EphA2 Agonists as Novel Inhibitors of Tumor Progression

该提案的总体目标是测试EphA2激酶激动剂是否可以通过在申请人的实验室中发现的新的负信号通路来抑制前列腺癌的进展。肿瘤转移有两个基本步骤：一是肿瘤细胞的扩散，二是肿瘤远端部位的生长。细胞运动是肿瘤细胞扩散的中心，而转移生长是由自分泌和旁分泌因子控制的，这些因子往往汇聚在RAS/MAPK信号通路上。因此，能够抑制细胞运动或抑制MAPK激活的药物是抗癌药物开发的潜在候选者。申请人的实验室证明，EphA2受体酪氨酸激酶(RTK)可以通过靶向整合素和粘着斑激酶来抑制细胞迁移，并通过降低RAS GTP负荷来减弱RAS/MAPK级联反应。此外，在人和狗的前列腺癌(PCA)中，EphA2的表达与肿瘤的进展相关。这些数据表明，EphA2是预防前列腺癌进展的新靶点。为此，我们从噬菌体展示文库中分离到了一个小的EphA2结合肽(EP1)。在功能上，EP1反映了ePhin-A1抑制细胞运动和生长的能力，确立了用小分子靶向EphA2的可行性。这项建议的目的是：1)在体内验证EphA2作为抗癌进展靶点。初步研究将评估EphA2激动剂如何影响绿色荧光蛋白(GFP)标记的PC-3细胞的皮下肿瘤生长。在第二阶段的研究中，GFP-PC-3细胞将被原位植入，以确定EphA2激动剂对转移的影响，这将部分通过活体动物的全身成像进行监测，并通过使用荧光立体显微镜的尸检器官成像进行监测。2)测定EphA2配体结合区与Ephin-A1或EP-1多肽复合物的X射线晶体结构。这些结构信息将用于指导选择更小和更有效的多肽激动剂。3)确定EphA2激活抑制Ras/MAPK通路的效应分子。拟议研究的完成可能会导致前列腺癌的新的基于机制的治疗药物和策略。

The overall objective of this proposal is to test whether EphA2 kinase agonists can inhibit prostate cancer progression through novel negative signaling pathways identified in the applicant's laboratory. There are two basic steps in tumor metastasis: one is tumor cell dissemination and the other is growth at distal sites. Cell motility is central to tumor cell dissemination, while metastatic growth is governed by autocrine and paracrine factors that frequently converge on Ras/MAPK signaling pathway. Therefore, agents that can inhibit either cell motility or suppress MAPK activation are potential candidates for anti-cancer drug discovery. The applicant's laboratory demonstrates that EphA2 receptor tyrosine kinase (RTK) can inhibit cell migration by targeting integrins and focal adhesion kinase, and attenuate Ras/MAPK cascade by lowering Ras GTP loading. Moreover, in both human and canine prostate cancer (PCa), EphA2 expression is correlated with tumor progression. These data suggest that EphA2 is a novel target to prevent progression of PCa. To this end, a small EphA2-binding peptide (EP1) has been isolated from phage display libraries. Functionally, EP1 mirrors ephrin-A1 in its ability to suppress cell motility and growth, establishing the feasibility of targeting EphA2 with small molecules. The aims of this proposal are: 1) To validate EphA2 as an anti-cancer progression target in vivo. Initial studies will assess how EphA2 agonists affect subcutaneous tumor growth of green fluorescence protein (GFP)- tagged PC-3 cells. In the second phase studies, GFP-PC-3 cells will be implanted orthotopically to determine the effects of EphA2 agonists on metastasis, which will be monitored in part by whole body imaging in live animals, and by organ imaging at necropsy using fluorescence stereomicroscope. 2) To determine X- ray crystallographic structure of EphA2 ligand-binding domain in complex with ephrin-A1 or EP-1 peptide. The structural information will be used to guide the selection of smaller and more potent peptide agonists. 3) To identify the effectors which mediate the inhibitory effects of EphA2 activation on Ras/MAPK pathway. Completion of the proposed studies can lead to new mechanism-based therapeutic agents and strategies for prostate cancer.