权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Targeting pancreatic cancer's metabolic addiction to HuR

针对胰腺癌对 HuR 的代谢成瘾

基本信息

批准号：
10611147
负责人：
Jordan M Winter
金额：
$ 43.17万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10611147
关键词：
3&apos Untranslated Regions Acute Antioxidants Binding Binding Proteins Binding Sites Biological Assay Biology CRISPR/Cas technology Cancer Etiology Cell Culture Techniques Cell Line Cell Nucleus Cell Survival Cells Cellular biology Cessation of life Chemistry Chemoresistance Clinical Data Clustered Regularly Interspaced Short Palindromic Repeats Computational Biology Culture Media Cytoplasm Dependence Diabetic mouse Elements Engraftment Enzymes Exhibits Fractionation Genes Genetic Glucose Glutamine Homeostasis Hour Hyperglycemia Hyperglycemic Mice Impairment Isocitrates Knock-out Knockout Mice Lead Maintenance Malignant Neoplasms Malignant neoplasm of pancreas Mediator Medical Messenger RNA Metabolic Metabolic stress Metabolism Mitochondria Modeling Molecular Molecular Biology Techniques Mus Mutation NADP Normal tissue morphology Nude Mice Nutrient Oxidation-Reduction Oxygen Consumption Pancreatic Ductal Adenocarcinoma Pathology Performance Pharmaceutical Chemistry Pharmaceutical Preparations Predisposition Property Proteins Publishing RNA Binding RNA Interference RNA Stability RNA-Binding Proteins Reactive Oxygen Species Refractory Regulation Research Resistance Role Streptozocin Stress Structure Testing Therapeutic Transcript Transfection Translations United States Withdrawal Work Xenograft procedure addiction alpha ketoglutarate alternative treatment antioxidant enzyme arm cancer cell chemotherapy cytotoxic drug testing gemcitabine improved in vivo inhibitor insight lead optimization mRNA Expression mortality mouse model mutant new therapeutic target novel novel therapeutics overexpression pancreatic cancer cells pancreatic ductal adenocarcinoma cell pancreatic ductal adenocarcinoma model posttranscriptional programs protein expression response statistics therapeutic target transcriptome transcriptome sequencing tumor tumor microenvironment tumorigenesis

项目摘要

Pancreatic ductal adenocarcinoma (PDA) is the 3rd leading cause of cancer death in the U.S. and is generally refractory to chemotherapy. We discovered that the harsh PDA microenvironment primes cancer cells against additional cytotoxic insults (e.g., chemotherapy) and promotes PDA aggressiveness. A better understanding of the molecular underpinnings behind this adaptive program would expose PDAs metabolic vulnerabilities. We identified the RNA binding protein, HuR (ELAVL1), as a major player in the acute pro-survival response. Upon stress, HuR translocates from the nucleus to the cytoplasm with key survival transcripts, like IDH1 (an NADPH generating enzyme). HuR enhances RNA stability and protein translation of target mRNAs, to rapidly adjust the transcriptome in response to stress. Our research highlights two metabolic processes in the HuR adaptive program: a) antioxidant defense and b) mitochondrial performance. HuR silencing in PDA cells produced excessive ROS and NADPH depletion under low glucose or chemotherapy stress. An unbiased RNA seq analysis of antioxidant genes in HuR deficient cells identified IDH1 as the leading antioxidant enzyme under HuR control. RNA binding and RNA stability assays showed that HuR regulates IDH1 post-transcriptionally, and HuR deficient PDA cells had markedly reduced IDH1 mRNA and protein expression. HuR-deficient cells failed to engraft in nude mice, while IDH1 overexpression rescued PDA engraftment. HuR-deficient cells also had dysfunctional mitochondria, reflected by reduced oxygen consumption, ATP, and mitochondrial abundance. Based on this body of work, we hypothesize that PDAs reliance on HuR under low nutrient conditions exposes new therapeutic opportunities. In Aim 1, we establish the survival impact of the HuR- IDH1 axis, by editing out HuR binding sites (CRISPR) in the IDH1 3’UTR. We generated a conditional IDH1 knockout mouse, and will cross it with an established PDA model to validate IDH1 as a therapeutic target. We will test an allosteric modulator of mutant IDH1 (GSK-321) as a novel wild type IDH1 inhibitor in PDA, and launch hit-to-lead optimization to improve drug properties. In Aim 2, we identify specific aspects of mitochondrial biology under HuR control through studies of mitochondrial structure and function in HuR- deficient PDA cells. The importance of the HuR-IDH1 axis on mitochondrial ROS levels will be demonstrated. Additional transcripts will impact HuR’s regulation of mitochondrial performance will be identified. A novel mitochondrial inhibitor, CPI-613, will be combined with HuR or IDH1 inhibition as a new synthetic lethal approach against PDAs adaptive metabolic program. In Aim 3, we will use a diabetic mouse model to show that a hyperglycemic state suppresses the HuR pro-survival network, and sensitizes PDA to chemotherapy. Successful engraftment of HuR-deficient cells in hyperglycemic mice would suggest that HuR is less important under these conditions. Our studies of HuR biology will improve understanding of PDA metabolic tendencies, and reveal therapeutic opportunities relevant to PDAs nutrient deprived microenvironment.

