BAP1及其突变体调控PTEN泛素化降解在肝癌侵袭转移及奥拉帕尼抗肿瘤效应中的作用及机制研究

Tumor recurrence and metastasis is the primary cause of death in patients with hepatocellular carcinoma (HCC); oncogene activation and antioncogene inactivation play a pivotal role in this process. Deubiquitinating enzyme BAP1 harbors gene mutations in HCC, but the function of BAP1 and its mutants in HCC remains unclear. In preliminary studies, we found that BAP1 was frequently mutated in HCC, and most of the mutations were predicted to be inactivating mutation by protein structural and functional analysis; BAP1 expression was significantly reduced in HCC, and was significantly correlated with the prognosis of HCC patients after curative resection; downregulation of BAP1 markedly promoted HCC invasion and metastasis as well as the anti-tumor effect of Olaparib; Co-immunoprecipitation and mass spectrometer proteomic analysis showed that BAP1 could form a complex with PTEN and inhibited the degradation of PTEN induced by the E3 ubiquitin ligase WWP2. Therefore, we hypothesized that BAP1 downregulation or inactivating mutation may promote HCC invasion and metastasis as well as the anti-tumor effect of Olaparib by regulating the ubiquitination and degradation of PTEN as well as the downstream signaling pathways. This study aims to confirm the role of BAP1 and its mutants in HCC invasion and metastasis as well as in the anti-tumor effect of Olaparib at molecule, cell, tissue and animal levels using HCC tumor models in vitro and in vivo as well as HCC pathological specimens from patients and mice, and explore the mechanism through immunoprecipitation, deubiquitination assay, etc. This study will enrich the mechanism underlying HCC invasion and metastasis, and provide a novel modality for the prevention and treatment of HCC recurrence and metastasis.

肿瘤复发转移是肝癌患者死亡的首要原因，癌基因激活和抑癌基因失活是该过程的关键因素。BAP1在肝癌中存在基因突变，但其功能目前尚不清楚。我们研究发现：BAP1在肝癌中频发突变，功能结构分析提示主要为失活突变；BAP1在肝癌中表达下调，并与患者预后显著相关；BAP1表达下调促进肝癌侵袭转移并增敏奥拉帕尼的抗肝癌活性；免疫共沉淀和蛋白质谱分析显示BAP1可与PTEN结合，并拮抗E3泛素连接酶WWP2介导的PTEN降解。据此我们提出假说：BAP1表达下调或失活突变可能通过调控PTEN泛素化降解及其下游信号通路促进肝癌侵袭转移、增敏奥拉帕尼的抗肝癌活性。本研究拟从分子、细胞、组织、动物水平，利用肝癌体内外模型、患者及小鼠肝癌病理标本，以免疫共沉淀、去泛素化实验等方法，研究BAP1及其突变体在肝癌侵袭转移及奥拉帕尼抗肿瘤效应中的作用和分子机制。本研究将进一步丰富肝癌复发转移机制，并为其防治提供新思路。

肿瘤复发转移是肝癌患者死亡的首要原因，癌基因激活和抑癌基因失活是该过程的关键因素。BAP1在肝癌中存在基因突变，但其功能目前尚不清楚。因此，本项目主要聚焦于肝癌中存在的BAP1基因突变并分析其对BAP1蛋白结构和功能的影响、评估BAP1及其突变体在预测肝癌复发转移中的临床价值、阐明BAP1及其突变体在肝癌侵袭转移和相关分子靶向药物抗肿瘤效应中的作用及分子机制。.我们研究发现：1、BAP1在肝癌中频发突变，突变频率为5.71%（6/105）。结构和功能分析提示，其中的4个突变（p.Phe50Ser、p.Glu125Val、p.Arg150Gly、p.Tyr189Cys）为失活突变，且均位于BAP1基因的保守序列。2、BAP1在临床HCC癌组织中呈低表达，在有肿瘤复发的HCC癌组织中呈更低表达。BAP1低表达的肿瘤呈现更强的侵袭性：肿瘤更大，数目更多，血管侵犯更严重，TNM分期更高。BAP1低表达的HCC患者术后总生存时间及无瘤生存时间显著低于BAP1高表达的HCC患者，是HCC患者术后预后的独立危险因素。3、BAP1的表达水平与肝细胞癌侵袭转移能力密切相关，BAP1表达下调促进肝细胞癌增殖、侵袭和转移并增敏奥拉帕尼的抗肝癌活性；同时，BAP1发生失活突变（p.Phe50Ser、p.Glu125Val、p.Arg150Gly、p.Tyr189Cys）后，其抑制HCC细胞增殖、迁移、侵袭的功能减弱，进而促进HCC细胞的增殖、侵袭转移。4、BAP1通过结合和稳定PTEN，抑制AKT/GSK-3β/Snail通路，抑制肝细胞癌的上皮-间质转化和侵袭转移；BAP1表达下调或失活突变后，BAP1稳定PTEN的功能减弱，AKT/GSK-3β/Snail通路激活，诱导肝细胞癌的上皮-间质转化和促进肝细胞癌的侵袭转移。.本研究立足已有研究基础，结合国内外最新研究进展，进一步明确了BAP1在我国肝癌患者中存在的基因突变，分析了BAP1突变对其蛋白结构和功能的影响，评估了BAP1及其突变体在预测肝癌复发转移中的临床价值，阐明了BAP1及其突变体在肝癌侵袭转移中的作用及相关分子机制，初步揭示了其在奥拉帕尼抗肝癌活性中的作用，从而为肝癌复发转移的防治提供了新思路，具有非常重要的理论和临床转化前景。