Cancer gene discovery using Sleeping Beauty transposon-mediated mutagenesis

描述(由申请人提供)：在这项提案中，斯塔尔博士将使用睡美人(SB)转座子为基础的突变来筛选新的癌症基因。已识别基因的蛋白质产物将通过改变它们在细胞系中的水平，并测量对增殖、凋亡、克隆形成和信号通路的影响，来分析它们在人类癌症中的作用。SB系统已被用于推动小鼠肿瘤的发生，并随后确定了驱动白血病、肉瘤、肝细胞癌和结直肠癌的已知和新的基因突变。然而，一些组织类型已被证明对SB诱变不敏感。为了成功筛选这些组织中的基因，斯塔尔博士提出了对SB系统的几项修改。 斯塔尔博士在明尼苏达大学共济会癌症中心(MCC)担任博士后研究员期间，对开发SB系统起到了重要作用。为了帮助斯塔尔博士过渡到独立的研究生涯，他组建了一个顾问委员会，由几个不同领域的专家组成，这些领域对他的研究和职业发展的成功至关重要。除了他的导师和咨询委员会提供的直接支持外，斯塔尔博士还将使用MCC可用的共享资源。这些核心设施包括生物成像、组织采购、流式细胞仪、比较病理学、细胞遗传学、生物信息学/生物统计学、RNA干扰、高通量测序，以及能够产生转基因、敲除和敲入小鼠的小鼠遗传学实验室。候选人打算继续他的博士后研究两年，同时致力于这项研究提案的目标1和2。斯塔尔博士的长期目标是在学术领域获得一份独立研究员的职位，他计划把自己的职业生涯献给研究癌症的遗传机制。 记录人类肿瘤基因组内遗传变化的研究证实，大量的遗传缺陷有助于肿瘤的生长，但只有一小部分被表征。此外，在同一种肿瘤类型中，不同肿瘤之间存在很大的异质性。这项提案中的研究解决了癌症研究领域中的一个关键需求，即开发更好的方法来识别实际上推动肿瘤生长的基因变化的子集。在特定的目标1和2中，斯塔尔博士在结直肠癌(WAC、Tcf12和Rsp2)和肺癌(Akap13、CUL3和Map4k3)筛查中发现的几个新的候选癌症驱动基因将被研究，以确定它们在人类癌症中的特定作用。这些蛋白质的水平将在人类细胞系中发生变化，无论是正常的还是转化的，以确定它们对生长、凋亡、在软琼脂中形成集落的能力以及关键信号通路的调节的影响。目的3提出了几个改进的SB系统，使用新开发的高活性转座酶和转座子，以发现新的肺癌基因。这些研究的结果可以用来开发更个性化的癌症治疗方法。 公共卫生相关性：该项目的目标是使用动物模型来了解肺癌和结直肠癌的遗传基础。使用这种动物模型发现的基因突变随后将在人类肺癌和结直肠癌组织和细胞系中进行功能测试。这些研究的结果将帮助我们决定如何更好地治疗这些常见的、致命的癌症。

DESCRIPTION (provided by applicant): In this proposal Dr. Starr will use Sleeping Beauty (SB) transposon-based mutagenesis to screen for novel cancer genes. The protein products of identified genes will be analyzed for their role in human cancer by altering their levels in cell lines and measuring the effect on proliferation, apoptosis, colony formation, and signaling pathways. The SB system has been used to drive tumorigenesis in mice and subsequently identify both known and novel genetic mutations that drive leukemias, sarcomas, hepatocellular carcinomas, and colorectal cancers. Some tissue types, however, have proven refractory to SB mutagenesis. To successfully screen for genes in these tissues Dr. Starr has proposed several modifications to the SB system. Dr. Starr has been instrumental in developing the SB system during his tenure as a post-doctoral fellow in the Masonic Cancer Center (MCC) at the University of Minnesota. To help Dr. Starr transition to an independent research career, he has assembled an advisory committee consisting of experts in several different areas critical for the success of his research and career development. In addition to the direct support provided by his Mentor and advisory committee, Dr. Starr will use the shared resources available in the MCC. These include core facilities in biological imaging, tissue procurement, flow cytometry, comparative pathology, cytogenetics, bioinformatics/biostatistics, RNA interference, high-throughput sequencing, and a Mouse Genetics Laboratory capable of generating transgenic, knock-out, and knock-in mice. The candidate intends to continue his post-doctoral fellowship for two years while working on Aims 1 and 2 of this research proposal. Dr. Starr's long-term goal is to secure a position as an independent investigator in an academic setting where he plans on dedicating his career to understanding the genetic mechanisms driving cancer. Studies documenting genetic changes within human tumor genomes confirm that a large number of genetic defects contribute to tumor growth, yet only a small subset have been characterized. Furthermore, within a single tumor type there is great heterogeneity between individual tumors. The studies in this proposal address a critical need in the field of cancer research to develop better methods of identifying the subset of genetic changes that are actually driving tumor growth. In Specific Aims 1 and 2, several novel candidate cancer-driving genes identified by Dr. Starr in a screen for colorectal cancer (Wac, Tcf12, and Rspo2) and lung cancer (Akap13, Cul3, and Map4k3) will be studied to determine their specific role in human cancer. The levels of these proteins will be altered in human cell lines, both normal and transformed, to determine their effect on growth, apoptosis, the ability to form colonies in soft agar, and regulation of key signaling pathways. Aim 3 proposes several improvements to the SB system, using newly developed highly active transposases and transposons, to uncover novel lung cancer genes. The results of these studies can be used to develop a more personalized approach to cancer therapy. PUBLIC HEALTH RELEVANCE: The goal of this project is to use an animal model to understand the genetic basis of lung and colorectal cancer. The genetic mutations discovered using this animal model will then be functionally tested in human lung and colorectal cancer tissue and cell lines. The results of these studies will help us decide how to better treat these common, deadly cancers.