Zhang Ruishan, Li Xiang, Liu Zhuangkai, Wang Yuying, Zhang Hao, Xu Hong
Department of Breast Cancer, Cancer Hospital of China Medical University, Liaoning Cancer Hospital and Institute, 44 Xiaoheyan Road, Dadong District, Shenyang, 110042 Liaoning People's Republic of China.
Cancer Cell Int. 2020 May 19;20:175. doi: 10.1186/s12935-020-01260-5. eCollection 2020.
Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer that lacks expression of estrogen receptor (ER) and progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2) gene. Chemotherapy remains the standard of care for TNBC treatment, but considerable patients are very resistant to chemotherapy. Mutations or aberrant upregulation of EZH2 occur frequently, and EZH2 inhibitor (EZH2i) showed some preclinic antitumor effects in TNBC.
RNA-seq data of 3 TNBC cell lines either treated with 2 μM GSK343, or stably transduced with shEHZ2, compared to untreated controls (GSE112378) were analyzed by Limma R package. The Kaplan-Meier plotter (KM plotter) database was used to assess the relevance of FOSB mRNA expression to relapse-free survival (RFS) in TNBC. Cell number counting and colony formation assays were used to detect the biological effect of FOSB on the growth of TNBC cells in vitro. The effect of FOSB on TNBC tumor growth in vivo was investigated in a mice tumor xenograft model. Luciferase reporter and chromatin immunoprecipitation (Chip) assays were used to determine the regulatory roles of C/EBPβ on FOSB expression.
We found that FOSB, a member of the activator protein-1 complex, was a direct downstream target of EZH2. FOSB was significantly decreased in TNBC samples and associated with better relapse-free survival (RFS). EZH2-mediated histone 3 trimethylated on lysine 27 (H3K27me3), a marker of silent chromatin conformation, at the FOSB promoter inhibited it expression. Depletion of FOSB in TNBC cells promoted cell proliferation in vitro and tumor growth in vitro by inactivating the p53 pathway and conferred resistant to EZH2 inhibitor (EZH2i). Mechanistically, EZH2i promotes the shift from H3K27me3 to H3K27ac at the FOSB promoter, and recruits the transcription factor C/EBPβ to activate FOSB gene transcription.
Together, our results suggest that EZH2-mediated epigenetic inactivation of FOSB promotes TNBC expression and demonstrate that reactivation of FOSB expression by C/EBPβ underlies the anti-TNBC action of EZH2is.
三阴性乳腺癌（TNBC）是最具侵袭性的乳腺癌亚型，缺乏雌激素受体（ER）、孕激素受体（PR）以及人表皮生长因子受体2（HER2）基因的表达。化疗仍是TNBC治疗的标准疗法，但相当一部分患者对化疗具有很强的抗性。EZH2频繁发生突变或异常上调，并且EZH2抑制剂（EZH2i）在TNBC中显示出一些临床前抗肿瘤作用。
使用Limma R软件包分析了3种TNBC细胞系的RNA测序数据，这些细胞系分别用2 μM GSK343处理，或用shEHZ2稳定转导，与未处理的对照相比（GSE112378）。使用Kaplan-Meier plotter（KM plotter）数据库评估FOSB mRNA表达与TNBC无复发生存期（RFS）的相关性。使用细胞计数和集落形成试验检测FOSB对TNBC细胞体外生长的生物学效应。在小鼠肿瘤异种移植模型中研究FOSB对TNBC肿瘤体内生长的影响。使用荧光素酶报告基因和染色质免疫沉淀（Chip）试验确定C/EBPβ对FOSB表达的调控作用。
我们发现激活蛋白-1复合物成员FOSB是EZH2的直接下游靶点。FOSB在TNBC样本中显著降低，并与更好的无复发生存期（RFS）相关。EZH2介导的FOSB启动子上赖氨酸27处组蛋白3三甲基化（H3K27me3），一种沉默染色质构象的标志物，抑制了它的表达。TNBC细胞中FOSB的缺失通过使p53通路失活促进体外细胞增殖和体内肿瘤生长，并赋予对EZH2抑制剂（EZH2i）的抗性。机制上，EZH2i促进FOSB启动子处从H3K27me3向H3K27ac的转变，并招募转录因子C/EBPβ以激活FOSB基因转录。
总之，我们的结果表明EZH2介导的FOSB表观遗传失活促进TNBC的发生发展，并证明C/EBPβ对FOSB表达的重新激活是EZH2i抗TNBC作用的基础。
