以HDAC5-LSD1基因表达调控轴为靶点的抗三阴性乳腺癌实验研究

Triple negative breast cancer (TNBC) refers to any breast cancer that lacks expression of estrogen receptor alpha (ER-a), progesterone receptor (PR) and HER2/neu proteins. Clinically, TNBC is more aggressive and has higher distant metastases rate in comparison with non-TNBC counterparts. Currently, the only treatment option for patients with TNBC is common chemotherapy because anti-ER or anti-HER-2 target therapy does not work for TNBC. Therefore, targeted approaches for TNBC treatment are urgently needed in clinic. Recent evidence showed that abnormal epigenetic regulation play a cirtical role in tumorigenesis and progression of breast cancer. Our previous study demonstrated that HDAC5 (histone deacetylase 5) can interact with LSD1 to form an HDAC5-LSD1 axis to promote TNBC proliferation and migration by silencing a group of tumor suppressor genes, suggesting that HDAC5-LSD1 axis is a potential target for TNBC treatment. The purposes of this study are: 1) Identify the regulation mechanism of HDAC5-LSD1 axis and its specific effect factors; 2) Analyze the structural basis of HDAC5/LSD1 interaction, then design and screen specific small-molecular inhibitors to block HDAC5/LSD1 interaction through structure-based computational analysis. 3) Evalute effects of small-molecular inhibitors on TNBC proliferation and migration in patient-derived tumor xenograft model and TNBC cells. This study aims to provide novel targets for molecularly directed therapies for TNBC, with important research value and potential prospect for clinical application.

三阴性乳腺癌（TNBC）恶性程度高、易发生肿瘤转移，目前无有效治疗方法，是临床抗乳腺癌治疗的难点。表观遗传调控机制紊乱导致基因表达异常是乳腺癌发生、发展的关键因素之一。本项目前期研究发现，组蛋白去乙酰化酶HDAC5与组蛋白去甲基化酶LSD1能直接相互作用并形成HDAC5-LSD1基因表达调控轴，通过抑制抑癌基因表达促进TNBC细胞增殖和转移，是潜在的TNBC治疗靶点。本研究拟进一步探索HDAC5-LSD1调控轴影响TNBC细胞增殖和转移的分子机制并筛选其下游关键效应分子、鉴定HDAC5和LSD1相互作用的结构基础，应用计算生物学辅助设计和筛选HDAC5/LSD1相互作用的特异性小分子抑制剂，在细胞和TNBC人肿瘤组织裸鼠移植瘤模型水平上验证用小分子抑制剂阻断HDAC5-LSD1轴对TNBC细胞增殖和转移能力的影响，由此探索建立靶向HDAC5-LSD1调控轴的抗TNBC治疗新方法和新途径。

本研究项目按批准的项目执行书进行，并大胆对研究内容进一步拓展，已经按照预算完成81.8%的直接研究经费支出；在研究目标、人才培养、学术交流方面都完成了计划，并正在进行拓展研究。具体如下：.1）.部分阐明了HDAC5-LSD1调控轴影响TNBC细胞增殖和侵袭转移的分子机制。通过多个CRISPR-Cas9介导的稳定基因敲除细胞系和小鼠模型研究发现：HDAC5-LSD1调控轴通过抑制P21、E-Cadherin、Spi-1表达和dsRNA的堆积，参与TNBC和ER+乳腺癌细胞中细胞增殖、迁移、RNA m6A修饰和免疫逃逸。完成了RNA-Seq、ATAC-Seq和DAN甲基化的基因组学建库和高通量测序工作，其测序结果正在分析，将在近期完成HDAC5-LSD1调控轴下游信号通路和关键效应分子鉴定。.2）完成了LSD1突变体表达质粒的构建、Co-IP和GST-Pull down实验，发现 LSD1通过其Tower结构域与HDAC5氨基末端200-498位氨基酸区域相互作用。.3） 建立了靶向HDAC5-LSD1基因表达调控轴的TNBC治疗新方法和新途径。利用前述基因敲除细胞模型和荷瘤小鼠模型研究表明：使用组蛋白去乙酰化酶抑制剂Sulforaphane、LSD1抑制剂优降宁或Akt抑制剂MK-2206和阿霉素、顺铂联合应用能够产生药物协同效应抑制乳腺癌细胞增殖。证明以HDAC5-LSD1基因表达调控轴为靶点的上述抑制剂与化疗药物联合应用具有抗TNBC治疗临床应用价值。通过rAAV（重组腺相关病毒）衣壳蛋白改造，实现了抗体靶向乳腺癌细胞的rAAV基因递送，建立了基于rAAV的乳腺癌基因治疗靶向系统。还使用rAAV2-CRISPR-saCas9为载体，进行新生小鼠颞静脉注射，获得肝脏LSD1基因敲除的成体C57BL/6小鼠。.总体上，本项目完成了预期目标，执行中建立了一系列新的、可直接应用的研究方法、工具和实验模型。未来将在此基础上，围绕本项目的核心科学问题深入研究。