NEDD4L泛素化调控AQP9与结直肠癌侵袭转移的关系研究

Colorectal Cancer (CRC) is the third most common cancer in China. After radical surgery, about 50% advanced CRC experience recurrence and metastasis. Biomarkers that can predict prognosis and clinical outcome are critical targets for CRC treatment. We have found that AQP9 expression level were significantly higher in the tumor tissue of CRC patients with metastasis compared to those without metastasis; Microarray analysis indicated that cell adhesion genes and chemokine signalings are enriched in AQP9-overexpressed cells. Further analysis showed that AQP9 could activate Wnt/β-Catenin signaling, which may subsequently contribute to the enhanced tumor cell migration. These preliminary results suggest that AQP9 might be an important marker associated with CRC metastasis. Furthermore, we found that E3 ubiquitin ligase NEDD4L can regulate AQP9 expression level in CRC cells. Therefore, we speculate that the deregulated AQP9 by NEDD4L regulates Wnt signaling thereby promoting tumor metastasis in colorectal cancer. In the present study, we will investigate the interaction between NEDD4L-AQP9-Wnt signaling. Moreover, we will utilize cell and animal models to investigate whether AQP9 would promote CRC metastasis through Wnt signaling activation. Finally, the interactions among NEDD4L, AQP9 and other relevant genes in AQP9-Wnt pathway will be verified in colorectal tissue specimens. The success of this study will highlight the role of AQP9 in CRC metastasis treatment.

中晚期结直肠癌(CRC)术后约有50%出现复发转移。我们前期研究发现AQP9在复发转移的CRC组织中高表达；其过表达使肿瘤转移相关通路显著富集。但AQP9在CRC中的调控机制尚不明确。我们预实验发现AQP9在CRC中存在泛素化修饰，泛素连接酶NEDD4L与AQP9存在相互作用并调控其表达；AQP9过表达可活化Wnt信号，促进肿瘤细胞迁移。因此我们提出NEDD4L泛素化调控AQP9表达，并通过Wnt通路影响CRC转移的假说。本课题拟通过突变质粒构建、蛋白质谱、免疫共沉淀等方法，在体外实验中研究NEDD4L-AQP9-Wnt通路间的相互作用机制；利用细胞和动物模型探讨AQP9泛素化对CRC肿瘤侵袭转移的影响；利用临床样本分析AQP9上下游通路与CRC临床病理特征和预后的关系。本研究的开展将阐明AQP9影响CRC转移的详细分子机制和临床意义，为基于AQP9的CRC靶向治疗提供实验依据和理论基础。

中晚期结直肠癌(CRC)术后约有50%出现复发转移。我们在前期研究中发现跨膜通道蛋白AQP9可以作为II/III期结直肠癌化疗敏感性的独立预测因子。AQP9高表达的CRC患者接受辅助化疗后，无病生存显著优于低表达组，进一步研究发现，AQP9可以促进化疗药物5-FU的跨膜转运，从而促进细胞凋亡。但是，在没有接受辅助化疗的CRC患者中，AQP9高表达组总生存较低表达组有更差的趋势。研究表明，水通道蛋白在细胞增殖、迁移以及癌变过程中发挥重要的作用，是疾病治疗和药物开发的潜在靶点，与结直肠癌的发生也关系密切。因此我们猜测，AQP9除了跨膜转运化疗药物以外，在CRC进展过程中是否还发挥了其他的功能？本项目中，我们通过公共数据库、临床样本和体内动物实验证实了AQP9高表达与结直肠癌转移和不良预后显著相关；通过RNA-seq和细胞模型证实了AQP9通过激活WNT信号通路，促进EMT从而促进CRC细胞迁移和侵袭能力显著增强；进一步证实了AQP9可以通过与Wnt 通路的关键分子DVL2结合，从而稳定DVL2激活下游Wnt/β-catenin信号通路；并且，我们发现AQP9的C端SVIM结构域是与Dvl2结合的关键位点；删除SVIM结构域，可以阻断两者的相互作用。证实了E3连接酶NEDD4L可以调控AQP9蛋白水平和泛素化。本研究的开展阐明了AQP9影响CRC转移的详细分子机制和临床意义，为基于AQP9的CRC靶向治疗提供实验依据和理论基础。