Apoptotic Pathway Defects in Neuroblastoma

神经母细胞瘤的凋亡途径缺陷

• 批准号: 6582100
• 负责人: VINCENT J. KIDD
• 金额: $ 33.66万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-15 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6582100
• 关键词: apoptosis; cysteine endopeptidases; enzyme activity; gene induction /repression; gene targeting; genetic models; genetically modified animals; human tissue; laboratory mouse; loss of heterozygosity; methylation; microarray technology; model design /development; neoplasm /cancer chemotherapy; neoplasm /cancer genetics; neoplastic growth; neural crest; neuroblastoma; oncogenes; xenotransplantation

DESCRIPTION (provided by applicant): Late stage neuroblastoma tumors, particularly those with amplified MYCN genes, have a poor prognosis, primarily due to their ability to survive treatment with multiple chemotherapeutic agents/irradiation. The continuing goal of this research program is to understand how genetic alterations, such as MYCN gene amplification and chromosome lp36 loss-of-heterozygosity (LOH), contribute to this tumor phenotype. During the last funding period we found that a critical apoptotic signaling molecule, caspase-8, is preferentially silenced by methylation in >60% of the stage 4 neuroblastoma patient tumors with simplified MYCN, whereas <4% of those stage 1-4 tumors without amplified MYCN silence expression of the CASP8 gene. A similar observation has now been made in N-Myc-induced neuroblastoma tumors from mice. We also found that reprogramed expression of caspase-8 in human NB cells that are normally caspase-8 null resensitized them to apoptosis induced by the chemotherapeutic drugs doxorubicin and cisplatin. This was observed in both cell culture and in vivo xenograft mouse models. Finally, we have reported, as have others, that caspase-8 is capable of functioning as both an initiator and executioner caspase, allowing it to amplify certain mitochondrial-mediated cell death signals. This function is somewhat unique among the caspases identified thus far, and could be one reason it is selectively silenced in certain tumors. Based upon this data we hypothesize that the silencing of CASP8 by methylation may provide a more permissive cellular environment that can tolerate the overexpression of N-Myc without undergoing cell death, and perhaps contribute to the ability of these tumor cells to survive treatment with certain chemotherapeutic drugs. To test this hypothesis we propose to develop mouse models by gene knockout or transgenic expression of an inactive, dominant negative form of caspase-8 that either totally eliminate, or down-regulate enzyme activity, and determine whether it contributes to accelerated tumor cell growth in the presence of N-Myc overexpression or the response of these tumors to chemotherapeutic drugs. These experiments will include the use of complementary approaches; namely the induction of oncogenes such as MYCN in cultured neural crest cells isolated from these mice, as well as the response of the various cells, xenografts, and in vivo tumors to therapy. Finally, we will examine these different mouse NB tumors and normal adrenal gland tissue, as well as human patient samples of various stages and matched-treated/untreated samples by microarray analysis to identify possible genes, other than CASP8, whose expression is significantly altered by N-Myc overexpression and/or drug treatment. Such studies will provide significant insight into how these tumor cells circumvent apoptosis and prolong their life.

描述（由申请人提供）：晚期神经母细胞瘤肿瘤，特别是那些具有扩增的 MYCN 基因的肿瘤，预后较差，主要是因为它们能够在多种化疗药物/放射治疗后存活。该研究计划的持续目标是了解遗传改变（例如 MYCN 基因扩增和染色体 lp36 杂合性丢失 (LOH)）如何导致这种肿瘤表型。在上一个资助期间，我们发现，在具有简化 MYCN 的 4 期神经母细胞瘤患者肿瘤中，超过 60% 的关键细胞凋亡信号分子 caspase-8 优先被甲基化沉默，而在没有放大 MYCN 的 1-4 期肿瘤中，CASP8 基因的表达沉默为 <4%。现在在 N-Myc 诱导的小鼠神经母细胞瘤中也进行了类似的观察。我们还发现，正常情况下 caspase-8 缺失的人 NB 细胞中 caspase-8 的重新编程表达使它们对化疗药物阿霉素和顺铂诱导的细胞凋亡重新敏感。在细胞培养和体内异种移植小鼠模型中都观察到了这一点。最后，我们和其他人一样报道了 caspase-8 能够充当启动者和执行者 caspase，使其能够放大某些线粒体介导的细胞死亡信号。这种功能在迄今为止鉴定的半胱天冬酶中有些独特，并且可能是它在某些肿瘤中选择性沉默的原因之一。基于这些数据，我们假设通过甲基化沉默 CASP8 可能会提供更宽松的细胞环境，能够耐受 N-Myc 的过度表达而不发生细胞死亡，并且可能有助于这些肿瘤细胞在某些化疗药物治疗后存活下来。为了检验这一假设，我们建议通过基因敲除或转基因表达无活性、显性失活形式的 caspase-8 来开发小鼠模型，完全消除或下调酶活性，并确定它是否有助于在 N-Myc 过度表达或这些肿瘤对化疗药物产生反应的情况下加速肿瘤细胞生长。这些实验将包括使用补充方法；即在从这些小鼠中分离出的培养神经嵴细胞中诱导致癌基因（例如 MYCN），以及各种细胞、异种移植物和体内肿瘤对治疗的反应。最后，我们将通过微阵列分析检查这些不同的小鼠 NB 肿瘤和正常肾上腺组织，以及不同阶段的人类患者样本和匹配治疗/未治疗的样本，以识别除 CASP8 之外的可能基因，其表达因 N-Myc 过表达和/或药物治疗而显着改变。此类研究将为了解这些肿瘤细胞如何避免细胞凋亡并延长其寿命提供重要的见解。