流场下CD44 介导的肿瘤滚动促进整合素亲和度上调的分子机制的研究

CD44 is a family of glycoproteins highly expressed on melanoma cell surface. We previously found that under blood flow conditions, engagement of CD44 with selectins was responsible for melanoma rolling and initial tethering onto endothelium, while integrin αvβ3 mediated firm adhesion of tumor to endothelium by assuming extended conformation and elevating its affinity. However, the molecular mechanisms of modulation of integrin affinity by CD44-mediated cell rolling remain elusive. Therefore, in the current proposal, we plan to investigate the mechanisms of integrin activation primed by CD44-selectin engagement through mechanotransduction. By employing flow chamber cell adhesion assay, western blotting and gene silencing, we will interrogate the effect of CD44-selectin binding on initiation of mechanotransduction involving PKC and p38 to facilitate integrin-mediated tumor firm adhesion. We will then dissect integrin αvβ3 affinity modulation by CD44-mediated cell rolling by using flow cytometry-based FRET techniques. Finally, by employing intravital microscopy, we will determine the role of CD44 rolling on blood vessel in regulating integrin activation-mediated tumor firm adhesion in vivo. This proposal will add new dimension of tumor mechanotransduction triggered by force-regulated bond formation to previous findings. Results from this proposal will shed light on the molecular mechanisms underlying melanoma metastasis, and provide insights into new therapeutic targets for treating melanoma.

CD44是黑色素瘤细胞表面高表达的糖基化蛋白，参与了血液流场下与选择素的相互作用并帮助肿瘤在内皮细胞表面滚动黏附。整合素αvβ3亲和度的提高可以增强肿瘤细胞的稳定黏附能力，但是CD44介导的细胞滚动对整合素亲和度的影响还不清楚。因此,本课题旨在探讨CD44-选择素结合所引起的力学信号转导所导致整合素亲和度增加的分子机制。本课题拟通过流动腔细胞黏附实验，蛋白水平的检测及RNA干扰，考察黑色素瘤CD44对选择素的黏附是否启动细胞内PKC及p38参与的力学信号通路，从而促进整合素介导的稳定粘附；并利用基于流式细胞仪的FRET，探讨CD44黏附对整合素αvβ3亲和度构象变化的影响；最后，通过活体成像，研究CD44介导的肿瘤细胞在血管壁上的滚动对细胞稳定黏附的作用。本课题将从肿瘤细胞黏附分子成键的力学调控所启动的胞内信号转导这个新的视角，揭示黑色素瘤转移的分子机制，对设计合理的治疗药物具有重要意义。

研究肿瘤与血管内皮细胞的相互作用是抑制肿瘤血道转移的前提。CD44是黑色素瘤表面高表达的糖基化蛋白。 项目研究黑色素瘤对内皮细胞黏附机制，采用平行板流动腔技术，FRET， 微管黏附实验，流式细胞仪技术，shRNA干扰，发现高侵袭力的肿瘤细胞高表达CD44v8-10。该剪辑型是由U2AF2剪接因子选择性剪接v8-10外显子产生的。 CD44v8-10相较CD44s对选择素有更高的亲和力，因此在流场下能有效介导黑色素瘤细胞在内皮细胞上滚动。CD44的黏附激活了细胞内的PKC及p38力学信号通路， 从而提高了整合素alphavbeta3的亲和度，介导了肿瘤的稳定黏附。通过spinning-disk confocal microscopy, 这一机制在体内得到验证。 通过共聚焦显微镜及共培养，项目证实了黑色素瘤能引起局部的凝血级联反应，生成的thrombin，能作用于内皮细胞上的PAR受体，磷酸化VE-cadherin细胞质端。引起内皮细胞连接的开放。该项目的完成为靶向抑制肿瘤血道转移奠定了基础。