乙酰肝素酶诱导的内皮细胞活化在恶性黑色素瘤凝血通路激活中的作用及机制研究

Human malignant melanoma is a highly metastatic tumor associated with poor prognosis and high resistance to treatment. To metastasize to distant organs, such as liver, lung, brain and bone, cancer cells must interact with vascular endothelial cells and migrate through the vessel wall. In our recent work, we could show that heparanase-1 (HPSE), the only known enzyme for degradation of the heparan sulfate (HS) side chains of the ECM, plays pivotal roles in the progression and metastasis of malignant melanoma. However, besides of the ECM remodeling, the activation of endothelium by tumor-derived secretagogues is also proven to be important for tumor cell extravasation. Interestingly, HPSE has.recently been demonstrated to be implicated in the coagulation cascade in several tumor entities, yet the detailed molecular mechanism remains poorly understood. Therefore, we hypothesize that tumor-derived HPSE is able to induce EC activation following by WPB exocytosis and a prompt luminal ULVWF secretion thereby facilitating coagulation progress in tumor microenvironment..In this project we will focus on the impact of HPSE in malignant melanoma related coagulation, in particular 1) HPSE-induced EC activation accompanied by WPB exocytosis in vitro; 2) different animal models will be established to confirm the hypothesis in vivo; 3) impact of HPSE on regulating the VWF degrading enzyme ADAMTS13 activity.

人类恶性黑色素瘤（MM）是一种高度转移性肿瘤，具有不良预后和高度耐药性。为了实现远处器官转移，例如肝、肺、脑和骨，癌细胞必须与血管内皮细胞（EC）相互作用并通过血管壁迁移。我们最近的工作表明乙酰肝素酶（HPSE）在MM的进展和转移中发挥着关键作用。此外，EC活化也被发现其对于肿瘤细胞的迁移是极其重要的。最近有研究表明HPSE涉及实体肿瘤的凝血级联反应，但其具体的分子机制仍未明确。基于此，我们假设肿瘤衍生的HPSE能够诱导EC活化，激发EC存储的Weibel-Palade小体（WPB）胞吐效应介导管腔血管性血友病因子（VWF）的迅速形成，从而促进肿瘤微环境中凝血通路的激活。本项目将聚焦HPSE在MM相关凝血通路中的分子作用，特别是：（1）明确HPSE对EC活化的诱导作用及相关机制；（2）建立不同的动物模型在体内验证科学假说；（3）研究HPSE对VWF特异降解酶ADAMTS13活性的调节作用。

肿瘤相关血栓形成是癌症患者常见的并发症，可促进癌细胞扩散并降低存活率。肿瘤内皮的促凝作用有助于微血管闭塞和增加转移的形成。在本研究中，我们分析了黑色素瘤来源的乙酰肝素酶(HPSE)激活内皮细胞和启动凝血通路的作用。在人和小鼠黑色素瘤细胞中，HPSE的敲除降低了细胞上清液中血管内皮生长因子A(VEGF-A)的可获得性。VEGF-A浓度的降低与内皮细胞活化的降低直接相关，内皮细胞活化的特征是促凝血剂血管性血友病因子(VWF)分泌减弱。在一个实验性黑色素瘤模型中，管腔VWF水平降低与血小板聚集性显著降低和肿瘤血管闭塞减少相关。此外，缺乏黑色素瘤的HPSE减少了纤维蛋白的形成，这也暗示了血浆凝血的参与。我们测量了组织因子(TF)在HPSE缺陷小鼠肿瘤中的表达减少，以及TF依赖的凝血酶生成减弱。在人类黑色素瘤细胞中，HPSE的敲除促进了TF的表达，并与增强的NF-kB信号相关。综上所述，我们发现HPSE影响肿瘤相关的高凝程度。我们的数据表明HPSE与VEGF-A或TF触发的内皮细胞激活之间存在功能联系。靶向HPSE的促凝血特性似乎是预防肿瘤相关血栓形成和凝血依赖性肿瘤进展的合理策略。