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

Huaier Polysaccharides Targets Cancer Stem Cells in Triple-Negative Breast Cancer via ASM-mediated Autophagy-dependent Ferroptosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Huaier Polysaccharides Targets Cancer Stem Cells in Triple-Negative Breast Cancer via ASM-mediated Autophagy-dependent Ferroptosis Lin-xi Zhou, Zi-wei Wu, Ke-fei Luo, Hong Zheng, Yuan Tian, Qin-wen Pan, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5706155/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. Triple-negative breast cancer Huaier cancer stem cell ferroptosis GPX4 ASM Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Breast cancer ranks as the most prevalent cancer threatening the health of Chinese females [ 1 ]. Currently, the conventional treatments of breast cancer mainly include surgical resection, postoperative chemotherapy, radiotherapy, and endocrine therapy [ 1 , 2 ]. Although current studies suggest that postoperative adjuvant therapy is an important strategy for preventing recurrence and prolong the survival period of breast cancer patients, conventional adjuvant therapy does not benefit all breast cancer patients [ 3 ]. Triple-negative breast cancer (TNBC) lacks of estrogen, progesterone, and human epidermal growth factor receptor 2 (HER-2) receptors [ 4 ]. These patients cannot receive adjuvant therapy, such as endocrine therapy or targeted therapy and are often prone to local recurrence and distant metastasis, thus having a poor prognosis [ 5 ]. Breast cancer stem cells (BCSCs) is the cancer lineages with the ability of tumorigenesis and self-renewal. Several studies have reported that BCSCs contribute to chemotherapy resistance and critical for the metastasis and relapse of breast cancer. Treatment with chemotherapy can effectively eliminate tumor cells in some patients; however, it can also increase the proportion of enriched BCSCs, which eventually raises the risk of recurrence and metastasis in TNBC patients [ 6 , 7 ]. Therefore, identifying new strategies that target and inhibit BCSCs is currently the focus of TNBC treatments [ 8 , 9 ]. Ferroptosis is a Fe 2+ -dependent and programmed cell death, accompanied massive ROS accumulation and excessive lipid oxidation [ 10 , 11 ]. A recent study conducted an integrated multi-omics analysis which identified significantly activated ferroptosis in breast tumors with high imaging intratumor heterogeneity (IITH). The study suggests that ferroptosis is a predisposing factor for breast cancer with high IITH, and that its inhibitors could be a promising option for refractory breast cancer [ 12 ]. In addition, iron is present at high concentrations in BCSCs, and iron metabolism is important for maintaining the stemness of BCSCs. The promotion of ferroptosis inhibits stem cell characteristics in breast, colorectal, and lung cancer [ 13 – 15 ]. Recently, some natural medicinal fungi and their extracts were found to have unique advantages in preventing tumorigenesis, inhibiting the formation of tumor foci, and preventing recurrence and metastasis. Among them, Huaier, whose main active ingredient is the polysaccharide of Trametes robiniophila Murr (PS-T), has been applied to the adjuvant therapy of malignancies, including breast cancer. Huaier has been reported to improve life quality for cancer patients with few side effects and high safety [ 16 , 17 ]. In a previous clinical study, we demonstrated that Huaier effectively inhibited the metastasis and recurrence of TNBC [ 18 ]. However, the effects of PS-T on TNBC stem cells and ferroptosis are yet to be investigated. Herein, we found for the first time that PS-T could promote ferroptosis of BCSCs in TNBC and that the underlying mechanism may involve PS-T-induced GPX4 degradation through the ASM-mediated autophagic lysosomal pathway. This study reveals a novel mechanism by which PS-T inhibits metastasis and recurrence in patients with TNBC and provides a theoretical basis and data support for identifying new molecular targets and strategies for TNBC treatment from the perspective of ferroptosis. Materials and Methods Chemicals We purchased the Huaier extract from Gaitianli Pharmaceutical Co., Ltd. (Qidong, China). The polysaccharides were prepared according to our previous work, including isolation, dialysis, extraction, lyophilisation, elution, and purification [ 19 ]. α-Tocopherol phosphate (HY-16686, 100 µM) was obtained from MedChemExpress (USA). Liproxstatin-1 (T2376, 200 mM), 3-methyladenine (T1879, 5 mM), and ASM-IN-1 (T74778, 2 µM) were obtained from TargetMol (USA). Lastly, Z-VAD-FMK (S7023, 30 µM), Necrostatin-1 (S8037, 20 µM), and LDC7559 (S9622, 5 µM) were acquired from Selleck (USA). Anti-human CD24 (PE, 311106, 5 µL/test) and anti-mouse/human CD44 (FITC, 103022, 5 µL/test) were acquired from Biolegend (USA). BODIPY™ 581/591 C11 (D3861, 5 µM/test), GPX4 (PA5-102521), and ASM antibody (PA5-77047) were obtained from Thermo Fisher (USA). The antibodies listed below were from Cell Signaling Technology (USA): GPX4 (52455), SLC7A11 (98051), FTH1 (3998), NRF2 (12721), DMT1 (15083), NCOA4 (66849), and β-actin (4970). Cell lines The MDA-MB-231, HCC1806, and HCC1937 cell lines were purchased via the FuHeng Cell Centre (Shanghai, China). MDA-MB-231 was maintained in L-15 medium (LA9510; Solarbio, Beijing, China) at 37°C in 100% air, HCC1806 and HCC1937 were cultured in RPMI-1640 medium (C11875500B; Gibco, USA) at 37°C in 5% CO 2 , with foetal bovine serum (10%) (10091148; Gibco, New Zealand) and L-glutamine (1%) (C0222; Beyotime, Shanghai, China)in the medium. Sorted stem cells remained in 3dGRO sphere medium at standard condition, and digested at a density of 90%. Flow cytometry and cell sorting The cells were exposed to distinct drugs for 24 h after adherence. After adjusted to 1 x 10 6 cells/mL using assay buffer, the cells were incubated with CD24 and CD44 at 37°C avoiding light for 30 min. Subsequently, the samples were supplemented with 300 µL of Buffer (70-S1001; MultiSciences, Zhejiang, China). When using the ALDEFLUOR Stem Cell Identification kit (01700; STEMCELL, Canada), every sample was added to activated ALDEFLUOR, mixed, and half of the mixture was transferred to a DEAB control tube before incubating the antibody. The samples were then analyzed with a BD LSR Fortessa or sorted using a BD FACS Aria III. Colony formation and mammosphere assays For colony formation, seed cells in 6-well plates with2,000 each, treat with drugs for 24 h, and cultured 7–10 days. Change medium every two days. Before observation, the plates were fixed (BL539A; Biosharp, Shanghai, China) and stained (C0121; Beyotime, Shanghai, China) according to the manufacture provided protocol. For mammosphere assays, cells were seeded in ultra-low attachment multi-well plates (CLS3471; Corning® Costar®, USA) with 2,000 cells per well. Drugs were added after 24 h, and the cells were cultured for 10 days. Within these days, half of the amount was added to the medium every two days. The mammospheres were then observed under a microscope. Xenograft assay The female nude mice used for this experiment were purchased from Byrness Weil Biotech Ltd. (Chongqing). Approximately 1 × 10 5 ALDH + MDA-MB-231 cells were injected into the mammary fat pads of the nude mice, including a control group and a group treated with PS-T. The PS-T-treated group was given a dose of 3 mg every two days via intragastric administration starting from the second day after injection. After 4 weeks, all mice were euthanized and the tumors were observed. CCK-8 assay Cells at 2 x 10 4 cells/mL were inoculated on 96-well plates and exposed to indicated drugs for 24 h after adherence. Add ten microliters of the CCK-8 cell counting kit (CK04, DOJINDO) in the medium and culture at 37°C for 1 h. Quantify the optical density (OD) with Varioskan Flash spectrophotometer (Thermo Scientific). DCF assay Seed the cells on coverslips in 6-well plates (2.5 mL), add the drug and culture 24 h after adherence, and incubated with DCFH-DA of ROS Assay Kit (S0033; Beyotime, Shanghai, China)in a medium without fetal bovine serum at 37°C. After 30 min of incubation, wash with a medium without fetal bovine serum and fixed in 4% paraformaldehyde. Treated with antifade mounting medium containing DAPI (P0131; Beyotime, Shanghai, China), observe under a fluorescence microscope (FV-1000; Olympus, Tokyo). Lipid oxidation experiments The amount of MDA in cells and tissues was quantified using a Lipid Peroxidation MDA Assay Kit (S0131; Beyotime, Shanghai, China). Following provided instruction, mix with MDA detecting