Enhancer/suppressor screens for angiogenic signaling

描述(申请人提供)：血管生成是人类肿瘤进展的限速步骤。参与肿瘤血管形成的关键蛋白是很好的治疗靶点，因为形成血管的细胞是由遗传稳定的细胞组成的，而不是肿瘤本身的恶性细胞。血管内皮生长因子受体被认为是胚胎和肿瘤血管生成的主要调节因子，因此，目前是抗血管生成治疗的靶点。斑马鱼胚胎的透明度使其成为血管生成研究的理想模型，因为活体动物很容易观察到血液流动。在初步研究中，人血管内皮生长因子受体抑制剂PTK787/ZK222584被用来诱导斑马鱼胚胎血管的有效抑制。这种化学遗传学方法允许检查血管内皮生长因子受体信号通路，因为下游效应器AKT/PKB的上调可以覆盖受体阻断以提供强大的血管救援(Chan等人，2002年)。在这项拨款提案中，化学抑制与已证实的正向遗传学相结合，以确定血管生成信号中的关键基因作为抗血管生成表型的增强器或抑制器。在PTK787的影响下，利用F2杂合子胚胎进行了中试筛选。在F3纯合子隐性胚胎中，血管生成缺陷已被遗传学证实。因此，大规模的筛查将使我们能够确定在血管内皮生长因子信号通路中与生理相关的角色，作为癌症治疗中抑制剂治疗的额外靶点。该应用程序有两个具体目标。目的1.在药物致敏的血管内皮生长因子受体作用下进行血管生成突变的增强子筛查。目的2.在斑马鱼胚胎中进行抑制基因筛查，寻找可以盖过药物诱导的抗血管生成作用的突变。

DESCRIPTION (provided by applicant): Angiogenesis is a rate-limiting step in the progression of human tumors. Key proteins involved in tumor vessel formation are good targets for therapy, since the forming vasculature is composed of genetically stable cells unlike the malignant cells in the tumor proper. The vascular endothelial growth factor (VEGF) receptors are thought to be master regulators of embryonic and tumor angiogenesis; thus, are currently targets for anti-angiogenic therapy. The transparency of the zebrafish embryo makes it an ideal model for angiogenic studies since blood flow can be easily observed in a living animal. In preliminary studies, a human VEGF receptor inhibitor, PTK787/ZK222584, was used to induce potent inhibition of embryonic vessels in the zebrafish. This chemcial genetic approach allowed an examination of the VEGF receptor signaling pathway as an upregulation of a downstream effector, AKT/PKB, can override the receptor block to provide a robust vessel rescue (Chan et al., 2002). In this grant proposal, chemical inhibition is combined with proven forward genetics to identify critical genes in angiogenic signaling as enhancers or suppressors of the anti-angiogenic phenotype. A pilot screen has been conducted using F2 heterozygous embryos under the influence of PTK787. Angiogenic defects have been confirmed genetically in F3 homozygous recessive embryos. Thus, a large scale screen will allow us to identify physiologically relevant players in the VEGF signaling pathway as additional targets for inhibitor therapy in cancer treatments. The application has two specific aims. Aim 1. To perform an enhancer screen for angiogenic mutations under drug-sensitized VEGF receptor function. Aim 2. To perform a suppressor screen for mutations that can override the drug-induced anti-angiogenics effects in zebrafish embryos.