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

• 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
• 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

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.

胰腺癌仍然是人类最致命的恶性肿瘤之一。在过去十年中，在确定这一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，现已证实这些突变发挥了重要的致病作用。然而，最近应用的全基因组缺失分析和高通量DNA测序加速了新的突变检测的速度远远超过任何能力，以功能评估确定的候选基因。基因发现和功能注释之间的这种不匹配只会随着胰腺癌基因组测序的完成而增加，这是约翰霍普金斯的研究人员目前正在进行的一项工作。为了缓解这一瓶颈，并提供一个系统的胰腺癌基因组的高通量注释，我们已经产生了第一个斑马鱼模型的外分泌胰腺癌。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， 生物体在评估人类癌症的分子基础方面提供了许多优势。当人类KRAS的致癌版本在发育中的斑马鱼胰腺中表达时，胰腺祖细胞不能进行正常的外分泌分化，导致随后形成侵袭性胰腺癌。斑马鱼胰腺癌的侵袭和转移，并表现出许多与人类形式的疾病共同的特征，包括刺猬信号的异常激活。除了建立第一个斑马鱼外分泌胰腺癌模型外，我们还成功地产生了转基因株系，其中修饰的Gal 4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，我们现在有机会在功能上评估各种遗传病变改变胰腺癌发生和/或进展的能力，实现在小鼠中技术上不可行的通量水平。使用这些技术，我们现在计划追求以下具体目标：首先，通过将其模块化引入斑马鱼肿瘤发生模型，功能性注释胰腺癌基因组中鉴定的候选显性突变;其次，使用靶向斑马鱼外分泌胰腺祖细胞的诱导型Gal 4/UAS系统，研究胰腺肿瘤发生中hMYC表达的分级变化的影响;第三，在斑马鱼中开发基于Cre的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，从而更快地开发有效的靶向治疗。