Third Department of Breast Surgery, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China.
National Clinical Research Center for Cancer, Tianjin, China.
J Exp Clin Cancer Res. 2021 Apr 6;40(1):120. doi: 10.1186/s13046-021-01901-1.
Not all breast cancer (BC) patients who receive neoadjuvant chemotherapy achieve a pathologic complete response (pCR), but the reasons for this are unknown. Previous studies have shown that exosomes produced in the tumor microenvironment in response to chemotherapy promote a chemotherapy-resistant phenotype in tumors. However, the role of BC chemotherapy-elicited exosomes in regulating chemoresistance is poorly understood.
Using commercial kits, serum exosomes were extracted from patients before neoadjuvant chemotherapy, after one cycle of chemotherapy and after four cycles of chemotherapy consisting of doxorubicin (DOX) and paclitaxel (PTX). Their miRNAs were sequenced, and the correlation between the sequencing results and chemotherapy effects was further verified by RT-qPCR using patient serum exosomes. Cell Counting Kit-8 (CCK-8) was used to detect chemosensitivity. Stemness was assessed by CD44+/CD24- population analysis and mammosphere formation assays. Chromatin immunoprecipitation (ChIP) experiments were performed to verify the binding of signal transducer and activator of transcription 3 (STAT3) to the promoter of miRNAs.
Here, we provide clinical evidence that chemotherapy-elicited exosomal miR-378a-3p and miR-378d are closely related to the chemotherapy response and that exosomes produced by BC cells after stimulation with DOX or PTX deliver miR-378a-3p and miR-378d to neighboring cells to activate WNT and NOTCH stemness pathways and induce drug resistance by targeting Dickkopf 3 (DKK3) and NUMB. In addition, STAT3, which is enhanced by zeste homolog 2 (EZH2), bound to the promoter regions of miR-378a-3p and miR-378d, thereby increasing their expression in exosomes. More importantly, chemotherapeutic agents combined with the EZH2 inhibitor tazemetostat reversed chemotherapy-elicited exosome-induced drug resistance in a nude mouse tumor xenograft model.
This study revealed a novel mechanism of acquired chemoresistance whereby chemotherapy activates the EZH2/STAT3 axis in BC cells, which then secrete chemotherapy-elicited exosomes enriched in miR-378a-3p and miR-378d. These exosomes are absorbed by chemotherapy-surviving BC cells, leading to activation of the WNT and NOTCH stem cell pathways via the targeting of DKK3 and NUMB and subsequently resulting in drug resistance. Therefore, blocking this adaptive mechanism during chemotherapy may reduce the development of chemotherapy resistance and maximize the therapeutic effect.
并非所有接受新辅助化疗的乳腺癌（BC）患者都能达到病理完全缓解（pCR），但原因尚不清楚。先前的研究表明，肿瘤微环境中对化疗产生的外泌体促进肿瘤产生化疗耐药表型。然而，BC 化疗诱导的外泌体在调节化疗耐药中的作用知之甚少。
使用商业试剂盒，从接受新辅助化疗前、化疗 1 周期后和包含多柔比星（DOX）和紫杉醇（PTX）的 4 周期化疗后患者的血清外泌体中提取外泌体。对其 miRNA 进行测序，并通过使用患者血清外泌体的 RT-qPCR 进一步验证测序结果与化疗效果之间的相关性。细胞计数试剂盒-8（CCK-8）用于检测化疗敏感性。通过 CD44+/CD24-群体分析和类乳腺球体形成测定评估干性。进行染色质免疫沉淀（ChIP）实验以验证信号转导和转录激活因子 3（STAT3）与 miRNA 启动子的结合。
在这里，我们提供了临床证据，表明化疗诱导的外泌体 miR-378a-3p 和 miR-378d 与化疗反应密切相关，并且 DOX 或 PTX 刺激后 BC 细胞产生的外泌体将 miR-378a-3p 和 miR-378d 递送至邻近细胞，通过靶向 Dickkopf 3（DKK3）和 NUMB 激活 WNT 和 NOTCH 干性途径并诱导耐药。此外，由 zeste 同源物 2（EZH2）增强的 STAT3 与 miR-378a-3p 和 miR-378d 的启动子区域结合，从而增加外泌体中它们的表达。更重要的是，化疗药物联合 EZH2 抑制剂 tazemetostat 在裸鼠肿瘤异种移植模型中逆转了化疗诱导的外泌体诱导的耐药性。
本研究揭示了一种新的获得性化疗耐药机制，即化疗激活 BC 细胞中的 EZH2/STAT3 轴，然后这些细胞分泌富含 miR-378a-3p 和 miR-378d 的化疗诱导的外泌体。这些外泌体被化疗存活的 BC 细胞吸收，通过靶向 DKK3 和 NUMB 激活 WNT 和 NOTCH 干细胞途径，从而导致耐药。因此，在化疗期间阻断这种适应性机制可能会降低化疗耐药的发展并最大限度地提高治疗效果。
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