working solution at 100°C for 15 min. After cooling and centrifugation, 200 µL supernatant of each was added into a 96-well plate. The absorbance of 532mn was detected with Varioskan Flash. As for the BODIPY™ 581/591 C11 probe, cells were incubated at 37°C in absence of light exposure for 30 min. After digestion, wash twice with buffer, re-suspended with buffer to 300 µL, measured their fluorescence with BD LSRFortessa. Western blotting Cells were harvested and their proteins were lysed with RIPA lysis buffer (P0013B; Beyotime, Shanghai, China). Measure the protein abundance using an enhanced BCA protein assay kit (P0010; Beyotime, Shanghai, China). Afterward, transfer to a polyvinylidene difluoride membrane. The membrane was blocked, incubated with primary and secondary antibodies, and visualized using the ECL detection system. RNA isolation and qPCR Isolate Total mRNA according to the instruction of the TaKaRa MiniBEST Universal RNA Extraction Kit (9767; Takara, Japan). Moreover, cDNA was obtained using the PrimeScript™ RT Master Mix (RR036A; Takara, Japan). The sequences and lengths of the PCR primers are listed in Table S1 . The reactions were detected with TB Green® Premix Ex Taq™ II FAST qPCR (CN830S; Takara) in an Eppendorf Mastercycler realplex. The mRNA expression was calculated according to the 2 −∆∆CT method. Immunohistochemistry (IHC) staining Implement IHC staining following the SP kit instructions (SP-9000; ZSGB-BIO, Beijing, China). Staining intensity was quantified: 0 = negative; 1 = weakly positive; 2 = positive; and 3 = strongly positive. The percentage of stained cells was counted as: 0, less than 5%; 1,6–25%; 2, 26–50%; 3, 51–75%; and 4, more than 75%. IHC staining was quantified by multiplying the signal value with the stained cells percentage. The mean fluorescence (MFI) was quantified using ImageJ. Bioinformatic analysis The TCGA database provided RNA-sequencing data and clinical information on patients with TNBC. LASSO regression was implemented using the R package glmnet. Nomogram model was constructed by the R package rms using Cox method. Sangerbox 3.0 was used to perform ROC curve and ssGSEA enrichment analysis [ 20 ]. The gene set files PECE_MAMMARY_STEM_CELL_UP, PECE_MAMMARY_STEM_CELL_DN, and WP_FERROPTOSIS were retrieved via the Molecular Signatures Database ( https://www.gsea-msigdb.org/gsea/msigdb ). During GSEA enrichment analysis, differentially expressed genes (DEGs) were analyzed and plotted using the GSEA 4.1 software. For survival analysis, Kaplan-Meier curve was visualized the Kaplan-Meier Plotter ( http://kmplot.com/analysis/ ) and the bc-GenExMiner v5.0 online tools ( http://bcgenex.ico.unicancer.fr/BC-GEM/GEM-Accueil.php?js=1 ). Statistical analysis All quantitative data are visualized using a mean ± standard deviation mode. The Student t-test and the Mann–Whitney U test were used to evaluate difference of two groups. For pairwise comparisons, one-way analysis of variance and post-hoc Bonferroni tests were used. The logistic regression was used to univariate and multivariate analysis. P < 0.05 was considered significant. All analyses were carried out on GraphPad Prism 8.0 and SPSS 26.0. Results High stem-related signature risk score predicts worse prognosis in TNBC We constructed a LASSO regression model based on 167 TNBC patients from the TCGA database to screen key genes associated with stemness and identified 17 genes that were predominant in the prognosis: CDC5L, IL4R, NDP, REXO2, TBCA, RGS17, RAB2B, ABHD6, PUM2, UQCR11, TCF7L2, SFPQ, TXN, EEF1D, SHQ1, FHL1, and PLBD1 (Fig. 1 A, B). Risk factor analysis of the 17 genes suggested that the risk factor score was significantly associated with the prognosis of patients with TNBC (Fig. 1 C). The areas under the time-dependent ROC curve at 1, 3, and 5 years were 0.80, 0.86, and 0.88, separately, indicating high prognostic performance (Fig. 1 D). The 167 samples were then separated into high- (n = 91) and low-risk group (n = 76) according to the risk factor score, among which high-risk group TNBC patients had higher mortality (HR = 22.89, 95%CI: 5.44–96.3, P = 5.8e-10) (Fig. 1 E). Furthermore, we explored the impact of these 17 genes on TNBC prognosis. Overall survival (OS), relapse-free survival (RFS), and distant metastasis-free survival (DMFS) data were collected and visualized using forest plots (Fig. 1 F, G, and Additional file 1: Fig. S1 ). Univariate analysis showed that primary tumor, Nodal status, TNM stage, and risk factor score were independent predictors of TNBC prognosis (Fig. 2 A). Multivariate analysis indicated that risk factor score was superior in predicting prognosis than conventional clinical features like TNM stage (Fig. 2 B). Besides, we constructed a nomogram model combining data on survival time, survival status, and other clinical characteristics to demonstrated that the risk score has good predictive value for mortality risk (C-index = 0.891, 95%CI: 0.85–0.94, P = 1.33e-62) (Fig. 2 C). The calibration curves indicate that the signature has favorable prediction accuracy for 3 and 5 years (Fig. 2 D). To validate our prognostic model, external validation sets (GSE20711, GSE58812, and GSE21653) was used. The areas under the ROC curve at 9 years were 0.78, 0.63, and 0.70 (Fig. 2 E). PS-T reduces the stemness characteristics of BCSCs in TNBC To explore the effect of PS-T on cell stemness characteristics in TNBC, we analyzed the enrichment level of the mammary stem cell pathway using ssGSEA and found decreased ssGSEA scores in the PS-T-treated group (Fig. 3 A). A dose-dependent decrease in the expression of the stem cell markers POU5F1, SOX2, and NAONG was observed after PS-T treatment (Fig. 3 B). The proportions of ALDH + and CD44 high CD24 low MDA-MB-231 cells were significantly decreased after PS-T treatment in a dose-dependent manner (Fig. 3 C). The ALDH + cell population was sorted for the colony formation and mammosphere assays. With an increase in PS-T concentration, the number and size of the colonies and mammospheres gradually decreased (Fig. 3 D, E). ALDH + MDA-MB-231 cells were transplanted into female nude mice to observe the effect of PS-T on breast tumor formation. The results showed that the probability of tumor formation was severely reduced by PS-T treatment (Fig. 3 F). These results suggest that PS-T could reduce the stemness of BCSCs in TNBC. PS-T restrains BCSCs by activating ferroptosis in TNBC To illustrate the mechanism underlying PS-T-mediated regulation of BCSCs in TNBC, we evaluated the viability of PS-T-treated ALDH + cells after treatment with cell death inhibitors. PS-T significantly inhibited cell proliferation. Furthermore, the inhibitory effect was only slightly restored after the addition of inhibitors of apoptosis, necrosis, or pyroptosis, but was significantly reversed with the assistance of ferroptosis inhibitors, α-Tocopherol phosphate (α-toc) and Liproxstatin-1 (Lip-1) (Fig. 4 A, Additional file 1: Fig. S2 A). In addition, GSEA enrichment results revealed that the ferroptosis pathway was more active in PS-T–treated TNBC cells compared to control cells (ES = 0.61, P = 0.024) (Fig. 4 B, Additional file 1: Fig. S2 B). Ferroptosis has been reported to promoted by the accumulation of reactive oxygen species (ROS) and excessive lipid oxidation [ 21 ]. We investigated changes in intracellular ROS levels and lipid oxidation after PS-T treatment. The mean fluorescence intensity (MFI) of DCF and BODIPY™ 581/591 C11 and the MDA concentration in ALDH + MDA-MB-231 cells decreased after PS-T treatment (Fig. 4 C-E). Subsequently, we verified the PS-T-induced reduction in lipid peroxidation in vivo using an MDA assay and flow cytometry (Fig. 4 F, G). Furthermore, we inhibited ferroptosis in TNBC cells using ferroptosis inhibitors α-tocopherol (α-toc) and liproxstatin-1 (Lip-1). The results showed that the lipid oxidation level and intracellular MDA concentration in PS-T-treated cells barely increased after the addition of the ferroptosis inhibitors (Fig. 5 A-C). To validate the role of PS-T-induced ferroptosis in the inhibitory effect on BCSCs, we co-treated cells with PS-T and ferroptosis inhibitors. The PS-T treatment did not significantly reduce the mRNA levels of POU5F1, SOX2, or NAONG, the percentage of ALDH + and CD44 high CD24 low cells, or the formation of colonies and mammospheres in the presence of ferroptosis inhibitors (Fig. 5 D-I). Collectively, these data suggest that PS-T inhibits BCSCs by activating ferroptosis in TNBC cells. PS-T promotes ferroptosis of BCSCs by inducing autophagic degradation of GPX4 The expression levels of ferroptosis