Occludin蛋白磷酸化在肺预转移小生境微血管完整性改变中的调控机制研究

Formation of premetastatic niche plays a critical role in the lung metastasis of malignant tumors. Vascular hyperpermeabilty in premetastatic lungs facilitates the extravasation and colonization of the cancer cells. Our previous studies showed that VEGF secreted from the primary tumor cells could enhance the vascular permeability not only via the destruction of adherent junctions between endothelial cells, but also through the downregulation of occludin, a key protein of the tight junctions. Treatment with the VEGF antibodies blocked the VEGF induced occludin downregulation and decreased the vascular permeability in vitro and vivo. Hereby, we hypothesize that VEGF could induce the phosphorylation of occludin, and ubiquitination, following degradation of occludin, leading to vascular hyperpermeability of the premetastatic niche. In order to test the hypothesis, murine melanoma cells were transplanted subcutaneously in mice to construct the xenograft tumor model; immunoprecipitation, site-directed mutagenesis, immuofluorescence, confocal microscope, electron microscope and in vitro transfection were used to verify that VEGF induced phosphorylation of serine residues of occludin, leading to ubiquitination and following internalization, degradation of occludin and then destruction of the tight junction between vascular endothelial cells. This study of vascualr hyperpermeabilty in premetastatic niche may shed novel light on the molecular mechanisms of vascular hyperpermeability in premetastatic lung and render for new strategies and sights of preventing lung metastasis.

肺预转移小生境（premetastatic niche）的形成是肿瘤肺转移的关键步骤。预转移小生境内微血管通透性增高，有利于癌细胞的穿出和定植。我们在前期研究中发现，原发肿瘤分泌的VEGF诱导肺血管内皮通透性增高，不仅与内皮细胞粘附连接复合物的分离有关，而且与紧密连接蛋白occludin磷酸化密切相关。我们据此推测，在肺预转移小生境形成过程中，VEGF 通过诱导血管内皮细胞紧密连接蛋白occludin磷酸化，进而引起其泛素化降解下调，参与调控肺血管内皮通透性。为证实这一假说，本研究拟通过小鼠肺预转移动物模型，应用免疫沉淀、基因定点突变技术、免疫荧光染色、激光共聚焦和透射电镜等技术，从体内外实验两个层面，系统研究紧密连接蛋白occludin磷酸化在VEGF诱导肺预转移小生境微血管完整性改变中的作用位点及调控机制，以期探寻肺预转移小生境血管通透性增高的分子发生机理，为防治肿瘤转移提供新思路。

肺预转移小生境（premetastatic niche）的形成是肿瘤肺转移的关键步骤。因此，探寻肺预转移小生境的分子发生机理，能够为防治肿瘤转移提供新思路。预转移小生境内微血管通透性增高，有利于癌细胞的穿出和定植。探讨微血管通透性升高的具体机制可能发现影响预转移小生境形成的关键环节。我们课题组按照项目书中的研究计划进行了肺预转移小生境微血管内皮完整性破坏及促进癌细胞渗出的研究以及VEGF诱导血管内皮通透性升高的具体机制研究。我们发现，在预转移阶段小鼠肺组织中，血管通透性升高，肺组织发生炎症反应。进一步研究证实，导致这种改变的细胞因子可能是VEGF。我们用VEGF的拮抗剂贝伐珠单抗处理荷瘤小鼠，发现贝伐珠单抗能够抑制转移前期肺组织血管通透性升高。体外实验证实，VEGF可以诱导紧密连接蛋白occludin下调，并且这一下调是通过诱导occludin发生磷酸化-泛素化进而经泛素-蛋白酶体途径降解来实现的。