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题名	转录因子KLF5去泛素化酶的筛选及其在乳腺癌中的功能与机制研究
作者	秦君英
学位类别	博士
答辩日期	2015-05
授予单位	中国科学院研究生院
授予地点	北京
导师	陈策实
关键词	去泛素化酶 KLF5 三阴性乳腺癌 BAP1 细胞增殖 USP3 肺转移
其他题名	Identification of the deubiquitinases for KLF5 transcription factor and studying their functions and mechanisms in breast cancer
中文摘要	转录因子KLF5去泛素化酶的筛选及其在乳腺癌中的功能与机制研究 摘 要 乳腺癌是一种严重危害全球女性健康的恶性肿瘤。三阴性乳腺癌（TNBC：ERα/PR/HER2均为阴性的乳腺癌）约占所有乳腺癌的15-20%；但因其缺乏有效的治疗靶点和远端器官转移使TNBC成为所有乳腺癌中预后最差的一种类型。因此，鉴定TNBC新的分子靶标，并阐述其作用机理迫在眉睫。 去泛素化酶（Deubiquitinating enzymes：DUBs）可以逆转蛋白质的泛素化修饰，是泛素-蛋白酶体途径的关键调控因子。进一步研究发现：DUBs与肿瘤发生息息相关并且具有开发成为药物靶标的巨大潜力。 KLF5属于锌指结构的转录因子，特异性地在ERα（以后简称ER）阴性的乳腺癌中高表达。研究发现，KLF5可以作为病人预后的分子标志物：KLF5高表达的病人生存时间显著短于KLF5低表达的病人。我们课题组前期研究证明KLF5主要是通过上调其下游靶基因FGF-BP和mPGES1来促进乳腺（癌）生长、生存和肿瘤发生。这些研究表明：KLF5是乳腺癌有效的治疗靶点。但是，从药物研发的角度考虑，转录因子本身并不是理想的直接药物靶点，所以找到正向调控KLF5的酶可能提供更好的药物干预靶点。我们实验室最早发现KLF5受到泛素化修饰后通过蛋白酶体途径降解。进一步，我们找到了两个控制KLF5降解的关键E3泛素连接酶 WWP1和SCFFbw7；最近美国一个研究小组发现Smurf2也是KLF5的E3泛素连接酶。目前，还没有关于KLF5去泛素化酶的任何报道。因此，我们的科学猜想是：KLF5的去泛素化酶应该可以正向调控KLF5的蛋白稳定性并且具有癌蛋白的功能。 通过扫描包含87个人类去泛素化酶的siRN文库去鉴定KLF5可能的去泛素化酶。经过几轮筛选，我们最终确定BAP1和USP3作为KLF5的候选去泛素化酶。 为了进一步确定BAP1是否可以增加KLF5蛋白的稳定性。我们转染针对BAP1不同位置的siRNA（避免脱靶效应）分别在MCF10A和HCC1806以及HCC1937 TNBC细胞株；发现敲低BAP1，内源KLF5及其下游靶基因FGF-BP的蛋白水平都会下降。同时我们还发现无论是敲低BAP1或KLF5，p27的蛋白水平上升都非常明显。BAP1可以拮抗多个E3泛素连接酶介导的KLF5的降解以及延长KLF5的蛋白半衰期。进一步在体内体外实验中，BAP1可以非常显著地降低KLF5的多聚泛素化水平，但其酶活性突变体BAP1C91S则不具有这种功能。 接下来，我们探索了BAP1稳定KLF5的作用机制。通过免疫共沉淀(IP)和Pull-down实验发现内源的BAP1和KLF5存在相互作；纯化的GST-BAP1和KLF5-6×His存在直接相互作用。同时我们构建了一系列GST-融合的BAP1或His-融合的KLF5截短突变体来检测其负责相互作用的区段，发现BAP1和KLF5的N-端和C-端负责参与相互作用。 我们研究发现，BAP1可以与HCF-1，OGT以及其他一些蛋白形成功能复合物。KLF5在BAP1/HCF-1复合物中；并且KLF5/BAP1/HCF-1复合物通过结合于p27基因的启动子上抑制p27基因的转录。进一步研究发现，KLF5/BAP1/HCF-1复合物通过抑制p27基因的表达，促进细胞周期的G1/S进程。 功能研究表明，敲低BAP1 或KLF5抑制细胞的增殖、生长、迁移和侵袭。动物实验表明，敲低BAP1导致的肿瘤抑制和肺转移减慢，瞬时过表达KLF5可以部分恢复BAP1敲低导致的表型。 另外，我们发现USP3也降低内源KLF5的蛋白水平。USP3可以拮抗E3泛素连接酶介导的KLF5的降解并延长其半衰期。同时，USP3可以降低KLF5的泛素化水平，并且是去泛素化酶活性依赖的。USP3和KLF5存在相互作用；KLF5的PY motif可能参与调控这一过程。功能实验提示USP3可能参与调控DNA damage 相关功能，但需要进一步证明。 综上，我们的研究不仅证明BAP1和USP3是KLF5的去泛素化酶，而且阐述了其调控机制。BAP1和USP3有可能作为TNBC或其他癌症的潜在治疗靶标。
英文摘要	Identification of the deubiquitinases for KLF5 transcription factor and studying their functions and mechanisms in breast cancer Abstract Breast cancer, as a malignant tumor, has become an increasing threat for women woridwide. ERα/PR/HER2 triple-negtive breast cancer (TNBC) comprises approximately 15-20% of breast cancesr and has the poorest prognosis because of lacking of effective therapeutic targets and distant vital organ metastasis. It is very important and urgent to identify novel therapeutic targets for TNBC and to understand their metastasis mechanisms. Ubiquitination is a protein post-translational modification which can be reversed by a large family of deubiquitinating enzymes (DUBs). DUBs are well established to regulate cancer development and novel targets for cancer therapy. KLF5 (Krüppel-like zinc finger transcription factor 5) is a transcription factor highly expressed in estrogen receptorα (ERα)-negative basal subtype breast cancers. High KLF5 mRNA and protein levels have been reported as a potent biomarker for unfavorable prognosis for