Syntenin调控YAP1影响胰腺癌侵袭迁移能力的机制研究

Pancreatic cancer is characterized by pronounced potential for migration, invasion, and epithelial-to-mesenchymal transition, which are the major reasons for the high mortality of this type of cancer. In our preliminary study, we identified syntenin, an oncogene specifically expressed in the tumor tissue of high metastasis patients, by high throughput sequencing of clinical tissue sampling of pancreatic cancer.. Syntenin (syndecan-binding protein, SDCBP) is a member of the PDZ domain-containing family and plays a vital role in diverse physiological processes, such as protein trafficking, nucleus import and export, and activation of transcription factors mainly through its two PDZ domains. However, little has been reported about the role of syntenin in pancreatic cancer. Stable cell lines with syntenin knockout and activation were set up through CRISPR/Cas9. In preliminary study, syntenin was found overexpressed in pancreatic cancer tissue and cell lines and promoted cancer cell invasion and migration. Furthermore, the expression of YAP1 was regulated by syntenin and can form protein complex structures with syntenin. Syntenin has two PDZ binding domains with large numbers of interacting ligands, and YAP1 has the domain of the PDZ-binding motif. We propose that syntenin can directly bind to YAP1 to stabilize YAP1 protein expression and regulate nuclear -cytoplasm localization. . The role of syntenin in inducing invasion, migration, and metastasis in pancreatic cancer is also investigated by in vivo and in vitro studies. Western, RT-QPCR, IHC, ICC, Co-IP, GST pulldown, Ch-IP, and 8×GTIIC-TEAD luciferase assay are used to determine the molecular mechanisms that regulate the interaction between syntenin and YAP1. This study can offer a new mechanism of pancreatic cancer metastasis and provide scientific basis for the development of novel treatment strategies for patients with pancreatic cancer.

胰腺癌的高转移性是其预后差的主要原因。通过对胰腺癌临床组织样本进行高通量测序，我们鉴定出一种在高转移人群中特异性高表达的蛋白syntenin。Syntenin是一种胞内衔接蛋白，主要依赖于其PDZ结构域影响胞内蛋白质转运、蛋白质定位、转录因子激活等多种生物功能；然而在胰腺癌中尚无syntenin相关报导。我们前期通过CRISPR/Cas9敲除及激活系统成功构建了syntenin敲除及过表达细胞系。通过预实验发现胰腺癌的侵袭迁移能力明显受syntenin调控。同时，我们发现YAP1这一促进胰腺癌恶性表型的蛋白其表达量显著受syntenin调控并与syntenin存在蛋白间相互结合，而YAP1蛋白结构中存在PDZ结合模体。基于测序及预实验结果，本项目拟通过临床样本分析，动物实验，细胞实验和分子实验等探讨syntenin在胰腺癌中的功能及分子机制，并为临床胰腺癌的治疗提供新策略。

背景和目的：胰腺癌高转移性是极其重要的临床问题，深入阐明胰腺癌的独特转移机制并寻找有效的治疗靶点，对胰腺癌的诊治具有重大推动作用。Syntenin/SDCBP(多配体聚糖结合蛋白)，又被称为黑色素瘤分化相关基因（MDA-9）。作为一类支架蛋白，syntenin主要通过其两个PDZ结构域发挥作用。.方法和结果：采用121胰腺癌癌与癌旁配对芯片对Syntenin进行免疫组化染色，发现Syntenin在胰腺癌中显著高表达，且表达水平与肿瘤大小和淋巴结转移呈显著负相关，其表达显著影响患者的总生存和无复发生存。在裸鼠皮下和胰腺原位异种移植瘤模型中发现，Syntenin可以促进小鼠胰腺肿瘤的增殖，促进肝脏、腹腔转移能力，减低小鼠总的生存期。体外细胞实验，通过Transwell、划痕、细胞运动、侵袭性伪足形成和明胶降解实验、Western检测EMT标记物发现Syntenin可以促进胰腺癌癌细胞的运动、侵袭迁移和转移能力。通过克隆形成实验、EDU、RTCA发现Syntenin可以促进胰腺癌细胞系的增殖能力。机制方面，Syntenin可以上调YAP1蛋白水平的表达，通过加CHX和MG132或氯喹实验，发现Syntenin可以稳定YAP1的表达，抑制YAP1通过蛋白酶体途径降解。Co-IP实验，发现syntenin与YAP1之间存在结合，进一步我们构建Syntenin带有HA标签的HA-PDZ1和HA-PDZ2的截断质粒，以及YAP1带有Flag标签的Flag-TAD、Flag-TID和Flag-YAP1/ΔTAD的截断质粒，分别进行外源的IP实验，发现HA-Syntenin可以通过PDZ1、PDZ2结构域和Flag-YAP1的TAD结构域结合。通过对YAP1进行泛素化IP实验，发现Syntenin和YAP1结合后抑制YAP1的泛素化水平，且Syntenin和YAP1结合后抑制YAP1的泛素连接酶β-TrCP和YAP1的结合从而抑制YAP1的泛素化。通过对临床常用药物化合物库进行筛选，我们发现雷贝拉唑钠可降低syntenin表达。雷贝拉唑钠可以抑制肿瘤生长、肝脏、腹腔的转移能力并使小鼠的总生存期延长。结论：Syntenin在胰腺癌中高表达可能是胰腺癌高转移的内在分子机制，syntenin通过稳定YAP1的表达来发挥促癌作用，可作为潜在的治疗靶点，改善胰腺癌患者的生存和预后。