Phenazine derivatives attenuate the stemness of breast cancer cells through triggering ferroptosis

Breast Cancer Stem Cells (BCSCs) are positively correlated with the metastasis, chemoresistance, and recurrence of breast Cancer. However, there are still no drugs targeting BCSCs in clinical using for breast Cancer treatment. Here, we tried to screen out small-molecule compounds targeting BCSCs from the phenazine library established by us before. We focused on the compounds without affecting cell viability and screened out three potential compounds (CPUL119, CPUL129, CPUL149) that can significantly attenuate the stemness of breast Cancer cells, as evident by the decrease of stemness marker expression, CD44⁺/CD24^- subpopulation, mammary spheroid-formation ability, and tumor-initiating capacity. Additionally, these compounds suppressed the metastatic ability of breast Cancer cells in vitro and in vivo. Combined with the transcriptome sequencing analysis, Ferroptosis was shown on the top of the most upregulated pathways by CPUL119, CPUL129, and CPUL149, respectively. Mechanistically, we found that these three compounds could trigger Ferroptosis by accumulating and sequestering iron in lysosomes through interacting with iron, and by regulating the expression of proteins (IRP2, TfR1, ferritin) engaged in iron transport and storage. Furthermore, inhibition of Ferroptosis rescued the suppression of these three compounds on breast Cancer cell stemness. This study suggests that CPUL119, CPUL129, and CPUL149 can specifically inhibit the stemness of breast Cancer cells through triggering Ferroptosis and may be the potential compounds for breast Cancer treatment.