Cholesterol Homeostasis in Malignant Glioma

恶性胶质瘤中的胆固醇稳态

基本信息

批准号：
8586908
负责人：
Elizabeth Anne Kolar
金额：
$ 4.27万
依托单位：
HUGO W. MOSER RES INST KENNEDY KRIEGER
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-12-01 至 2014-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8586908
关键词：
1-Phosphatidylinositol 3-Kinase Accounting Acyl Coenzyme A Adult Affect Apoptosis Behavior Binding Proteins Biological Brain Neoplasms Cell Line Cell Nucleus Cell membrane Cells Child Cholesterol Cholesterol Homeostasis Coenzyme A Coenzyme A Ligases Defect Doxycycline Enzymes Fatty Acids Gene Targeting Genetic Transcription Glioma Growth Human Hydroxymethylglutaryl-CoA reductase Immunofluorescence Immunologic In Vitro Intracranial Neoplasms Knowledge LDL Cholesterol Lipoproteins Lead Life Lipids Low Density Lipoprotein Receptor Low-Density Lipoproteins Malignant - descriptor Malignant Glioma Malignant Neoplasms Measures Membrane Membrane Microdomains Microarray Analysis Morphology Mus Neuroglia Nude Mice Pathway interactions Phospholipids Plasma Property Proteins Receptor Protein-Tyrosine Kinases Reporter Research Response Elements Role SCAP protein Signal Transduction Signal Transduction Pathway Source Sphingolipids Steroids Sterols Structure System Therapeutic Tissues Transcript Very Long Chain Fatty Acid Western Blotting Xenograft procedure aggressive therapy cancer cell cell growth cellular imaging fatty acid metabolism glioma cell line inhibitor/antagonist knock-down lipid metabolism meetings membrane synthesis neoplastic cell novel therapeutics outcome forecast research study small hairpin RNA subcutaneous transcription factor tumor tumorigenic uptake vector

项目摘要

The Watkins Lab has observed that when U87 human malignant glioma cells are deficient for a particular acyl- CoA synthetase, ACSVL3, the cells had significantly lower cholesterol levels than did wild-type U87 cells. Depleting U87 cells of ACSVL3 decreased their malignant growth properties in vitro and decreased their tumorigencity in mice. Because there is no known role for any acyl-CoA synthetase in cholesterol metabolism, exactly how ACSVL3 affects cholesterol homeostasis and how this in turn affects malignancy is unknown. I hypothesize that depletion of ACSVL3 affects the sterol response element binding proteins (SREBPs) that control the transcription of genes important in cholesterol homeostasis and fatty acid synthesis, and the lack of cholesterol disrupts lipid raft formation and cellular signaling from these rafts. To answer this, I propose to 1) determine the effect of ACSVL3 depletion on cholesterol levels in human glioma cells, 2) determine how ACSVL3 regulates cholesterol homeostasis in cancer cells, and 3) determine how ACSVL3 depletion affects lipid raft formation and signaling. I plan on accomplishing the first aim by measuring intracellular cholesterol levels, and see how the growth and the tumorigenic properties are affected by repleting or depleting the cells of cholesterol. In the second aim, I use qRT-PCR to observe changes in transcription levels of SREBP target genes in the control U87 cells and compare them to ACSVL-deficient cells. I will also assess translocation of SREBPs to the nucleus and the potential involvement of SCAP and Insig proteins. Aim 3 will be accomplished by using fluorescent cholesterol markers to observe the changes in lipid raft morphology and Western Blotting and immunofluorescence will allow me to look at differences in c-Met levels and raft marker proteins. Reporter constructs will be used in live cell imaging studies to assess effects on signaling via c-Met, PI3 kinase, and Akt. This research may lead to new therapeutic strategies for human cancers and further our understanding of the role of lipid metabolism in malignancy.

沃特金斯实验室（Watkins Lab）观察到，当U87人类恶性神经胶质瘤细胞缺乏特定的酰基-COA合成酶ACSVL3时，这些细胞的胆固醇水平明显低于野生型U87细胞。 ACSVL3的U87细胞耗尽，在体外降低了其恶性生长特性，并降低了小鼠的肿瘤性。由于在胆固醇代谢中，任何酰基辅酶A合成酶都没有已知的作用，因此ACSVL3如何影响胆固醇稳态以及这种情况如何影响恶性肿瘤。我假设ACSVL3的耗竭会影响固醇反应元件结合蛋白（SREBPS），该蛋白控制了在胆固醇稳态和脂肪酸合成中重要的基因的转录，并且缺乏胆固醇破坏了这些系列的脂质筏的形成和细胞信号。要回答这一点，我建议1）确定ACSVL3耗竭对人胶质瘤细胞中胆固醇水平的影响，2）确定ACSVL3如何调节癌细胞中的胆固醇稳态，以及3）确定ACSVL3耗竭如何影响脂质筏的形成和信号。我计划通过测量细胞内胆固醇水平来实现第一个目标，并查看生长和肿瘤特性如何受到补充或耗尽胆固醇细胞的影响。在第二个目标中，我使用QRT-PCR观察对照U87细胞中SREBP靶基因转录水平的变化，并将其与ACSVL缺陷型细胞进行比较。我还将评估SREBPS向细胞核的易位以及SCAP和Insig蛋白的潜在参与。 AIM 3将通过使用荧光胆固醇标记来观察脂质筏形态的变化，蛋白质印迹和免疫荧光将使我能够研究C-MET水平和RAFT标记蛋白的差异。记者构建体将用于实时细胞成像研究中，以评估通过C-MET，PI3激酶和AKT对信号的影响。这项研究可能导致针对人类癌症的新治疗策略，并进一步了解我们对脂质代谢在恶性肿瘤中的作用的理解。