Functional Evaluation of Human Pancreatic Cancer Genes in a Zebrafish System

胰腺癌仍然是人类最致命的恶性肿瘤之一。在过去的十年里， 在确定这种疾病的遗传基础方面取得了前所未有的进展，包括 一些常见的体细胞突变的发现现在被证实起着重要的致病作用。 然而，近年来全基因组缺失分析和高通量DNA测序的应用 加快了新突变检测的速度，远远超过了从功能上评估识别的能力 候选基因。基因发现和功能注释之间的这种不匹配只会随着 完成已经在进行中的胰腺癌基因组测序，这是目前的一项工作 被约翰霍普金斯大学的调查人员追捕。为了缓解这一瓶颈，并提供 系统用于更高吞吐量的胰腺癌基因组注释，我们已经生成了第一个 斑马鱼胰腺外分泌癌模型的建立。基于低成本和适中的占地面积 保持成年斑马鱼以及快速产生大量转基因品系的能力，这 生物体在评价人类癌症的分子基础方面具有许多优势。当一个致癌性的 人类KRAS版本在斑马鱼发育中的胰腺中表达，胰腺前体细胞未能表达 经历正常的外分泌分化，导致随后形成侵袭性胰腺癌。 斑马鱼胰腺癌侵袭和转移，表现出许多与人类相似的特征。 疾病的形式，包括刺猬信号的异常激活。此外，还创建了第一个 斑马鱼外分泌型胰腺癌模型的建立，我们成功地获得了转基因品系，在其中 修饰的Gal4转录激活子在胰腺祖细胞中表达。使用转座子 将UAS调控的转基因插入斑马鱼基因组的技术，我们现在有机会 对多种基因损伤改变胰腺癌始发的能力进行功能性评估 和/或进展，达到在鼠标中技术上不可行的吞吐量水平。使用这些 技术，我们现在计划追求以下具体目标：第一，对候选对象进行功能注释 在胰腺癌基因组中发现的显性突变，通过将它们模块化地引入 斑马鱼肿瘤发生模型的建立；第二，研究hMYC基因表达的渐进性变化对 利用可诱导的Gal4/UAS系统靶向斑马鱼的祖细胞进行胰腺肿瘤的发生 第三，建立KRAS介导的小鼠胰腺肿瘤模型。 斑马鱼。综上所述，这些研究将提供重要的新信息，关于遗传基础的 胰腺癌，使有效的靶向治疗得以更快地发展。 相关性(请参阅说明)： 胰腺癌是人类最致命的恶性肿瘤之一，五年存活率为 低于5%，而且这一数字在过去40年中没有变化。通过确定人类的遗传基础 这种疾病，我们的计划将产生临床相关信息，很可能直接影响 化学预防、早期发现和治疗的战略。

Pancreatic cancer remains one of the most deadly human malignancies. During the past decade, unprecedented progress has been made identifying the genetic basis for this disease, including the discovery of a number of common somatic mutations now confirmed to play important pathogenic roles. However, the recent application of whole genome deletion analysis and high throughput DNA sequencing has accelerated the rate of novel mutation detection well beyond any ability to functionally evaluate identified candidate genes. This mismatch between gene discovery and functional annotation will only increase with the completion of the already in-progress sequencing of the pancreatic cancer genome, an effort currently being pursued by investigators here at Johns Hopkins. In order to alleviate this bottleneck, and provide a system for higher throughput annotation of the pancreatic cancer genome, we have generated the first zebrafish model of exocrine pancreatic cancer. Based on the low costs and modest floorspace required to maintain adult zebrafish, as well as the ability to rapidly generate large numbers of transgenic lines, this organism offers many advantages in evaluating the molecular basis of human cancer. When an oncogenic version of human KRAS is expressed in developing zebrafish pancreas, pancreatic progenitor cells fail to undergo normal exocrine differentiation, leading to the subsequent formation of invasive pancreatic cancer. Zebrafish pancreatic cancers invade and metastasize, and exhibit many features in common with the human form of the disease, including abnormal activation of hedgehog signaling. In addition creating the first zebrafish model of exocrine pancreatic cancer, we have successfully generated transgenic lines in which a modified Gal4 transcriptional activator is expressed in pancreatic progenitor cells. Using transposon technology to insert UAS-regulated transgenes into the zebrafish genome, we now have the opportunity to functionally evaluate a wide variety of genetic lesions for their ability to modify pancreatic cancer initiation and/or progression, achieving a level of throughput not technically feasible in the mouse. Using these techniques, we now plan to pursue the following Specific Aims: First, to functionally annotate candidate dominant mutations identified in the pancreatic cancer genome, through their modular introduction into the zebrafish tumorigenesis model; second, to study the effects of graded changes in hMYC expression in pancreatic tumorigenesis, using an inducible Gal4/UAS system targeting progenitor cells in zebrafish exocrine pancreas; and third, to develop Cre-based models of KRAS-mediated pancreatic neoplasia in zebrafish. Together, these studies will provide important new information regarding the genetic basis for pancreatic cancer, allowing for the more rapid development of effective targeted therapies. RELEVANCE (Seeinstructions): Pancreatic cancer represents one of the most deadly human malignancies, with five year survival rates of less than 5% and no change in this figure over the past four decades. By determining the genetic basis for this disease, our program will generate clinically relevant information that is likely to directly impact on strategies for chemoprevention, early detection and treatment.