Role of Polyamines in MYCN-Amplified Neuroblastoma

神经母细胞瘤(NB)是儿童时期第三常见的恶性肿瘤，约占儿童恶性肿瘤的15% 儿童中与癌症相关的死亡。在NB肿瘤中发现的最显著的异常之一是 MYCNo癌基因的扩增，与更具侵袭性的肿瘤密切相关 晚期疾病的预后。完全缺乏对高危(晚期)NB的有效治疗 患者表明需要新的治疗方法，包括阻断MYCN表达的药物。 我们以前工作的主要目标是评估多胺生物合成途径作为一种 NB治疗的替代靶点。我们研究的中心是评估两个经过临床测试的 多胺抑制剂，DFMO和SAM486A，特异性抑制多胺生物合成酶 鸟氨酸脱羧酶和S腺苷蛋氨酸脱羧酶。自.以来 MYCN已被证明可以激活ODC基因的表达，我们假设ODC和多胺可能 在MVC/V扩增的神经母细胞瘤发生中起一定作用。我们的初步结果表明，多胺的消耗 在MVC/V扩增的NB细胞中，G！细胞周期停滞与p27Klp1水平、视网膜母细胞瘤蛋白 Rb的磷酸化，最有趣的是，阻止了MYCN的表达，从而为 多胺抑制剂在晚期NB患者中的潜在应用。在目前的提案中，我们概述了 研究MYCN、ODC和多胺如何调节NB中重要的细胞周期和细胞生存途径 细胞。在特定的目标1中，我们将研究在NB细胞中MYCN的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。这些研究将使用三个 独特的NB细胞系，其中MYCN的表达可以通过添加四环素来开启/关闭。具体而言 目的探讨多胺在细胞周期调控和PI3K/Akt细胞存活激活中的作用。 Nb细胞。具体地说，我们将确定单个多胺(腐胺， 精胺和精胺)对关键信号蛋白的调节和磷酸化/激活 包括MYCN、p27Kip1、CDK2、Akt和mTOR。在特定的目标3中，我们将阐明ODC在细胞中的作用 通过检测它与两种新的细胞蛋白的相互作用来传递信号。它们的生理相关性 相互作用将通过突变分析和一系列基于细胞的生物检测来验证。这个 提出的进行这项工作的方法和技术有：Northern印迹、RT-PCR、核Run-on、 信使RNA半衰期研究，Western印迹，酵母双杂交，免疫共沉淀，GST-下拉， 定点突变，DNA克隆，激光共聚焦显微镜，酶和体外激酶分析，Flow 细胞仪和高效液相色谱检测。在这项研究的结论中，我们将揭示ODC和个人 多胺调节重要的分子事件，控制细胞周期和细胞生存途径的NB细胞。 我们的研究将有助于开发治疗最具侵袭性形式的 儿童癌症神经母细胞瘤。

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 MYCNoncogene, 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 MVC/V-amplified NB tumorigenesis. Our initial results showed that the depletion of polyamines in MVC/V-amplified NB cells caused G! cell cycle arrest and affected p27Klp1 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.