Endogenous synthesis of TRAIL by glioma cancer stem cells and resistance to TRAIL therapy

Cleveland Clinic Lerner Com-Cwru, Cleveland OH

Investigators

Abstract

PROJECT SUMMARY/ABSTRACT Our preliminary data indicate that human glioma cancer stem-like cells (CSLCs) synthesize and secrete tumor necrosis related-apoptosis inducing ligand (TRAIL). Consistent with this, other investigators have demonstrated endogenous-TRAIL expression in glioblastoma (GBM) biopsies. Despite the synthesis of endogenous-TRAIL the CSLCs continue to proliferate, suggesting endogenous-TRAIL is signaling a pro-tumor function(s). We found that downregulation of endogenous-TRAIL inhibited proliferation in CSLCs, suggesting TRAIL may signal for proliferation. This is supported by reports in the literature that TRAIL can signal for proliferation, migration and inflammation in cancer cells. One potential mechanism for the failure of TRAIL in clinical trials is that TRAIL-DRs are sequestered intracellularly effectively reducing TRAIL-apoptotic signaling initiated at the cell surface. In non-brain cancer cells, clathrin-mediated endocytosis (CME) of TRAIL-DR inhibits TRAIL-induced apoptosis. CME of TRAIL-DR is induced by activated dynamin-1, a large GTPase that promotes membrane scission. In non-brain cancer cells, dynamin-1 was recently shown to be activated by dephosphorylation by calcineurin - a protein phosphatase. In an effort to inhibit dynamin-1 activation in GBM and block the CME of TRAIL-DR, and the resultant inhibition of apoptosis, we searched for a natural compound that inhibits dynamin-1 or the upstream activator-calcineurin, and found that quercetin (a natural flavonoid) directly inhibits calcineurin. Consistent with quercetin inhibition of calcineurin and thereby inhibition of dynamin-1, our preliminary data show that quercetin and an inhibitor of dynamin-1, sensitize glioma CSLCs to TRAIL-apoptosis. We hypothesize that: (1) an un-explored mechanism for the resistance of GBM tumors to TRAIL is the synthesis/expression of endogenous-TRAIL that promotes pro-tumorigenic functions in CSLCs; and (2) increased dynamin-1 protein in GBM results in increased levels of activated dynamin-1 promoting CME of TRAIL-DR that inhibits TRAIL-apoptosis, and that quercetin inhibition of calcineurin (calcineurin activates dynamin-1) will sensitize CSLCs to TRAIL-apoptosis. Aim 1) will determine whether knock-out (KO) or downregulation of endogenous-TRAIL in CSLCs and mouse glioma cells alters cell proliferation, migration, chemokine/cytokine secretion or sensitivity to TRAIL-induced apoptosis, and will dissect the necessary signaling pathway(s) for the pro-tumorigenic effect of endogenous- TRAIL. This will be followed by studies of TRAIL-KD CSLCs/mouse glioma cells in the athymic nude or C57BL/6 mouse brain to confirm the pro-tumorigenic effect(s) of endogenous-TRAIL found in vitro. Aim 2) will determine whether the flavonoid quercetin decreases activation of dynamin-1 in human glioma CSLCs through inhibition of calcineurin, thereby decreasing CME of TRAIL-DR and increasing TRAIL- apoptosis in CSLCs propagated in vitro, and when propagated in vivo in the athymic nude mouse brain.