About 10-20% of breast cancers are triple-negative breast cancers (TNBC). Immunotherapy blocking PD-L1 and PD-1 interaction reactivates TILs, which has shown promising clinical effects. However, identifying new immune check-point targets to improve the efficacy or safety of PD-1/PD-L1 blockade therapy is still urgently needed. Dihydroartemisinin (DA) is widely used as a clinical antimalarial drug. In this study, we found effective anticancer effect of DA in TNBC in vitro and in vivo. It showed DA significantly induced cell death in TNBC. The treatment of DA also increased oxidative stress, cell apoptosis and cell migration and invasion ability inhibition of MDA-MB-231 cells. In MDA-MB-231 xenograft nude mice model, DA administration significantly retarded the tumor growth. More importantly, we found that treating TNBC cells with DA significantly reduced cellular PD-L1 mRNA and protein level in cell lines and xenograft mice tissue. The PD-L1 level of MDA-MB-231 cells decreased by DA reduced the binding of PD-1 protein and sensitized cells to activated T cells. In details, DA reduced FoxO3a levels in MDA-MB-231 cells, in line with the increase of IRE1/IKK/FoxO3a-S644 phosphorylation. Furthermore, FoxO3a overexpression or knockdown increased or decreased PD-L1 protein levels accordingly. Dual luciferase reporter gene assay and ChIP-PCR results showed that FoxO3a, as a transcription factor, bound the promoter region of PD-L1 and improved its transcriptional activity, while DA treatment also reduced the PD-L1 gene promoter activity. In conclusion, these findings suggest that DA can exert an antitumor effect in TNBC cells and sensitize tumor cells to activated T cells, probably by regulating IRE1/IKK/FoxO3a/PD-L1 pathway. Therefore, DA could be a safe promising antitumor and PD-L1 regulatory drug.

Supported by NFSC (31771128 and 31371088), FDCT (0127/2019/A3, 0113/2018/A3 and 0038/2020/AMJ), Guangdong Provincial Funding Committee for Basic and Applied Fundamental Research (2022-Natural Science Foundation), University of Macau (File No. MYRG2018-00134-FHS and MYRG2020-00158-FHS) *Corresponding Author