Abstract 1057: Co-treatment with fenretinide and safingol induced p38 MAPK and/or FOXO3a activated pro-survival autophagy in glioblastoma multiforme cells
Authors: Vad Nikhil , Wang Dong , Linch Charlie , Maurer Barry J. 
Published: July 2017

Abstract
Cytotoxicity of the synthetic retinoid, fenretinide (4-HPR), is associated with increased reactive oxygen species and/or de novo synthesized D-erythro-dihydroceramides. Co-treatment with L-threo-sphinganine (safingol, S), synergistically increased fenretinide cytotoxicity in cell lines of many cancer types coincident with increase of safingol-derived, L-threo-dihydroceramides. Previously, we showed that 4-HPR+S induced ER stress, unfolded protein response (UPR), pro-survival autophagy, and mixed non-apoptotic and apoptotic cell death in glioblastoma multiforme (GBM) cells in vitro. However, the mechanisms regulating such activity remained unclear, including the role of ceramide-activated signal transducers, such as ASK1. We now report that 4-HPR+S activated stress kinase ASK1 via phosphorylation at Thr845 (+1-6 hrs), an event known to induce p38 MAPK phosphorylation. Furthermore, 4-HPR+S induced an increase in the nuclear localization of transcription factor FOXO3a coincident with decreased Akt signaling and increased phosphorylated levels of JNK1/2, p38 MAPK, ERK1/2, and autophagy regulator, AMPK (+1-6 hrs). 4-HPR+S caused a time-dependent (+6-24 hrs) increase of GRP78 protein levels, a key regulator of ER stress response and UPR, and increased levels of pro-apoptotic transcription factor, CHOP. GRP78 increase was p38 MAPK-dependent, as p38 MAPK inhibitor, SB203580, greatly reduced GRP78 increase. Interestingly, 4-HPR+S induced cytotoxicity was unchanged in the presence of an inhibitor of ERK 1/2 (PD98059) but significantly decreased when cells were concurrently treated with p38 MAPK inhibitor, SB203580, or JNK1/2 inhibitor, SP600125 (+24-72 hrs, p ≤ 0.05). Significantly, co-treatment with dorsomorphin, an AMPK inhibitor, increased and accelerated 4-HPR+S cytotoxicity (+24-72 hrs, p ≤ 0.05). Moreover, 4-HPR+S treatment induced FOXO3a Ser7 phosphorylation (+1-6 hrs) and a corresponding decrease in levels of nuclear E3-Ub protein ligase, Skp2. The decrease of SKp2 protein was coincident with increased levels of CARM1 protein, a known transcriptional activator of autophagy-related genes. Activation of ASK1, JNK1/2, p38 MAPK and ERK1/2 by 4-HPR+S occurred earlier, and to a greater extent, than single agent 4-HPR and S exposures at all time points (+1-24 hrs). Together, these results support that 4-HPR+S induced both pro-survival autophagy and cell death at least partly through activation of FOXO3a and ASK1-dependent activation of p38 MAPK, possibly in response to the known progressive increase of de novo synthesized D-erythro and L-threo dihydroceramides. A Phase I trial of intravenous 4-HPR+S in adult solid tumors (ClinicalTrials.gov Identifier:NCT0155307) is currently open in the Texas State-supported, South Plains Oncology Consortium (SPONC.org).
Citation Format: Nikhil Vad, Dong Wang, Charlie Linch, Barry J. Maurer. Co-treatment with fenretinide and safingol induced p38 MAPK and/or FOXO3a activated pro-survival autophagy in glioblastoma multiforme cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1057. doi:10.1158/1538-7445.AM2017-1057