pathway markers were detected by western blotting. GPX4 (Glutathione Peroxidase 4) was found to be downregulated after PS-T treatment (Fig. 6 A). Immunohistochemical staining also confirmed the PS-T-induced downregulation of GPX4 protein in vivo (Fig. 6 B, C). Additionally, the overall survival of high-GPX4 TNBC patients was significantly shorter (HR 2.78, 95%CI: 1.45–5.31, P = 0.0012; HR 3.29, 95%CI: 1.03–10.53, P = 0.0444) (Fig. 6 D, E, and Additional file 1: Fig. S3 A). These results suggested that PS-T promotes ferroptosis by downregulating GPX4, thereby inhibiting BCSCs in TNBC. Subsequently, we examined the regulatory mechanism through which PS-T impacts GPX4 protein expression. There was no statistically significant difference in the GPX4 mRNA levels in TNBC cells treated with or without PS-T, indicating that PS-T does not regulate GPX4 expression at the transcriptional level (Additional file 1: Fig. S3 B, C). Our previous study found that PS-T promotes autophagic flux in TNBC cells and inhibits the invasion and migration ability of Snail protein [ 16 ], suggesting that PS-T may degrade GPX4 protein through the autophagy-lysosomal pathway. The autophagy inhibitor 3-methyladenine (3-MA) was used for further validation. As expected, when 3-MA inhibited autophagosome formation, GPX4 protein and cell membrane lipid oxidation levels in PS-T-treated ALDH + MDA-MB-231 cells were comparable to untreated cells (Fig. 6 F-I). Furthermore, after the inhibition of autophagy, PS-T treatment did not reduce the mRNA levels of stem cell markers, the proportion of ALDH + and CD44 high CD24 low cells, or the formation of colonies and mammospheres (Fig. 6 J-M). Collectively, these results suggest that PS-T may degrade the GPX4 protein through the autophagy-lysosomal pathway and induce autophagy-dependent ferroptosis in TNBC stem cells. PS-T promotes the autophagic degradation of GPX4 by upregulating ASM in BCSCs Acid sphingomyelinase (ASM; gene name: SMPD1 ) is activated when tumor cells encounter extracellular stimulation (antineoplastic drugs, oxidative stress, radiation, etc.) [ 22 ]. The absence of ASM contributes to the resistance of tumor cells to antitumor drug-induced apoptosis [ 23 , 24 ]. Interestingly, ASM participates in the occurrence of autophagy by regulating the formation of autophagosomes and lysosomal membranes [ 25 , 26 ]. The ferroptosis inducer erastin triggers ferroptosis by mediating ASM-dependent autophagic degradation of GPX4 [ 27 ]. Western blotting and qPCR indicate that ASM was upregulated in ALDH + MDA-MB-231 cells after PS-T treatment (Fig. 7 A, B). The promotion of ASM expression by PS-T was verified using 4T-1 xenograft (Fig. 7 C, D). Histological examination revealed a positive correlation between PS-T-induced ASM upregulation and autophagic GPX4 degradation in the TNBC stem cells (Fig. 7 D). We performed immunohistochemical analysis of 12 TNBC samples and found a significant increased LC3 abundance, whereas the expression of GPX4 protein decreased in the high-ASM group (Fig. 7 E). Furthermore, the ability of PS-T to induce autophagy was significantly inhibited by ASM-IN-1. In addition, ASM-IN-1 treatment reversed the PS-T-induced GPX4 degradation in ALDH + MDA-MB-231 cells (Fig. 7 F). These results indicate that ASM is required for PS-T-induced GPX4 degradation in TNBC stem cells. Discussion As the most lethal breast cancer subtype, triple-negative breast cancer (TNBC) patients cannot benefit from endocrine therapy and anti-HER-2 targeted therapy, as a result of the lack of hormone receptors. Chemotherapy remains an important systemic therapy for TNBC; however, not all patients benefit from chemotherapy, and many patients often experience recurrence and metastasis within a short period after the completion of chemotherapy, leading to a very poor prognosis [ 1 , 2 ]. Breast cancer stem cells (BCSCs) are a subpopulation of tumor cells with self-renewal abilities and multidirectional differentiation potential. Compared to ordinary tumor cells, BCSCs have stronger chemotherapy resistance, invasion, and metastasis abilities, which may be the seed of recurrence and metastasis after chemotherapy in patients with TNBC [ 6 , 7 ]. Therefore, finding a new strategy to target and inhibit BCSCs is the focus of the current clinical treatment of TNBC [ 8 , 9 ]. Studies have indicated that BCSCs is necessary for chemotherapy resistance, metastasis, and recurrence in TNBC [ 6 ]. According to the LASSO regression analysis using TCGA data, we found that the abundance of stem cell-related genes was associated with adverse prognosis in patients with TNBC (Fig. 1 , 2 ). Our research group is committed to studying the effects and mechanisms of Huaier on the treatment. In a previous clinical study, Huaier granules significantly improved the overall survival rate and delayed disease progression in patients with TNBC [ 18 ]. To investigate whether PS-T plays an anticancer role by inhibiting BCSCs, we purified polysaccharide of Huaier (PS-T), the main active component of Huaier granules, and performed further experiments [ 19 ]. ssGSEA enrichment analysis indicated the suppression of the mammary stem cell pathway after PS-T treatment (Fig. 3 A). Quantitative PCR and flow cytometry analysis of PS-T-treated cells showed that PS-T reduced the level of stem cell markers and the proportion of ALDH + and CD44 high CD24 low cells in a dose-dependent manner (Fig. 3 B, C). The ALDH + MDA-MB-231 cells were sorted for subsequent experiments. Based on colony formation and mammosphere assays, PS-T suppressed the proliferation and stemness of ALDH + cells (Fig. 3 D, E). Consistently, PS-T significantly limited tumor formation in ALDH + cells (Fig. 3 F). Taken together, PS-T reduced the stemness characteristics of TNBC stem cells, in vitro and in vivo. Based on the cell viability experiments and GSEA enrichment analysis, we hypothesized that PS-T inhibits TNBC stem cells via stimulating the ferroptosis pathway (Fig. 4 A, B, Additional file 1: Fig. S2 ). Ferroptosis was reported as a programmed cell death type, triggered by massive ROS accumulation and excessive lipid oxidation [ 28 ]. Recent studies have reported that there is a high concentration of iron in cancer stem cells and that iron metabolism is conducive to maintaining the stemness of cancer stem cells [ 13 , 29 ]. Promotion of ferroptosis can inhibit the stem cell characteristics of breast, colorectal, and lung cancer [ 13 – 15 ]. As detected by immunofluorescence and colorimetry, PS-T induced intracellular ROS and lipid oxidation in TNBC cells in vitro and in vivo (Fig. 4 C-G). The inhibition effect of PS-T on BCSCs was reversed by the addition of ferroptosis inhibitors. (Fig. 5 ). GPX4 is the key regulator for ferroptosis, and acts by reducing lipid peroxidation in the cell membrane, thereby inhibiting ferroptosis [ 11 , 30 ]. Both in vitro and in vivo , PS-T downregulates GPX4 protein level (Fig. 6 A-C). This reduction further confirmed that PS-T promotes ferroptosis in TNBC stem cells. In addition, our data demonstrated that PS-T downregulated GPX4 levels by inducing autophagy (Fig. 6 F, G). When autophagy was pharmacologically inhibited, PS-T no longer promoted ferroptosis or inhibited the stemness of BCSCs in TNBC (Fig. 6 H-M). Furthermore, PS-T promotes autophagic GPX4 degradation by upregulating ASM expression (Fig. 7 ), a key enzyme in regulating the sphingomyelin cycle that has a crosstalk with autophagy and GPX4 [ 27 ]. However, the key PS-T transcription factors that upregulate ASM need further investigation. These results suggest that PS-T inhibits BCSCs by promoting the ASM-mediated autophagic degradation of GPX4 and by inducing ferroptosis, ultimately improving the prognosis of patients with TNBC. Exploring and confirming this scientific problem can not only reveal a new mechanism by which PS-T inhibits metastasis and recurrence in TNBC patients, but also provide a theoretical basis and data support for discovering new molecular targets and strategies for TNBC treatment from the perspective of ferroptosis. Conclusion PS-T induces autophagy-dependent GPX4 degradation by upregulating ASM in BCSCs and promotes ferroptosis to inhibit the stemness characteristics, thereby improving the prognosis of patients with TNBC. Declarations Acknowledgments We would like to thank Editage (www.editage.cn) for the English language editing. Author contributions Conceptualization: JJ, HLM and MHW; Methodology: LXZ, ZWW, KFL, ZH and TY; Data analysis: LXZ, ZWW, ZH and PQW; Writing—original draft preparation: LXZ and ZWW; Writing—reviewing and editing: