Nuclear IRS-1-DNA repair and mutagenesis in medulloblastoma

描述(由申请人提供)：这是RO1项目“IRS-1JCVT抗原在小脑肿瘤中的相互作用”的竞争性延续。早期的研究结果表明，胰岛素受体底物1(IRS-1)是IGF-I受体信号系统的主要胞浆成分，可以转运到细胞核。重要的是，我们在髓母细胞瘤细胞系和髓母细胞瘤临床标本中检测到核IRS-1，这些标本对多瘤病毒JC大T抗原呈阳性。我们的进一步研究表明，核IRS-1可以通过在DNA损伤部位检测到IRS-1与RAD51之间的直接相互作用来抑制DNA修复的忠实组成部分(同源重组DNA修复，HRR)。我们还证明，在没有JCVT抗原的情况下，核IRS-1对DNA修复的干扰以及随后的突变积累可能会发生。在这方面，在髓母细胞瘤中高表达的雌激素受体β(ER？)可以将IRS-1转位到JCVT抗原阴性细胞的核中。这些发现表明，IRS-1和ER之间的相互作用可能参与了细胞对DNA损伤的反应。这也表明，核IRS-1抑制DNA忠实修复是一种常见事件，并不局限于JCV阳性的髓母细胞瘤。因此，我们制定了一项研究计划，将分析导致髓母细胞瘤基因组不稳定性发展的基本机制。这一建议建立在这样的假设基础上，即在髓母细胞瘤中发现的高活性内质网介导IRS-1移位到细胞核，导致HRR抑制、不忠诚的DNA修复以及活跃生长的髓母细胞瘤细胞基因组的不稳定发展。为了验证这一假设，我们开发了一系列实验，这些实验概括为三个具体目标。在目标1中，我们将评估ER对HRR的IGF-I依赖和IGF-I非依赖成分的影响，并将表征ER？、IRS-1和RAD51在人髓母细胞瘤细胞系中的结合和亚细胞定位。在第二个目标中，我们将评估IGF-IR-IRS-1-E信号轴的药理学和分子操作如何影响体外DNA修复的保真度和RCAS/TV-a转基因小鼠的肿瘤进展。最后，在目标3中，我们将分析从髓母细胞瘤患者的临床样本中获得的蛋白水平、亚细胞定位以及IRS-1、ER？和RAD51之间的共定位。由于DNA损伤剂经常被用来消除包括髓母细胞瘤在内的中枢神经系统肿瘤，DNA修复保真度的缺陷可能会产生强烈的突变后果。这反过来可能导致肿瘤进展或在基因毒性治疗后肿瘤复发。更好地了解IRS-1、ER和DNA修复蛋白之间的信号串扰有望为未来针对基因组不稳定性的治疗策略提供新的分子靶点，这种不稳定性在髓母细胞瘤患者中很常见。髓母细胞瘤是一种儿童小脑肿瘤，肿瘤细胞在中枢神经系统内的扩散与预后不良有关。由于DNA损伤剂经常被用来消除髓母细胞瘤，DNA修复机制的缺陷被强烈怀疑会导致导致肿瘤进展或肿瘤复发的突变积累。与细胞信号通路相关的DNA修复机制的研究对于开发新的治疗策略以防止这些小脑肿瘤在中枢神经系统中的恶性扩散至关重要。

DESCRIPTION (provided by applicant): This is a competing continuation of a RO1 project "IRS-1 JCV T-antigen interaction in Cerebellar Tumors". The results from previous founding period demonstrate that Insulin Receptor Substrate 1 (IRS-1), which is the major cytosolic component of the IGF-I receptor signaling system, can translocate to the nucleus. Importantly, we have detected nuclear IRS-1 in medulloblastoma cell lines and in medulloblastoma clinical samples, which were positive for polyomavirus JC large T-antigen. Our further studies demonstrate that nuclear IRS-1 can inhibit faithful component of DNA repair (homologous recombination DNA repair, HRR) via a direct interaction between IRS-1 and Rad51 detected at the sites of damaged DNA. We also demonstrated that the interference with DNA repair by nuclear IRS-1, and subsequent accumulation of mutations may happen in the absence of JCV T-antigen. In this respect, estrogen receptor beta (ER¿), which is highly expressed in medulloblastomas, can translocate IRS-1 to the nucleus in JCV T-antigen negative cells. These findings indicate that the interaction between IRS-1 and ER¿ could be engaged in cellular responses to DNA damage. It also suggests that inhibition of the faithful DNA repair by nuclear IRS-1 is a common event, not restricted to the medulloblastoma cases which are JCV positive. Therefore, we have developed a research plan which will analyze fundamental mechanisms leading to the development of genomic instability in medulloblastomas. The proposal is founded on the hypothesis that highly active ER¿ found in medulloblastoma mediates translocation of IRS-1 to the nucleus causing inhibition of HRR, unfaithful DNA repair, and the development of genomic instability in actively growing medulloblastoma cells. To test this hypothesis, we have developed a series of experiments, which are outlined in three Specific Aims. In Aim #1, we will evaluate ER¿ effects on IGF-I dependent and IGF-I -independent components of HRR, and will characterize binding and subcellular localizations among ER¿, IRS-1 and Rad51 in human medulloblastoma cell lines. In Aim#2, we will evaluate how pharmacological and molecular manipulations with the IGF-IR-IRS-1-E¿ signaling axis affect DNA repair fidelity in vitro and tumor progression in RCAS/tv-a transgenic mice. Finally, in Aim #3, we will analyze protein levels, subcellular localization and co-localization among IRS-1, ER¿, and Rad51 in clinical samples obtained from patients with medulloblastoma. Since DNA damaging agents are frequently used to eliminate CNS tumors including medulloblastoma, defects in DNA repair fidelity may have strong mutagenic consequences. This in turn may result in tumor progression or tumor recurrences after genotoxic treatment. Better understanding of the signaling cross-talk between IRS-1, ER¿ and DNA repair proteins is expected to provide new molecular targets for future therapeutic strategies against genomic instability, which is common in medulloblastoma patients. PUBLIC HEALTH RELEVANCE Medulloblastomas are cerebellar tumors of the childhood, in which the spread of tumor cells within central nervous system (CNS) is associated with poor prognosis. Since DNA damaging agents are frequently used to eliminate medulloblastoma, defects in DNA repair mechanisms are strongly suspected to cause accumulation of mutations contributing to tumor progression or tumor recurrences. The proposed studies of DNA repair mechanisms in association with cell signaling pathways are critically important for the development of new therapeutic strategies against malignant dissemination of these cerebellar tumors in CNS.