsigargin (Tg) was used to induce extrinsic ER-stress. Cytotoxic effects and GSC targeting were determined by MTS and neurosphere formation assays; apoptosis was studied by caspase activation and use of pan-caspase inhibitor ZVAD; necrostatin-1 was applied for demonstrating necroptosis. Various known chemical modulators of the PERK and IRE1 branches of the UPR were employed. ATF6 and PERK expression was blocked by RNAi and CRIPSR/Cas9, respectively. Expression of genes was studied by RT-qPCR and western blotting. RNA sequencing was used for UPR gene transcriptional profiling. Results: Tg effectively reduced viability of the GBM neurospheres that was accompanied by induction of both apoptosis and necroptosis. Modulation of ATF6, IRE1 and PERK activity showed that the PERK branch is mediating cytotoxicity. This was confirmed by genetic ablation of PERK expression. Interestingly, Tg potently reduced neurosphere formation indicating targeting of GSCs, which coincided with down-regulation of the stem cell transcription factor SOX2. The PERK branch was found to be instrumental for this effect. Serum-differentiated neurospheres appeared to be more resistant for Tg. Transcriptional profiling of GBM neurospheres and differentiated counterparts overall revealed enhanced expression of key UPR branch genes in neuropspheres that may be linked to enhanced sensitivity for ER stress. Genes displaying a more than 2-fold differential expression included sterol regulatory element-binding proteins (SREBPs) known to modulate for example ATF6 activity. Conclusions: ER stress induced by Tg effectively reduces viability of GBM neurospheres and targets the self-renewal capacity of GSCs. The PERK branch of the UPR is instrumental in these cytotoxic effects. ER stress aggravation as well as PERK modulation appear to be promising approaches for further exploration as possible therapeutic strategies in GBM. Citation Format: Frank A. Kruyt, Natalia M. Peñaranda-Fajardo, Coby Meijer. The unfolded protein response sensor PERK mediates extrinsic ER stress-induced inhibition of glioblastoma stem cell self-renewal [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 445.