Role of Polyamines in MYCN-Amplified Neuroblastoma

多胺在 MYCN 扩增神经母细胞瘤中的作用

基本信息

批准号：
7448548
负责人：
ANDRE S BACHMANN
金额：
$ 21.47万
依托单位：
UNIVERSITY OF HAWAII AT MANOA
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-08-01 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7448548
关键词：
Accounting Adenosylmethionine Decarboxylase Affect Apoptosis Biological Assay Cell Cycle Cell Cycle Arrest Cell Cycle Progression Cell Cycle Regulation Cell Line Cell Proliferation Cell Survival Cells Cessation of life Child Childhood Cloning Co-Immunoprecipitations Communication DL-alpha-Difluoromethylornithine DNA Development Disease Employee Strikes Enzymes Evaluation Event Flow Cytometry Gene Expression Genetic Transcription Half-Life High Pressure Liquid Chromatography Hydrogen Peroxide In Vitro Individual Investigation Laser Microscopy MYCN gene Malignant Childhood Neoplasm Malignant Neoplasms Mammalian Cell Measures Messenger RNA Metabolic Metabolism Molecular Mutation Neuroblastoma Nitric Oxide Northern Blotting Nuclear Oncogenes Ornithine Decarboxylase Pathogenesis Pathway interactions Patients Pharmaceutical Preparations Phosphorylation Phosphotransferases Physiological Play Polyamines Production Protein Overexpression Proteins Putrescine Rate Regulation Research Retinoblastoma Protein Reverse Transcriptase Polymerase Chain Reaction Risk Role Running SAM486A SAT gene Sepiapterin reductase Series Signal Pathway Signal Transduction Signaling Protein Site-Directed Mutagenesis Spermidine Spermidine/Spermine N1-Acetyltransferase Spermine Staging System Techniques Testing Tetracycline Tetracyclines Translating Western Blotting Work Yeasts base enzyme activity human FRAP1 protein inhibitor/antagonist insight migration novel novel therapeutics outcome forecast response tumor tumorigenesis urea cycle yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Neuroblastoma (NB) is the third most common malignancy in childhood and accounts for about 15% of cancer-related deaths in children. One of the most striking abnormalities found in NB tumors is the amplification of the MYCN oncogene, which strongly correlates with more aggressive tumors and poor prognosis of late stage disease. The complete absence of effective treatments for high-risk (late stage) NB patients indicates the need for novel therapeutic approaches, including drugs that block MYCN expression. The primary objective of our previous work has been to evaluate the polyamine biosynthetic pathway as an alternative target for NB therapy. Central to our investigation was the evaluation of two clinically tested polyamine inhibitors, DFMO and SAM486A, which specifically inhibit the polyamine biosynthetic enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), respectively. Since MYCN has been shown to activate ODC gene expression, we hypothesized that ODC and polyamines might play a role in MYCN-amplified NB tumorigenesis. Our initial results showed that the depletion of polyamines in MYCN-amplified NB cells caused G1 cell cycle arrest and affected p27Kip1 levels, retinoblastoma protein Rb phosphorylation, and most intriguingly, blocked MYCN expression, thus providing a rationale for the potential use of polyamine inhibitors in late-stage NB patients. In the current proposal we outline studies that examine how MYCN, ODC, and polyamines regulate important cell cycle and cell survival pathways in NB cells. In Specific Aim 1 we will study whether MYCN overexpression in NB cells impacts endogenous ODC and other metabolic enzymes of polyamine metabolism, and whether this translates into changes in polyamine pools, cell cycle progression, and cell proliferation. These studies will be performed using three unique NB cell lines, in which MYCN expression can be switched on/off by adding tetracycline. In Specific Aim 2 we will examine the role of polyamines in cell cycle regulation and PI3K/Akt cell survival activation in NB cells. Specifically, we will determine the differential effects of individual polyamines (putrescine, spermidine, and spermine) on the regulation and phosphorylation/activation of key signaling proteins including MYCN, p27Kip1, Cdk2, Akt, and mTOR. In Specific Aim 3 we will elucidate the role of ODC in cell signaling by examining its interaction with two new cellular proteins. The physiological relevance of these interactions will be verified by mutational analyses and a series of cell-based biological assays. The proposed methods and techniques to pursue this work are: Northern blot, RT-PCR, nuclear run-ons, messenger RNA half-life studies, Western blot, yeast two-hybrid, co-immunoprecipitation, GST-pull down, site-directed mutagenesis, DNA cloning, confocal laser microscopy, enzyme and in vitro kinase assays, flow cytometry, and HPLC. At the conclusion of this research, we will have uncovered how ODC and individual polyamines regulate important molecular events that control cell cycle and cell survival pathways of NB cells. Our research will be useful in the development of novel treatments for the most aggressive forms of the childhood cancer neuroblastoma.

