IGF SIGNAL TRANSDUCTION PATHWAY IN MEDULLOBLASTOMA

项目3：髓母细胞瘤中的IGF-信号转导通路 大量证据表明，IGF-I自身/旁分泌系统是脑肿瘤发生发展过程中的重要组成部分。尽管如此，胰岛素样生长因子-I受体(IGF-IR)在原始神经外胚层肿瘤/髓母细胞瘤(PNETs/MBS)中的作用及其信号通路却鲜有人关注，PNETs/MBS约占所有儿童脑肿瘤的25%。这项研究建议建立在IGF-IR系统和JCVT抗原在髓母细胞瘤的发生和/或进展中协同作用的假设之上。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%的人类人口；(Ii)JCV T抗原在包括PNET/MBS在内的人类肿瘤中被发现；(Iii)JCV T抗原的异位表达可在培养中转化细胞，并在实验动物中产生肿瘤；(Iv)JCV T抗原及其小鼠的对应物SV40 T抗原不能转化不具有IGF-IR功能的细胞。为了进一步支持我们的假设，我们开发了新的证据来证明 主要的IGF-IR底物IRS-1在人和小鼠髓母细胞瘤细胞系中的过表达，以及IGF-I系统的激活，包括髓母细胞瘤患者活检组织中IGF-IR蛋白的结构性磷酸化。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培养条件下生存和生长所必需的，最后，我们证明了 JCVT抗原与IRS-1相互作用。我们提出了三个具体的目标来直接验证IGF-1信号通路在髓母细胞瘤发生中的功能作用的假说；在第一个目标中，将利用IGF-IR的突变分析来确定IGF-IR和JCVT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；在第二个目标中，将利用JCVT抗原阳性和阴性的髓母细胞瘤细胞株来确定一组独特的IGF-IR通路是否参与了JCVT抗原介导的转化。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通路，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，第三个目标是在体内测试针对IGF-IR和IRS-1功能的显性否定策略。这些研究的结果在 体内研究将使我们能够评估将胰岛素样生长因子-IR信号通路/S从其与JCVT抗原的功能协同中解偶联是否能够消除小脑组织中的髓母细胞瘤。

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.