Huaier Polysaccharides Targets Cancer Stem Cells in Triple-Negative Breast Cancer via ASM-mediated Autophagy-dependent Ferroptosis

Background Breast cancer stem cells (BCSCs) is critical in multiple progression of triple-negative breast cancer (TNBC). The iron concentration has been found to be higher in BCSCs. The inhibition of ferroptosis is conducive to maintaining the stemness of tumor stem cells. Despite preliminary reports indicating the effectiveness of (polysaccharides of Huaier) PS-T in the treatment of TNBC, its role and specific mechanisms in BCSCs have not been systematically studied, Methods LASSO regression was used to screen for stem cell-related genes that are predictive of TNBC prognosis. The effects of PS-T on TNBC stem cells were examined using ssGSEA, flow cytometry, qPCR, colony formation assays, mammosphere assays, and xenograft models. CCK-8 and GSEA analyses were performed to investigate the mechanism by which PS-T inhibited BCSCs in TNBC. PS-T-induced ferroptosis was assessed using DCF and lipid oxidation assays. Ferroptosis inhibitors were used to verify that PS-T inhibits BCSCs by activating ferroptosis. We investigated the ASM-mediated autophagic degradation of GPX4 induced by PS-T through both in vitro and in vivo assays and samples from patients with TNBC. Additionally, we confirmed these findings by inhibiting autophagy and ASM expression, Results A high stem-related signature risk score predicts a worse prognosis for TNBC. The proportions of ALDH ⁺ , CD44 ^high CD24 ^low cells, and cells with stem cell markers decreased proportionally to the dose post-PS-T therapy for TNBC. ALDH ⁺ cells were flow-sorted and exhibited impaired stemness characteristics induced by PS-T. Moreover, the ferroptotic pathway was more active in PS-T-treated TNBC cells than in control cells. PS-T increased reactive oxygen species (ROS) and lipid oxidation levels of BCSCs. However, in the presence of ferroptosis inhibitors, PS-T did not significantly affect the growth of BSCSs. During in vitro and in vivo experiments, PS-T induces autophagic degradation of GPX4. Inhibition of autophagy reverses downregulation of GPX4, activation of ferroptosis, and suppression of BCSCs in TNBC. In addition, ASM was upregulated by PS-T and its expression was related to PS-T-induced autophagic GPX4 degradation, Conclusions Taken together, PS-T induced autophagy-dependent GPX4 protein degradation by upregulating ASM, thereby promoting ferroptosis and inhibiting the stemness of BCSCs, ultimately benefiting patients with TNBC.