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

胶质瘤干细胞内源合成 TRAIL 及对 TRAIL 治疗的耐药性

• 批准号: 10863308
• 负责人: Candece L Gladson
• 金额: $ 47.8万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-18 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10863308
• 关键词: Apoptosis; Apoptotic; Biological Assay; Biopsy; Brain; C57BL/6 Mouse; Calcineurin; Calcineurin inhibitor; Calmodulin; Cell Proliferation; Cell Survival; Cell surface; Cells; Cellular Indexing of Transcriptomes and Epitopes by Sequencing; Clathrin; Clinical Trials; Data; Dissection; Down-Regulation; Dynamin I; Endocytosis; Enzyme-Linked Immunosorbent Assay; Event; FK506; Failure; Flavonoids; Flow Cytometry; Gene Expression; Genes; Glioblastoma; Glioma; Guanosine Triphosphate Phosphohydrolases; Human; Immunophenotyping; In Situ Hybridization; In Vitro; Induction of Apoptosis; Inflammation; Inhibition of Apoptosis; Inhibition of Cell Proliferation; Injections; Integral Membrane Protein; Invaded; Kinetics; Knock-out; Label; Ligands; Literature; Luciferases; Malignant Glioma; Malignant Neoplasms; Mediating; Membrane; Messenger RNA; Mus; Natural Compound; Necrosis; Nude Mice; PPP3CA gene; Plasma; Proliferating; Protein Dephosphorylation; Protein phosphatase; Proteins; Quantitative Reverse Transcriptase PCR; Quercetin; Reporting; Research Personnel; Resistance; Role; Sampling; Signal Pathway; Signal Transduction; TNFRSF10A gene; TNFRSF10B gene; Technology; Testing; Xenograft procedure; blood-brain barrier crossing; cancer cell; cancer stem cell; chemokine; coated pit; cytokine; genetic signature; in vitro testing; in vivo; in vivo evaluation; inhibitor; knock-down; migration; mouse model; overexpression; pharmacologic; resistance mechanism; stem-like cell; transcriptome sequencing; transcriptomics; tumor; tumorigenic

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.

项目摘要/摘要 我们的初步数据表明，人神经胶质瘤干细胞（CSLC）合成并分泌 肿瘤坏死相关 - 凋亡诱导配体（TRAIL）。与此一致，其他调查人员也 在胶质母细胞瘤（GBM）活检中表现出内源性轨道表达。尽管合成了 内源性拖网CSLC继续增殖，表明内源性轨迹是在肿瘤的信号 功能。我们发现内源性轨迹的下调抑制了CSLC中的增殖，这表明 步道可能会发出增殖的信号。文献中的报告支持这一点 癌细胞的增殖，迁移和炎症。导轨失败的一种潜在机制 临床试验是，TRAIL-DRS是隔离的细胞内有效降低轨道凋亡信号传导的 在细胞表面启动。在非脑癌细胞中，网格蛋白介导的TRAIL-DR的内吞作用（CME） 抑制跟踪诱导的凋亡。 TRAIL-DR的CME由激活的Dynamin-1诱导，这是一个大的GTPase 促进膜分裂。在非脑癌细胞中，最近显示Dynamin-1被激活 钙调神经酶 - 一种蛋白质磷酸酶的去磷酸化。为了抑制GBM中的Dynamin-1激活 并阻止TRAIL-DR的CME，以及由此产生的凋亡抑制作用，我们搜索了自然 抑制Dynamin-1或上游激活剂 - 钙调蛋白的化合物，发现槲皮素（一种天然 类黄酮）直接抑制钙调蛋白。与槲皮素抑制钙调神经酶一致，从而抑制 dynamin-1的初步数据表明，槲皮素和Dynamin-1的抑制剂，使神经胶质瘤CSLC敏感 进行凋亡。我们假设：（1）一种未探索GBM电阻的机制 trail的肿瘤是内源性轨道的合成/表达，可促进促肿瘤 CSLC的功能； （2）GBM中增加的Dynamin-1蛋白会增加水平 激活的Dynamin-1促进trail-DR的CME抑制trail-凋亡，该槲皮素 抑制钙调蛋白（钙调神经酶激活dynamin-1）将使CSLC敏感到trail-凋亡。目的 1）将确定CSLC和小鼠中内源性拖网的敲除（KO）还是下调 神经胶质瘤细胞改变细胞的增殖，迁移，趋化因子/细胞因子分泌或对踪迹诱导的敏感性 凋亡，并将解剖必要的信号通路，以实现内源性的效果效应 踪迹。紧随 C57BL/6小鼠大脑确认体外发现的内源性trail型促肿瘤作用。目标2）会 确定类黄酮槲皮素是否通过人神经胶质瘤CSLC中的dynamin-1激活 抑制钙调神经酶，从而减少了TRAIL-DR的CME并增加CSLC中的Trail-凋亡 在体外传播，并在无胸腺裸鼠大脑中体内传播时。