TGF-β1-YAP-CTGF功能轴调控胰腺癌细胞与胰腺星状细胞交互作用促进胰腺癌进展的机制研究

Desmoplastic reaction is one of the most important pathological features of pancreatic cancer. Recently, we have found that YAP was increased in pancreatic cancer tissue and YAP could promote the growth, invasion and metastasis of pancreatic cancer cells, at the same time it could also promote PSC activation through secretion of CTGF and YAP was positively correlated with the degree of desmoplastic reaction. The YAP level was increased significantly in tumor cells after treated with TGF-β1. So we speculated that overexpression of YAP in pancreatic cancer cells could promote stellate cell activation through paracrine CTGF, and the TGF-β1 secreted by activated stellate cells will increase YAP expression in adjacent pancreatic cancer cells. So there may exist a TGF-β1-YAP-CTGF function axis regulating pancreatic cancer cell and pancreatic stellate cell interactions. To further address this hypothesis, clinical pathology information, co-culture model in vitro, nude mouse model and LSL-KrasG12D/+; Trp53fl/+; Pdx1-Cre (KPC) transgenic mouse model would be designed and lots of technologies such as molecular biology technology, Laser capture microscopic cutting, Optical in vivo Imaging technology will be conducted. This study will help us to delineate the key role of the TGF-β1-YAP-CTGF function axis in pancreatic cancer cells and pancreatic stellate cell and the molecular mechanisms of pancreatic cancer cell and pancreatic stellate cell co-couple interaction.

间质增生反应是胰腺癌的重要病理特征之一。本课题组前期研究结果显示，YAP在胰腺癌中高表达，促进胰腺癌细胞的生长及侵袭转移，同时激活下游信号通路分泌CTGF促进星状细胞活化，与间质纤维化程度呈正相关；而体外给予TGF-β1后肿瘤细胞的YAP表达显著提高。因此我们推测胰腺癌细胞中YAP通过旁分泌CTGF促进星状细胞活化，活化的星状细胞TGF-β1分泌增多作用于临近的胰腺癌细胞，使YAP表达上升，提示存在以YAP为核心的TGF-β1-YAP-CTGF功能轴调控胰腺癌细胞与胰腺星状细胞交互作用。本研究将从临床病理、细胞培养、裸鼠胰腺癌模型、LSL-KrasG12D/+; Trp53fl/+; Pdx1-Cre (KPC)转基因动物模型4个方面入手，应用分子生物学，体外细胞共培养，小动物活体成像等技术探索TGF-β1-YAP-CTGF功能轴调控胰腺癌细胞与胰腺星状细胞交互作用的机制。

胰腺癌是一种恶性度极高的消化系统肿瘤，诊断时约80%的胰腺癌患者合并有局部或远处转移，.手术切除率低，并且对常规化疗、放疗均不敏感，其发病率和死亡率几乎相等。间质增生反应是胰腺癌的重要病理特征之一，与胰腺癌的不良预后，化疗抵抗相关。本研究从临床病理、细胞培养、裸鼠胰腺癌模型、LSL-KrasG12D/+; Trp53fl/+; Pdx1-Cre (KPC)转基因动物模型4个方面入手，应用分子生物学，体外细胞共培养，等技术探索TGF-β1-YAP-CTGF功能轴调控胰腺癌细胞与胰腺星状细胞交互作用的机制。结果显示，免疫组织化学染色及Western-blot结果说明正常胰腺组织和胰腺癌组织中均存在YAP的表达，与正常胰腺组织相比，胰腺癌组织的YAP表达量高于正常胰腺组织，且YAP的表达与胰腺癌的分期相关，GEPIA数据库显示胰腺癌组织中YAP的高表达与胰腺癌患者的不良预后相关；慢病毒转染技术成功构建稳定敲除YAP的胰腺癌细胞；敲除YAP抑制了胰腺癌细胞的增殖和克隆成球能力；敲除YAP抑制了胰腺癌细胞的侵袭能力和EMT转化； 敲除YAP抑制了胰腺癌细胞的CTGF表达水平，且胰腺癌组织中YAP的表达水平与CTGF及间质纤维化相关指标α-SMA表达呈正相关；敲除胰腺癌细胞的YAP抑制共培养体系中胰腺星状细胞的活化和胰腺星状细胞TGF-β1的表达与分泌；敲除YAP抑制TGF-β1诱导的胰腺癌细胞侵袭转移和EMT，并且抑制TGF-β1诱导SMAD2入核，预先给与SMAD2信号通路抑制剂SB431542干预细胞可显著逆转TGF-β1诱导的胰腺癌细胞YAP表达升高；在裸鼠皮下移植瘤实验中，敲除YAP抑制胰腺癌细胞的皮下成瘤能力和促胰腺星状细胞活化和间质纤维化反应。使用YAP抑制剂Verteporfin能显著抑制KPC鼠胰腺癌肿瘤的生长，Verteporfin通过抑制YAP抑制了胰腺癌的增值和间质纤维化水平。研究胰腺癌微环境对胰腺癌发生发展的影响，探究胰腺癌的间质纤维化具体机制，可以对胰腺癌的治疗以及预防复发转移提供强有力的理论支持，对胰腺癌的精准治疗有重要意义。