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，包括发现了一些常见的体细胞突变，现在被证实发挥了重要的致病作用。然而，最近全基因组缺失分析和高通量DNA测序的应用已经大大加快了新突变检测的速度，远远超过了对已识别的候选基因进行功能评估的能力。随着已经在进行中的胰腺癌基因组测序的完成，基因发现和功能注释之间的这种不匹配只会增加，约翰·霍普金斯大学的研究人员目前正在进行这项工作。为了缓解这一瓶颈，并提供一个更高吞吐量的胰腺癌基因组注释系统，我们建立了第一个胰腺外分泌癌斑马鱼模型。基于维持成年斑马鱼所需的低成本和有限的占地面积，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。当人类KRAS的致癌版本在发育中的斑马鱼胰腺中表达时，胰腺前体细胞无法进行正常的外分泌分化，导致随后形成侵袭性胰腺癌。斑马鱼胰腺癌侵袭和转移，并表现出许多与人类形式的疾病相似的特征，包括刺猬信号的异常激活。除了建立第一个斑马鱼胰腺外分泌癌模型外，我们还成功地获得了转基因系，其中修饰的Gal4转录激活因子在胰腺前体细胞中表达。利用转座子技术将UAS调控的转基因插入斑马鱼基因组，我们现在有机会从功能上评估各种遗传损伤改变胰腺癌启动和/或进展的能力，实现在小鼠中技术上不可行的吞吐水平。利用这些技术，我们现在计划追求以下具体目标：第一，通过将它们模块化地引入斑马鱼肿瘤发生模型中，从功能上注释在胰腺癌基因组中确定的候选显性突变；第二，利用可诱导的Gal4/UAS系统，靶向斑马鱼外分泌胰腺的前体细胞，研究hMYC表达的分级变化在胰腺肿瘤发生中的作用；以及第三，建立基于CRE的斑马鱼KRAS介导的胰腺肿瘤模型。总之，这些研究将提供有关胰腺癌遗传基础的重要新信息，使有效的靶向治疗得以更快地发展。

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.