乳腺癌（BC）是全世界女性中最常见的癌症。 BC 干细胞 (BCSC) 已知参与乳腺癌发生并导致治疗耐药。 BC 的程序性死亡配体 1 (PD-L1) 表达与不良预后相关。以 PD-L1 为靶点的免疫疗法具有巨大的潜力，并且在应用于癌症治疗时已取得成功。然而，PD-L1是否调节BCSC的形成尚不清楚。BCSC通过无血清悬浮培养进行富集。通过乳腺球形成测定、CD44/Cd24-、乙醛脱氢酶（ALDH）测定、CSC标记物分析和乳腺球生长测定来检查BCSC的特性。为了阐明含溴结构域蛋白 4 (BRD4)、核 PD-L1 和 RelB 蛋白在 BCSC 干性中的功能，使用 BRD4 抑制剂和降解剂、PD-L1 降解剂和 RelB 抑制剂检查了乳腺球的形成。通过体内肿瘤模型和小鼠研究，研究了特异性 BRD4 抑制剂 3',4',7,8-四羟基黄酮 (THF) 的抗肿瘤功能，以及 c-Myc、PD-L1 和 RelB 的蛋白水平在THF处理下的肿瘤模型中进行了检查。BRD4在乳腺CSC中上调并调节BC的干性。使用 BRD4 PROTAC、ARV-825 和 BRD4 抑制剂 ()-JQ1 下调 BRD4，可抑制乳腺球形成并降低乳腺 CSC 标记物（CD44 /CD24- 和 ALDH1）、干细胞标记基因和乳腺球生长的水平。 BRD4 抑制剂 (JQ1) 和降解剂 (ARV825) 通过抑制 PD-L1 转录水平来下调 PD-L1 的膜和核部分。 PD-L1 的敲低会抑制乳腺球的形成。 Verteporfin 是一种 PD-L1 降解剂，抑制 PD-L1 的转录本和蛋白质水平，并下调 RelB 的转录本和蛋白质水平。骨化三醇（一种 RelB 抑制剂）和使用 si-RelB 敲低 RelB 可通过白细胞介素 6 (IL-6) 表达调节乳腺球形成。 THF 是一种天然产物，是一种有效的选择性 BRD4 抑制剂，抑制乳腺球形成，并通过下调 c-Myc、PD-L1 和 RelB 来降低 CD44 /CD24- 水平和乳腺球生长。在使用 4T1 和 MC38 细胞的小鼠肿瘤模型中，3',4',7,8-THF 显示出杀肿瘤活性以及肿瘤和肿瘤引流淋巴结 (TDLN) 中 CD3 CD4 和 CD3 CD8 T 细胞水平的增加。结果首次证明 BRD4/核 PD-L1/RelB 轴在乳腺 CSC 形成中的重要作用。细胞核 PD-L1 调节 RelB，RelB/p65 复合物诱导 IL6 和乳腺 CSC 形成。靶向细胞核 PD-L1 代表了顽固性 BC 免疫治疗的一种潜在且新颖的工具。视频摘要。© 2023。作者。

Breast cancer (BC) is the most common cancer diagnosed in women worldwide. BC stem cells (BCSCs) have been known to be involved in the carcinogenesis of the breast and contribute to therapeutic resistance. The programmed death-ligand 1 (PD-L1) expression of BC correlated with a poor prognosis. Immunotherapies that target PD-L1 have great potential and have been successful when applied to cancer treatment. However, whether PD-L1 regulates BCSC formation is unknown.BCSCs were enriched by serum-free suspension culture. The properties of BCSCs were examined by mammosphere formation assay, CD44+/Cd24-, aldehyde dehydrogenase (ALDH) assay, CSC marker analysis, and mammosphere growth assay. To elucidate the functions of bromodomain-containing protein 4 (BRD4), nuclear PD-L1, and RelB proteins in the stemness of BCSCs, mammosphere formation was examined using BRD4 inhibitor and degrader, PD-L1 degrader, and RelB inhibitor. The antitumor function of 3',4',7,8-tetrahydroxyflavone (THF), a specific BRD4 inhibitor, was studied through in vivo tumor model and mouse studies, and the protein levels of c-Myc, PD-L1, and RelB were examined in tumor model under THF treatment.BRD4 was upregulated in breast CSCs and regulates the stemness of BCs. The downregulation of BRD4 using BRD4 PROTAC, ARV-825, and BRD4 inhibitor, (+)-JQ1, inhibits mammosphere formation and reduces the levels of breast CSC markers (CD44+/CD24- and ALDH1), stem cell marker genes, and mammosphere growth. BRD4 inhibitor (JQ1) and degrader (ARV825) downregulate membrane and nuclear fractions of PD-L1 through the inhibition of PD-L1 transcript levels. The knockdown of PD-L1 inhibits mammosphere formation. Verteporfin, a PD-L1 degrader, inhibits the transcripts and protein levels of PD-L1 and downregulates the transcript and protein levels of RelB. Calcitriol, a RelB inhibitor, and the knockdown of RelB using si-RelB regulate mammosphere formation through interleukin-6 (IL-6) expression. THF is a natural product and a potent selective BRD4 inhibitor, inhibits mammosphere formation, and reduces the levels of CD44+/CD24- and mammosphere growth by downregulating c-Myc, PD-L1, and RelB. 3',4',7,8-THF shows tumoricidal activity and increased levels of CD3+CD4+ and CD3+CD8+ T-cells in the tumor and tumor-draining lymph nodes (TDLNs) in the murine tumor model using 4T1 and MC38 cells.The results show the first evidence of the essential role of the BRD4/nuclear PD-L1/RelB axis in breast CSC formation. The nuclear PD-L1 regulates RelB, and the RelB/p65 complex induces IL6 and breast CSC formation. Targeting nuclear PD-L1 represents a potential and novel tool for immunotherapies of intractable BC. Video Abstract.© 2023. The Author(s).