REGULATION OF TUMOR SUPPRESSION BY ARF

 描述(由申请人提供)：ARF通过p53依赖和非独立的途径防止肿瘤发生。ARF通过负调控MDM2对癌基因的反应来激活p53的能力已经得到了很好的研究。然而，最近对ARF的P53非依赖功能的分析揭示了许多细胞过程中涉及的似乎不同数量的蛋白质靶点。为了克服这一限制，我们试图在功能上连接ARF的几个不依赖于p53的靶点。我们已经证明，ARF限制了miRNA DROSHA-DDX5处理复合体的活性，以防止细胞转化，并且我们已经确定，在双链RNA传感增强的情况下，ARF抑制先天性免疫信号，以抑制增强的肿瘤发生。我们假设ARF的p53非依赖性靶点包含一个可用于抑制肿瘤形成的共性。我们处于解决这一问题的理想位置，在ARF领域拥有丰富的经验，并将这些知识应用于癌症生物学研究。我们已经建立的记录包括ARF识别MDM2核仁隔离以激活P53，发现ARF的P53不依赖的功能，以及靶向NPM，DDX5，DROSHA，和干扰素-β作为ARF的P53不依赖的靶标。我们将确定看似独特的ARF靶点如何通过胞质miRNA或双链RNA传感机制刺激干扰素-β的产生。我们还将确定干扰素-β的激活是否是胞质RNA普遍增加的结果，并确定胞质RNA积聚的机制。在缺乏ARF和P53的情况下，识别负责产生干扰素-β的特定miRNA或累积的胞质RNA将是确定增强肿瘤发生机制的关键。我们将识别ISGylated的蛋白质以及它们是如何驱动肿瘤发生的。在这种背景下，ISG15修饰蛋白在肿瘤形成中的发现是完全新颖的，并将表明该途径在肿瘤细胞生物学中的重要作用。我们将确定哪些ARF靶点可以作为人类乳腺癌ARF/P53功能丧失的有效生物标志物，以及其中是否有任何一个可以预测患者的生存。对同一人类乳腺癌中的arf/p53、其靶点和干扰素-β途径成分的全面分析将为研究界提供第一个急需的预后工具，以预测这一新的抑制者和驱动因素网络。最后，我们将使用JAK1/2抑制剂在体外抑制已建立的和同基因的人类癌细胞系以及体内自发和异种移植的肿瘤的发生。这些临床前研究有可能测试JAK1/2抑制剂 在我们已经表现出对该途径的遗传依赖性的环境中。此外，我们的检测还将能够识别可能对JAK1/2抑制提供抗性的互补途径。

 DESCRIPTION (provided by applicant): ARF prevents tumorigenesis through p53-dependent and -independent pathways. ARF's ability to activate p53 by negatively regulating MDM2 in response to oncogenes has been well studied. However, recent analysis of ARF's p53-independent functions has uncovered a seemingly disparate number of protein targets involved in numerous cellular processes. To overcome this limitation, we have sought to functionally link several of the p53-independent targets of ARF. We have shown that ARF limits the activity of the miRNA Drosha-DDX5 processing complex to prevent cellular transformation and we have established that ARF inhibits innate immune signaling in the face of increased double-stranded RNA sensing to suppress enhanced tumorigenesis. We hypothesize that ARF's p53-independent targets contain a commonality that can be used to inhibit tumorigenesis. We are ideally positioned to solve this problem, having extensive experience in both the ARF field and in applying this knowledge to the study of cancer biology. Our established track record involves the identification of Mdm2 nucleolar sequestration by ARF to activate p53, the discovery of the p53-independent function of ARF, and the targeting of NPM, DDX5, Drosha, and IFN-β as p53-independent targets of ARF. We will determine how seemingly unique ARF targets act to stimulate IFN-β production through a cytosolic miRNA or double-stranded RNA sensing mechanism. We will also establish whether IFN-β activation is a result of a more generalized increase in cytosolic RNAs and determine the mechanism for cytosolic RNA accumulation. The identification of specific miRNAs or accumulated cytosolic RNAs responsible for IFN-β production will be key in determining the mechanism of enhanced tumorigenesis in the absence of ARF and p53. We will identify proteins that are ISGylated and how they drive tumorigenesis. The discovery of ISG15-modified proteins in tumor formation is completely novel in this context and would indicate a prominent role for this pathway in tumor cell biology. We will establish which ARF targets serve as valid biomarkers of ARF/p53 loss of function in human breast cancer and whether any of these predict patient survival. A comprehensive analysis of ARF/p53, its targets, and IFN-β pathway components within the same human breast cancers will provide the research community with the first much-needed prognostic tool for this novel network of suppressors and drivers. Finally, we will use JAK1/2 inhibitors to inhibit the tumorigenesis of established and isogenic human cancer cell lines in vitro as well as spontaneous and xenografted in vivo tumors. These pre-clinical studies have the potential to test JAK1/2 inhibitors in a setting where we have shown a genetic dependence on the pathway. Additionally, our assays will also be able to identify complimentary pathways that might provide resistance to JAK1/2 inhibition.