描述（由申请人提供）：神经母细胞瘤 (NB) 是儿童期第三大常见恶性肿瘤，约占儿童癌症相关死亡的 15%。 NB 肿瘤中发现的最显着的异常之一是 MYCN 癌基因的扩增，这与更具侵袭性的肿瘤和晚期疾病的不良预后密切相关。高危（晚期）NB 患者完全缺乏有效的治疗方法，这表明需要新的治疗方法，包括阻断 MYCN 表达的药物。我们之前工作的主要目标是评估多胺生物合成途径作为 NB 治疗的替代靶点。我们研究的核心是评估两种经过临床测试的多胺抑制剂 DFMO 和 SAM486A，它们分别特异性抑制多胺生物合成酶鸟氨酸脱羧酶 (ODC) 和 S-腺苷甲硫氨酸脱羧酶 (AdoMetDC)。由于 MYCN 已被证明可以激活 ODC 基因表达，我们假设 ODC 和多胺可能在 MYCN 扩增的 NB 肿瘤发生中发挥作用。我们的初步结果表明，MYCN 扩增的 NB 细胞中多胺的消耗导致 G1 细胞周期停滞并影响 p27Kip1 水平、视网膜母细胞瘤蛋白 Rb 磷酸化，最有趣的是，阻断 MYCN 表达，从而为多胺抑制剂在晚期 NB 患者。在当前的提案中，我们概述了研究 MYCN、ODC 和多胺如何调节 NB 细胞中重要的细胞周期和细胞存活途径。在具体目标 1 中，我们将研究 NB 细胞中 MYCN 过度表达是否影响内源性 ODC 和多胺代谢的其他代谢酶，以及这是否会转化为多胺池、细胞周期进程和细胞增殖的变化。这些研究将使用三种独特的 NB 细胞系进行，其中可以通过添加四环素来打开/关闭 MYCN 表达。在具体目标 2 中，我们将研究多胺在 NB 细胞中细胞周期调节和 PI3K/Akt 细胞存活激活中的作用。具体来说，我们将确定各种多胺（腐胺、亚精胺和精胺）对关键信号蛋白（包括 MYCN、p27Kip1、Cdk2、Akt 和 mTOR）的调节和磷酸化/激活的不同影响。在特定目标 3 中，我们将通过检查 ODC 与两种新细胞蛋白的相互作用来阐明 ODC 在细胞信号传导中的作用。这些相互作用的生理相关性将通过突变分析和一系列基于细胞的生物测定来验证。开展这项工作的拟议方法和技术包括：Northern 印迹、RT-PCR、核连载、信使 RNA 半衰期研究、蛋白质印迹、酵母双杂交、免疫共沉淀、GST 下拉、定点诱变、DNA 克隆、共聚焦激光显微镜、酶和体外激酶测定、流式细胞术和 HPLC。在这项研究结束时，我们将揭示 ODC 和单个多胺如何调节控制 NB 细胞的细胞周期和细胞存活途径的重要分子事件。我们的研究将有助于开发针对最具侵袭性的儿童癌症神经母细胞瘤的新疗法。