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，人们认为正常的人类细胞需要大约五个突变才能转化为癌细胞。然而，对于大多数人类癌症，发生这些转化事件的细胞群体的身份仍然不确定。构成大多数上皮组织（如表皮）的终末分化细胞不断被替换，使得它们不太可能积累肿瘤形成所需的遗传命中数。有趣的是，体干细胞本身具有癌细胞的一些标志性特征，这增加了肿瘤可能通过更少或甚至单个突变事件由干细胞产生的可能性。然而，由于操作和诱变干细胞群体的困难，这种“癌症干细胞假说”的直接证据缺乏。最近，我们的实验室证明了piggyBac（PB）转座子在哺乳动物细胞和小鼠中的高效转座。通过将转座酶（PBase）从PB中分离，我们产生了一个二分系统，其中细胞中两种组分的存在对于转座是必要的。空间调节的PB转座可以通过从组织特异性启动子表达PBase来实现，使得该系统特别适合于靶向干细胞群体进行诱变。该提案的总体目标是使用PB插入诱变来突变表皮干细胞，筛选促进肿瘤发生的突变，并鉴定受影响的基因。我们将通过以下方法完成这一工作：（1）我们将诱变培养的人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，以进行锚定非依赖性生长。(2)我们将通过在表皮干细胞中特异性表达转基因PBase来体内诱变这些细胞，以动员小鼠中的PB。然后将筛选这些小鼠的皮肤肿瘤形成。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：该提案旨在开发新的研究方法，以便能够确定对许多人类疾病具有重要意义的基因。具体来说，这项研究的结果将有助于鉴定参与人类癌症起始的新基因。这些信息将增加对癌症如何发展的一般理解，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.