JJ, HLM, and MHW. All authors have read and agreed to the published version of the manuscript. Funding This study was funded by the National Natural Science Foundation of China [Grant No. 82474127], the Natural Science Foundation of Chongqing [Grant No. CSTB2023NSCQ-MSX0520], and Military Key Clinical Specialty [Project No. 41561Z23612]. Availability of data and materials Additional Figures and associated Figure legends are provided in the Supplementary Text and are available online. Raw RNA sequencing data were uploaded to the GEO database (accession number GSE253683). Ethics approval and consent to participate All experiments were approved by the Laboratory Animal Welfare and Ethics Committee of the Army Medical University (SYXK-20170002) and the Ethics Committee of the Southwest Hospital of the Army Medical University (KY2021118). Consent for publication Not applicable. Competing interests The authors declare no conflict of interest. References Li Y, Zhang H, Merkher Y, Chen L, Liu N, Leonov S, Chen Y: Recent advances in therapeutic strategies for triple-negative breast cancer. J Hematol Oncol 2022, 15(1):121. Leon-Ferre RA, Goetz MP: Advances in systemic therapies for triple negative breast cancer. Bmj 2023, 381:e071674. Joensuu H, Gligorov J: Adjuvant treatments for triple-negative breast cancers. 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Hu B, Yan W, Wang M, Cui X, Hu Y, Chen Q et al: Huaier polysaccharide inhibits the stem-like characteristics of ERα-36(high) triple negative breast cancer cells via inactivation of the ERα-36 signaling pathway. Int J Biol Sci 2019, 15(7):1358-1367. Shen W, Song Z, Zhong X, Huang M, Shen D, Gao P et al: Sangerbox: A comprehensive, interaction-friendly clinical bioinformatics analysis platform. iMeta 2022, 1(3):e36. Dixon SJ, Stockwell BR: The role of iron and reactive oxygen species in cell death. Nat Chem Biol 2014, 10(1):9-17. Hannun YA, Obeid LM: Sphingolipids and their metabolism in physiology and disease. Nat Rev Mol Cell Biol 2018, 19(3):175-191. van Hell AJ, Haimovitz-Friedman A, Fuks Z, Tap WD, Kolesnick R: Gemcitabine kills proliferating endothelial cells exclusively via acid sphingomyelinase activation. Cell Signal 2017, 34:86-91. Grammatikos G, Teichgräber V, Carpinteiro A, Trarbach T, Weller M, Hengge UR, Gulbins E: Overexpression of acid sphingomyelinase sensitizes glioma cells to chemotherapy. Antioxid Redox Signal 2007, 9(9):1449-1456. Perrotta C, Cervia D, De Palma C, Assi E, Pellegrino P, Bassi MT, Clementi E: The emerging role of acid sphingomyelinase in autophagy. Apoptosis 2015, 20(5):635-644. Lee JK, Jin HK, Park MH, Kim BR, Lee PH, Nakauchi H et al: Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer's disease. J Exp Med 2014, 211(8):1551-1570. Thayyullathil F, Cheratta AR, Alakkal A, Subburayan K, Pallichankandy S, Hannun YA, Galadari S: Acid sphingomyelinase-dependent autophagic degradation of GPX4 is critical for the execution of ferroptosis. Cell Death Dis 2021, 12(1):26. Cao JY, Dixon SJ: Mechanisms of ferroptosis. Cell Mol Life Sci 2016, 73(11-12):2195-2209. Pandrangi SL, Chittineedi P, Chalumuri SS, Meena AS, Neira Mosquera JA, Sánchez Llaguno SN et al: Role of Intracellular Iron in Switching Apoptosis to Ferroptosis to Target Therapy-Resistant Cancer Stem Cells. Molecules 2022, 27(9). Yang WS, SriRamaratnam R, Welsch ME, Shimada K, Skouta R, Viswanathan VS et al: Regulation of ferroptotic cancer cell death by GPX4. Cell 2014, 156(1-2):317-331. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigures20241224.docx SupplementaryTableS120241224.xlsx OriginalDataFerro20241010.pdf RawDataFerro20241010.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5706155","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":395157104,"identity":"fd76eafc-384d-493c-9898-ec0fd802ddf6","order_by":0,"name":"Lin-xi Zhou","email":"","orcid":"","institution":"First Affiliated Hospital of The Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lin-xi","middleName":"","lastName":"Zhou","suffix":""},{"id":395157105,"identity":"e8055e09-1aff-4484-b912-59a24d0ff7d9","order_by":1,"name":"Zi-wei Wu","email":"","orcid":"","institution":"First Affiliated Hospital of The Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zi-wei","middleName":"","lastName":"Wu","suffix":""},{"id":395157106,"identity":"493753d8-0246-415a-8982-5f63e46b1e7a","order_by":2,"name":"Ke-fei Luo","email":"","orcid":"","institution":"First Affiliated Hospital of The Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ke-fei","middleName":"","lastName":"Luo","suffix":""},{"id":395157107,"identity":"a1412b89-a9d9-4b04-b5c9-a0ca2be5a22a","order_by":3,"name":"Hong Zheng","email":"","orcid":"","institution":"Xinqiao Hospital Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Zheng","suffix":""},{"id":395157108,"identity":"b52cc8a6-cb18-4976-bc94-47012c4a5caa","order_by":4,"name":"Yuan Tian","email":"","orcid":"","institution":"First Affiliated Hospital of The Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Tian","suffix":""},{"id":395157109,"identity":"a9d0d719-5c8b-45a9-baed-61615e9be278","order_by":5,"name":"Qin-wen Pan","email":"","orcid":"","institution":"First Affiliated Hospital of The Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qin-wen","middleName":"","lastName":"Pan","suffix":""},{"id":395157110,"identity":"7fe9e2e5-6291-4870-8cb5-3713f71ba9f1","order_by":6,"name":"Jun Jiang","email":"","orcid":"","institution":"First Affiliated Hospital of The Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Jiang","suffix":""},{"id":395157111,"identity":"f40ba64d-79cf-4106-b642-865b62a2321a","order_by":7,"name":"Ling-mi Hou","email":"","orcid":"","institution":"Affiliated Hospital of North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Ling-mi","middleName":"","lastName":"Hou","suffix":""},{"id":395157112,"identity":"004f18e9-e632-44fd-a8fe-96aa6da09b2f","order_by":8,"name":"Ming-hao Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYDACZjCCgwNybOztB0jTYszHcyaBGIsQWhLnSTgY4FWu2857+HVBjU1i/+z2C4w/2+6kt0kwJDD8qNiGU4vZYb406xnH0hJn3DlTwMzb9iy3TbrxAGPPmdt4tPCYGfOwHc5tuJGTwMy47XBum8wBIKONkJZ/h3PnA7Uw/tx2OJ1NIsGAkBbjx7xth3M33Eg/wMC77XACMVrMmHn70uo33shhYOb9d9iwDRjIB/H65fwZ488832yM5W6kP2D8ceawvHx7+8EHPypwawECNgkIzWP+AyZ0AJ96IGD+AKHZHxBQOApGwSgYBSMVAADq7lygfJk5XgAAAABJRU5ErkJggg==","orcid":"","institution":"First Affiliated Hospital of The Army Medical University","correspondingAuthor":true,"prefix":"","firstName":"Ming-hao","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-12-24 12:23:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5706155/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5706155/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72612291,"identity":"d02d6a47-0922-4b2f-a1e5-36cb4cdf0662","added_by":"auto","created_at":"2024-12-30 10:28:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2651525,"visible":true,"origin":"","legend":"\u003cp\u003eHigh stem-related signature risk score predicts worse prognosis in TNBC\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-B) \u003c/strong\u003eLASSO regression prognostic model for 167 TNBC patientsbased on TCGA database.\u003cstrong\u003e (C) \u003c/strong\u003eRisk factor analysis of TNBC patients in TCGA database (n = 167).\u003cstrong\u003e (D)\u003c/strong\u003e Validation of the prognostic model by the time-dependent ROC curves. \u003cstrong\u003e(E) \u003c/strong\u003eKaplan-Meier survival analysis of TNBC patients in the high (\u003cem\u003eblue\u003c/em\u003e) and low-risk group (\u003cem\u003ered\u003c/em\u003e). \u003cstrong\u003e(F-G)\u003c/strong\u003e TNBC prognostic forest plot of 17 stem-related genes from the TCGA database.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5706155/v1/fb3da529bb311f0427330ac3.png"},{"id":72612290,"identity":"184f8b8f-9142-457b-8266-58f6dcf4360e","added_by":"auto","created_at":"2024-12-30 10:28:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2301107,"visible":true,"origin":"","legend":"\u003cp\u003eValidation of the stem-related prognostic risk model\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-B) \u003c/strong\u003eUnivariate and multivariate analysis of the stem-related prognostic risk model. \u003cstrong\u003e(C) \u003c/strong\u003eThe nomogram for the prognostic risk model based on TCGA database. \u003cstrong\u003e(D)\u003c/strong\u003eBootstrap analysis forindicating the probability of 3 and 5 years based on the prognostic model. \u003cstrong\u003e(E) \u003c/strong\u003eValidation of the prognostic model in external datasets (GSE20711, GSE58812, and GSE21653) by using the ROC curves at 9 years.