Project 1

项目1

• 批准号: 8744370
• 负责人: DAN THEODORESCU
• 金额: $ 45.23万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-23 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8744370
• 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; Image; 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; clinically relevant; clinically significant; dehydroxylation; deprivation; human FRAP1 protein; human tissue; immunocytochemistry; mutant; novel; prostate cancer cell; protein expression; response; sensor; stoichiometry; tumor

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CD 24的表达平行。我们确定，在CD 24基因5'区的缺氧诱导因子1a（HlF 1a）反应元件是这种效应的基础。自从H！F1 a的表达是前列腺癌（PCa）转移所必需的，其翻译受mTOR调节，我们评估了H1 F1 a蛋白的表达，发现RalA和RalB的表达是缺氧和AD中最大H1 F1 a反应所必需的。我们还发现，与HIF 1a一样，RalA在缺氧期间经历脯氨酸脱羟基化，这促进与其效应物磷脂酶D（PLD）（mTOR的调节剂）的接合。此外，我们鉴定了与mTOR结合的eIF 3b翻译起始组分。作为RalB的直接结合伴侣。因此，提出假设，在PCa。Ral GTP酶是氧和雄激素生物传感器，并且它们的活性允许转移性PCa细胞通过维持mTOR介导的HIFla翻译来克服缺氧和AD的自然和治疗逆境。具体目的将检验这一假设：目的1将研究翻译后Ral修饰对HIF 1a表达的作用。我们将绘制RalA中的脯氨酸羟基化位点，并确定它们在RalA活性和HIF 1a表达中的作用。我们还将评估RalB磷酸化对HIF 1a表达的作用，因为我们已经发现RalB通过PKC（一种缺氧激活的激酶）在S198处磷酸化而被激活。在目的2中，我们确定Ral在缺氧和AD中对全局和HIF 1a翻译和组装的表达的性质以及帽依赖性翻译复合物的活性。我们还研究了RalB突变体与eIF 3b相互作用缺陷的这些特征。目的3将使用人异种移植物和新的转基因模型来确定PCa中对Ral的需求。与Ral的翻译后修饰相关的基因表达特征将在来自通过直肠切除术治疗的患者的肿瘤中进行评估，以确定其预测复发的能力。我们的项目通过显示Ral GTP酶是氧和雄激素传感器提供了一种新的范例，其调节HIFla，同时通过与其他项目的共同目标和使用所有核心整合在POl中。