Nuclear IRS-1-DNA repair and mutagenesis in medulloblastoma

描述（由申请人提供）：这是RO 1项目“IRS-1 JCV T抗原在小脑肿瘤中的相互作用”的竞争性延续。前期的研究结果表明胰岛素受体底物1（IRS-1）是IGF-I受体信号转导系统的主要胞质组分，它可以转位到细胞核。重要的是，我们已经在髓母细胞瘤细胞系和髓母细胞瘤临床样本中检测到了核IRS-1，这些样本对多瘤病毒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IRS-1和在受损DNA位点检测到的Rad 51之间的直接相互作用来抑制DNA修复的忠实成分（同源重组DNA修复，HRR）。我们还证明了核IRS-1对DNA修复的干扰，以及随后的突变积累可能在JCV T抗原不存在的情况下发生。在这方面，在髓母细胞瘤中高度表达的雌激素受体β（ER <$）可以将IRS-1转运到JCV T抗原阴性细胞的细胞核中。这些发现表明IRS-1和ER？之间的相互作用可能参与细胞对DNA损伤的反应。这也表明，核IRS-1抑制DNA的忠实修复是一个常见的事件，不限于髓母细胞瘤的情况下，是JCV阳性。因此，我们制定了一项研究计划，将分析导致髓母细胞瘤基因组不稳定性发展的基本机制。该建议是基于这样的假设，即在成神经管细胞瘤中发现的高活性ER？介导IRS-1易位到细胞核，导致HRR抑制，不忠实的DNA修复，以及活跃生长的成神经管细胞瘤细胞中基因组不稳定性的发展。为了验证这一假设，我们开发了一系列实验，这些实验在三个具体目标中概述。在目标#1中，我们将评估ER <$对HRR的IGF-I依赖性和IGF-I非依赖性组分的影响，并将表征ER <$、IRS-1和Rad 51在人髓母细胞瘤细胞系中的结合和亚细胞定位。在目标#2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RCAS/tv-a转基因小鼠的体外DNA修复保真度和肿瘤进展。最后，在目标#3中，我们将分析从髓母细胞瘤患者获得的临床样本中IRS-1、ER和Rad 51之间的蛋白水平、亚细胞定位和共定位。由于DNA损伤剂经常用于消除中枢神经系统肿瘤，包括髓母细胞瘤，DNA修复保真度的缺陷可能具有强烈的致突变后果。这反过来可能导致遗传毒性治疗后肿瘤进展或肿瘤复发。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，这在髓母细胞瘤患者中很常见。 髓母细胞瘤是儿童小脑肿瘤，其中肿瘤细胞在中枢神经系统（CNS）内扩散与不良预后相关。由于DNA损伤剂经常用于消除髓母细胞瘤，DNA修复机制的缺陷被强烈怀疑会导致突变的积累，从而导致肿瘤进展或肿瘤复发。与细胞信号通路相关的DNA修复机制的拟议研究对于开发新的治疗策略对抗CNS中这些小脑肿瘤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.