乙酰华蟾毒精靶向抑制HSP110发挥抗TNBC的作用机制研究

Triple-negative breast cancer (TNBC) is the main concern and a challenging part in the field of breast cancer research and currently lacks corresponding therapeutic targets. Heat shock protein 110 (HSP110) was highly expressed in a variety of malignancies, and expression levels are associated with clinical stage and metastasis. The role of HSP110 in TNBC still remains unclear. Our previous research indicated that HSP110 was overexpressed in TNBC cells and tissues. Additionally, we synthesized a novel prodrug of CBF, named acetyl-cinobufagin (Ac-CBF), with stronger anti-tumor effect and higher bioavailability. Subsequently we used a biotinylated Ac-CBF (Bio Ac-CBF), and a proteome microarray identifies the direct targets and discovered that Ac-CBF bound 35 proteins containing HSP110. Moreover, the result of computational molecular docking and SPR analysis indicated that Ac-CBF could directly bind to HSP110. And the cell experiments confirmed that it directly targets HSP110. Subsequently, we found that HSP110 specific inhibitor Ac-CBF significantly inhibited cell proliferation and migration, as well as induced apoptosis in TNBC cells. In this research, we design a series of experiments in the cellular, molecular, animal and clinical levels and try to confirm our hypothesis. This project will not only have a high scientific impact on the molecular mechanism of Ac-CBF in the development of TNBC but also will provide a novel promising target for the treatment of TNBC.

三阴性乳腺癌（TNBC）是乳腺癌研究领域的热点和难点，目前临床上缺乏相应的治疗靶点。热休克蛋白110（HSP110）在多种恶性肿瘤中高表达，且表达水平与临床分期和转移有关，但HSP110在TNBC发生发展中的作用尚不清楚。项目组前期发现HSP110在TNBC细胞和患者癌组织中高表达，发现乙酰华蟾毒精具有极强的抑制TNBC细胞增殖的作用，经蛋白质组芯片筛选药物靶点，计算机模拟、SPR实验及细胞实验均证实其直接靶向HSP110。随后以此作为工具化合物，发现乙酰华蟾毒精能有效抑制TNBC细胞迁移，诱导细胞凋亡。本项目将进一步设计分子、细胞、动物和临床实验，深入阐明乙酰华蟾毒精抗TNBC的分子机制，同时为HSP110作为TNBC治疗的新靶点提供理论依据。

本项目在基金委员会的资助下共完成了三部分的内容：第一部分，测试乙酰华蟾毒精在体外对TNBC细胞的抑制效果，考察乙酰华蟾毒精作为三阴性乳腺癌抗肿瘤治疗新药的可能性。实验结果表明，乙酰华蟾毒精在体内外均具有极强的TNBC细胞抑制能力，且对正常细胞毒性很小。第二部分，探索乙酰华蟾毒精抑制TNBC细胞的分子机制。研究表明，乙酰华蟾毒精可以靶向抑制STAT3，降低STAT3分子的磷酸化水平，并阻止STAT3胞质到胞核的转移，从而诱导TNBC细胞凋亡。其次，本项目创新性的发现乙酰华蟾毒精通过抑制STAT3通路抑制TNBC的上皮间充质转换（EMT），确证了STAT3在三阴性乳腺癌EMT中的关键介导作用。第三部分，评估了乙酰华蟾毒精在动物PDX模型上的安全性以及机制验证，乙酰华蟾毒精在体内抑制TNBC增大且无组织毒性表现。这些证据都表明乙酰华蟾毒精有望成为新的抗三阴性乳腺癌治疗药物。.总的来说，负责人以药物化学为学科基础，瞄准了中国传统中药天然成分的抗肿瘤能力，结合药理学和化学生物学，改良传统的中药有效成分，寻找对三阴性乳腺癌有良好治疗作用的新化合物。阐明中药天然成分的药理作用机制、发现其直接作用靶点，并确证该靶点在三阴性乳腺癌中的作用和功能。项目合计发表SCI论文7篇，获得授权发明专利4项。