去泛素化酶POH1调控E2F1表达及其在肝癌发生发展中的作用

Ubiquitination and deubiquitination are two opposite and reversible processes that control protein stability and biological activities, thereby playing a fundamental role in maintaining cellular homeostasis. Deregulation of certain deubiquitinating enzymes (DUB) that usually leads to the loss of the dynamic balance of ubiquitination and deubiquitination of the substrates is associated with cancer development and malignant progression...The master transcriptional factor E2F1 hyperactivation occurs frequently in certain human cancer, including hepatocelluar carcinoma (HCC), and contributes to malignant progression. E2F1 activity is regulated by proteolysis mediated by the ubiquitin-proteasome system. However, the deubiquitinase that regulates E2F1 ubiquitination and stability remains undefined. Our preliminary data based on RNAi screening of DUBs and biochemistry experiments strongly suggest that the deubiquitinase POH1 may stabilize E2F1 protein through binding to and deubiquitylating E2F1. In addition, the POH1 expression may efficiently facilitate tumor growth of liver tumor cells. The aim of the present study includes the follow aspects: 1) to delineate the molecular mechanisms underlying POH1-mediated deubiquitination of E2F1, including the details about the physical interaction between the two proteins and the main lysine sites of E2F1 as well as the cellular location responsible for POH1-mediated deubiquitination; 2) to examine the biological consequence of POH1 regulation of E2F1 in HCC cells; 3) to investigate the effects of oncogenic c-Myc activation on POH1 and E2F1 expression, and the mechanism and significance of POH1 regulation of E2F1 in c-Myc- driven liver tumor cell growth; 4) to explore the association of the POH1, E2F1 and c-Myc expression in HCC tissues and their clinical relevance in HCC development and progression. The study will provide novel insight into whether and how POH1-mediated deubiquitination of E2F1 in the development and progression of HCC, and potentially be informative for uncovering new targets for HCC manipulation.

蛋白的泛素化修饰和去泛素化是对立、呈动态平衡的生物学事件，调控蛋白的稳定和功能活性。已知去泛素化酶功能的改变将导致蛋白底物的泛素化水平的异常和肿瘤发生。.在肝癌等多种肿瘤中，转录因子E2F1蛋白水平异常上调并与肿瘤的进程相关。E2F1表达受泛素-蛋白酶体系统调控；目前，能够稳定E2F1蛋白的去泛素化酶仍未被发现。我们前期数据表明，去泛素化酶POH1可调控E2F1的稳定性和泛素化水平并能促进肝癌细胞的增殖。本项目目标于阐明POH1去泛素化调控E2F1稳定性的分子机制；分析POH1影响E2F1表达的生物学效应和促肿瘤生长的机制；观察癌基因c-myc对POH1-E2F1的调节作用；在临床肝癌中明确POH1、E2F1和c-Myc蛋白表达特点，证明这些调控关系的存在及其与疾病进展的关联。本研究将为认识POH1介导的去泛素化事件在肝癌发生、发展中的作用提供依据，并可能为肝癌的治疗提供潜在的分子靶点。

蛋白泛素化修饰和去泛素化修饰的失衡参与肿瘤的发生和发展。转录因子E2F1蛋白水平异常上调且与肝癌的恶性进程相关。调控E2F1蛋白稳定性的去泛素化酶有待于发现。我们的研究表明， POH1通过去泛素化作用而上调E2F1的稳定性、促进肝癌细胞的增殖和成瘤能力；该过程涉及E2F1提高FOXM1和Survivin的表达。在机制上，POH1能够下调E2F1蛋白的泛素-K63多聚链和泛素-K11多聚链的修饰，从而提升E2F1的蛋白水平；POH1和E2F1的相互作用主要依赖于POH1（181-310aa）区域和E2F1(201-437aa)区域。临床肝癌样本和相关数据库分析结果表明，肝癌中POH1和E2F1、FOXM1和Survivin的表达水平存在正相关性并和疾病的进展有关。在癌基因c-Myc/N-Ras或AKT/N-Ras驱动的鼠肝肿瘤模型中，相对于对照肝组织，肝肿瘤中的POH1表达水平显著提高。我们的研究还表明，POH1可以和TGF-β受体1和受体2 （TGFBR1和R2）相互作用,促进它们的表达并上调TGF-β信号的活化；在临床肝癌标本中，POH1表达和TGF-β信号效应蛋白Smad3的磷酸化水平具有相关性。以上结果支持POH1通过调控E2F1和TGF-β信号促进肝癌发生和发展。本研究揭示POH1介导的去泛素化事件在调控肝癌发生和疾病进程中的作用，为肝癌的治疗提供潜在的作用靶标。