E3泛素连接酶UBE3B通过HIF-2α调控乳腺癌生长与转移的分子机制研究

Breast cancer has highest incidence among female malignancy in China. Transcriptional factor HIF-2 is a master player which promotes breast tumor progression and metastasis in the tumor hypoxic microenvironment, but the underlying molecular mechanism remains poorly understood. Our preliminary data indicated that E3 ligase UBE3B is highly expressed in estrogen receptor positive (ER+) breast tumors and high levels of UBE3B are significantly correlated with poor clinical outcomes of patients with ER+ breast cancer. UBE3B augments HIF-2α stability and transcriptional activity by associating with its C-terminus. Knockdown of UBE3B via shRNA dramatically decreases the abilities of ER+ breast cancer cells in proliferation, migration, invasion and colony formation. All these exciting results led us to hypothesize that UBE3B promotes ER+ breast tumor growth and metastasis through stabilizing HIF-2α. To test this hypothesis, we will use molecular, biochemistry and cell biology approaches in cell-based assays to determine how UBE3B stabilizes HIF-2α, then utilize xenograft mouse model to elucidate the molecular mechanism by which UBE3B promotes ER+ breast tumor growth and metastasis. The successful completion of the proposed research will further uncover the fundamental mechanism of ER+ breast tumor growth and metastasis, and provide a novel therapeutic target for treatment of ER+ breast cancer.

乳腺癌发病率位列中国女性恶性肿瘤之首。转录因子HIF-2是促进乳腺癌在肿瘤低氧环境下进一步恶化与转移的关键因子，但其确切机制尚未完全阐明。在前期研究中，我们发现E3泛素连接酶UBE3B在雌激素受体阳性（ER+）乳腺癌中显著高表达，且其高表达与ER+乳腺癌患者临床存活率呈显著负相关。UBE3B与HIF-2α 的C末端接合并促进HIF-2α的稳定性和转录活性。敲低UBE3B则显著抑制ER+乳腺癌细胞增殖、迁移、侵袭和形成克隆的能力。我们推测“UBE3B通过增强HIF-2α的稳定性促进ER+乳腺癌的生长与转移”。本项目将首先利用生物化学，细胞生物学与分子生物学等技术在细胞水平明确UBE3B对HIF-2α稳定性的调节。其次利用小鼠模型重点关注UBE3B促进ER+乳腺癌生长与转移的分子机制。完成本课题将进一步揭示ER+乳腺癌生长与转移的分子机制，为其防治提供新思路和新靶点。

E3泛素连接酶因其在Kaufman眼脑面综合征（KOS）中的重要作用而引起科研人员的注意。近年来，越来越多的研究表明，UBE3B在肿瘤发生发展中也可能扮演重要角色，但是其具体作用以及发挥作用的分子机制还鲜有报道。通过亲和纯化结合质谱的方法我们筛选到UBE3B可能结合的蛋白HIF-2α，而Co-IP、Western、pull-down等实验证实UBE3B的确直接接合HIF-2α。接下来，UBE3B被证实促进HIF-2α第394、497和503位赖氨酸的K63连接多泛素化，进而抑制E3泛素连接酶VHL介导的HIF-2α蛋白酶体途径的降解，因而促进HIF-2α的稳定性和下游靶基因的转录，最终促进乳腺癌的生长与转移。值得注意的是，UBE3B对HIF-1α似乎无影响。免疫组化显示，与癌旁正常乳腺组织相比较，乳腺癌组织中UBE3B的蛋白质水平明显升高且与HIF-2α的蛋白质水平呈正相关。Kaplan-Meier分析表明UBE3B高表达显著降低乳腺癌患者的临床存活率。综上所述，UBE3B是新的HIF-2α正调控因子，在乳腺癌中发挥促癌作用。本研究有多方面的意义：（1）首次于体内证实UBE3B调控瘤生长与转移的功能，拓展了UBE3B的功能；（2）丰富了HIF信号通路的调控机制；（3）为乳腺癌提供了新的诊断标记物和分子靶标；（4）深化了我们对乳腺癌生物学特性的认知。