The Role of Caspase-8 in Neuroblastoma Tumorigenesis

描述（由申请人提供）：神经母细胞瘤（NB）是儿童最常见的颅外实体瘤，占儿童癌症死亡的15%。所有NB患者中超过50%存在侵袭性转移性疾病。值得注意的是，在诊断时患有转移性疾病的患者的死亡率仍然保持在约30- 50%，尽管积极治疗和我们对疾病的理解显着增加。在这种疾病中已经发现了几种遗传异常。然而，尽管N-myc扩增与侵袭性转移性疾病相关，但对这些遗传改变如何促进肿瘤形成和转移知之甚少。我们的实验室发现，NB细胞系和患者经常表现出caspase-8（C8）表达的损失，无论是通过表观遗传机制，或在少数情况下，通过基因缺失。我们还表明，半胱天冬酶-8的丢失通过阻止整合素介导的细胞死亡和降低肿瘤对凋亡刺激的反应性来促进肿瘤起始和增强转移。本研究旨在验证caspase-8在N-myc诱导的NB中起重要调节作用的假说。我们进一步假设C8在神经母细胞瘤形成和转移中具有凋亡和非凋亡作用，并且caspase-8表达和/或活性的调节与其他遗传变化（如肿瘤形成中MYCN过表达）合作。为了验证这个假设，我们计划：1。开发小鼠模型系统，其重现在大多数人类患者中观察到的MYCN表达的增加和胱天蛋白酶-8表达的减少，并使用该系统来确定减少C8表达如何改变MYCN过表达对细胞凋亡、增殖和存活的影响，以及2.）确定caspase-8是否在保留C8表达的NB肿瘤的肿瘤发生和转移中发挥非凋亡作用。这些研究将检查C8的凋亡功能是否在这些细胞中被废除，通过降低C8水平低于诱导凋亡所需的阈值和/或通过翻译后修饰，降低酶活性或改变亚细胞定位。我们还将描述参与C8在生存和增殖中的非凋亡功能的途径，并确定在人类NB患者中是否观察到C8表达或翻译后修饰的类似降低。在完成这些研究后，我们将获得对这种复杂疾病的生物学的重要新见解，可用于开发更有针对性的治疗方法，并理想地改善诊断为侵袭性转移性疾病的NB患者的预后。此外，由于C8的丢失与成神经管细胞瘤和复发性侵袭性胶质母细胞瘤的不良预后相关，这些肿瘤也表现出N-myc扩增和/或过表达，因此这些研究的数据可能会提供对其他人类肿瘤生物学的深入了解。 公共卫生相关性：神经母细胞瘤是儿童时期最常见的颅外实体瘤，约占儿童癌症的7%至10%，占15岁以下儿童癌症死亡的15%。重要的是，>50%的神经母细胞瘤患者存在转移性疾病。尽管进行了积极的治疗，这些患者中仍有30%至50%死亡。本研究旨在确定caspase-8和N-myc在神经母细胞瘤中表达改变的两种蛋白在肿瘤发生、转移和化疗反应中的作用。

DESCRIPTION (provided by applicant): Neuroblastoma (NB), the most frequent extracranial solid tumor in children, causes 15% of pediatric cancer deaths. Greater than 50% of all NB patients present with aggressive metastatic disease. Significantly, the death rate for patients that have metastatic disease at the time of diagnosis remains at ~30-50%, despite aggressive treatment and significant increases in our understanding of the disease. Several genetic abnormalities have been identified in this disease. However, little is known about how these genetic alterations contribute to tumor formation and metastasis, although N-myc amplification correlates with aggressive metastatic disease. Our laboratory discovered that NB cell lines and patients frequently exhibit loss of caspase-8 (C8) expression either by epigenetic mechanisms or in a few cases through gene deletion. We have also shown that the loss of caspase-8 facilitates tumor initiation and enhances metastasis via the prevention of integrin mediated cell death and decreases the responsiveness of tumors to apoptotic stimuli. The studies in this proposal are designed to test the t the hypothesis that caspase-8 plays an important modifier role in N-myc induced NB. We further hypothesize that C8 has both apoptotic and nonapoptotic roles in neuroblastoma formation and metastasis and that modulation of caspase-8 expression and/or activity cooperate with other genetic changes, such as MYCN over-expression in tumor formation. To test this hypothesis we plan to: 1.) develop a mouse model system that recapitulated the increase in MYCN expression and the decrease in caspase-8 expression that is seen in most human patients and to use this system to determine how reducing C8 expression alters the effects of MYCN over-expression on apoptosis, proliferation and survival and 2.) To determine whether caspase-8 plays nonapoptotic roles in tumorigenesis and metastasis in NB tumors that retain C8 expression. These studies will examine whether the apoptotic functions of C8 are abrogated in these cells, by reducing C8 levels below the threshold needed for induction of apoptosis and/or by posttranslational modification that decrease enzymatic activity or alter subcellular localization. We will also delineate that pathways involved in the nonapoptotic functions of C8 in survival and proliferation and determine whether similar decreases in C8 expression or posttranslational modifications are seen in human NB patients. Upon completion of these studies we will have obtained significant new insight into the biology of this complex disease that can be used to develop more targeted treatments and ideally improve the prognosis of NB patients diagnosed with aggressive metastatic disease. In addition, since loss of C8 has been correlated with poor prognosis in medulloblastoma and with relapsed aggressive glioblastoma, which also exhibit N-myc amplification and/or overexpression, the data from these studies may provide insight into the biology of other human tumors. PUBLIC HEALTH RELEVANCE: Neuroblastoma is the most common extracranial solid tumor in childhood, accounting for approximately 7% to10% of pediatric cancers and 15% of all pediatric cancer deaths in patients less than 15 years old. Importantly, >50% of all neuroblastoma patients present with metastatic disease. Despite aggressive treatment 30% to 50% of these patients die. The studies in this proposal are designed to determine the role of caspase-8 and N-myc, two proteins whose expression is altered in neuroblastoma, in tumorigenesis, metastasis and chemotherapeutic responsiveness.