Y-Box 1 and normoxic HIF1 in Sonic hedgehog medulloblastoma tumor stem cell radiation resistance

项目摘要 髓母细胞瘤是小儿中枢神经系统最常见的恶性肿瘤。当前 髓母细胞瘤的治疗范例包括手术切除、肿瘤部位的放射和 颅脊髓放疗和化疗。虽然这种疗法的治愈率约为70%， 幸存者受到长期神经副作用的困扰，肿瘤复发是致命的。因此需要 开发治疗方法，减少对放射的需求，减少复发的发生率， 并且可以成功地应用于复发的患者。髓母细胞瘤可分为4个分子 亚群，其中之一，Sonic hedgehog（SHH）亚群，进一步分为具有野生型p53的SHH或 突变型p53，预后最差。SHH亚组的特点是最常见的局部复发。 然而，对导致复发的机制的理解有限，最近的研究表明， 复发的遗传驱动因素可能与原始肿瘤驱动因素完全不同， 针对原发肿瘤驱动因素的治疗毫无意义。使用Shh髓母细胞瘤和 小脑颗粒神经元祖细胞（CGNP）的原代培养物，被认为是Shh髓母细胞瘤 我们已经观察到致癌DNA和RNA结合蛋白YB1促进了 辐射后的DNA修复我们还观察到YB1定位于血管周围龛（PVN）肿瘤 小鼠和人髓母细胞瘤中的细胞。这些细胞以其抗辐射特性而闻名。我们 还观察到HIF 1a，其mRNA翻译由YB1促进，同样在PVN中发现， 我们假设它促进了干细胞样表型的维持，这也赋予了辐射 阻力HIF 1a通常在常氧条件下降解，但我们的初步研究表明， 它以活性氧（ROS）和NADPH氧化酶（Nox4）依赖的方式稳定。在这里， 我们建议使用小鼠Shh成神经管细胞瘤模型来检验这一假设， YB1在体内可诱导肿瘤细胞在血管周围龛中死亡， 使用ROS或NADPH氧化酶抑制剂的髓母细胞瘤将使HIF 1a不稳定，并类似地增加辐射 反应最后，我们提出了一个翻译的目标，其中我们将利用一种新的微流体药物输送 测试YB1和ROS抑制对新鲜切除的人肿瘤细胞中细胞活力的联合作用的平台 髓母细胞瘤肿瘤切片，沿着分析原发性和复发性髓母细胞瘤中的HIF 1a、YB 1和Nox 4定位。 髓母细胞瘤标本来自亚特兰大儿童医院新建立的生物储存库。我们 研究有可能验证YB1活性和HIF1a稳定性作为新的治疗靶点， 神经管母细胞瘤，并建立一个先例，筛选患者样本，以预测对药物的反应， 在复发的情况下给予，或改善原发性肿瘤的放射反应。

Project Summary Medulloblastomas are the most common malignant pediatric tumor of the central nervous system. The current treatment paradigm for medulloblastomas includes surgical resection, irradiation of the tumor site and craniospinal irradiation as well as chemotherapy. While this regimen is associated with a ~70% cure rate, survivors are beset with long-term neurological side effects, and tumor recurrence is fatal. Thus, there is a need to develop therapeutic approaches that reduce the requirement for irradiation, reduce the incidence of recurrence, and may be successfully applied to patients that relapse. Medulloblastomas can be divided into 4 molecular subgroups, one of which, the Sonic hedgehog (SHH) subgroup, is further divided into SHH with wild type p53 or mutant p53, which bears the worst prognosis. The SHH subgroup features the most frequent local recurrence. However, there is a limited understanding of mechanisms causing recurrence, and recent studies have shown that genetic drivers of recurrence may be completely different than the original tumor drivers, rendering targeted therapies against the primary tumor drivers pointless. Using mouse models for Shh medulloblastoma and primary cultures of cerebellar granule neuron progenitor (CGNPs) cells, proposed to be Shh medulloblastoma cells-of-origin, we have made the observation that the oncogenic DNA- and RNA-binding protein YB1 promotes DNA repair after radiation. We have also observed that YB1 localizes to the perivascular niche (PVN) tumor cells in mouse and human medulloblastoma. These cells are known for their radiation resistant properties. We have also observed that HIF1a, whose mRNA translation is promoted by YB1, is likewise found in the PVN, where we hypothesize that it promotes maintenance of the stem cell-like phenotype, which also confers radiation resistance. HIF1a is normally degraded under normoxic conditions, however our preliminary studies indicate that it is stabilized in a reactive oxygen species (ROS) and NADPH oxidase (Nox4)-dependent manner. Here, we propose to use mouse Shh medulloblastoma models to test the hypothesis that pharmacologically targeting YB1 in vivo can induce tumor cell death in the perivascular niche, and that in vitro and in vivo treatment of mouse medulloblastoma with inhibitors of ROS or NADPH oxidase will destabilize HIF1a and similarly increase radiation response. Finally, we propose a translational aim wherein we will utilize a novel microfluidic drug delivery platform to test the combined effects of YB1 and ROS inhibition on cell viability in freshly resected human medulloblastoma tumor slices, along with analysis of HIF1a, YB1, and Nox4 localization in primary and recurrent medulloblastoma specimens from a newly established biorepository at Children’s Hospital of Atlanta. Our studies have the potential to validate YB1 activity and HIF1a stabilization as novel therapeutic targets in medulloblastoma, and to set a precedent for screening patient samples to predict response to drugs that could be given in the event of relapse, or to improve radiation response in the primary tumor.