EphA2-Induced Angiogenesis in Ewing Sarcoma Cells Works through bFGF Production and Is Dependent on Caveolin-1

Angiogenesis is the result of the combined activity of the tumor microenvironment and signaling molecules. The angiogenic switch is represented as an imbalance between pro- and anti-angiogenic factors and is a rate-limiting step in the development of tumors. Eph receptor tyrosine kinases and their membrane-anchored ligands, known as ephrins, constitute the largest receptor tyrosine kinase (RTK) subfamily and are considered a major family of pro-angiogenic RTKs. Ewing sarcoma (EWS) is a highly aggressive bone and soft tissue tumor affecting children and young adults. As other solid tumors, EWS are reliant on a functional vascular network for the delivery of nutrients and oxygen and for the removal of waste. Based on the biological roles of EphA2 in promoting angiogenesis, we explored the functional role of this receptor and its relationship with caveolin-1 (CAV1) in EWS angiogenesis. We demonstrated that lack of CAV1 results in a significant reduction in micro vascular density (MVD) on 3 different in vivo models. In vitro, this phenomenon correlated with inactivation of EphA2 receptor, lack of AKT response and downregulation of bFGF. We also demonstrated that secreted bFGF from EWS cells acted as chemoattractant for endothelial cells. Furthermore, interaction between EphA2 and CAV1 was necessary for the right localization and signaling of the receptor to produce bFGF through AKT and promote migration of endothelial cells. Finally, introduction of a dominant-negative form of EphA2 into EWS cells mostly reproduced the effects occurred by CAV1 silencing, strongly suggesting that the axis EphA2-CAV1 participates in the promotion of endothelial cell migration toward the tumors favoring EWS angiogenesis.

血管生成（Angiogenesis）是肿瘤微环境（tumor microenvironment）与信号分子协同作用的共同结果。血管生成开关（angiogenic switch）表现为促血管生成因子与抗血管生成因子间的失衡，是肿瘤发生发展中的限速步骤。Eph受体酪氨酸激酶（Eph receptor tyrosine kinases）及其膜锚定配体（membrane-anchored ligands）——即Ephrin（ephrins）——构成了规模最大的受体酪氨酸激酶（receptor tyrosine kinase, RTK）亚家族，被认为是促血管生成受体酪氨酸激酶的主要家族之一。尤因肉瘤（Ewing sarcoma, EWS）是一类侵袭性极强的骨与软组织肿瘤，好发于儿童及青年群体。与其他实体瘤类似，尤因肉瘤依赖功能性血管网络以获取营养物质与氧气，并清除代谢废物。鉴于EphA2（EphA2）在促进血管生成过程中的生物学功能，本研究探究了该受体及其与小窝蛋白1（caveolin-1, CAV1）的关联在尤因肉瘤血管生成中的作用。实验证实，在3种不同的体内模型（in vivo models）中，小窝蛋白1的缺失会显著降低微血管密度（microvascular density, MVD）。体外实验中，该现象与EphA2受体失活、AKT通路应答缺失以及碱性成纤维细胞生长因子（basic fibroblast growth factor, bFGF）表达下调密切相关。本研究同时证实，尤因肉瘤细胞分泌的碱性成纤维细胞生长因子可作为内皮细胞（endothelial cells）的趋化因子。进一步研究发现，EphA2与小窝蛋白1的相互作用是受体正确定位及信号传导的必要条件，该过程可通过AKT通路介导碱性成纤维细胞生长因子的产生，并促进内皮细胞迁移。最后，向尤因肉瘤细胞中导入显性负突变形式的EphA2，基本重现了小窝蛋白1基因沉默所产生的生物学效应，这强烈提示EphA2-CAV1信号轴参与介导内皮细胞向肿瘤组织的迁移，从而促进尤因肉瘤的血管生成。