IGF SIGNAL TRANSDUCTION PATHWAY IN MEDULLOBLASTOMA

项目#3：成神经管细胞瘤中的IGF信号转导通路 大量证据表明IGF-I自分泌/旁分泌系统是脑肿瘤发生和发展的重要组成部分。尽管如此，很少有人关注IGF-I受体（IGF-IR）及其信号通路在原始神经外胚层肿瘤/髓母细胞瘤（PNTR/MB）中的作用，PNTR/MB约占所有儿科脑肿瘤的25%。本研究建议建立在IGF-IR系统和JCV T抗原在髓母细胞瘤的发展和/或进展中合作的假设基础上。这一假设的重要性得到了以下几个发现的支持：（i）JC病毒（JCV）感染超过80%的人群;（ii）在包括PNET/MB在内的人类肿瘤中发现了JCV T抗原;（iii）JCV T抗原的异位表达转化了培养物中的细胞，并且在实验动物中是致瘤的;和（iv）JCV T-抗原，以及其鼠对应物-SV 40 T-抗原，不能转化不具有功能性IGF-IR的细胞。 人和小鼠髓母细胞瘤细胞系中主要IGF-IR底物IRS-1的过表达，以及髓母细胞瘤患者活组织检查中IGF-I系统的激活，包括IGF-IR蛋白的组成性磷酸化。我们还发现，JCV-T-抗原和IGF-IR都是髓母细胞瘤细胞系在非贴壁依赖性培养条件下生存和生长所必需的，最后，我们证明， JCV T抗原和IRS-1相互作用。提出了三个具体目标来直接验证IGF-1信号通路在髓母细胞瘤发生中的功能作用的假设：在第一个目标中，IGF-IR的突变分析将用于确定IGF-IR和JCV T抗原之间的功能相互作用是否有助于髓母细胞瘤的恶性转化;在第二个目的中，将使用JCV T抗原阳性和阴性成神经管细胞瘤细胞系来确定在JCV T抗原介导的转化中是否涉及一组独特的IGF-IR途径。代谢抑制剂结合信号分子的突变分析，包括IRS-1和PTEN磷酸酶，将被用来改变IGF-IR途径，并测试它们在T抗原和非T抗原介导的转化中的重要性;最后，在第三个目标中，将在体内测试针对IGF-IR和IRS-1功能的显性阴性策略。这些结果在 体内研究将使我们能够评估IGF-IR信号通路与JCV T抗原的功能协同作用的解偶联是否将从小脑组织中消除成神经管细胞瘤肿瘤。

Project #3: IGF-Signal Transduction Pathway in Medulloblastoma A considerable line of evidence points at the IGF-I auto/paracrine system as an important component in the development and progression of brain tumors. Despite this, little attention has been paid to the role of IGF-I receptor (IGF-IR), and its signaling pathways, in primitive neuroectodermal tumors/ medulloblastomas (PNETs/MBs) that represent about 25% of all pediatric brain tumors. This research proposal is founded on the hypothesis that the IGF-IR system and the JCV T-antigen cooperate in the development and/or progression of medulloblastomas. Importance of this hypothesis is supported by several findings: (i) JC virus (JCV) infects greater than 80% of the human population; (ii) JCV T-antigen was found in human tumors including PNET/MBs; (iii) ectopic expression of JCV T-antigen transforms cells in culture and is tumorogenic in experimental animals; and (iv) JCV T-antigen, as well as, its murine counterpart - SV40 T-antigen, are not able to transform cells that do not possess functional IGF-IR. To further support our hypothesis, we have developed new evidence demonstrating overexpression of the major IGF-IR substrate, IRS-1, in both human and mouse medulloblastoma cell lines, and the activation of IGF-I system including constitutive phosphorylation of the IGF-IR protein in biopsies from patients with medulloblastoma. We have also found that both JCV-T-antigen and IGF-IR are necessary for medulloblastoma cell lines to survive and grow in anchorage-independent culture condition, and finally, we have demonstrated that JCV T-antigen and IRS-1 interact with each other. Three specific aims are proposed to directly test the hypothesis on the functional role for IGF-1 signaling pathway in the genesis of medulloblastoma; in the first aim mutational analysis of the IGF-IR will be utilized to determine whether functional interaction between the IGF-IR and JCV T-antigen contributes to malignant transformation in medulloblastomas; in the second aim JCV T-antigen positive and negative medulloblastoma cell lines will be employed to determine whether a unique set of IGF-IR pathways is involved in JCV T-antigen mediated transformation. Metabolic inhibitors in combination with mutational analysis of signaling molecules, including IRS-1 and PTEN phosphatase, will be employed to alter IGF-IR pathways and to test their importance in both T-antigen and non T-antigen mediated transformation; finally in the third aim dominant negative strategies against the IGF-IR and IRS-1 function will be tested in vivo. The results of these in vivo studies will allow us to evaluate whether uncoupling of the IGF-IR signaling pathway/s from its functional synergy with JCV T-antigen will eliminate medulloblastoma tumors from cerebellar tissues.