类泛素化蛋白水解酶I（SENP1）调控肝细胞癌生长转移的作用和机制研究

Hepatocellular carcinoma, HCC is a major malignancy worldwide and the second commonest fatal cancer in China. Despite definite improvements in the outcome of patients with HCC, the overall prognosis of this cancer is still unsatisfactory because of late presentation and frequent tumor recurrence after surgical resection. New adjuvant treatment modalities for HCC are much awaited. In this regard, knowledge of the molecular and cellular targets underlying the development and progression of HCC is of importance as this can provide novel opportunities for therapeutic interventions for this cancer. The molecular mechanisms of HCC are however unclear. Post-translational protein modifications by members of the ubiquitin family are widely recognized as important regulatory control systems for a variety of biological pathways. Post-translational modification of proteins involves conjugation by members of the small ubiquitin-related modifier (SUMO)/sentrin family. SUMO chains facilitate the recruitment of ubiquitin ligases, which subsequently target poly-ubiquitinated and/or poly-SUMOylated proteins for proteasomal degradation. SUMO conjugation (SUMOylation) is a dynamic process in that it can be readily reversed by a family of Sentrin/SUMO-specific proteases (SENPs) (de-SUMOylation. Six SENP proteins (SENP1, SENP2, SENP3, SENP5, SENP6, and SENP7) have been identified in human. Each of these SENPs has distinct subcellular localization and substrate specificity, suggesting that they are non-redundant. SENP1 is essential for stabilization of HIF1α during hypoxia. Hypoxia is frequently found in HCC due to the rapid growing nature of HCC. Cells adapt to hypoxia through hypoxia-inducible factors (HIFs) which are composed of the constitutively expressed HIF-1β subunit and the oxygen sensitive subunit HIF-1/2α. .In this proposal, we hypothesize that SENP1 plays an important role in hepatocarcinogenesis by regulating HIFs via deSUMOylation, particularly in a hypoxic tumor microenvironment commonly seen in HCC. We propose to 1) evaluate the expression and clinical significance of SENP1 in HCC; 2) characterize the functional role of SENP1 in HCC; and 3) delineate the molecular mechanism by which SENP1 works in HCC.

SUMO化修饰是近年来研究较多的一种小类泛素化修饰方式。SUMO化过程是一个可逆的动态循坏过程，其逆反应过程（称作去SUMO化）依赖于SUMO特异性蛋白酶（SUMO-specific proteases，SENPs）的作用,但其在肝细胞癌发生发展中的作用和机制还不明确。我们的初步研究表明SENP1在肝癌组织中高表达，且与肝癌分期和转移相关。近年来的研究发现， SUMO化修饰可能是调控HIF-1α稳定性及转录活性的重要方式。本课题一方面要结合临床数据和功能研究，确定SENP1参与肝癌发生发展的作用和机制，另一方面拟从两个层面揭示SENP1调控肝癌细胞生物学特性的分子机制：一是阐明SENP1调控的HIFα去SUMO化在肝癌发生发展中作用和机制，二是利用SILAC技术鉴定SENP1直接靶向的SUMO化蛋白，进而为寻找和发现可用于肝癌治疗的靶点或药物提供线索或基础。

小类泛素化（Small Ubiquitin like Modification，SUMOylation）是近年来研究较多的一种蛋白质翻译后修饰方式，能通过改变底物蛋白的稳定性、生物活性、细胞内定位以及与其它分子之间的相互作用，调控细胞的生长、迁移、代谢等多种生命活动。SUMO化过程是一个可逆的动态循坏过程，其逆反应过程（称作去SUMO化）依赖于SUMO特异性蛋白酶（SUMO-specific proteases，SENPs）的作用,但其在肝细胞癌发生发展中的作用和机制还不明确。近年来的研究发现， SUMO化修饰可能是调控缺氧诱导因子HIF-1α稳定性及转录活性的重要方式。缺氧是实体瘤内部的常见现象，缺氧微环境与肝细胞癌的发生和发展密切相关。本项目在分子、细胞 、动物模型、临床样本等多个层面，系统研究了SENP1抑制HIF-1α的SUMO化修饰增强肝癌细胞干性，促进肝癌发生的作用和机制。我们发现SENP1在人肝癌样本中表达上调，且与病人的病理分期及瘤内血管浸润密切相关。在人肝癌样本中，SENP1 mRNA 水平与VEGF、LOX，LOXL2等HIF 靶基因的表达呈显著正相关，且证实SENP1作为HIF-α的靶基因，其表达受到缺氧信号的调控。功能研究显示，SENP1 敲低能够显著抑制缺氧诱导的肝癌细胞干性增强和肝癌细胞的成瘤率，而SENP1 过表达则能够增强这种作用。SENP1 敲低能够显著降低缺氧诱导的HIF-1α 的蛋白水平和转录活性，但对HIF-2α影响不大。且SENP1对肝癌干细胞的作用依赖于其对HIF-1α的去SUMO化。由此，我们认为SENP1作为一个去SUMO化酶，能够与HIF-1α信号形成正反馈调节环，进而促进缺氧条件下肝癌细胞干性的维持和肝癌形成。该研究为探索肝癌发生机制，寻找肝癌治疗靶点提供了线索。