Transposon mutagenesis screen for mammalian cancer gene discovery

描述(申请人提供)：癌症研究主要集中在将正常人类细胞转化为恶性肿瘤所需的基因变化上。因此，人们认为正常的人类细胞大约需要五个突变才能转化为癌细胞。然而，对于大多数人类癌症来说，发生这些转化事件的细胞群体(S)的身份仍然不确定。构成大多数上皮组织的终末分化细胞，如表皮，不断被替换，因此它们不太可能积累形成肿瘤所需的基因命中数量。有趣的是，体细胞干细胞天生就具有癌细胞的一些特征，这增加了肿瘤可能是由干细胞通过较少的甚至可能是单一的突变事件而产生的可能性。然而，由于操纵和诱变干细胞种群的难度，这一“癌症干细胞假说”缺乏直接证据。最近，我们的实验室发现了在哺乳动物细胞和小鼠中高效的转座子。通过将转座酶(Pbase)从PB中分离出来，我们产生了一个两部分系统，在这个系统中，细胞中两个组分的存在是转座所必需的。通过表达组织特异性启动子的PBase，可以实现空间调控的PB转座，使该系统特别适合于靶向干细胞群进行突变。这项建议的总体目标是使用PB插入突变来突变表皮干细胞，筛选促进肿瘤发生的突变，并识别受影响的基因。我们将完成以下工作：(1)我们将对培养的人和小鼠表皮角质形成细胞进行诱变，并利用细胞转化实验进行体外恶性转化筛选，以实现锚定非依赖性生长。(2)体内诱变表皮干细胞，通过在表皮干细胞中特异性表达转基因PBase来动员小鼠外周血淋巴细胞。然后将对这些小鼠进行皮肤肿瘤形成的筛查。这项研究的结果将允许直接评估癌症干细胞假说，并有助于识别在癌症发生和发展中重要的基因和途径。相关性：这项提案寻求开发新的研究方法，以识别对许多人类疾病重要的基因。具体地说，这项研究的结果将有助于识别与人类癌症启动有关的新基因。这些信息将增加对癌症如何发展的总体了解，并确定癌症治疗的潜在靶点。

DESCRIPTION (provided by applicant): The study of cancer has focused primarily on the genetic changes required to transform normal human cells into malignant cancers. Accordingly, it is thought that normal human cells require approximately five mutations to transform into a cancer cell. However, the identity of the cell population(s) in which these transforming events occurs remains undefined for most human cancers. The terminally differentiated cells that constitute the majority of epithelial tissues, such as the epidermis, are continually replaced making it unlikely that they would accumulate the number of genetic hits required for tumor formation. Interestingly, somatic stem cells inherently possess some of the hallmark traits of cancer cells raising the possibility that tumors may arise from stem cells through fewer or perhaps even a single mutational event. However, direct evidence for this "cancer stem cell hypothesis" is lacking due to the difficulty of manipulating and mutagenizing stem cell populations. Recently, our lab demonstrated highly efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. By separating the transposase (PBase) from PB, we generated a bipartite system in which the presence of both components in a cell is necessary for transposition. Spatially regulated PB transposition can be achieved by expressing PBase from a tissue- specific promoter, making this system particularly amenable to targeting stem cell populations for mutagenesis. The overall aim of this proposal is to use PB insertional mutagenesis to mutate epidermal stem cells, screen for mutations that promote tumorigenesis, and identify the affected genes. We will accomplish this as follows: (1) We will mutagenize cultured epidermal keratinocytes from both human and mouse and screen for in vitro malignant transformation utilizing a cell transformation assay for anchorage independent growth. (2) We will mutagenize epidermal stem cells in vivo by expressing transgenic PBase specifically in these cells to mobilize PB in mice. These mice will then be screened for skin tumor formation. Results from this study will allow direct evaluation of the cancer stem cell hypothesis as well as facilitate the identification of genes and pathways important in the initiation and progression of cancer. Relevance: This proposal seeks to develop new research methods that will allow for the identification of genes important for many human diseases. Specifically, results from this study will facilitate the identification of new genes involved in the initiation of human cancers. This information will increase the general understanding of how cancer develops as well as identify potential targets for cancer therapy.