Interplay between low levels of GAS7 expression and MYCN overexpression and its impact on neuroblastoma metastasis

神经母细胞瘤（NB）是儿童最常见的颅外实体瘤，通常在诊断时表现为 广泛转移的证据，特别是在MYCN癌基因扩增的患者中。这 亚组有非常高的治疗失败和死亡的风险，尽管接受了大大加强化疗。 由于缺乏详细的治疗方法， 了解这种复杂形式的肿瘤的多步骤细胞和分子发病机制。因此，在本发明中， 迫切需要新的机制见解，从而导致对转移性NB的更安全和更有效的治疗。 该建议源于对135名患者的原发性NB样本的全基因组测序分析， 我们发现了一个新的缺失生长停滞特异性基因7（GAS 7，位于染色体上 17p13.1），在MYCN扩增定义的高风险NB患者亚组中。进一步分析显示 低水平的GAS 7表达与晚期、MYCN扩增和低表达相关。 临床结果，而在MYCN过表达的NB斑马鱼模型中敲除gas 7促进了 肿瘤细胞的血行转移到这种疾病患者中常见的部位。我们还发现 MYCN可以间接结合GAS 7启动子区域，导致其转录抑制，而 GAS 7表达的降低可增加MYCN蛋白水平。因此，这个问题的核心假设 应用是，除了具有17 p杂合缺失的情况之外， MYCN可以下调GAS 7的表达，并维持在这种衔接蛋白的缺陷水平。这反过来又 似乎维持MYCN蛋白的高水平表达，促进肿瘤细胞从肿瘤细胞中解离。 原发肿瘤及其随后的扩散。这一假设将在三个具体目标中得到检验：1） 探讨转移性NB中MYCN过表达和低水平GAS 7表达之间的相互作用，2） 剖析MYCN诱导的下游转录和信号传导变化的级联反应， GAS 7缺陷导致高危NB转移的作用; 3）评估GAS 7缺陷的作用 NB传播的时间和GAS 7过表达阻断或减弱MYCN驱动的细胞凋亡的能力， 肿瘤转移，并在体内评估MYCN过表达-GAS 7缺陷对NB的影响 转移使用不同的小鼠模型。该提案的主要创新之处在于使用了新兴的 在高通量动物模型中前瞻性整合宿主和肿瘤基因组学的技术， 分析NB转移中低水平GAS 7表达和MYCN过表达之间的相互作用。的 这项研究的意义有三个方面：（i）它将揭示MYCN对早期NB的贡献 通过其对粘附、运动和侵袭的影响，（ii）阐明其潜在的机制 MYCN与NB转移中低水平GAS 7表达的合作，以及（iii）确定有希望的 转移性信号级联中的分子靶点，可以用于治疗。

Neuroblastoma (NB), the most common extracranial solid tumor in children, typically presents at diagnosis with evidence of widespread metastasis, especially in patients with amplification of the MYCN oncogene. This subgroup has a very high risk of treatment failure and death despite receiving greatly intensified chemotherapy. Attempts to improve the treatment of disseminated NB have been impeded by the lack of a detailed understanding of the multistep cellular and molecular pathogenesis of this complex form of the tumor. Thus, new mechanistic insights leading to safer and more effective treatments for metastatic NB are urgently needed. This proposal grew from whole-genome sequencing analyses of primary NB samples from 135 patients, in which we identified a novel deletion of a growth arrest-specific gene 7 (GAS7, located on chromosome 17p13.1), in a subset of patients with high-risk NB defined by MYCN amplification. Further analysis showed that low levels of GAS7 expression are associated with advanced-stage, MYCN amplification and a poor clinical outcome, while knockout of gas7 in a zebrafish model of NB with MYCN overexpression promotes hematogenous metastasis of tumor cells to sites commonly seen in patients with this disease. We also found that MYCN can bind indirectly to the GAS7 promoter region, leading to its transcriptional repression, while reduced expression of GAS7 can increase MYCN protein levels. Hence, the central hypothesis of this application is that, in addition to cases with the heterozygous deletion of 17p, over- or amplified expression of MYCN can downregulate GAS7 expression and maintain at deficient levels of this adaptor protein. This, in turn, appears to maintain MYCN protein expression at high levels, facilitating dissociation of tumor cells from the primary tumor and their subsequent dissemination. This hypothesis will be tested in three specific aims: 1) To probe the interplay between MYCN overexpression and low levels of GAS7 expression in metastatic NB, 2) To dissect the MYCN-induced cascade of downstream transcriptional and signaling changes in the context of GAS7 deficiency that lead to metastasis in high-risk NB, and 3) To assess the contribution of GAS7 deficiency to the timing of NB dissemination and the ability of GAS7 overexpression to block or attenuate MYCN-driven tumor metastasis, and to evaluate in vivo the effect of MYCN overexpression-GAS7 deficiency on NB metastasis using different murine models. The major innovation of this proposal is the use of emerging technology with a forward-looking integration of host and tumor genomics in a high-throughput animal model to dissect the interplay between low levels of GAS7 expression and MYCN overexpression in NB metastasis. The significance of this study is threefold: it will (i) shed new light on the contributions of MYCN to early NB metastasis via its effects on adhesion, motility, and invasion, (ii) clarify the mechanism(s) that underlie MYCN’s cooperation with low levels of GAS7 expression in NB metastasis, and (iii) identify promising molecular targets within metastatic signaling cascades that could be exploited therapeutically.