BVES调控“癌细胞挤出”在肝癌转移中的作用及机制研究

Recent studies have found that crowding induces extrusion of live cells to maintain epithelial homeostasis, normally live extruded cells eventually die through a process termed anoikis. This intresting finding suggests that cancer cells, resistant to anoikis, may initiate metastasis from the primary tumor by cancer cell extrusion. Our previous researches demonstrated that extruded HCC cells induced by overcrowding could continue to survive, the number of extruded cells is closely related to the metastasis potential of HCC cell lines; BVES, a novel cell adhesion molecule, was downregulated in HCC. Downregulation of BVES promoted invasion and metastasis of HCC; we also found that BVES was downregulated in extruded cells, and ZO-1 was detached from membrane to the cytoplasm of the extruded cells, overexpression of BVES regulated the expression of ZO-1 and RhoA activity, decreased F-actin assembly, and inhibited the extrusion of HCC cells. Base on the present theories and our previous results, we plan to investigate the role of BVES in the regulation of cancer cell extrusion using silicone membrane stretch method and 2-D/3-D cell cultures; we would further explore the underline mechanism of ZO-1/RhoA and other signaling pathways on BVES regulated cancer cell extrusion; we intend to verify the effect of BVES in the regulation of cancer cell extrusion in HCC metastasis by in vivo imaging technology in zebrafish xenograft model and indirect orthotopic liver transplantation tumor model in mice. In conclusion, this project which aims at clarifying the role and molecular mechanism of BVES in the regulation of cancer cell extrusion in the metastases of HCC will provide new clues and ideas to inhibit HCC metastasis.

研究发现，正常上皮细胞通过活细胞挤出方式减轻增殖产生的上皮层拥挤，被挤出的细胞发生失巢凋亡。该现象启发我们，具备失巢凋亡抵抗的肿瘤细胞能否通过该方式从原发灶转移? 本项目前期研究表明，拥挤环境中肝癌细胞可被挤出，且能继续存活，挤出数与细胞转移潜能相关；肝癌中BVES表达减少，抑制其表达促进细胞侵袭转移；进一步研究发现，挤出细胞中BVES表达减少，细胞浆游离ZO-1增多；过表达BVES增加ZO-1表达及细胞膜定位，降低RhoA活性，减少F-actin聚集，进而抑制肝癌细胞挤出。本课题拟借助硅胶膜拉伸法和水凝胶2D/3D模型深入研究BVES对肝癌细胞挤出作用的调控；阐明下游ZO-1/RhoA等信号通路在肝癌细胞挤出中的作用机制；斑马鱼和裸鼠体内模型证实BVES对肝癌细胞挤出和转移的影响。期望通过本研究阐明BVES调控癌细胞挤出在肝癌转移中的作用及机制，为抑制肝癌转移提供新的思路。

最近研究发现，正常上皮细胞通过将活细胞挤出的方式减轻增殖产生的上皮层拥挤，被挤出的细胞发生失巢凋亡。该现象启发我们，具备失巢凋亡抵抗的肿瘤细胞能否通过该方式从原发灶转移?本研究通过2D及3D细胞挤出培养模型、大小皿倒扣模型、细胞拉伸模型等多种方法观察细胞挤出现象及BVES对细胞挤出的调控。我们观察到在100%融合度的培养状态下，部分LO2肝细胞被周围细胞所包围，受到“集体攻击”，且细胞变圆。同样，标记GFP的Huh7和SK-Hep1细胞也受到周围正常的LO2细胞“排挤”。高尔基体GM130特异性染色和激光共聚焦扫描三维重建更直观地观察到肝癌细胞被LO2细胞挤出现象。计数硅胶膜拉伸模型中挤出细胞，发现肝癌细胞挤出数明显多于LO2细胞，且高转移的SK-Hep1细胞多于低转移潜能的Huh7细胞。大小皿倒扣模型中，大皿中挤出的细胞克隆数SK-Hep1＞Huh7> LO2,与细胞侵袭潜能一致，通过GFP荧光标记发现，小皿中SK-Hep1细胞较Huh7更容易被LO2细胞挤出。三维细胞培养模型中， L02 细胞呈现为边界清楚的结节样结构，而在Huh7 和SK-Hep-1 细胞形成的细胞团周围，可见部分细胞从边缘脱离，向周围迁移。在即将脱落的细胞及其相邻细胞间，骨架蛋白F-actin 染色明显增强。在肝癌组织标本中我们也发现了部分肝癌细胞挤出现象。BVES蛋白表达于细胞膜和胞浆，肝癌组织中BVES较癌旁组织明显减少，BVES表达与肝癌细胞系转移潜能呈负相关，通过细胞挤出模型发现，被挤出细胞其BVES表达减少。抑制Huh7细胞BVES表达促进细胞挤出，而在SK-Hep1细胞中过表达BVES则抑制细胞挤出。裸鼠人肝癌原位移植瘤模型中过表达BVES抑制肝癌细胞肝内外转移，在肝内原位瘤边缘，过表达BVES的肝癌细胞挤出明显减少。深入研究BVES调控肝癌细胞挤出机制，我们发现，BVES与ZO1和GEFT共定位，且调控ZO1的表达和定位，影响GEFT亚细胞定位，进而抑制RhoA蛋白活性。我们还筛选出部分BVES下游信号分子和通路，对进一步的机制研究提供参考。