Enhancer/suppressor screens for angiogenic signaling

描述（由申请人提供）：血管生成是人类肿瘤进展中的限速步骤。参与肿瘤血管形成的关键蛋白质是治疗的良好靶点，因为形成的脉管系统由遗传稳定的细胞组成，与肿瘤中的恶性细胞不同。血管内皮生长因子（VEGF）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。斑马鱼胚胎的透明性使其成为血管生成研究的理想模型，因为在活体动物中可以很容易地观察到血流。在初步研究中，使用人VEGF受体抑制剂PTK 787/ZK 222584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VEGF受体信号传导途径，因为下游效应物AKT/PKB的上调可以超越受体阻断以提供稳健的血管拯救（Chan等人，2002年）。在这项资助提案中，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子或抑制子。在PTK 787的影响下，使用F2杂合胚胎进行了中试筛选。在F3纯合子隐性胚胎中，血管生成缺陷已在遗传学上得到证实。因此，大规模筛选将使我们能够鉴定VEGF信号通路中的生理相关参与者作为癌症治疗中抑制剂疗法的额外靶点。该申请有两个具体目标。目标1.在药物致敏的VEGF受体功能下进行血管生成突变的增强子筛选。目标二。在斑马鱼胚胎中进行抑制突变的筛选，以克服药物诱导的抗血管生成作用。

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.