The Role of Caspase-8 in Neuroblastoma Tumorigenesis

描述(申请人提供)：神经母细胞瘤(NB)是儿童最常见的颅外实体肿瘤，占儿童癌症死亡的15%。超过50%的NB患者存在侵袭性转移性疾病。值得注意的是，尽管积极的治疗和我们对这种疾病的了解显著增加，但在诊断时患有转移性疾病的患者的死亡率仍保持在~30%-50%。在这种疾病中已经发现了几种基因异常。然而，尽管N-myc扩增与侵袭性转移性疾病相关，但人们对这些基因改变如何促进肿瘤的形成和转移知之甚少。我们的实验室发现，Nb细胞株和患者经常表现出caspase-8(C8)的表达缺失，或者是通过表观遗传机制，或者在少数情况下是通过基因缺失。我们还表明，caspase-8的缺失通过阻止整合素介导的细胞死亡来促进肿瘤的启动和转移，并降低肿瘤对凋亡刺激的反应性。这项研究的目的是检验caspase-8在N-myc诱导的NB中起重要调节作用的假设。我们进一步假设C8在神经母细胞瘤的形成和转移中既有凋亡的作用也有非凋亡的作用，caspase-8的表达和/或活性的调节与其他遗传变化，如MYCN在肿瘤形成中的过度表达是协同的。为了验证这一假设，我们计划：1.)建立一种小鼠模型系统，概括大多数人类患者中MYCN表达增加和caspase-8表达下降的情况，并使用该系统来确定C8表达减少如何改变MYCN过表达对细胞凋亡、增殖和存活的影响。目的：探讨caspase-8在C8表达的NB肿瘤的发生和转移中是否发挥非凋亡性作用。这些研究将通过将C8水平降低到诱导细胞凋亡所需的阈值以下和/或通过降低酶活性或改变亚细胞定位的翻译后修饰来检验C8在这些细胞中的凋亡功能是否被取消。我们还将描述与C8在生存和增殖中的非凋亡功能有关的途径，并确定在人类NB患者中是否存在类似的C8表达减少或翻译后修饰。这些研究完成后，我们将对这种复杂疾病的生物学有了重大的新见解，可用于开发更有针对性的治疗方法，理想地改善被诊断为侵袭性转移疾病的NB患者的预后。此外，由于C8的缺失与髓母细胞瘤和复发性侵袭性胶质母细胞瘤的预后不良相关，这些肿瘤也表现出N-myc扩增和/或过表达，这些研究的数据可能为其他人类肿瘤的生物学提供洞察力。 公共卫生相关性：神经母细胞瘤是儿童最常见的颅外实体肿瘤，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。尽管这些患者接受了积极的治疗，但仍有30%至50%的患者死亡。这项研究旨在确定caspase-8和N-myc这两种蛋白在神经母细胞瘤中的表达变化，在肿瘤的发生、转移和化疗反应中所起的作用。

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.