Abstract 4656: ER stress, unfolded protein response, and autophagy are pro-survival responses to fenretinide + safingol treatment in GBM cells
Authors: Vad Nikhil M. , Wang Dong , Maurer Barry J. 
Published: July 2016

The cytotoxicity of the synthetic retinoid, fenretinide (4-HPR), is associated with increased dihydroceramide and reactive oxygen species (ROS) levels and enhanced by co-treatment with safingol (S), an L-threo-dihydroceramide precursor. The ER stress response displays dichotomic effects wherein mild short-term stressors activate responses to neutralize or adapt to stress, but severe or long-lasting stressors activate cell death cascades. Here, we report that ER stress and autophagy are pro-survival responses in glioblastoma multiforme (GBM) cells to unfolded proteins-related cytotoxicity induced by 4-HPR+S in vitro. In T98G and A172 cells, 4-HPR+S induced mixed cell death associated with time-dependent increase of TUNEL-positivity (+12-36 h) and cleavage of caspase-3 (+12-36 h), but pan- caspase inhibitor, boc-D-fmk, did not rescue cell death. Cytotoxicity (+6-24 h) was associated with minimal mitochondrial depolarization or dual-staining of Annexin V/PI. Assays for known non-apoptotic mechanisms were negative. There was time-dependent (+6-24 h) increase of GRP78 protein levels, a key regulator of ER stress response; ER stress sensors, PERK, IRE1α, and ATF6, were activated (+6-24 h); levels of pro-apoptotic transcription factor, CHOP, increased (+6-24 h). Cytotoxicity was increased by PERK pathway inhibitors, salubrinal (+24-72 h, p≤0.05), GSK 2606414 (+24-72 h, p≤0.05), and GSK 2656157 (+48-72 h, p≤0.05), indicating that PERK activation was pro-survival. Treatment increased levels of CHIP, a key co-chaperone targeting unfolded proteins for degradation (+6-24 h). There was strong activation of the ubiquitin-proteasome system (UPS) and autophagy preceding or concurrent with the accumulation of poly-ubiquitinated proteins (+12-24 h). Autophagy was evidenced by increased conversion of LC3B-I to LC3B-II (+6-24 h). LC3B-II levels were increased by lysosomal degradation inhibitor, bafilomycin A1, indicating increased autophagic flux. Cytosolic levels of HDAC6, a coordinator of protein turnover via UPS and autophagy through trafficking of protein aggregates into pre-lysosomal structures (aggresomes) prior to autophagosome engulfment, increased (+6-24 h). siRNA knockdown of CHIP enhanced cytotoxicity (+24-72 h, p≤0.05). siRNA ablation of autophagy (ATG7, BECN1)(+24-36 h, p≤0.05), or pharmacological disruption by mefloquine, increased cytotoxicity (+24-72 h, p≤0.05), indicating that autophagy was pro-survival. Cytotoxicity was increased by siRNA knockdown of HDAC6 (+24-72 h, p≤0.05) and selective HDAC6 inhibitor, ACY1215, (+24-72 h, p≤0.05). Together, these results support that ER stress/unfolded protein response (UPR) is pro-survival in GBM cells exposed to 4-HPR+S. This suggests that co-agents that reduce ER stress response, UPR, or autophagy processes may further sensitize GBM cells to 4-HPR+S.

Citation Format: Nikhil M. Vad, Dong Wang, Barry J. Maurer. ER stress, unfolded protein response, and autophagy are pro-survival responses to fenretinide + safingol treatment in GBM cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4656.