Modeling glioma with sporadic co-LOH of p53 and NF1 using a mouse genetic mosaic

癌症是美国第二大死因。癌症研究的一个最终目标是获得知识，开发早期发现肿瘤的工具，并开发在肿瘤变得恶性和转移之前进行有效治疗的药物。为了实现这些目标，彻底了解肿瘤的启动过程是至关重要的。首先，与晚期肿瘤相比，在没有额外基因改变的情况下启动肿瘤的研究应该能够清楚地阐明目标基因突变的因果影响。第二，早期肿瘤相对正常的组织结构使得研究肿瘤起始细胞与其周围环境之间的关系成为可能。尽管目前可用的基因工程小鼠模型已经阐明了癌症机制的许多方面，但主要关注的是具有病理可识别特征的肿瘤，提供进展的知识，而不是起始肿瘤。在这项资助中，我们建议使用一种新的小鼠遗传模型来研究P53肿瘤抑制基因在胶质瘤肿瘤起始过程中的作用。虽然对P53的研究已经有20多年的历史，但P53突变对体内细胞的最早影响仍然难以捉摸。为了克服早期缺乏突变细胞鉴定方法而造成的研究肿瘤发生的障碍，我们将使用称为MADM的小鼠遗传镶嵌系统(MADM)。MADM系统可以灭活正常小鼠体内极少数细胞中的P53，这与人类癌症的克隆起源非常相似。同样重要的是，MADM系统明确地用绿色荧光蛋白标记突变细胞，使我们能够在p53丢失后几个小时内调查肿瘤的起源。利用MADM提供的单细胞分辨率，我们将研究p53突变细胞的肿瘤起始过程作为一系列克隆进化事件，重点关注内在细胞信号通路和外在细胞-生态位通讯的变化。我们提出的研究将有助于对体内p53在肿瘤启动过程中的作用有价值的基础了解。此外，我们的遗传镶嵌系统提供的细胞分辨率将揭示肿瘤细胞和它们的微环境之间的共同进化过程。对早期肿瘤发生过程的基本认识将为发展肿瘤早期检测的分子标志物提供基础，并为合理的治疗策略提供指导。最后但并非最不重要的是，我们提出的工作将探索肿瘤起始的未知领域，并为完善小鼠模型以从机制上理解人类癌症提供概念基础。人们对p53抑癌基因的研究已有二十多年的历史。然而，该基因的突变如何导致体内突变细胞的最初选择优势仍有待了解。利用小鼠基因马赛克系统，可以在p53基因丢失后数小时内标记突变细胞，我们建议研究脑肿瘤启动过程中分子和细胞水平的早期变化，作为p53基因丢失的直接和即时后果。拟议的研究将清楚地揭示P53在预防脑内肿瘤发生中的作用，并将导致对癌症机制的基本了解，并为制定癌症检测和治疗策略提供基础。

Cancer is the second leading cause of death in the United States. An ultimate goal of cancer research is to gain knowledge to develop tools for early detection of tumors, and to develop drugs for effective treatment before tumors become malignant and metastasize. To achieve these goals, it is critical to thoroughly understand the tumor initiation process. First, compared to late stage tumors, studies of initiating tumors free of additional genetic alterations should lead to a clear elucidation of the causal effects by the mutation of gene of interest. Second, the relatively normal tissue organization in early stage tumors allows the investigation of the relationship between tumor-initiating cells and their neighboring environment. Though currently available genetic engineered mouse models have elucidated many aspects of cancer mechanisms, the main focus has been on tumors with pathological identifiable characteristics, rendering knowledge of progressed rather than initiating tumors. In this grant, we propose to use a novel mouse genetic model to study the role of the p53 tumor suppressor gene in the tumor initiation process of glioma. Although p53 has been studied for more than 20 years, the earliest impact of p53 mutation on cells in vivo remains elusive. To overcome the hurdle of studying tumor initiation due to the lack of identification method for mutant cells at the early stage, we will use the mouse genetic mosaic system termed MADM (mosaic analysis with double markers). The MADM system can inactivate p53 in a very small number of cells within an otherwise normal mouse, closely mimicking the clonal origin of human cancers. As importantly, the MADM system unambiguously labels the mutant cells with green fluorescence protein, enabling us to investigate tumor initiation within hours after p53 loss. Taking advantage of the single cell resolution provided by MADM, we will investigate the tumor initiation process of p53 mutant cells as a series of clonal evolution events, focusing on the changes in both intrinsic cell signaling pathways and extrinsic cell-niche communications. Our proposed studies will lead to the valuable basic understanding of in vivo p53 function during tumor initiation. Furthermore, the cellular resolution provided by our genetic mosaic system will reveal the process of co-evolution between tumor cells and their microenvironment. The basic understanding of the early tumorigenesis process will provide the basis for the development of molecular markers for early detection of tumors, and serve as the guidance for rationale treatment strategies. Last but not least, our proposed work will explore the uncharted territory of tumor initiation, and provide conceptual groundwork for the refinement of mouse models for mechanistic understanding of human cancers. The p53 tumor suppressor gene has been studied intensively for more than two decades. However, it remains to be understood how mutation in this gene results in the initial selective advantage of mutant cells in the body. Using a mouse genetic mosaic system that can mark the mutant cells within hours after loss of the p53 gene, we propose to study the early changes at molecular and cellular level during brain tumor initiation as a direct and immediate consequence of p53 loss. The proposed studies will clearly reveal the p53 action in preventing tumorigenesis in the brain, and should lead to basic understanding of cancer mechanisms and provide the basis to develop strategies for cancer detection and treatment.