EphA2 Agonists as Novel Inhibitors of Tumor Progression

本提案的总体目标是测试EphA 2激酶激动剂是否可以通过申请人实验室中鉴定的新型负信号传导途径抑制前列腺癌进展。 肿瘤转移有两个基本步骤：一个是肿瘤细胞播散，另一个是在远端部位生长。 细胞运动是肿瘤细胞播散的核心，而转移生长是由自分泌和旁分泌因子控制的，这些因子经常聚集在Ras/MAPK信号通路上。 因此，可以抑制细胞运动或抑制MAPK活化的药剂是抗癌药物发现的潜在候选者。 申请人的实验室证明EphA 2受体酪氨酸激酶（RTK）可以通过靶向整联蛋白和粘着斑激酶来抑制细胞迁移，并通过降低Ras GTP负载来减弱Ras/MAPK级联。 此外，在人和犬前列腺癌（PCa）中，EphA 2表达与肿瘤进展相关。这些数据表明EphA 2是预防PCa进展的新靶标。 为此，已经从噬菌体展示文库中分离出小的EphA 2结合肽（EP 1）。在功能上，EP 1反映了肝配蛋白A1抑制细胞运动和生长的能力，建立了用小分子靶向EphA 2的可行性。 该提议的目的是：1）验证EphA 2作为体内抗癌进展靶标。 初步研究将评估EphA 2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。 在第二阶段研究中，将原位植入GFP-PC-3细胞以确定EphA 2激动剂对转移的影响，这将部分地通过活体动物中的全身成像以及通过尸检时使用荧光立体显微镜的器官成像来监测。2)确定与肝配蛋白-Al或EP-1肽复合的EphA 2配体结合结构域的X射线晶体学结构。 结构信息将用于指导更小和更有效的肽激动剂的选择。3)鉴定EphA 2激活对Ras/MAPK通路的抑制作用的效应物。 完成拟议的研究可能会导致新的机制为基础的治疗药物和前列腺癌的战略。

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.