CRL4B E3泛素连接酶在骨肉瘤细胞中的功能解析及其致病机理研究

Osteosarcoma is one of the most common types of primary bone malignancies, and its genesis and progression are regulated by numerous factors. The ubiquitin-proteasome system (UPS) is tightly associated with genesis of multiple cancer types and promotes the timely degradation of short-lived proteins with key regulatory roles in a vast array of biological processes, such as cell cycle progression, DNA damage and repair, transcription of oncogenes and apoptosis. However, it remains unclear about the involvement of UPS in the pathogenesis of osteosarcoma. Our previous results have demonstrated that the expression of CUL4B is significantly increased in osteosarcoma tissues from patients and in osteosarcoma cell lines. Additionally, knockdown of CUL4B can cause severe effects on osteosarcoma cells, including inhibition of proliferation and clonability, induction of apoptosis and cell cycle arrest. In this project, we will further study the molecular mechanism of CUL4B in the pathogenesis of osteosarcoma cells. Firstly, we will try to identify the components of CRL4B E3 ligase and its substrate(s) in osteosarcoma cells by using immunoprecipitation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. We will further identify the ubiquitination site(s) of substrate(s) and test their biological functions. These studies will contribute to understanding of osteosarcoma progression and chemoresistance, and provide opportunities to develop CUL4B as a biomarker in the diagnosis and prognosis of osteosarcoma. Moreover, it may provide potential target(s) in the therapy of osteosarcoma in the future.

骨肉瘤是高度恶性的实体肿瘤, 其形成及恶化受多种因素影响或调控。泛素化－蛋白酶体选择性降解底物蛋白途径与多种类型癌症的发生密切相关, 其参与癌细胞周期调控、DNA损伤修复、癌基因转录和细胞凋亡等过程。但是到目前为止, 关于泛素化介导的蛋白修饰降解途径是否对骨肉瘤致病发挥作用还不得而知。本课题组前期研究发现CUL4B在骨肉瘤细胞中高表达, 且干扰CUL4B表达能抑制骨肉瘤细胞的增殖和形成克隆能力, 诱导细胞凋亡和导致细胞周期阻滞等。因此本项目拟在前期研究基础上, 开展CUL4B对骨肉瘤致病的分子机理研究。采用免疫沉淀和LC-MS/MS质谱分析鉴定骨肉瘤中CRL4B E3泛素连接酶各蛋白成员, 并找到其所识别的特异底物、底物的泛素化位点和对其生物学功能进行研究。本研究将为探索CUL4B作为骨肉瘤诊断分子标记及其在预后判断中的价值提供理论依据, 以及为骨肉瘤的靶向治疗提供潜在的干预靶点。

本项目根据前期的研究成果即CUL4B在骨肉瘤细胞中高表达利用，通过体内体外免疫沉淀方法、LC-MS/MS质谱分析和体外蛋白免疫共沉淀等技术，发现CUL4B可以和DDB1和RBX1形成复合物，DDB1又分别和DCAF11和DCAF13相互作用，从而形成CRL4BDCAF11和CRL4BDCAF13两个泛素连接酶。CRL4BDCAF11能识别细胞周期调控蛋白p21作为底物，而CRL4BDCAF13则识别肿瘤抑制因子PTEN作为底物。敲低CRL4BDCAF11和CRL4BDCAF13两个泛素连接酶成员能分别抑制p21和PTEN的泛素化水平，造成其积累，从而在体内和体外均能抑制骨肉瘤细胞的生长。对造成CUL4B过表达的分子机理进行研究发现，转录因子NF-κB和微小RNA miR-300均能影响CUL4B的表达。促炎性细胞因子TNF-α激活NF-κB信号通路，NF-κB从细胞质转运到细胞核中，从而激活CUL4B的表达。DNA甲基化介导的miR-300低表达解除了其对CUL4B的抑制，造成CUL4B的高表达。另外，我们也通过体外高通量筛选获得一个特异抑制DDB1-CUL4B相互作用的天然小分子化合物（TSC01131），其在体内体外实验中均展现出良好的抑制肿瘤细胞生长的能力。综上所述，我们发现了两种造成CUL4B高表达的分子机制，发现了CUL4B能形成CRL4BDCAF11和CRL4BDCAF13两个泛素连接酶，分别对p21和PTEN进行泛素化，从而通过影响细胞周期蛋白和肿瘤抑制因子的表达来调节癌细胞的生长。本研究深入阐释了CUL4B如何参与骨肉瘤的致病过程，为骨肉瘤的检测和治疗提供了更多靶点和指标，同时也为研究其它由CUL4B过表达造成的肿瘤提供了借鉴。我们发现的小分子化合物具有良好的应用价值，将为CUL4B过表达的癌症靶向治疗提供可能。