Sensitized screen to identify cooperating genes involved in pancreatic cancer

描述(申请人提供)：癌症是一个多步骤的过程，需要积累大量的基因突变。识别参与正常细胞转化的基因是开发新的生物标记物和新的治疗方案的第一步。胰腺癌的发展仍然是一种致命的疾病。在北美，5年存活率不到5%。现有的胰腺癌模型系统的数量已经大大扩展到包括动物和细胞培养模型，这些模型能够确定特定的基因改变在胰腺癌发展中的作用。诊断的关键失误之一是通常在疾病进展的后期，当症状第一次被注意到时。在识别癌细胞内发生的导致胰腺上皮内瘤变(Panin)导致导管腺癌(PDAC)的致病突变方面取得了进展。动物模型的使用一直局限于研究已知基因及其相互作用。然而，动物模型的主要优势之一是将它们用作基因发现工具。这个试点项目的目的是建立一个系统，该系统将有助于识别在胰腺癌发展过程中与肿瘤启动和进展有关的基因。我们最近在实验室引进了睡美人转座子系统。睡美人转座子是一种可转座的可移动元件，需要两个互不相连的遗传位点，已被证明可在小鼠中诱导肿瘤发生。利用条件转座酶基因座，我们可以特异性地在胰腺谱系中诱导可转座元件的动员。我们假设，睡美人(SB)对胰腺的特异性诱导将加速小鼠胰腺肿瘤的发生和发展。携带胰腺特异致癌基因Kras等位基因的小鼠表现出完全外显性胰腺上皮内肿瘤(Panins)，这是PDAC的前驱症状。我们建议启动一项敏化筛查，以确定在携带KrasG12D等位基因的小鼠中，Sb在胰腺谱系内的转位是否会导致潜伏期缩短和更快地进展为PDAC。在胰腺系和衰老小鼠中，转座子和激活突变将被激活。将对发生的胰腺肿瘤进行采集和分析。病理分析将提供与肿瘤类型和进展有关的数据。分子分析将包括转座子插入位点的分离和测序。与KrasG12D协同作用的基因组位点将被确定。此外，我们还将确定可能在野生型小鼠体内诱发PDAC的基因座。这项建议将1)建立睡美人转座子系统，作为分离和分析协同致癌基因的有价值的工具，以及2)特异性地分离与胰腺癌相关的基因。与公共卫生相关：我们将建立一个新的癌基因和肿瘤抑制基因的筛查，这些基因与与90%的人类胰腺癌相关的癌基因特异性地协同作用。我们将确定发生在胰腺内的基因事件，这些事件表达激活的Kras等位基因，导致肿瘤转化。这些基因的识别将提供新的生物标记物，用于监测人类胰腺癌的发展，以及识别靶向新治疗方案的分子途径。

DESCRIPTION (provided by applicant): Cancer is a multistep process, which requires the accumulation of numerous mutations in genes. The identification of the genes involved in the transformation of normal cells is a first step in the development of new biomarkers and new therapeutic regimens. The development of pancreatic cancer is still a deadly disease. The 5 year survival rate is less than 5% in North America. The number of available model systems of pancreatic cancer has greatly expanded to include both animal and cell culture models amenable to define the role of specific genetic alterations in the development of pancreatic cancer. One of the critical failings of diagnosis is the usual late stage of disease progression when symptoms are first noted. Advances have been made in the identification of the causative mutations that occur within the cancer cell that contributes to the initiation of pancreatic intraepithelial neoplasia (PanIN) leading to ductal adenocarcinoma (PDAC). The use of animal models has been limited to the study of known genes and their interactions. However, one of the main advantages of animal models is their use as a gene discovery tool. The aim of this pilot project is to establish a system which would facilitate the identification of genes that cooperate in tumor initiation and progression in the development of pancreatic cancer. We have recently imported into our laboratory the Sleeping Beauty transposon system. The sleeping beauty transposon is a transposable mobile element that requires two unlinked genetic loci and has been shown to induce tumorigenesis in the mouse. Making use of a conditional transposase locus we can specifically induce mobilization of the transposable element within the pancreatic lineage. We are hypothesizing that the pancreatic specific induction of Sleeping Beauty (SB) will accelerate the development and progression of pancreatic tumorigenesis in mice. Mice harboring a pancreatic specific oncogenic Kras allele exhibit complete penetrance of developing pancreatic intraepithelial neoplasm (PanINs) which are precursors to PDAC. We are proposing to initiate a sensitized screen to determine if SB transposition within the pancreatic lineage results in a decreased latency and a more rapid progression to PDAC in mice harboring the KrasG12D allele. The transposon and activating mutation will be activated in the pancreatic lineage and the mice aged. Pancreatic tumors that develop will be harvested and analyzed. Pathological analysis will provide the data relating to tumor type and progression. Molecular analysis will encompass the isolation and sequencing of the transposon insertion sites. The genomic loci that cooperate with the KrasG12D will be identified. In addition, we will be identifying loci that can potentially induce PDACs within wildtype mice as well. This Proposal will 1) establish the Sleeping Beauty Transposon System as a valuable tool for the isolation and analysis of cooperating oncogenic loci as well as 2) specifically isolate genes associated with pancreatic cancer. PUBLIC HEALTH RELEVANCE: We will be establishing a screen for novel oncogenes and tumor suppressor genes that specifically cooperate with an oncogene associated with >90% of human pancreatic cancer. We will identify the genetic events that occur within the pancreas that express the activated Kras allele that results in neoplastic transformation. The identification of these genes would provide new biomarkers with which to monitor the development of pancreatic cancer in humans as well as to identify molecular pathways with which to target new therapeutic regimens.