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5706155/v1/ba6fbb63ec60a722c1a042e2.png"},{"id":72612293,"identity":"15a19b01-164e-450d-9c45-c3658fdc98b1","added_by":"auto","created_at":"2024-12-30 10:28:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3591483,"visible":true,"origin":"","legend":"\u003cp\u003ePS-T reduces the stemness characteristics of BCSCs in TNBC\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eThe ssGESA scores of MAMMARY-STEM-CELL gene set in MDA-MB-231 cells treated with or without PS-T. \u003cstrong\u003e(B) \u003c/strong\u003eThe mRNA levels of POU5F1, SOX2, and NAONG in 0, 10, and 50 μg/mL PS-T–treated MDA-MB-231 cells were detected by quantitative PCR (n = 9). \u003cstrong\u003e(C) \u003c/strong\u003eThe proportion of ALDH\u003csup\u003e+\u003c/sup\u003e (\u003cem\u003eup\u003c/em\u003e) and CD44\u003csup\u003ehigh\u003c/sup\u003eCD24\u003csup\u003elow\u003c/sup\u003e MDA-MB-231 cells (\u003cem\u003edown\u003c/em\u003e) treated with 0, 10, 20, and 50 μg/mL PS-T was determined by flow cytometry (n = 9). \u003cstrong\u003e(D-E)\u003c/strong\u003e Clonal formation (D) and mammosphere assays (E) of ALDH\u003csup\u003e+\u003c/sup\u003e MDA-MB-231 cells treated with indicated concentration of PS-T. Scale bars = 50 μm. \u003cstrong\u003e(F) \u003c/strong\u003eXenograft assay using ALDH\u003csup\u003e+\u003c/sup\u003e fraction upon PS-T treatment in MDA-MB-231 cells. (mean ± standard deviation; ns, not significant; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5706155/v1/a77fd64141322f5343450555.png"},{"id":72612296,"identity":"93c599a5-e118-4837-ac85-d277c07edb6c","added_by":"auto","created_at":"2024-12-30 10:28:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2119626,"visible":true,"origin":"","legend":"\u003cp\u003ePS-T restrains BCSCs by inducing ferroptosis in TNBC\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eCell viability heatmap of the effects of different cell death inhibitors on PS-T–induced cell death. ALDH\u003csup\u003e+\u003c/sup\u003e MDA-MB-231 cells were treated with indicated concentration of PS-T with or without Z-VAD-FMK (30 µM), Necrostatin-1 (Necro-1, 20 µM), LDC7559 (5 µM), α-tocopherol (α-toc, 100 µM) or liproxstatin-1 (Lip-1, 200 nM) for 24 h (n = 9). \u003cstrong\u003e(B) \u003c/strong\u003eGSEA result of MDA-MB-231 cells treated with or without PS-T using WP_FERROPTOSIS gene set.\u003cstrong\u003e (C-E)\u003c/strong\u003e The ROS fluorescence (C), C11 BODIPY fluorescence (D), and relative MDA concentration (E) of 0, 10, 20, and 50 μg/mL PS-T–treated ALDH\u003csup\u003e+\u003c/sup\u003e MDA-MB-231 cells (n = 9). Scale bars = 20 μm.\u003cstrong\u003e (F-G)\u003c/strong\u003e The relative MDA concentration (F) and C11 BODIPY fluorescence (G) of breast cancer tissue of xenograft models treated with or without PS-T (n = 9). (mean ± standard deviation; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5706155/v1/ee009ea62f5277af3edc85d2.png"},{"id":72612718,"identity":"bb3cfaf3-1178-48fa-906c-09472b0a3706","added_by":"auto","created_at":"2024-12-30 10:36:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3762841,"visible":true,"origin":"","legend":"\u003cp\u003eFerroptosis inhibitors reverse the effect of PS-T on BCSCs\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-C) \u003c/strong\u003eThe ROS fluorescence (A), C11 BODIPY fluorescence (B), and relative MDA concentration (C) in ALDH\u003csup\u003e+\u003c/sup\u003e MDA-MB-231 cells that were treated alone or in combination with PS-T (10 μg/mL) and ferroptosis inhibitors α-tocopherol (α-toc, 100 µM) or liproxstatin-1 (Lip-1, 200 nM) for 24 h (n = 9). Scale bars = 20 μm. \u003cstrong\u003e(D-E)\u003c/strong\u003e The mRNA levels of POU5F1, SOX2, and NAONG in MDA-MB-231 cells that treated with PS-T combined with α-toc (D) or Lip-1 (E) (n = 9). \u003cstrong\u003e(F-G)\u003c/strong\u003e The proportion of ALDH\u003csup\u003e+ \u003c/sup\u003e(\u003cem\u003eup\u003c/em\u003e) and CD44\u003csup\u003ehigh\u003c/sup\u003eCD24\u003csup\u003elow\u003c/sup\u003e cells (\u003cem\u003edown\u003c/em\u003e) in PS-T–treated MDA-MB-231 cells that co-treated with or without α-toc (F) or Lip-1 (G) (n = 9). \u003cstrong\u003e(H-I)\u003c/strong\u003e Clonal formation (H) and mammosphere assays (I) of ALDH\u003csup\u003e+\u003c/sup\u003e MDA-MB-231 cells treated with PS-T, ferroptosis inhibitors, and a combination of both. Scale bars = 50 μm. (mean ± standard deviation; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5706155/v1/25d56e17daec65945e587a8a.png"},{"id":72612310,"identity":"d65a1cab-1b70-4d80-b3b7-d51586c33256","added_by":"auto","created_at":"2024-12-30 10:28:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":487526,"visible":true,"origin":"","legend":"\u003cp\u003ePS-T promotes ferroptosis of BCSCs by inducing autophagic degradation of GPX4\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eExpression levels of ferroptosis-related markers in ALDH\u003csup\u003e+ \u003c/sup\u003eMDA-MB-231 cells treated with 10 μg/mL PS-T for 24 h (n = 3). The grayscales were quantified using ImageJ software and calculated relative to β-actin levels. \u003cstrong\u003e(B-C)\u003c/strong\u003e Representative images of GPX4 protein expression in the control (\u003cem\u003eup\u003c/em\u003e) and PS-T–treated mice (\u003cem\u003edown\u003c/em\u003e) (n = 9). Scale bars = 100 and 50 μm. \u003cstrong\u003e(D-E) \u003c/strong\u003eOverall survival (OS) of low- and high-GPX4 group TNBC patients from GEO (D) and TCGA database (E). \u003cstrong\u003e(F)\u003c/strong\u003e Expression of GPX4 and LC3 protein in ALDH\u003csup\u003e+ \u003c/sup\u003eMDA-MB-231 that were divided into four groups, including control, PS-T (10 μg/mL), 3-MA (5 mM), 3-MA+PS-T (n = 3).\u003cstrong\u003e (G-I) \u003c/strong\u003eThe ROS fluorescence (G), C11 BODIPY fluorescence (H), and relative MDA concentration (I) in ALDH\u003csup\u003e+\u003c/sup\u003e MDA-MB-231 cells that were treated alone or in combination with PS-T and 3-MA for 24 h (n = 9). Scale bars = 20 μm.\u003cstrong\u003e (J)\u003c/strong\u003e The mRNA levels of POU5F1, SOX2, and NAONG in MDA-MB-231 cells treated with PS-T and 3-MA alone or in combination (n = 9).\u003cstrong\u003e (K)\u003c/strong\u003e The proportion of ALDH\u003csup\u003e+ \u003c/sup\u003e(\u003cem\u003eup\u003c/em\u003e) and CD44\u003csup\u003ehigh\u003c/sup\u003eCD24\u003csup\u003elow\u003c/sup\u003e cells (\u003cem\u003edown\u003c/em\u003e) in MDA-MB-231 cells treated with DMSO, PS-T, 3MA, and 3-MA+P-ST (n = 9).\u003cstrong\u003e (L-M)\u003c/strong\u003e Clonal formation (L) and mammosphere assays (M) of PS-T–treated ALDH\u003csup\u003e+\u003c/sup\u003e MDA-MB-231 cells that co-treated with or without treated with PS-T, 3-MA, and a combination of both. Scale bars = 50 μm. (mean ± standard deviation; ns, not significant; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5706155/v1/772870bbfaafcbce23ac0aeb.png"},{"id":72612313,"identity":"22f96914-8e27-4e33-a264-7e84b9d150e3","added_by":"auto","created_at":"2024-12-30 10:28:24","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":500043,"visible":true,"origin":"","legend":"\u003cp\u003ePS-T promotes autophagic GPX4 degradation by upregulating ASM in BCSCs\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-B) \u003c/strong\u003eThe expression level of SMPD1 in 0, 10, and 50 μg/mL PS-T–treated ALDH\u003csup\u003e+ \u003c/sup\u003eMDA-MB-231 cells was detected by western blotting (A) (n = 3) and quantitative PCR (B) (n = 9).\u003cstrong\u003e (C-D)\u003c/strong\u003e Immunofluorescence (C) and Immunohistochemistry (D) staining of ASM, LC3, and GPX4 protein in the control (\u003cem\u003eup\u003c/em\u003e) and PS-T–treated mice (\u003cem\u003edown\u003c/em\u003e) (n = 9). Scale bars = 50 μm.\u003cstrong\u003e (E)\u003c/strong\u003e The levels of ASM, LC3, and GPX4 in TNBC patients were assessed by immunohistochemistry staining (n = 6). Scale bars = 50 μm. \u003cstrong\u003e(F)\u003c/strong\u003e Expression level of ASM, LC3, and GPX4 were photographed after treatment with DMSO (control), PS-T (10 μg/mL), or ASM-IN-1 (2 μM) alone or in combination with both (n = 3). Relative gray density was measured and analyzed statistically and are presented as a histogram\u003cem\u003e \u003c/em\u003e(\u003cem\u003edown\u003c/em\u003e). (mean ± standard deviation; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5706155/v1/31683161bb8d98c8267ae22b.png"},{"id":74201124,"identity":"6d75e34b-ffb4-42ed-a55f-da25535d6d0e","added_by":"auto","created_at":"2025-01-20 02:16:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16302393,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5706155/v1/e6f26906-0a60-49ef-9cab-150a4c73de70.pdf"},{"id":72612294,"identity":"d71549ce-7878-4a50-8b7a-e3f707e13abc","added_by":"auto","created_at":"2024-12-30 10:28:23","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":581103,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures20241224.docx","url":"https://assets-eu.researchsquare.com/files/rs-5706155/v1/88f74760fe453d6fbe1f6837.docx"},{"id":72612288,"identity":"c35682a3-02dc-416f-9115-88efaa74e0fd","added_by":"auto","created_at":"2024-12-30 10:28:22","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10176,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS120241224.