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

项目总结/摘要 我们的初步数据表明，人类胶质瘤癌干细胞（CSLCs）合成和分泌 肿瘤坏死相关凋亡诱导配体（TRAIL）。与此相一致的是，其他调查人员 在胶质母细胞瘤（GBM）活检中证实了内源性TRAIL表达。尽管综合了 CSLCs继续增殖，表明内源性TRAIL是促肿瘤细胞增殖的信号传导。 function（s）.我们发现内源性TRAIL的下调抑制了CSLCs的增殖，提示 TRAIL可能是增殖的信号。这一点得到了文献报道的支持，即TRAIL可以发出信号， 增殖、迁移和炎症。TRAIL失败的一个潜在机制是， 在临床试验中，TRAIL-DR被隔离在细胞内，有效地减少了TRAIL-凋亡信号传导。 从细胞表面开始。在非脑癌细胞中，TRAIL-DR的网格蛋白介导的内吞作用（CME） 抑制TRAIL诱导的凋亡。TRAIL-DR的CME由激活的发动蛋白-1诱导，发动蛋白-1是一种大的GT3， 促进细胞膜断裂。在非脑癌细胞中，发动蛋白-1最近被证明是由 通过钙调神经磷酸酶-一种蛋白磷酸酶去磷酸化。为了抑制GBM中发动蛋白-1的活化， 并阻断TRAIL-DR的CME，以及由此产生的对凋亡的抑制，我们寻找了一种天然的 抑制发动蛋白-1或上游激活剂-钙调神经磷酸酶的化合物，并发现槲皮素（天然 类黄酮）直接抑制钙调磷酸酶。与槲皮素抑制钙调神经磷酸酶从而抑制 对于发动蛋白-1，我们的初步数据表明槲皮素和发动蛋白-1的抑制剂使胶质瘤CSLC增敏 TRAIL-凋亡。我们推测：（1）GBM的耐药机制尚未被探索 肿瘤与TRAIL的相互作用是内源性TRAIL的合成/表达，其促进促肿瘤发生 GBM中动力蛋白-1蛋白的增加导致CSLCs中动力蛋白-1水平的增加。 激活的发动蛋白-1促进TRAIL-DR的CME，抑制TRAIL-凋亡，槲皮素 钙调磷酸酶的抑制（钙调磷酸酶激活发动蛋白-1）将使CSLC对TRAIL-凋亡敏感。目的 1)将确定CSLC和小鼠中内源性TRAIL的敲除（KO）或下调 神经胶质瘤细胞改变细胞增殖、迁移、趋化因子/细胞因子分泌或对TRAIL诱导的 细胞凋亡，并将剖析必要的信号通路的促肿瘤作用的内源性- 小道。随后将进行TRAIL-KD CSLC/小鼠神经胶质瘤细胞在无胸腺裸或无胸腺裸小鼠中的研究。 C57 BL/6小鼠脑中的肿瘤细胞，以证实在体外发现的内源性TRAIL的促肿瘤发生作用。（2）将 确定类黄酮槲皮素是否通过以下途径降低人脑胶质瘤CSLC中发动蛋白-1的活化： 抑制钙调磷酸酶，从而降低CSLC中TRAIL-DR的CME并增加TRAIL-凋亡 在体外繁殖时，以及在无胸腺裸鼠脑中体内繁殖时。

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.