Functional Evaluation of Human Pancreatic Cancer Genes in a Zebrafish System

斑马鱼系统中人类胰腺癌基因的功能评估

基本信息

批准号：
8464656
负责人：
Steven D Leach
金额：
$ 33.07万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-04-01 至 2015-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8464656
关键词：
Adult Cancer Biology Cancer Model Candidate Disease Gene Chemoprevention DNA Sequence Data Development Disease Early Diagnosis Early treatment Embryo Evaluation Event Exhibits Exocrine pancreas Gene Dosage Gene Expression Genetic Genome Human Invaded KRAS2 gene Laboratories Lesion Malignant Neoplasms Malignant neoplasm of pancreas Mediating Methods MicroRNAs Modeling Molecular Mus Mutation Mutation Detection Neoplasm Metastasis Oncogenes Oncogenic Organism Pancreas Participant Phenotype Physiology Play Program Research Project Grants Reagent Regulatory Element Research Personnel Role Somatic Mutation Stem cells Survival Rate System Techniques Technology Transcription Coactivator Transgenes Transgenic Organisms Zebrafish base cancer genome cancer initiation clinically relevant cost deletion analysis gain of function gene discovery genome sequencing human disease loss of function novel pancreatic neoplasm pancreatic tumorigenesis positional cloning progenitor programs second grade smoothened signaling pathway transgene expression tumorigenesis zebrafish genome

项目摘要

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.

胰腺癌仍然是最致命的人类恶性肿瘤之一。在过去的十年中，在确定这种疾病的遗传基础方面取得了前所未有的进展，包括发现了许多常见的体细胞突变，现已证实发挥重要的致病作用。然而，最近全基因组缺失分析和高通量 DNA 测序的应用加快了新突变检测的速度，远远超出了对已识别候选基因进行功能评估的能力。随着约翰霍普金斯大学研究人员目前正在进行的胰腺癌基因组测序工作的完成，基因发现和功能注释之间的不匹配只会增加。为了缓解这一瓶颈，并提供一个更高通量的胰腺癌基因组注释系统，我们生成了第一个外分泌胰腺癌斑马鱼模型。基于维持成年斑马鱼所需的低成本和适度的占地面积，以及快速产生大量转基因品系的能力，这有机体在评估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，胰腺祖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。斑马鱼胰腺癌会侵袭和转移，并表现出许多与人类疾病相同的特征，包括刺猬信号的异常激活。除了创建第一个外分泌胰腺癌斑马鱼模型外，我们还成功生成了转基因株系，其中修饰的 Gal4 转录激活因子在胰腺祖细胞中表达。使用转座子技术将 UAS 调节的转基因插入斑马鱼基因组中，我们现在有机会对多种遗传病变进行功能评估，了解它们改变胰腺癌发生和/或进展的能力，从而达到技术上不可行的通量水平。鼠标。利用这些技术，我们现在计划实现以下具体目标：首先，通过将胰腺癌基因组中确定的候选显性突变模块化引入斑马鱼肿瘤发生模型中，对它们进行功能注释；其次，使用针对斑马鱼外分泌胰腺祖细胞的诱导型 Gal4/UAS 系统，研究 hMYC 表达的分级变化对胰腺肿瘤发生的影响；第三，开发基于 Cre 的 KRAS 介导的斑马鱼胰腺肿瘤模型。总之，这些研究将提供有关胰腺癌遗传基础的重要新信息，从而更快地开发有效的靶向治疗。