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5706155/v1/226a779b74055a952a3ec537.xlsx"},{"id":72612292,"identity":"ae1e1fe9-f488-42ff-943a-a37225aa3340","added_by":"auto","created_at":"2024-12-30 10:28:23","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":268143,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalDataFerro20241010.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5706155/v1/f85338898afaafaf67f76d3a.pdf"},{"id":72612299,"identity":"bd5ee6a9-a46a-457e-9fac-70fba72e2285","added_by":"auto","created_at":"2024-12-30 10:28:23","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":5622121,"visible":true,"origin":"","legend":"","description":"","filename":"RawDataFerro20241010.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5706155/v1/5e8c2d88f4dc71570a075934.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Huaier Polysaccharides Targets Cancer Stem Cells in Triple-Negative Breast Cancer via ASM-mediated Autophagy-dependent Ferroptosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer ranks as the most prevalent cancer threatening the health of Chinese females [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Currently, the conventional treatments of breast cancer mainly include surgical resection, postoperative chemotherapy, radiotherapy, and endocrine therapy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although current studies suggest that postoperative adjuvant therapy is an important strategy for preventing recurrence and prolong the survival period of breast cancer patients, conventional adjuvant therapy does not benefit all breast cancer patients [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Triple-negative breast cancer (TNBC) lacks of estrogen, progesterone, and human epidermal growth factor receptor 2 (HER-2) receptors [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These patients cannot receive adjuvant therapy, such as endocrine therapy or targeted therapy and are often prone to local recurrence and distant metastasis, thus having a poor prognosis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBreast cancer stem cells (BCSCs) is the cancer lineages with the ability of tumorigenesis and self-renewal. Several studies have reported that BCSCs contribute to chemotherapy resistance and critical for the metastasis and relapse of breast cancer. Treatment with chemotherapy can effectively eliminate tumor cells in some patients; however, it can also increase the proportion of enriched BCSCs, which eventually raises the risk of recurrence and metastasis in TNBC patients [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, identifying new strategies that target and inhibit BCSCs is currently the focus of TNBC treatments [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFerroptosis is a Fe\u003csup\u003e2+\u003c/sup\u003e-dependent and programmed cell death, accompanied massive ROS accumulation and excessive lipid oxidation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. A recent study conducted an integrated multi-omics analysis which identified significantly activated ferroptosis in breast tumors with high imaging intratumor heterogeneity (IITH). The study suggests that ferroptosis is a predisposing factor for breast cancer with high IITH, and that its inhibitors could be a promising option for refractory breast cancer [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In addition, iron is present at high concentrations in BCSCs, and iron metabolism is important for maintaining the stemness of BCSCs. The promotion of ferroptosis inhibits stem cell characteristics in breast, colorectal, and lung cancer [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently, some natural medicinal fungi and their extracts were found to have unique advantages in preventing tumorigenesis, inhibiting the formation of tumor foci, and preventing recurrence and metastasis. Among them, Huaier, whose main active ingredient is the polysaccharide of \u003cem\u003eTrametes robiniophila Murr\u003c/em\u003e (PS-T), has been applied to the adjuvant therapy of malignancies, including breast cancer. Huaier has been reported to improve life quality for cancer patients with few side effects and high safety [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In a previous clinical study, we demonstrated that Huaier effectively inhibited the metastasis and recurrence of TNBC [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, the effects of PS-T on TNBC stem cells and ferroptosis are yet to be investigated.\u003c/p\u003e \u003cp\u003eHerein, we found for the first time that PS-T could promote ferroptosis of BCSCs in TNBC and that the underlying mechanism may involve PS-T-induced GPX4 degradation through the ASM-mediated autophagic lysosomal pathway. This study reveals a novel mechanism by which PS-T inhibits metastasis and recurrence in patients with TNBC and provides a theoretical basis and data support for identifying new molecular targets and strategies for TNBC treatment from the perspective of ferroptosis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003eWe purchased the Huaier extract from Gaitianli Pharmaceutical Co., Ltd. (Qidong, China). The polysaccharides were prepared according to our previous work, including isolation, dialysis, extraction, lyophilisation, elution, and purification [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. α-Tocopherol phosphate (HY-16686, 100 \u0026micro;M) was obtained from MedChemExpress (USA). Liproxstatin-1 (T2376, 200 mM), 3-methyladenine (T1879, 5 mM), and ASM-IN-1 (T74778, 2 \u0026micro;M) were obtained from TargetMol (USA). Lastly, Z-VAD-FMK (S7023, 30 \u0026micro;M), Necrostatin-1 (S8037, 20 \u0026micro;M), and LDC7559 (S9622, 5 \u0026micro;M) were acquired from Selleck (USA).\u003c/p\u003e \u003cp\u003eAnti-human CD24 (PE, 311106, 5 \u0026micro;L/test) and anti-mouse/human CD44 (FITC, 103022, 5 \u0026micro;L/test) were acquired from Biolegend (USA). BODIPY\u0026trade; 581/591 C11 (D3861, 5 \u0026micro;M/test), GPX4 (PA5-102521), and ASM antibody (PA5-77047) were obtained from Thermo Fisher (USA). The antibodies listed below were from Cell Signaling Technology (USA): GPX4 (52455), SLC7A11 (98051), FTH1 (3998), NRF2 (12721), DMT1 (15083), NCOA4 (66849), and β-actin (4970).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell lines\u003c/h3\u003e\n\u003cp\u003eThe MDA-MB-231, HCC1806, and HCC1937 cell lines were purchased via the FuHeng Cell Centre (Shanghai, China). MDA-MB-231 was maintained in L-15 medium (LA9510; Solarbio, Beijing, China) at 37\u0026deg;C in 100% air, HCC1806 and HCC1937 were cultured in RPMI-1640 medium (C11875500B; Gibco, USA) at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e, with foetal bovine serum (10%) (10091148; Gibco, New Zealand) and L-glutamine (1%) (C0222; Beyotime, Shanghai, China)in the medium. Sorted stem cells remained in 3dGRO sphere medium at standard condition, and digested at a density of 90%.\u003c/p\u003e\n\u003ch3\u003eFlow cytometry and cell sorting\u003c/h3\u003e\n\u003cp\u003eThe cells were exposed to distinct drugs for 24 h after adherence. After adjusted to 1 x 10\u003csup\u003e6\u003c/sup\u003e cells/mL using assay buffer, the cells were incubated with CD24 and CD44 at 37\u0026deg;C avoiding light for 30 min. Subsequently, the samples were supplemented with 300 \u0026micro;L of Buffer (70-S1001; MultiSciences, Zhejiang, China). When using the ALDEFLUOR Stem Cell Identification kit (01700; STEMCELL, Canada), every sample was added to activated ALDEFLUOR, mixed, and half of the mixture was transferred to a DEAB control tube before incubating the antibody. The samples were then analyzed with a BD LSR Fortessa or sorted using a BD FACS Aria III.