背景:乳腺癌(BC)是全球女性中最常见的癌症。已知BC干细胞(BCSC)参与乳腺的癌变并有助于治疗抗性。BC的程序性死亡配体1(PD-L1)表达与不良预后相关。靶向PD-L1的免疫疗法具有巨大的潜力,并且在应用于癌症治疗时已经成功。然而,PD-L1是否调节BCSC的形成尚不清楚。
方法:采用无血清悬浮培养法富集BCSCs。BCSCs的性质通过乳腺球形成试验进行检测,CD44+/Cd24-,醛脱氢酶(ALDH)测定,CSC标记分析,和乳腺球生长测定。为了阐明含溴结构域蛋白4(BRD4)的功能,BCSC的核PD-L1和RelB蛋白,使用BRD4抑制剂和降解剂检查乳腺球形成,PD-L1降解器,和RelB抑制剂。3'的抗肿瘤功能,4\',7,8-四羟基黄酮(THF),一种特定的BRD4抑制剂,通过体内肿瘤模型和小鼠研究,和c-Myc的蛋白质水平,在THF处理下在肿瘤模型中检查PD-L1和RelB。
结果:BRD4在乳腺CSC中上调,调节BCs的干性。使用BRD4PROTAC下调BRD4,ARV-825和BRD4抑制剂,(+)-JQ1,抑制乳腺球形成并降低乳腺CSC标志物(CD44+/CD24-和ALDH1)的水平,干细胞标记基因,和乳腺球生长。BRD4抑制剂(JQ1)和降解剂(ARV825)通过抑制PD-L1转录物水平下调PD-L1的膜和核部分。PD-L1的敲低抑制乳腺球形成。Verteporfin,PD-L1降解器,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,RelB抑制剂,使用si-RelB敲除RelB通过白细胞介素-6(IL-6)的表达调节乳腺球的形成。THF是一种天然产物,是一种有效的选择性BRD4抑制剂,抑制乳腺球的形成,并通过下调c-Myc来降低CD44+/CD24-和乳腺球的生长水平,PD-L1和RelB。3\',4\',在使用4T1和MC38细胞的鼠肿瘤模型中,7,8-THF显示肿瘤活性和肿瘤和肿瘤引流淋巴结(TDLN)中CD3+CD4+和CD3+CD8+T细胞水平增加。
结论:结果显示BRD4/核PD-L1/RelB轴在乳腺CSC形成中的重要作用。核PD-L1调节RelB,和RelB/p65复合物诱导IL6和乳腺CSC形成。靶向核PD-L1代表了难治性BC免疫治疗的潜在和新颖工具。视频摘要。
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.