出核转运去泛素化酶在结直肠癌发生和治疗中的作用

Ubiquitination is a major mechanism to control the quality, expression level, and function of various proteins. As alterations in the ubiquitin- proteasome system (UPS) play a critical role in the development and progress of various cancers, including colorectal cancers (CRC), it has become an important target for developing new therapeutics. Proteasome inhibitor Bortezomib are the first of its class targeting UPS and has been used for the treatment of myeloma and leukemia. We found recently that the expression of many deubiquitinating enzymes (Dubs) is significantly changes in CRCs, and CRC cells are sensitive to a number of Dubs inhibitors. We also found that Bortezomib induced translocation of proteins from nucleus to cytoplasm, and blocking the translocation with a small molecular inhibitor of nuclear-exporting protein Crm1 enhanced the cytotoxic action of Bortezomib. Therefore, it is hypothesized that translocation of Dubs from nucleus to cytoplasm is critical for the survival and sensitivity to Bortezomib. To further demonstrate the role of nuclear exportation in the response to proteasome inhibitors, we propose in this study to delete Crm1 in CRC cells using the Crispr/Cas9 system and examine the interaction between Crm1 and all the 22 Dubs containing nuclear-exporting sequence (NES), and its regulation by Bortezomib. We will further systematically knockdown these Dubs using a shRNA library against them, and screen for Dubs that specifically affect the survival, proliferation, and sensitivity to cytotoxic drugs of CRC cells, but not that of immortalized colon epithelial cells. Meanwhile, we will evaluate whether inhibition of Crm1 could synergize with proteasome inhibitor in inhibiting the growth of xenografted CRC, which would lay a foundation for further clinic studies with molecules targeting Dubs.

泛素化系统是细胞内控制蛋白质量、水平和功能的主要机制,在肿瘤发生、发展中起关键作用,蛋白酶体抑制剂硼替佐米是首个作用于该系统的抗肿瘤药。我们发现:(1)肠癌中有多个去泛素化酶(Dubs)表达改变,癌细胞对Dubs抑制剂高度敏感;(2)在蛋白酶体抑制剂(PrI)作用下,肠癌细胞中许多核蛋白转位至胞浆, 阻断蛋白的出核使细胞对PrI更敏感。鉴于多个Dubs带有出核序列,我们提出Dubs的转运出核与肠癌细胞生存和对PrI的敏感性有关。本研究拟用Crispr/Cas9敲除出核转运蛋白Crm1, 证明出核转运与对PrI敏感性的关系。然后采用shRNA敲减Dubs表达及检测Crm1和PrI对它们的转位调节,发现影响肠癌细胞生存、生长和对PrI敏感性的Dubs。我们也将用结直肠癌的小鼠移植瘤,检验抑制Crm1是否增强硼替佐米的作用, 为用将来用特异的Dubs抑制剂治疗肠癌奠定基础。

结直肠癌是发生率、死亡率和带瘤患者数量均居前列的重大疾病。由于大多数患者确诊时已为临床II- IV期，有效和特异的化疗方法是改善预后、提高患者生存质量的重要方法。.泛素化系统是细胞内调节蛋白质水平、定位及相互作用的重要途径，在肿瘤的发生、发展中起关键作用。因此，靶向泛素化系统是发现新型抗肿瘤药物的重要方向。.鉴于去泛素化酶(Dubs)在泛素化中的重要作用、底物的相对特异性和催化特征，我们研究了带有出核信号的去泛素化酶(NES_Dubs)在结直肠癌细胞中的作用。通过筛选NES_Dubs的shRNA 文库，我们发现USP1、USP5和OTUB2的敲减导致细胞活力显著减低。进一步的分析发现，(1)结直肠癌中高表达的OTUB2能够结合和调控b-catenin的泛素化和降解，敲减OTUB2有效抑制APC/b-catenin通路的功能。由于APC/b-catenin通路的持续激活是结直肠癌发生的主要原因(>70% 的肿瘤)，我们的研究为开发OTUB2的特异抑制剂治疗结直肠癌提供了实验证据。(2)USP5在结直肠癌中表达增高，且和临床分期密切相关，在IV期肿瘤中表达最高。我们发现了参与蛋白质翻译延长的TUFM 和USP5结合，是USP5的重要底物，而转录因子EBF1调节USP5的表达。敲减USP1导致肠癌细胞生长抑制和凋亡。这些结果说明，USP5在结直肠癌的发展过程中起关键作用，可作为阻止肿瘤发展的有效靶点。(3)USP1在结直肠癌中表达增高，和患者的预后密切相关。抑制或敲减USP1 USP1，使得肠癌细胞对作用于DNA的抗肿瘤药物更敏感，但不影响作用于肿瘤细胞代谢的药物。因此，降低USP1功能以干扰肿瘤细胞的DNA修复，是结直肠癌治疗的新策略。.总之，我们发现了参与结直肠癌发生、发展和治疗的去泛素化酶OTUB2、USP5和USP，研究了他们的作用机制，为研发相应的小分子抑制剂进行抗肿瘤治疗奠定了基础。