Project 1

我们发现RalA和RalB的表达和活性在体外受缺氧和雄激素剥夺（AD）诱导的同时也受CD24表达的影响。我们确定CD24基因5'区域的缺氧诱导因子1a （HlF1a）反应元件是这种作用的基础。因为H !F1a的表达是前列腺癌（PCa）转移所必需的，其翻译受mTOR的调节，我们评估了HlF1a蛋白的表达，发现RalA和RalB的表达是缺氧和AD中最大的HlF1a反应所必需的。我们还发现，像HlF1a一样，RalA在缺氧时经历脯氨酸去羟基化，这促进了与其效应物磷脂酶D （PLD）的结合，PLD是mTOR的调节剂。此外，我们确定了与mTOR结合的elF3b翻译起始成分。作为直接的RalB结合伙伴。因此提出假设，在PCa。rgtpase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。具体目的将验证这一假设：目的1将研究翻译后Ral修饰对HlF1a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是由PKC（一种缺氧激活的激酶）在S198位点磷酸化激活的。在Aim 2中，我们确定了在缺氧和AD中，Ral在全局和HlF1a翻译中的表达以及帽依赖翻译复合物的组装和活性的性质。我们还研究了缺乏与elF3b相互作用的RalB突变体的这些特征。目的3将确定使用人类异种移植物和新型转基因模型的PCa对Ral的需求。与翻译后修饰相关的基因表达特征将在前列腺切除术患者的肿瘤中进行评估，以确定其预测复发的能力。我们的项目提供了一个新的范例，通过与其他项目共享目标和使用所有核心，显示Ral GTPases是调节HlF1a的氧和雄激素传感器，同时集成在P01中。

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.