Modulation of CCAAT/Enhancer Binding Protein Homologous Protein (CHOP)-dependent DR5 Expression by Nelfinavir Sensitizes Glioblastoma Multiforme Cells to Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)

Abstract

Human glioblastoma multiforme cells demonstrate varying levels of sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Endoplasmic reticulum (ER) stress has been shown to trigger cell death through apoptosis. We therefore pursued a strategy of integrating clinically relevant investigational agents that cooperate mechanistically through the regulation of ER stress and apoptosis pathways. Nelfinavir belongs to the protease inhibitor class of drugs currently used to treat patients with HIV and is in clinical trials as an anti-tumor agent. We found that Nelfinavir treatment led to ER stress-induced up-regulation of the DR5 receptor. This transactivation was mediated by the transcription factor CCAAT/enhancer binding protein homologous protein (CHOP). We also determined that ER stress-induced ATF4 up-regulation was responsible for modulation of CHOP. In contrast, DR4 receptor expression was unchanged by Nelfinavir treatment. Combining Nelfinavir with TRAIL led to a significantly enhanced level of apoptosis that was abrogated by siRNA silencing of DR5. We provide evidence that Nelfinavirinduced ER stress modulates DR5 expression in human glioblastoma multiforme cells and can enhance TRAIL efficacy. These studies provide a potential mechanistic rationale for the use of the Food and Drug Administration-approved agent Nelfinavir in combination with DR5 agonists to induce apoptosis in human malignancies.Malignant gliomas account for ϳ70% of the 22,500 new cases of malignant primary brain tumors that are diagnosed in adults in the United States each year (1). Glioblastoma multiforme is a high-grade, highly lethal, and frequent brain tumor in adults. Traditional treatments such as DNA damaging agents and radiotherapy have demonstrated modest effects on patient survival, likely because in part of an inherent high-level expression of anti-apoptotic proteins such as Bcl-2, Bcl-X L , and sFLIP (2, 3). Frequent gene amplification and overexpression of the death decoy receptor DcR3 have also been reported in glioblastoma (4). Therefore, new strategies to induce apoptosis may help to overcome therapeutic resistance and improve efficacy.Nelfinavir is an HIV protease inhibitor and has been used to treat HIV/AIDS patients for over a decade. Recently, several groups reported the anticancer activity of Nelfinavir in a wide range of human cancer cell lines and tumor xenografts. Nelfinavir induces ER 2 stress, the unfolded protein response, autophagy, apoptosis, and caspase-independent cell death in various cancer cells (5-9). Multiple mechanisms have been proposed to explain the anticancer activities of the drug, such as inhibition of AKT/protein kinase B (PKB) activation (10), the proteasome (9), or signal transducer and activator of transcription (STAT) 3 (11), and down-regulation of hypoxia-inducible factor 1␣ (HIF-1␣)/VEGF expression (8). However, the exact molecular mechanisms of Nelfinavir efficacy in human cancer cells remains unclear and may be cell type-specific. We currently have an...

ER stress regulates myeloid-derived suppressor cell fate through TRAIL-R–mediated apoptosis

“…RNA was extracted with total RNA extraction kit (Omega Bio-tek). cDNA was synthesized (cDNA reverse transcriptase kit; Applied Biosystems), and PCR was performed in duplicate for each tor of ER stress response-induced apoptosis (26) and is a known transcription factor able to directly regulate DR5 expression (10,27). Our experiments demonstrated that treatment of mouse IMCs and PMNs with the ER stress inducers tunicamycin and thapsigargin resulted in upregulation of DR5, recapitulating the effects observed in MDSCs.…”

“…We confirmed that, at selected concentrations, those cytokines activated their respective signaling pathways: NF-κB, STAT1, and STAT3 (Supplemental Figure 7). DR5 expression in tumor cells can also be induced by ER stress via spliced XBP1, a major mediator of the IRE1 pathway, and C/EBP homologous protein (CHOP), a downstream molecule of the PERK pathway (24,(26)(27)(28)(29). The ER stress inducer tunicamycin caused significantly increased DR5 expression in BM PMNs and monocytes ( Figure 6, A and B).…”

Endoplasmic reticulum proteostasis in glioblastoma—From molecular mechanisms to therapeutic perspectives

“…RDC11-induced CHOP activation is associated with the up-regulation of proapoptotic downstream molecules, Tribbles homolog 3 (TRB3) and ChaC glutathione-specific g-glutamylcyclotransferase 1 (CHAC1) (66). Nelfinavir (a drug belonging to protease inhibitor class), when combined with TNF-related apoptosis-inducing ligand (TRAIL), increases GBM apoptosis through CHOP and DR5 up-regulation (67). Finally, IRE1 activation is also involved in GBM cell apoptosis induced by a nitric oxide (NO) donor, S-nitroso-N-acetyl penicillamine, leading to the activation of TRAF2 and JNK and the phosphorylation of CREB, a transcription factor involved in NO-mediated cell death (68).…”

“…As such, a large number (>55) of small molecules have been tested on GBM cell lines and induced cell death by a mechanism that indirectly perturbs ER proteostasis, hence highlighting their potential use as therapeutic agents (149,150) [ Table 2 (22,(65)(66)(67)]. The impact of these molecules on ER proteostasis imbalance was in most cases (85%) inferred by the up-regulation of GRP78 and/or CHOP mRNA or protein abundance, but generally, the causality of ER stress in the cell death process was not demonstrated.…”

Inhibition of IRE1 modifies effect of glucose deprivation on the expression of TNF?-related genes in U87 glioma cells

“…It is possible that TRAILR4/TNFRSF10D as well as TRAILR2/ TNFRSF10B has relation to regulation of endoplasmic reticulum stress-mediated apoptosis. Moreover, modulation of CCAAT/enhancer binding protein homologous protein (ChoP)-dependent TRAILR2/ TNFRSF10B/DR5 expression by nelfinavir sensitizes glioblastoma multiform cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) [52]. Therefore, a better understanding of the mechanisms underlying TRAILR4/TNFRSF10D as well as TRAILR2/TNFRSF10B is required.…”