Mechanisms of Cell Cycle Associated Neoplasia

描述(由申请人提供)：调节细胞周期的基因突变是肿瘤发生的基本机制。这些突变涉及固有的细胞周期成分本身(细胞周期蛋白、CDK抑制剂)以及影响细胞周期机制的癌基因(如c-Myc)和肿瘤抑制基因(如P53、Fbw7)。这项建议的重点是这些癌症相关细胞周期通路的功能和调控。 Fbw7肿瘤抑制因子以细胞周期蛋白E、Notch和c-jun为靶点，在它们被磷酸化后进行降解。在初步研究中，我们发现Fbw7还调节依赖于磷酸化的c-Myc的周转，并且Fbw7的三种亚型(Fbw7pha、Fbw7beta、Fbw7Gamma)表现出独特的亚细胞定位。在目标1中，我们将通过在小鼠中开发Fbw7pha和Fbw7Gamma的条件零突变来测试Fbw7亚型在这些隔室中执行不同生物功能的假设。我们将研究每种异构体在调节特定Fbw7底物中的作用，并检验Fbw7Gamma调节核仁中c-Myc功能的假设。在目标2中，我们将确定Fbw7pha或Fbw7Gamma是否是肿瘤抑制因子。这些实验将定义Fbw7Gamma和Fbw7pha的正常和肿瘤功能。 影响苏氨酸58(T58)的突变是淋巴瘤中最常见的c-Myc突变，T58磷酸化调节c-Myc的稳定性。我们发现GSK-3对T58的磷酸化调节Fbw7介导的c-Myc翻转。在目标3中，我们将测试假设，即T58突变通过阻止c-Myc与Fbw7的相互作用而促进c-Myc相关肿瘤的发生。我们将建立T58突变的敲入小鼠，然后用于研究T58磷酸化在调节c-Myc丰度和功能中的作用，并确定其在c-Myc相关肿瘤发生中的作用。 P27 CDK抑制物在人类癌症中的表达降低意味着预后不良，而p27在小鼠中是一种肿瘤抑制因子。在上一个资助阶段，我们对p27基因缺失的小鼠进行了插入突变，以确定与p27-1oss协同作用的癌基因，并确定了三个候选癌基因(c-myc、Jdp1、GPC3/XpcI1)。目的4的目的是利用小鼠移植模型来确定血细胞中Jdp2和GPC3/Xpcl1表达缺失的后果，研究Jdp2或GPC3/Xpcl1激活和p27缺失之间的协同作用，并了解这种协同作用的机制。

DESCRIPTION (provided by applicant): Mutation of genes that regulate the cell cycle is a fundamental mechanism underlying tumorigenesis. These mutations involve intrinsic cell cycle components themselves (cyclins, Cdk inhibitors) as well as oncogenes (e.g., c-Myc) and tumor suppressors (e.g. p53, Fbw7) that impact upon the cell cycle machinery. This proposal focuses on the functions and regulation of these cancer-associated cell cycle pathways. The Fbw7 tumor suppressor targets cyclin E, Notch, and c-Jun for degradation after they have been phosphorylated. In preliminary studies, we found that Fbw7 also regulates phosphorylation-dependent c-Myc turnover and that the three Fbw7 isoforms (Fbw7alpha, Fbw7beta, Fbw7gamma) exhibit unique subcellular Iocalizations. In Aim 1 we will test the hypothesis that the Fbw7 isoforms perform distinct biologic functions in these compartments by developing conditional-null mutations of Fbw7alpha and Fbw7gamma in the mouse. We will examine the roles of each isoform in regulating specific Fbw7 substrates, and we will test the hypothesis that Fbw7gamma, regulates c-Myc function in the nucleolus. In Aim 2 we will determine if Fbw7alpha or Fbw7gamma are tumor suppressors. These experiments will define the normal and neoplastic functions of Fbw7gamma and Fbw7alpha. Mutations affecting threonine 58 (T58) are the most common c-Myc mutations in lymphomas and T58 phosphorylation regulates c-Myc stability. We have found that T58 phosphorylation by GSK-3 regulates Fbw7-mediated c-Myc turnover. In Aim 3 we will test the hypothesis that T58 mutations contribute to c-Myc associated neoplasia by preventing the interaction of c-Myc with Fbw7. We will develop knock-in mice in which T58 is mutated that will than be used to study the role of T58 phosphorylation in regulating c-Myc abundance and function, and to determine its role in c-Myc-associated tumorigenesis. Reduced expression of the p27 Cdk inhibitor in human cancers connotes poor prognosis, and p27 is a tumor suppressor in mice. In the last funding period we used insertional mutagenesis in p27-null mice to identify oncogenes that cooperate with p27-1oss, and identified three candidate oncogenes (c-myc, Jdpl, GPC3/XpcI1). The goals of Aim 4 are to utilize murine transplant models to determine the consequences of deregulated Jdp2 and GPC3/Xpcl1 expression in blood cells, to study the cooperativity between Jdp2 or GPC3/Xpcl1 activation and p27-loss, and to understand the mechanisms of this cooperativity.