Project 1

项目1

• 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
• 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

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，体外低氧和雄激素剥夺(AD)诱导的Rala和RalB的表达和活性与CD24的表达平行。我们确定CD24基因5‘端的低氧诱导因子1a(H1F1a)反应元件是这种作用的基础。由于H！F1a的表达是前列腺癌转移所必需的，而且它的翻译受mTOR的调节，我们对HlF1a蛋白的表达进行了研究，发现在低氧和AD中，Rala和RalB的表达是最大的HlF1a反应所必需的。我们还发现，与HlF1a一样，Rala在缺氧时经历了脯氨酸脱羟化，这促进了与其效应因子磷脂酶D(PLD)的结合，磷脂酶D是mTOR的调节因子。此外，我们还鉴定了与mTOR结合的elF3b翻译起始组件。作为RalB的直接结合伙伴。因此提出假设，在主成分分析中。Ral GTP酶是氧和雄激素生物传感器，其活性允许转移性PCA细胞通过维持mTOR介导的HlF1a翻译来克服缺氧和AD的自然和治疗逆境。特定的AIMS将检验这一假设：Aim 1将调查翻译后修饰对HlF1a表达的作用。我们将定位Rala中的Pro羟基化位点，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，因为我们已经发现RalB通过S198处的PKC(一种低氧激活的激酶)的磷酸化而被激活。在目标2中，我们确定了在整体和H1F1a翻译和组装上Ral的表达的性质，以及在缺氧和AD中帽子依赖的翻译复合体的活性。我们还研究了与elF3b相互作用不足的RalB突变体的这些特征。目的3将确定在使用人类异种移植和新的转基因模型的PCa中对ral的需求。将在接受前列腺切除术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，以确定他们预测复发的能力。我们的项目提供了一种新的范式，表明Ral GTP酶是氧和雄激素传感器，调节HlF1a，同时通过与其他项目的共同目标和所有核心的使用整合到P01中。