\u003c/p\u003e\n\u003ch3\u003eColony formation and mammosphere assays\u003c/h3\u003e\n\u003cp\u003eFor colony formation, seed cells in 6-well plates with2,000 each, treat with drugs for 24 h, and cultured 7\u0026ndash;10 days. Change medium every two days. Before observation, the plates were fixed (BL539A; Biosharp, Shanghai, China) and stained (C0121; Beyotime, Shanghai, China) according to the manufacture provided protocol. For mammosphere assays, cells were seeded in ultra-low attachment multi-well plates (CLS3471; Corning\u0026reg; Costar\u0026reg;, USA) with 2,000 cells per well. Drugs were added after 24 h, and the cells were cultured for 10 days. Within these days, half of the amount was added to the medium every two days. The mammospheres were then observed under a microscope.\u003c/p\u003e\n\u003ch3\u003eXenograft assay\u003c/h3\u003e\n\u003cp\u003eThe female nude mice used for this experiment were purchased from Byrness Weil Biotech Ltd. (Chongqing). Approximately 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e ALDH\u003csup\u003e+\u003c/sup\u003e MDA-MB-231 cells were injected into the mammary fat pads of the nude mice, including a control group and a group treated with PS-T. The PS-T-treated group was given a dose of 3 mg every two days via intragastric administration starting from the second day after injection. After 4 weeks, all mice were euthanized and the tumors were observed.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCCK-8 assay\u003c/h2\u003e \u003cp\u003eCells at 2 x 10\u003csup\u003e4\u003c/sup\u003e cells/mL were inoculated on 96-well plates and exposed to indicated drugs for 24 h after adherence. Add ten microliters of the CCK-8 cell counting kit (CK04, DOJINDO) in the medium and culture at 37\u0026deg;C for 1 h. Quantify the optical density (OD) with Varioskan Flash spectrophotometer (Thermo Scientific).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDCF assay\u003c/h3\u003e\n\u003cp\u003eSeed the cells on coverslips in 6-well plates (2.5 mL), add the drug and culture 24 h after adherence, and incubated with DCFH-DA of ROS Assay Kit (S0033; Beyotime, Shanghai, China)in a medium without fetal bovine serum at 37\u0026deg;C. After 30 min of incubation, wash with a medium without fetal bovine serum and fixed in 4% paraformaldehyde. Treated with antifade mounting medium containing DAPI (P0131; Beyotime, Shanghai, China), observe under a fluorescence microscope (FV-1000; Olympus, Tokyo).\u003c/p\u003e\n\u003ch3\u003eLipid oxidation experiments\u003c/h3\u003e\n\u003cp\u003eThe amount of MDA in cells and tissues was quantified using a Lipid Peroxidation MDA Assay Kit (S0131; Beyotime, Shanghai, China). Following provided instruction, mix with MDA detecting working solution at 100\u0026deg;C for 15 min. After cooling and centrifugation, 200 \u0026micro;L supernatant of each was added into a 96-well plate. The absorbance of 532mn was detected with Varioskan Flash. As for the BODIPY\u0026trade; 581/591 C11 probe, cells were incubated at 37\u0026deg;C in absence of light exposure for 30 min. After digestion, wash twice with buffer, re-suspended with buffer to 300 \u0026micro;L, measured their fluorescence with BD LSRFortessa.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eCells were harvested and their proteins were lysed with RIPA lysis buffer (P0013B; Beyotime, Shanghai, China). Measure the protein abundance using an enhanced BCA protein assay kit (P0010; Beyotime, Shanghai, China). Afterward, transfer to a polyvinylidene difluoride membrane. The membrane was blocked, incubated with primary and secondary antibodies, and visualized using the ECL detection system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation and qPCR\u003c/h2\u003e \u003cp\u003eIsolate Total mRNA according to the instruction of the TaKaRa MiniBEST Universal RNA Extraction Kit (9767; Takara, Japan). Moreover, cDNA was obtained using the PrimeScript\u0026trade; RT Master Mix (RR036A; Takara, Japan). The sequences and lengths of the PCR primers are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The reactions were detected with TB Green\u0026reg; Premix Ex Taq\u0026trade; II FAST qPCR (CN830S; Takara) in an Eppendorf Mastercycler realplex. The mRNA expression was calculated according to the 2\u003csup\u003e\u0026minus;∆∆CT\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC) staining\u003c/h2\u003e \u003cp\u003eImplement IHC staining following the SP kit instructions (SP-9000; ZSGB-BIO, Beijing, China). Staining intensity was quantified: 0\u0026thinsp;=\u0026thinsp;negative; 1\u0026thinsp;=\u0026thinsp;weakly positive; 2\u0026thinsp;=\u0026thinsp;positive; and 3\u0026thinsp;=\u0026thinsp;strongly positive. The percentage of stained cells was counted as: 0, less than 5%; 1,6\u0026ndash;25%; 2, 26\u0026ndash;50%; 3, 51\u0026ndash;75%; and 4, more than 75%. IHC staining was quantified by multiplying the signal value with the stained cells percentage. The mean fluorescence (MFI) was quantified using ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatic analysis\u003c/h2\u003e \u003cp\u003eThe TCGA database provided RNA-sequencing data and clinical information on patients with TNBC. LASSO regression was implemented using the R package glmnet. Nomogram model was constructed by the R package rms using Cox method. Sangerbox 3.0 was used to perform ROC curve and ssGSEA enrichment analysis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The gene set files PECE_MAMMARY_STEM_CELL_UP, PECE_MAMMARY_STEM_CELL_DN, and WP_FERROPTOSIS were retrieved via the Molecular Signatures Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.gsea-msigdb.org/gsea/msigdb\u003c/span\u003e\u003cspan address=\"https://www.gsea-msigdb.org/gsea/msigdb\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). During GSEA enrichment analysis, differentially expressed genes (DEGs) were analyzed and plotted using the GSEA 4.1 software. For survival analysis, Kaplan-Meier curve was visualized the Kaplan-Meier Plotter (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://kmplot.com/analysis/\u003c/span\u003e\u003cspan address=\"http://kmplot.com/analysis/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the bc-GenExMiner v5.0 online tools (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bcgenex.ico.unicancer.fr/BC-GEM/GEM-Accueil.php?js=1\u003c/span\u003e\u003cspan address=\"http://bcgenex.ico.unicancer.fr/BC-GEM/GEM-Accueil.php?js=1\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll quantitative data are visualized using a mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation mode. The Student t-test and the Mann\u0026ndash;Whitney U test were used to evaluate difference of two groups. For pairwise comparisons, one-way analysis of variance and post-hoc Bonferroni tests were used. The logistic regression was used to univariate and multivariate analysis. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant. All analyses were carried out on GraphPad Prism 8.0 and SPSS 26.0.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHigh stem-related signature risk score predicts worse prognosis in TNBC\u003c/h2\u003e \u003cp\u003eWe constructed a LASSO regression model based on 167 TNBC patients from the TCGA database to screen key genes associated with stemness and identified 17 genes that were predominant in the prognosis: CDC5L, IL4R, NDP, REXO2, TBCA, RGS17, RAB2B, ABHD6, PUM2, UQCR11, TCF7L2, SFPQ, TXN, EEF1D, SHQ1, FHL1, and PLBD1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Risk factor analysis of the 17 genes suggested that the risk factor score was significantly associated with the prognosis of patients with TNBC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The areas under the time-dependent ROC curve at 1, 3, and 5 years were 0.80, 0.86, and 0.88, separately, indicating high prognostic performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The 167 samples were then separated into high- (n\u0026thinsp;=\u0026thinsp;91) and low-risk group (n\u0026thinsp;=\u0026thinsp;76) according to the risk factor score, among which high-risk group TNBC patients had higher mortality (HR\u0026thinsp;=\u0026thinsp;22.89, 95%CI: 5.44\u0026ndash;96.3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8e-10) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Furthermore, we explored the impact of these 17 genes on TNBC prognosis. Overall survival (OS), relapse-free survival (RFS), and distant metastasis-free survival (DMFS) data were collected and visualized using forest plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G, and Additional file 1: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eUnivariate analysis showed that primary tumor, Nodal status, TNM stage, and risk factor score were independent predictors of TNBC prognosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Multivariate