Project 1

项目1

基本信息

批准号：
8916609
负责人：
DAN THEODORESCU
金额：
$ 47.73万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-08-23 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8916609
关键词：
Affect Androgens Animal Model Binding Biosensor Cessation of life Characteristics Chimera organism Complex Cyclic AMP-Dependent Protein Kinases Data Analyses Disease Figs - dietary Funding Gene Expression Profile Generations Genes Growth Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Histology Human Hydroxylation Hypoxia Hypoxia Inducible Factor In Vitro Knockout Mice Link Malignant neoplasm of prostate Maps Mass Spectrum Analysis Mediating Membrane Glycoproteins Metastatic Neoplasm to the Bone Metastatic Prostate Cancer Modification Molecular Mus Nature Neoplasm Metastasis Oxygen PTEN gene Pathway interactions Patients Phospholipase D Phosphorylation Phosphorylation Site Phosphotransferases Post-Translational Protein Processing Proline Prostatectomy Radical Prostatectomy Recurrence Response Elements Role Signal Pathway Signal Transduction Site Site-Directed Mutagenesis Testing Therapeutic Tissue Microarray Tissues Transgenic Mice Transgenic Model Translation Initiation Translations Work Xenograft procedure bone cancer imaging clinically relevant clinically significant dehydroxylation deprivation human FRAP1 protein human tissue immunocytochemistry mutant novel prostate cancer cell protein expression response sensor stoichiometry tumor

项目摘要

We found that expression and activity of RalA & RalB are induced by hypoxia and androgen deprivation (AD) in vitro in parallel with CD24 expression. We determined that a hypoxia-Inducible factor 1a (HlF1a) response element in the 5' region ofthe CD24 gene underlies this effect. Since H!F1a expression is necessary for metastasis in prostate cancer (PCa) and its translation is regulated by mTOR, we evaluated HlF1a protein expression and discovered that expression of RalA and RalB Is required for maximal HlF1a response in hypoxia and AD. We also found that, like HlF1a, RalA undergoes proline dehydroxylation during hypoxia and this promotes engagement with its effector, phospholipase D (PLD), a regulator of mTOR. Further, we identified the elF3b translation initiation component that binds to mTOR. as a direct RalB binding partner. Hence propose the hypothesis that in PCa. Ral GTPases are oxygen and androgen biosensors and their activity allows metastatic PCa cells to overcome the natural and therapeutic adversity of hypoxia and AD by maintaining mTOR-mediated translation of HlF1a. Specific Aims will test this hypothesis: Aim 1 will investigate the role of post-translational Ral modifications on HlF1a expression. We will map proline hydroxylation sites in RalA and determine their role in RalA activity and HlF1a expression. We will also evaluate the role of RalB phosphorylation on HIF1a expression, since we have discovered that RalB is activated by phosphorylation at S198 by PKC, a hypoxia activated kinase. In Aim 2 we determine the nature of Ral expression on global and HlF1a translation and assembly and activity of cap-dependent translational complexes in hypoxia and AD. We also examine these characteristics in RalB mutants deficient in interaction with elF3b. Aim 3 will determine the requirement for Ral in PCa using human xenografts and novel transgenic models. Gene expression signatures associated with post-translational modifications of Ral will be evaluated in tumors from patients treated by prostatectomy to determine their ability to predict recurrence. Our project provides a new paradigm by showing Ral GTPases are oxygen and androgen sensors that regulate HlF1a while integrating in the P01 by shared aims with other projects and use of all cores.

我们发现，RalA 和 RalB 的表达和活性在体外由缺氧和雄激素剥夺 (AD) 诱导，与 CD24 表达平行。我们确定 CD24 基因 5' 区域的缺氧诱导因子 1a (HlF1a) 反应元件是这种效应的基础。由于 H!F1a 表达对于前列腺癌 (PCa) 的转移是必需的，并且其翻译受 mTOR 调节，因此我们评估了 HlF1a 蛋白表达，发现 RalA 和 RalB 的表达是缺氧和 AD 中最大 HlF1a 反应所必需的。我们还发现，与 HlF1a 一样，RalA 在缺氧期间经历脯氨酸脱羟基，这促进了与其效应器磷脂酶 D (PLD)（mTOR 的调节剂）的结合。此外，我们还鉴定了与 mTOR 结合的 elF3b 翻译起始组件。作为直接 RalB 结合伙伴。因此提出PCa中的假设。 Ral GTPases 是氧和雄激素生物传感器，其活性使转移性 PCa 细胞能够通过维持 mTOR 介导的 HlF1a 翻译来克服缺氧和 AD 的自然和治疗逆境。具体目标将检验这一假设：目标 1 将研究翻译后 Ral 修饰对 HlF1a 表达的作用。我们将绘制 RalA 中脯氨酸羟基化位点的图谱，并确定它们在 RalA 活性和 HlF1a 表达中的作用。我们还将评估 RalB 磷酸化对 HIF1a 表达的作用，因为我们发现 RalB 是通过 PKC（一种缺氧激活激酶）在 S198 处磷酸化而激活的。在目标 2 中，我们确定了缺氧和 AD 中 Ral 表达对全局和 HlF1a 翻译和装配以及帽依赖性翻译复合物活性的性质。我们还检查了与 elF3b 相互作用缺陷的 RalB 突变体的这些特征。目标 3 将使用人类异种移植物和新型转基因模型确定 PCa 中对 Ral 的需求。将在接受前列腺切除术治疗的患者的肿瘤中评估与 Ral 翻译后修饰相关的基因表达特征，以确定其预测复发的能力。我们的项目提供了一个新的范例，表明 Ral GTPases 是调节 HlF1a 的氧和雄激素传感器，同时通过与其他项目的共同目标和使用所有核心来集成到 P01 中。