凝血酶抑制剂对肺癌血管生成拟态形成的抑制作用及分子机制研究

Angiogenesis is a key condition for tumor growth and metastasis. Vasculogenic mimicry (VM) is closely associated with poor prognosis of tumor patients. However, the current anti-angiogenic drugs are mainly based on the inhibition of endothelial cell-dependent angiogenesis, ignoring the existence of VM. Our previous work found that thrombin could promote the formation of VM in lung cancer, but the role and mechanism of thrombin in vasculogenic mimicry have not been reported. PAR-1 is the main receptor of thrombin. It is speculated that thrombin promotes epithelial mesenchymal transformation by activating PAR-1, thereby promoting the formation of VM. This subject intends to carry out the following research: 1. Human lung cancer specimens were collected to clarify the relationship between thrombin, PAR-1, VM and prognosis of patients, so as to reveal their clinical significance. 2. To study the effect and molecular mechanism of thrombin on the formation of VM in lung cancer at the cellular and molecular levels; 3. The effects of DTIP and r-hirudin on VM of lung cancer and the combined use of anti-endothelial cell-dependent vascular drugs on the development of lung cancer were studied in a mouse tumor model. Through this study, the role and mechanism of thrombin in VM formation were clarified, which provided a theoretical basis for the development of direct thrombin inhibitors for anti-cancer therapy and combination with anti-endothelial cell dependent vascular drugs.

血管生成是肿瘤生长转移的关键条件。血管生成拟态（vasculogenic mimicry，VM）与患者预后密切相关，但目前抗血管生成药物主要以抑制内皮细胞依赖性血管生成为基础，忽略了VM的存在。前期工作发现凝血酶可以促进肺癌VM形成，但凝血酶在VM中的作用机制还未见相关报道。PAR-1是凝血酶主要受体，我们推测凝血酶通过激活PAR-1，促进上皮间质转化，促进VM形成。本课题拟开展以下研究：1.收集人肺癌标本，明确凝血酶、PAR-1、VM及预后之间的关系，揭示其临床意义；2.在细胞和分子水平研究凝血酶对肺癌VM形成的作用及分子机制；3.通过小鼠肺癌模型研究直接凝血酶抑制剂短肽（DTIP）和重组水蛭素（r-hirudin）对VM的作用机制及与抗内皮细胞依赖性血管药物联用对肺癌发生发展的作用。通过此研究明确凝血酶对VM形成的作用和机制，为直接凝血酶抑制剂拓展为抗肿瘤治疗以及联合用药提供理论基础。

在上个世纪，人们就已经意识到肿瘤和凝血系统之间存在着一定的关系。在肿瘤患者中，血液呈现高凝状态，但这其中的机制目前还不是很清楚。肿瘤的侵袭、转移离不开血液提供营养物质，但传统的血管生成药物在实体肿瘤治疗中也并不理想，这提示了可能存在另外一种相对独立的系统参与肿瘤血供。血管生成拟态(vasculogenic mimicry)，简称VM，这种血液通道内没有内皮细胞参与，而全部由肿瘤细胞组成。但至目前为止，肿瘤的抗血管生成治疗还是以传统的抑制血管新生为基础，忽略了VM的存在。我们通过搜集人肺癌组织标本，发现在人非小细胞肺癌组织中凝血酶高表达，凝血酶表达越高，患者预后越差，越容易形成VM，实验结果说明凝血酶与VM的形成和肿瘤的发生发展密切相关。直接凝血酶抑制剂短肽(DTIP)是我们实验室研发的凝血酶抑制剂。体外划痕试验和Transwell实验结果显示DTIP可以抑制肺癌细胞的侵袭和转移。体外3D细胞培养中，凝血酶可以促进肺癌细胞形成管腔样结构，而DTIP可以抑制管腔的形成。在小鼠肺癌模型中，DTIP和r-hirudin可以显著抑制肿瘤的发生发展，提高小鼠生存时间。我们还发现在蛋白酶活化受体1(Protease activated receptor 1，PAR-1)高表达的患者中，与凝血酶阴性患者相比，凝血酶阳性患者预后最差，且更容易形成VM。然而，PAR-1低表达的患者中，两者没有差异。研究发现凝血酶通过PAR-1激活NF-κB信号通路，促进EMT，从而促进VM形成。将DTIP和吉非替尼联用可以增强抗肿瘤疗效，并抑制了吉非替尼的耐药性。以上结果说明直接凝血酶抑制剂DTIP可用于抗肿瘤治疗，将DTIP与EGFR抑制剂联合治疗可获得更好的抗肿瘤疗效。