breast cancer patients. Furthermore, our previous studies demonstrated that KLF5 promotes cell proliferation, survival and tumor growth partially through inducing transcription of downstream target genes, such as FGF-BP and mPGES1. These findings collectively define KLF5 as a potent therapeutic target for basal TNBC and other cancers. However, transcriptional factors are not ideal targets for drug development. It is significant to identify KLF5 upstream positive regulators, which may provide better therapeutic targets for cancer treatment. KLF5 has been identified as an unstable protein, which is ubiquitinated by WWP1, SCFFbw7, and Smurf2 E3 ligases for degraded. So far, no DUBs for KLF5 have been identified. Hence, we hypothesize that the DUBs of KLF5 should stabilize the KLF5 protein and possess oncogenic functions in breast cancer. To identify DUBs for KLF5, we unbiasedly screened a siRNA library against 87 human DUBs. After several rounds of screening, we finally identify BAP1 and USP3 as candidate DUBs for KLF5. To further confirm whether BAP1 increases KLF5 protein stability, we transfected siRNAs targeting different regions of BAP1 into MCF10A and HCC1806 and HCC1937 TNBC cell lines. Knockdown of BAP1decreased the endogenous protein levels of KLF5 and its downstream target gene FGF-BP. Interestingly, knockdown of either KLF5 or BAP1 upregulated the p27 protein levels in these breast cell lines. BAP1 can antagonize the E3 ligase-mediated KLF5 degradation. Furthermore, the KLF5 protein half-life was extended by BAP1 but not BAP1C91S (a catalytic inactive BAP1 mutant) overexpression. BAP1 dramatically decreased KLF5 protein polyubiquitination in vivo and in vitro, in its enzyme activity dependent manner. Next, we explored the mechanisms by which BAP1 stabilizes KLF5. Endogenous BAP1 interacts with endogenous KLF5. Purified GST-BAP1 and KLF5-6×His directly interact with each other. Furthermore, we generated a series of GST fused BAP1 deletion mutants and His tagged KLF5 truncations to identify the regions of BAP1 or KLF5 responsible for the interaction. We found that the interaction is mediated by N-terminus and C-terminus of both BAP1 and KLF5. BAP1 has been reported to form a protein complex with HCF-1, OGT and other several other protein. We found that KLF5 was in the BAP1/HCF-1complex. The KLF5/BAP1/HCF-1 complex inhibited p27 expression through binding to the p27 gene promoter. Moreover, the KLF5/BAP1/HCF-1 protein complex promotes cell cycle G1/S progression partially through inhibiting the p27 gene transcription. Functional studies revealed that knockdown of BAP1 or KLF5 inhibited breast cancer cell proliferation, cell migration and invasion in vitro. Furthermore, depletion of BAP1 inhibited tumorigenicity and lung metatssis, which can be partially rescued by ectopic KLF5 expression. Additionally, we found that depletion of USP3 also dreased KLF5 levels. USP
语种	中文
内容类型	学位论文
源URL	[http://159.226.149.26:8080/handle/152453/10181]
专题	昆明动物研究所_肿瘤生物学
推荐引用方式 GB/T 7714	秦君英. 转录因子KLF5去泛素化酶的筛选及其在乳腺癌中的功能与机制研究[D]. 北京. 中国科学院研究生院. 2015.

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