胰腺导管腺癌 (PDA) 是美国癌症死亡的第三大原因，通常是对化疗耐药。我们发现恶劣的 PDA 微环境会促使癌细胞抵抗额外的细胞毒性损伤（例如化疗）并促进 PDA 侵袭性。更好地理解这种适应性程序背后的分子基础将暴露 PDA 的代谢脆弱性。我们确定 RNA 结合蛋白 HuR (ELAVL1) 是急性促生存反应的主要参与者。之上应激时，HuR 会从细胞核转移到细胞质，并携带关键的生存转录本，如 IDH1（一种 NADPH）产生酶）。 HuR 增强目标 mRNA 的 RNA 稳定性和蛋白质翻译，以快速调节应激反应的转录组。我们的研究强调了 HuR 适应性中的两个代谢过程计划：a) 抗氧化防御和 b) 线粒体性能。 PDA 细胞中 HuR 沉默低血糖或化疗应激下 ROS 和 NADPH 过度消耗。无偏见的 RNA 测序对 HuR 缺陷细胞中抗氧化基因的分析确定 IDH1 是主要的抗氧化酶胡尔控制。 RNA 结合和 RNA 稳定性测定表明 HuR 在转录后调节 IDH1， HuR缺陷的PDA细胞的IDH1 mRNA和蛋白表达显着降低。 HuR缺陷细胞未能在裸鼠中植入，而 IDH1 过表达可挽救 PDA 植入。 HuR 缺陷细胞也线粒体功能障碍，表现为耗氧量、ATP 和线粒体减少丰富。基于这项工作，我们假设 PDA 在低营养条件下依赖 HuR 条件暴露了新的治疗机会。在目标 1 中，我们确定了 HuR- 的生存影响 IDH1 轴，通过编辑 IDH1 3’UTR 中的 HuR 结合位点 (CRISPR)。我们生成了一个条件 IDH1 敲除小鼠，并将其与已建立的 PDA 模型进行杂交，以验证 IDH1 作为治疗靶点。我们将在 PDA 中测试突变 IDH1 (GSK-321) 的变构调节剂作为新型野生型 IDH1 抑制剂，以及启动从命中到先导的优化以改善药物特性。在目标 2 中，我们确定了以下具体方面通过研究 HuR 中线粒体的结构和功能，了解 HuR 控制下的线粒体生物学 PDA 细胞缺陷。 HuR-IDH1 轴对线粒体 ROS 水平的重要性将得到证明。将确定影响 HuR 对线粒体性能的调节的其他转录本。一本小说线粒体抑制剂 CPI-613 将与 HuR 或 IDH1 抑制结合作为新的合成致死剂针对 PDA 适应性代谢程序的方法。在目标 3 中，我们将使用糖尿病小鼠模型来展示高血糖状态会抑制 HuR 促生存网络，并使 PDA 对化疗敏感。 HuR 缺陷细胞在高血糖小鼠中的成功植入表明 HuR 不太重要在这些条件下。我们对 HuR 生物学的研究将增进对 PDA 代谢倾向的理解，并揭示与 PDA 营养匮乏的微环境相关的治疗机会。