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

IRS-1 - JC T 抗原在小脑肿瘤中的相互作用

• 批准号: 6623329
• 负责人: Krzysztof Reiss
• 金额: $ 26.79万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-06 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6623329
• 关键词: Polyomavirus hominis 2; binding sites; biological signal transduction; cell line; chimeric proteins; disease /disorder model; genetically modified animals; genome; growth factor receptors; immunocytochemistry; immunoprecipitation; insulinlike growth factor; intracellular transport; laboratory mouse; medulloblastoma; molecular cloning; neoplastic transformation; pediatric neoplasm /cancer; protein localization; protein protein interaction; protein transport; site directed mutagenesis; tissue /cell culture; tumor antigens

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.

髓母细胞瘤约占所有小儿颅内肿瘤的 25%。 这些高度恶性肿瘤起源于小脑，主要影响五岁至十五岁的儿童。 尽管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) 可能有助于这些肿瘤的发生。 我们最近的研究表明，超过一半的人类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) 在这些肿瘤中强烈过表达，并且仅在 JCV T 抗原阳性髓母细胞瘤细胞系和 JCV 中观察到 IRS-1 易位至细胞核T 抗原阳性人髓母细胞瘤活检。 利用 JC 病毒 (JCV) 早期基因组的转基因动物模型提供了一个实验系统，可以在髓母细胞瘤中实际研究 IRS-1。 这些小鼠产生自发性小脑肿瘤，其组织学上与人类髓母细胞瘤非常相似。 该病毒的早期基因组编码调节蛋白 JCV T 抗原，该蛋白在细胞培养物中具有转化特性，并且在实验动物中具有致瘤性。 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关联，以及 JCV T 抗原在一些但不是全部人类肿瘤细胞中的表达。 这与转基因小鼠模型中的观察结果相当，其中并非所有含有 JCV 早期基因组的髓母细胞瘤细胞都表达 T 抗原。 尽管 JCV T 抗原与人髓母细胞瘤之间的因果关系仍有待确定，但 JCV T 抗原阳性和阴性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 核易位的作用可能是在不同的 T 抗原背景下进行研究。提出了三个具体目标来检验 IRS-1 核易位导致髓母细胞瘤恶性生长的假设。 第一个目标是对 IRS-1 和 JCV T 抗原进行突变分析，以确定参与这两个分子之间物理相互作用的结合域。 在第二个目标中，通过靶向 IRS-1 和 JCV T 抗原结合位点，我们将开发能够干扰 IRS-1-JCV T 抗原结合的新显性失活突变体。 这些新突变体将在 JCV T 抗原阳性和阴性髓母细胞瘤细胞系中进行测试，以确定 IRS-1 - JCV T 抗原相互作用是否有助于髓母细胞瘤中的转化表型。 最后，在第三个目标中，我们将表征 JCV T 抗原介导的 IRS-1 转定位到细胞核中的生物学意义，并确定 IRS-1 - JCV T 抗原相互作用的靶向破坏是否会减弱发展和/或实验动物原始神经外胚层肿瘤/髓母细胞瘤的进展。