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，人脑胶质瘤肿瘤干细胞(CSLCs)能够合成和分泌 肿瘤坏死相关凋亡诱导配体(TRAIL)。与此一致的是，其他调查人员已经 在胶质母细胞瘤(GBM)活检组织中发现内源性TRAIL的表达。尽管合成了 内源性TRAIL CSLCs继续增殖，提示内源性TRAIL是促肿瘤信号 函数(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。TRAIL可以发出信号的文献中的报告支持了这一点 癌细胞的增殖、迁移和炎症。TRAIL失败的一个潜在机制 临床试验表明，TRAIL-DR被隔离在细胞内，有效地减少了TRAIL-凋亡信号 在细胞表面启动。在非脑癌细胞中，TRAIL-DR的网状蛋白介导的内吞作用 抑制TRAIL诱导的细胞凋亡。TRAIL-DR的CME是由激活的Dynamin-1诱导的，Dynamin-1是一种大的GTP酶， 促进膜的断裂。在非脑癌细胞中，动力蛋白-1最近被证明是由 钙调神经磷酸酶--一种蛋白磷酸酶去磷酸化。为了抑制GBM中Dynamin-1的激活 并阻断TRAIL-DR的CME，从而抑制细胞凋亡，我们寻找一种天然的 一种抑制动力蛋白-1或上游激活剂-钙调神经磷酸酶的化合物，并发现槲皮素(一种天然的 黄酮类)直接抑制钙调神经磷酸酶。与Quercetin抑制钙调神经磷酸酶一致，从而抑制 对于Dynamin-1，我们的初步数据显示，栎素和Dynamin-1的抑制剂可以使胶质瘤CSLCs增敏 到TRAIL-细胞凋亡。我们的假设是：(1)一种尚未探索的GBM抗性机制 肿瘤至TRAIL是内源性TRAIL的合成/表达，促进促肿瘤发生 CSLCs的功能；以及(2)GBM中Dynamin-1蛋白的增加导致 激活的Dynamin-1促进TRAIL-DR的CME，抑制TRAIL-凋亡，以及槲皮素 抑制钙调神经磷酸酶(钙调神经磷酸酶激活Dynamin-1)将使CSLCs对TRAIL-凋亡敏感。目标 1)将确定CSLCs和小鼠内源性TRAIL的敲除(KO)或下调 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诱导的敏感性 细胞凋亡，并将剖析必要的信号通路(S)的促肿瘤作用的内源性- 小路。随后将对TRAIL-KD CSLCs/小鼠胶质瘤细胞在裸鼠裸鼠体内进行研究 体外发现内源性TRAIL对C57BL/6小鼠脑内的促肿瘤作用(S)。目标2)意志 黄酮类槲皮素是否通过以下途径降低人脑胶质瘤CSLCs中Dynamin-1的活性 钙调神经磷酸酶抑制CSLCs TRAIL-DR的CME，增加TRAIL-凋亡 在体外繁殖，当在体内繁殖时，在裸鼠脑中繁殖。