Role of ubiquitylation in renal cancer

描述(申请人提供)：肾癌占美国成人恶性肿瘤的3%，是癌症死亡的第六大原因。在这一应用中，我们建议研究导致最常见的肾癌形式--肾透明细胞癌(RCC)的致癌过程的分子基础。肾细胞癌的发生与von Hippel-Lindau肿瘤抑制蛋白(PVHL)功能丧失之间存在着明确的联系。已知，这种致癌效应可以通过干扰HIFA泛素化来介导。然而，在这一应用中，我们将扩展我们之前的工作，即在肾癌的发展中涉及一种新的pVHL依赖的信号通路：RNA聚合酶II(RNAPII)大亚基Rpb1的羟化，以及随后的pVHL肿瘤抑制复合体的泛素化。我们的工作假设是，P1465羟化和依赖pVHL的Rpb1泛素化提供了RNAPII复合体转录活性的微调，这导致了基因表达模式，促进了对环境变化(如氧化应激)的适当反应。我们认为，失去这种微调会导致RNAPII功能障碍，从而导致肿瘤发生。这项工作的总体目标是了解pVHL依赖的Rpb1羟化和泛素化的机制，并评估这一过程的中断在肿瘤发生中所起的作用。为了实现这一目标，我们将1)鉴定泛素和Rpb1中的赖氨酸，它们参与了P1465依赖的氧化应激反应中依赖pVHL的Rpb1多泛素化；2)鉴定参与Rpb1羟化的Pro羟基酶；3)确定未经历pVHL依赖羟化和泛素化的Rpb1突变体的致癌特性；以及4)评估与正常肾组织相比，人肾细胞癌中Rpb1羟化和泛素化的状态。对于肾癌患者，如果他们的癌症在早期被诊断出来，他们的预后要好得多；然而，在RCC中，早期诊断是不常见的，因为患者通常直到疾病晚期才会出现症状。由于这项研究探索了疾病发展的一种新的生化机制，它可以通过两种方式改善肾癌患者的预后：1)通过促进治疗肾癌的新药的开发；2)通过识别新的遗传标记，可以用来识别发生肾癌的风险个体。

DESCRIPTION (provided by applicant): Renal cancer accounts for 3% of adult malignancies in the US, and is the 6th leading cause of cancer mortality. In this application, we propose to examine the molecular basis of the oncogenic process leading to the most common form of kidney cancer, renal clear cell carcinoma (RCC). There is a well established link between RCC oncogenesis and the loss of function of the von Hippel-Lindau tumor suppressor protein (pVHL). It is known that this oncogenic effect can be mediated through disruption of HIFa ubiquitylation. However, in this application, we will expand on our previous work implicating a novel pVHL-dependent signaling pathway in the development of RCC: the hydroxylation of the large subunit of RNA Polymerase II (RNAPII), Rpb1, and its subsequent ubiquitylation by the pVHL tumor suppressor complex. Our working hypothesis is that P1465 hydroxylation and pVHL-dependent ubiquitylation of Rpb1 provide fine tuning of the RNAPII complex's transcriptional activity, which results in patterns of gene expression that facilitate appropriate responses to environmental changes (such as oxidative stress). We propose that loss of that fine-tuning results in dysfunctional activity of RNAPII leading to oncogenesis. The overall goal of this work is to understand the mechanism of pVHL-dependent Rpb1 hydroxylation and ubiquitylation, and to assess the role that disruption of this process plays in oncogenesis. To achieve this, we will 1) identify the lysines within ubiquitin and Rpb1 that participate in pVHL-dependent polyubiquitylation of Rpb1 in a P1465-dependent manner in response to oxidative stress; 2) identify the prolyl hydroxylases involved in hydroxylation of Rpb1; 3) determine the oncogenic properties of Rpb1 mutants that do not undergo pVHL-dependent hydroxylation and ubiquitylation; and 4) assess the status of Rpb1 hydroxylation and ubiquitylation in human RCC tumors as compared to normal kidney tissue. The prognosis for patients with RCC is much better if their cancer is diagnosed in early stages; however, early diagnosis is uncommon in RCC because patients often do not experience symptoms until advanced stages of the disease. Because this research explores a novel biochemical mechanism for disease development, it could improve the prognosis for RCC patients in two ways: 1) by contributing to the development of new drugs to treat RCC, and 2) by identifying new genetic markers that could be used to identify individuals at risk for developing RCC.