Transposon mutagenesis screen for mammalian cancer gene discovery

描述（由申请人提供）：癌症研究主要集中在将正常人类细胞转化为恶性癌症所需的遗传变化上。因此，人们认为正常的人体细胞大约需要5次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的身份仍然不明确。构成大多数上皮组织（如表皮）的终末分化细胞不断被替换，这使得它们不太可能积累肿瘤形成所需的基因命中数量。有趣的是，体细胞本身就具有癌细胞的一些标志性特征，这提高了肿瘤可能通过更少甚至是单一突变事件从干细胞中产生的可能性。然而，由于操纵和诱变干细胞群的困难，缺乏这种“癌症干细胞假说”的直接证据。最近，我们的实验室在哺乳动物细胞和小鼠中证明了piggyBac （PB）转座子的高效转座。通过将转座酶（PBase）从PB中分离出来，我们生成了一个两部分系统，其中细胞中两种成分的存在对于转座是必要的。空间调节的PB转位可以通过组织特异性启动子表达PBase来实现，使该系统特别适合靶向干细胞群体进行突变。本建议的总体目标是利用PB插入突变使表皮干细胞突变，筛选促进肿瘤发生的突变，并确定受影响的基因。我们将实现以下目标：(1)我们将诱变培养的人类和小鼠表皮角质形成细胞，并利用细胞转化试验筛选体外恶性转化，用于锚定独立生长。(2)我们将通过在表皮干细胞中特异性表达转基因PBase，在体内诱变表皮干细胞，从而调动小鼠体内的PB。然后将对这些小鼠进行皮肤肿瘤形成筛查。这项研究的结果将允许直接评估癌症干细胞假说，并促进识别在癌症发生和发展中重要的基因和途径。相关性：本提案旨在开发新的研究方法，以鉴定对许多人类疾病重要的基因。具体来说，这项研究的结果将有助于识别与人类癌症起始有关的新基因。这些信息将增加对癌症如何发展的总体理解，并确定癌症治疗的潜在目标。

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.