IRS-1 - JC T-antigen Interaction in Cerebellar Tumors

髓母细胞瘤约占所有儿童颅内肿瘤的25%。这些高度恶性的肿瘤发生在小脑，主要影响5到15岁的儿童。虽然髓母细胞瘤的病因尚不清楚，但一些报告表明胰岛素样生长因子I(IGF-1)可能与这些肿瘤的发展有关。我们最近的研究表明，超过一半的人髓母细胞瘤活检组织中存在磷酸化(活性)IGF-I受体(IGF-IR)。主要的IGF-IR信号分子胰岛素受体底物1(IRS-1)在这些肿瘤中高表达，并且IRS-1易位到细胞核中仅在JCV T抗原阳性的髓母细胞瘤细胞系和JCV T抗原阳性的人髓母细胞瘤活检组织中观察到。利用JC病毒(JCV)早期基因组的转基因动物模型提供了一种实验系统，在该系统中可以在髓母细胞瘤中实际研究IRS-1。这些小鼠患上自发性小脑肿瘤，在组织学上与人类髓母细胞瘤相似。这种病毒的早期基因组编码调节蛋白JCVT抗原，该蛋白在细胞培养中具有转化特性，并在实验动物中产生肿瘤。有趣的是，最近的研究揭示了JCV基因组与人类自发性髓母细胞瘤的相关性，并且JCV T抗原在一些但不是全部人类肿瘤细胞中表达。这与在转基因小鼠模型中观察到的结果相当，在转基因小鼠模型中，并不是所有含有JCV早期基因组的髓母细胞瘤细胞都表达T抗原。虽然JCVT抗原与人髓母细胞瘤之间的因果关系仍未确定，但JCVT抗原阳性和阴性的髓母细胞瘤细胞系的可用性提供了一个独特的实验系统，其中IRS-1核转位的作用可以在不同的T抗原背景下进行研究。为了验证IRS-1核易位导致髓母细胞瘤恶性生长的假说，我们提出了三个特定的目标。在第一个目标中，将应用IRS-1和JCVT抗原的突变分析来确定这两个分子之间物理相互作用所涉及的结合结构域。在第二个目标中，通过靶向IRS-1和JCVT抗原结合位点，我们将开发能够干扰IRS-1-JCVT抗原结合的新的显性负性突变体。这些新的突变体将在JCVT抗原阳性和阴性的髓母细胞瘤细胞系中进行测试，以确定IRS-1-JCVT抗原相互作用是否有助于髓母细胞瘤的转化表型。最后，在第三个目标中，我们将鉴定JCV T抗原介导的IRS-1转位到核内的生物学意义，并确定靶向阻断IRS-1-JCV T抗原相互作用是否减缓实验动物原始神经外胚层肿瘤/髓母细胞瘤的发生和/或进展。

Medulloblastomas represent about 25 percent of all pediatric intracranial neoplasms. These highly malignant tumors arise from the cerebellum and affect mainly children between ages five and fifteen. Although the etiology of medulloblastoma remains unknown, several reports suggest that insulin-like growth factor I (IGF-1) may contribute to the development of these tumors. Our recent studies revealed the presence of the phosphorylated (active) IGF-I receptor (IGF-IR) in more than half of human medulloblastoma biopsies examined. The major IGF-IR signaling molecule, insulin receptor substrate 1 (IRS-1) is strongly overexpressed in these tumors, and the IRS-1 translocation to the nucleus has been observed exclusively in JCV T-antigen positive medulloblastoma cell lines, and in JCV T- antigen positive human medulloblastoma biopsies. A transgenic animal model utilizing the JC virus (JCV) early genome provides an experimental system, in which the IRS-1 could be actually studied in medulloblastomas. These mice develop spontaneous cerebellar tumors that histologically are close parallels to human medulloblastomas. The early genome of this virus encodes regulatory protein, JCV T-antigen, that has transforming properties in cell culture, and is tumorogenic in experimental animals. Interestingly, recent studies revealed association of JCV genome with spontaneous medulloblastomas in humans, and the expression of JCV T-antigen in some but not all human tumor cells. This is comparable with the observation in transgenic mouse model where not all medulloblastoma cells containing JCV early genome, express T-antigen. Although a cause and effect relationship between JCV T-antigen and human medulloblastoma remains to be established, the availability of JCV T-antigen positive and negative medulloblastoma cell lines provides an unique experimental system, in which the role of IRS-1 nuclear translocation could be studied in a different T-antigen context. Three specific aims are proposed to test the hypothesis that IRS- 1 nuclear translocation contributes to the malignant growth in medulloblastoma. In the first aim, mutational analysis of the IRS-1 and JCV T-antigen will be applied to determine binding domains involved in the physical interaction between these two molecules. In the second aim, by targeting IRS-1 and JCV T- antigen binding sites we will develop new dominant negative mutants capable of interfering with the IRS-1- JCV T-antigen binding. These new mutants will be tested in both JCV T-antigen positive and negative medulloblastoma cell lines to determine whether the IRS-1 - JCV T-antigen interaction contributes to the transformed phenotype in medulloblastomas. Finally in the third aim, we will characterize biological significance of the JCV T- antigen -mediated translocalization of IRS-1 into the nucleus, and determine whether targeted disruption of the IRS-1 - JCV T- antigen interaction attenuates the development and/or progression of primitive neuroectodermal tumors/medulloblastomas in experimental animals.