Department of Radiation Oncology, Department of Thoracic Oncology, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China.
Advanced Medical Research Institute and Key Laboratory for Experimental Teratology of the Ministry of Education, Cheeloo College of Medicine, Shandong University, Jinan, Shandong 250012, China.
Theranostics. 2022 Feb 28;12(6):2598-2612. doi: 10.7150/thno.70581. eCollection 2022.
Triple-negative breast cancer (TNBC) is characterized by its unique molecular profile, aggressive nature and lack of targeted therapy. Chemotherapy induces expression of pluripotency factors and mediates an active induction of breast cancer stem cells (BCSCs) in TNBC, which potentiates the risk of tumor recurrence and metastasis and increases patient mortality. Adenosine receptor 2B (A2BR) expression and activation of its downstream signaling pathway has been implied to promote breast cancer metastasis. This study is to investigate the role of A2BR in the regulation of chemotherapy-induced BCSC enrichment. We generated shRNA-mediated A2BR knockdown subclones in TNBC cell lines and evaluated the effect on the BCSC phenotype by Aldefluor and mammosphere assays in vitro. We performed chromatin immunoprecipitation (ChIP) assay to investigate recruitment of transcription factor FOXO3 and histone modification enzymes KDM6A and p300 to the regulatory regions of pluripotency factors, as well as levels of histone modification marks H3K27ac and H3K27me3 on these regions. We employed both xenograft model and genetically engineered, autochthonous breast cancer model to evaluate the effect of A2BR on chemotherapy-induced BCSC enrichment in vivo. We demonstrated that chemotherapy increased protein level of A2BR, which contributed to chemotherapy-induced pluripotency factor expression and BCSC enrichment in TNBC. A2BR mediated activation of p38 MAPK and nuclear translocation of chromatin remodeling factor SMARCD3, which interacted and recruited histone demethylase KDM6A and histone acetyltransferase p300 specifically to the pluripotency factor genes , and . Recruitment of KDM6A and p300 decreased histone H3K27me3 and increases H3K27ac marks, and increased transcription factor FOXO3 binding to , and genes, leading to transcriptional activation of these genes and BCSC specification. Genetic or pharmacological inhibition of A2BR blocked chemotherapy-mediated epigenetic activation of pluripotency factor genes and BCSC enrichment in vitro and in vivo, and delayed tumor recurrence after chemotherapy was discontinued. Chemotherapy-induced A2BR expression mediates epigenetic activation of pluripotency factors and promotes breast cancer stemness. Targeting A2BR in combination with chemotherapy may block BCSC enrichment and improve outcome in TNBC.
三阴性乳腺癌（TNBC）的特征是其独特的分子谱、侵袭性和缺乏靶向治疗。化疗诱导多能性因子的表达，并介导 TNBC 中乳腺癌干细胞（BCSCs）的活性诱导，从而增加肿瘤复发和转移的风险，并增加患者死亡率。腺苷受体 2B（A2BR）的表达及其下游信号通路的激活被暗示可促进乳腺癌转移。本研究旨在探讨 A2BR 在调节化疗诱导的 BCSC 富集中的作用。我们在 TNBC 细胞系中生成了 shRNA 介导的 A2BR 敲低亚克隆，并通过 Aldefluor 和乳腺球体测定法在体外评估了对 BCSC 表型的影响。我们进行了染色质免疫沉淀（ChIP）实验，以研究转录因子 FOXO3 以及组蛋白修饰酶 KDM6A 和 p300 募集到多能性因子调节区域的情况，以及这些区域上组蛋白修饰标记 H3K27ac 和 H3K27me3 的水平。我们既采用了异种移植模型，又采用了遗传工程的、自发发生的乳腺癌模型，以评估体内 A2BR 对化疗诱导的 BCSC 富集的影响。我们证明了化疗增加了 A2BR 的蛋白水平，这有助于 TNBC 中化疗诱导的多能性因子表达和 BCSC 富集。A2BR 介导的 p300 募集和 p38 MAPK 的激活，以及染色质重塑因子 SMARCD3 的核转位，这些都特异性地与组蛋白去甲基酶 KDM6A 和组蛋白乙酰转移酶 p300 相互作用，并募集到多能性因子基因、和，募集的 KDM6A 和 p300 降低了组蛋白 H3K27me3 并增加了 H3K27ac 标记，增加了转录因子 FOXO3 与、和基因的结合，导致这些基因的转录激活和 BCSC 特化。体内和体外，A2BR 的遗传或药理学抑制阻断了化疗介导的多能性因子基因的表观遗传激活和 BCSC 富集，并在化疗停止后延迟了肿瘤复发。化疗诱导的 A2BR 表达介导了多能性因子的表观遗传激活，并促进了乳腺癌干细胞特性。在化疗中靶向 A2BR 可能会阻断 BCSC 富集并改善 TNBC 的预后。
