E3泛素连接酶HRD1/脂肪酸合酶（FASN）在乳腺癌发生发展中的分子机制研究

Breast cancer is one of the main diseases that threaten women health. The incidence of breast cancer is increasing year by year. In recent years, people gradually realize that a lot of diseases including malignant tumors, are related with the expression and function of the E3 ubiquitin ligases. In our previous study, it was found that the levels of the E3 ubiquitin ligase HRD1 in breast cancer tissues was significantly lower than that of the corresponding cancer adjacent tissues; overexpression of HRD1 could significantly inhibit breast cancer cell proliferation and migration, and inhibit the growth and metastasis of breast cancer. There is increasing evidence that fatty acid synthase (FASN) expression in human breast cancer cells is significantly higher than that of normal cells. FASN can activate the PI3K/Akt/mTOR signaling axis, enhance the activity of mTOR, which can in turn enhance transcription factor SREBP-1 activity, increasing FASN expression level of mRNA , which may be closely related to the invasion and metastasis of breast cancer cells. And, through the mass spectrometry analysis results show that FASN is one of the substrate of HRD1. So this topic idea, E3 ubiquitin ligase HRD1 interacted with FASN and promoted its ubiquitination and degradation by the proteasome，which inhibit the occurrence and development of breast cancer, but the specific mechanism remains unclear. The purpose of this study is to explore the mechanism of the interaction between HRD1 and FASN in breast cancer cells, and to find a new drug target for clinical treatment of breast cancer.

乳腺癌是威胁女性健康的主要疾病之一，在国内外妇女人群中的发病率逐年上升。近年来，人们认识到，包括乳腺癌在内的很多疾病都与不同E3泛素连接酶的表达与功能相关。我们前期研究结果发现，E3泛素连接酶HRD1在乳腺癌组织中的表达水平显著低于相应癌旁组织；过表达HRD1能显著抑制乳腺癌细胞的增值、生长与转移。而脂肪酸合酶（FASN）在人类乳腺癌细胞中的表达水平显著高于正常乳腺上皮细胞，并可激活PI3K/Akt/mTOR轴信号通路，增强mTOR活性，反过来可依次增强转录因子SREBP-1活性，导致FASN mRNA表达水平的增加，进而参与乳腺癌细胞的侵袭和转移。质谱分析结果表明，FASN是HRD1的作用底物之一。所以，本课题设想HRD1可介导FASN的降解进而抑制乳腺癌的发生发展，旨在探讨乳腺癌细胞中HRD1与FASN相互作用的机制，期待为临床治疗乳腺癌找到新的药物靶点。

背景：我们的前期研究表明E3泛素连接酶HMG-CoA还原酶降解蛋白1的过表达可抑制乳腺癌的增殖和侵袭，因此可作为肿瘤抑制因子。然而，其在乳腺癌细胞葡萄糖代谢过程中的作用尚不清楚。本研究旨在揭示HRD1在调节乳腺癌糖酵解有氧过程中的作用和分子机制。.主要研究内容：本研究初级阶段主要集中研究脂肪酸合酶FASN与程序性死亡配体PD-L1、微卫星不稳定相关蛋白（MLH1、MSH2、PMS2、MSH6）、PD-1等表达水平之间的相关关系，最终发现PD-L1的表达与FASN表达水平之间存在正相关性，期待为临床治疗乳腺癌找到相关靶点。.紧接着，我们又研究了HRD1对乳腺癌细胞糖酵解的影响，评估乳腺癌细胞的增殖、集落形成能力、侵袭和迁移能力等。通过质谱分析、免疫荧光及免疫共沉淀等方法验证了HRD1与PFKP的关系。运用泛素化降解实验检测PFKP泛素化水平。我们发现了过表达HRD1明显降低了有氧糖酵解、接着抑制乳腺癌的增殖和侵袭。质谱分析结果发现较多HRD1相互作用物，包括PFKP（磷酸果糖激酶的血小板异构体），是一种参与乳腺癌的Warburg效应过程的关键酶。从机制方面出发，HRD1与PFKP相互作用以及在细胞质中共定位表达，对PFKP进行泛素化和降解，最终降低PFKP在乳腺癌细胞中的表达和活性。此外，还观察了小鼠异种移植物的肿瘤生长和转移。.重要结果：我们发现PD-L1的表达与FASN表达水平之间存在正相关性；过表达HRD1明显降低了有氧糖酵解、接着抑制乳腺癌的增殖和侵袭。HRD1与PFKP相互作用以及在细胞质中共定位表达，对PFKP进行泛素化和降解，最终降低PFKP在乳腺癌细胞中的表达和活性。.科学意义：我们揭示了HRD1在Warburg效应中的新调控作用，并发现了乳腺癌代谢过程