去泛素化酶OTUD6B维持SOX9蛋白稳定性促进肝癌细胞干性的机制研究

Cancer stem cell (CSC) has been proposed to be the cellular origin of tumorigenesis and drug resistance in hepatocellular carcinoma (HCC). The stem-like properties of CSC are controlled by a core transcription factor SOX9, however, it remains not completely understood how SOX9 expression is activated in human HCC tissues. Deubiquitinase (DUB) plays important role in stabilizing protein substrates by inhibiting substrate ubiquitination and proteasomal degradation. We have applied siRNA screening of human DUBs and identified OTUD6B as a potential DUB for SOX9. Accordingly, knockdown of OTUD6B down-regulated SOX9 protein abundance, leading to impaired self-renewal of CSC in vitro. In searching for upstream regulatory mechanisms controlling OTUD6B expression, we have observed that OTUD6B gene is highly amplified in various cancers including HCC, leading to elevated expression of OTUD6B human HCC tissues. Moreover, we have further identified OTUD6B as a potential Wnt target. In light of these findings, we propose that OTUD6B expression may be up-regulated by both gene amplification and Wnt signaling, thereby stabilizing SOX9 protein to enhance stemness of HCC cells. We will apply various molecular, cellular and genetic techniques to critically challenge our hypothesis, through which we may identify a novel therapeutic target for eliminating liver CSCs.

肝癌组织中一群具有干细胞样特性（干性）的肝癌干细胞（CSC），是肿瘤演进和耐药种子细胞，其干性受SOX9调控，但维持SOX9蛋白高表达的机制不明。去泛素化酶（DUB）抑制底物泛素化降解，增加底物蛋白稳定性；我们采用siRNA筛选发现OTUD6B可能是SOX9的一个新DUB，下调OTUD6B则降低SOX9表达、抑制肝癌细胞干性。OTUD6B在肝癌组织表达升高，探讨其上游调控机制发现肝癌组织OTUD6B基因高度扩增，与经典癌基因类似；此外，OTUD6B可能是Wnt信号靶基因。我们因此提出假说：基因扩增和Wnt信号均可上调去泛素化酶OTUD6B，后者维持SOX9蛋白稳定性，促进肝癌细胞干性。本项目拟采用分子、细胞和遗传学技术，深入研究OTUD6B调控SOX9蛋白稳定性的上游机制，寻找针对CSC的治疗新靶点。

肿瘤干细胞是恶性肿瘤发生发展的根源细胞，其干性特征受SOX9等关键转录因子调控；蛋白翻译后修饰如泛素化在维持底物蛋白表达和活性中发挥重要作用，但尚不清楚泛素化修饰如何调控干性转录因子表达，进而影响肿瘤干性特征。针对这个科学问题，本项目通过建立无偏移的高通量CRISPR Screen筛选和验证，发现去泛素化酶OTUD6A而不是OTUD6B，特异性识别并下调SOX9的泛素化酶修饰，系统阐明OTUD6A上调SOX9蛋白稳定性的分子机制，证明OTUD6A-SOX9信号轴促进肝癌发生发展的重要作用，及其高表达与人类肝癌恶性进展的临床相关性；本项目还发现表观调控因子BRD4通过非经典途径，抑制E3泛素连接酶（FBXL14和β-Trcp1）介导干性转录因子Snail蛋白酶体降解，促进肿瘤侵袭转移；在线性泛素化修饰方面，揭示SHARPIN通过与β-Trcp1竞争性结合干性转录因子β-catenin而抑制其泛素蛋白酶体降解，进而在促进肿瘤细胞恶性增殖的作用机制。综上所述，本项目发现肿瘤干性转录因子的泛素化调控新机制，相关关键泛素化修饰酶如OTUD6A和SHARPIN等，有望成为肿瘤分子靶向治疗候选靶标，有清除肿瘤干细胞的抗癌策略提供理论新依据。