analysis indicated that risk factor score was superior in predicting prognosis than conventional clinical features like TNM stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Besides, we constructed a nomogram model combining data on survival time, survival status, and other clinical characteristics to demonstrated that the risk score has good predictive value for mortality risk (C-index\u0026thinsp;=\u0026thinsp;0.891, 95%CI: 0.85\u0026ndash;0.94, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.33e-62) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The calibration curves indicate that the signature has favorable prediction accuracy for 3 and 5 years (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). To validate our prognostic model, external validation sets (GSE20711, GSE58812, and GSE21653) was used. The areas under the ROC curve at 9 years were 0.78, 0.63, and 0.70 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePS-T reduces the stemness characteristics of BCSCs in TNBC\u003c/h2\u003e \u003cp\u003eTo explore the effect of PS-T on cell stemness characteristics in TNBC, we analyzed the enrichment level of the mammary stem cell pathway using ssGSEA and found decreased ssGSEA scores in the PS-T-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). A dose-dependent decrease in the expression of the stem cell markers POU5F1, SOX2, and NAONG was observed after PS-T treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The proportions of ALDH\u003csup\u003e+\u003c/sup\u003e and CD44\u003csup\u003ehigh\u003c/sup\u003eCD24\u003csup\u003elow\u003c/sup\u003e MDA-MB-231 cells were significantly decreased after PS-T treatment in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The ALDH\u003csup\u003e+\u003c/sup\u003e cell population was sorted for the colony formation and mammosphere assays. With an increase in PS-T concentration, the number and size of the colonies and mammospheres gradually decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). ALDH\u003csup\u003e+\u003c/sup\u003e MDA-MB-231 cells were transplanted into female nude mice to observe the effect of PS-T on breast tumor formation. The results showed that the probability of tumor formation was severely reduced by PS-T treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). These results suggest that PS-T could reduce the stemness of BCSCs in TNBC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePS-T restrains BCSCs by activating ferroptosis in TNBC\u003c/h2\u003e \u003cp\u003eTo illustrate the mechanism underlying PS-T-mediated regulation of BCSCs in TNBC, we evaluated the viability of PS-T-treated ALDH\u003csup\u003e+\u003c/sup\u003e cells after treatment with cell death inhibitors. PS-T significantly inhibited cell proliferation. Furthermore, the inhibitory effect was only slightly restored after the addition of inhibitors of apoptosis, necrosis, or pyroptosis, but was significantly reversed with the assistance of ferroptosis inhibitors, α-Tocopherol phosphate (α-toc) and Liproxstatin-1 (Lip-1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Additional file 1: Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). In addition, GSEA enrichment results revealed that the ferroptosis pathway was more active in PS-T\u0026ndash;treated TNBC cells compared to control cells (ES\u0026thinsp;=\u0026thinsp;0.61, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.024) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Additional file 1: Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). Ferroptosis has been reported to promoted by the accumulation of reactive oxygen species (ROS) and excessive lipid oxidation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. We investigated changes in intracellular ROS levels and lipid oxidation after PS-T treatment. The mean fluorescence intensity (MFI) of DCF and BODIPY\u0026trade; 581/591 C11 and the MDA concentration in ALDH\u003csup\u003e+\u003c/sup\u003e MDA-MB-231 cells decreased after PS-T treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-E). Subsequently, we verified the PS-T-induced reduction in lipid peroxidation in vivo using an MDA assay and flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, G).\u003c/p\u003e \u003cp\u003eFurthermore, we inhibited ferroptosis in TNBC cells using ferroptosis inhibitors α-tocopherol (α-toc) and liproxstatin-1 (Lip-1). The results showed that the lipid oxidation level and intracellular MDA concentration in PS-T-treated cells barely increased after the addition of the ferroptosis inhibitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). To validate the role of PS-T-induced ferroptosis in the inhibitory effect on BCSCs, we co-treated cells with PS-T and ferroptosis inhibitors. The PS-T treatment did not significantly reduce the mRNA levels of POU5F1, SOX2, or NAONG, the percentage of ALDH\u003csup\u003e+\u003c/sup\u003e and CD44\u003csup\u003ehigh\u003c/sup\u003eCD24\u003csup\u003elow\u003c/sup\u003e cells, or the formation of colonies and mammospheres in the presence of ferroptosis inhibitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-I). Collectively, these data suggest that PS-T inhibits BCSCs by activating ferroptosis in TNBC cells.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ePS-T promotes ferroptosis of BCSCs by inducing autophagic degradation of GPX4\u003c/h2\u003e \u003cp\u003eThe expression levels of ferroptosis pathway markers were detected by western blotting. GPX4 (Glutathione Peroxidase 4) was found to be downregulated after PS-T treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Immunohistochemical staining also confirmed the PS-T-induced downregulation of GPX4 protein in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C). Additionally, the overall survival of high-GPX4 TNBC patients was significantly shorter (HR 2.78, 95%CI: 1.45\u0026ndash;5.31, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0012; HR 3.29, 95%CI: 1.03\u0026ndash;10.53, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0444) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, E, and Additional file 1: Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA). These results suggested that PS-T promotes ferroptosis by downregulating GPX4, thereby inhibiting BCSCs in TNBC.\u003c/p\u003e \u003cp\u003eSubsequently, we examined the regulatory mechanism through which PS-T impacts GPX4 protein expression. There was no statistically significant difference in the GPX4 mRNA levels in TNBC cells treated with or without PS-T, indicating that PS-T does not regulate GPX4 expression at the transcriptional level (Additional file 1: Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB, C). Our previous study found that PS-T promotes autophagic flux in TNBC cells and inhibits the invasion and migration ability of Snail protein [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], suggesting that PS-T may degrade GPX4 protein through the autophagy-lysosomal pathway. The autophagy inhibitor 3-methyladenine (3-MA) was used for further validation. As expected, when 3-MA inhibited autophagosome formation, GPX4 protein and cell membrane lipid oxidation levels in PS-T-treated ALDH\u003csup\u003e+\u003c/sup\u003e MDA-MB-231 cells were comparable to untreated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-I). Furthermore, after the inhibition of autophagy, PS-T treatment did not reduce the mRNA levels of stem cell markers, the proportion of ALDH\u003csup\u003e+\u003c/sup\u003e and CD44\u003csup\u003ehigh\u003c/sup\u003eCD24\u003csup\u003elow\u003c/sup\u003e cells, or the formation of colonies and mammospheres (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ-M). Collectively, these results suggest that PS-T may degrade the GPX4 protein through the autophagy-lysosomal pathway and induce autophagy-dependent ferroptosis in TNBC stem cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003ePS-T promotes the autophagic degradation of GPX4 by upregulating ASM in BCSCs\u003c/h2\u003e \u003cp\u003eAcid sphingomyelinase (ASM; gene name: \u003cem\u003eSMPD1\u003c/em\u003e) is activated when tumor cells encounter extracellular stimulation (antineoplastic drugs, oxidative stress, radiation, etc.) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The absence of ASM contributes to the resistance of tumor cells to antitumor drug-induced apoptosis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Interestingly, ASM participates in the occurrence of autophagy by regulating the formation of autophagosomes and lysosomal membranes [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The ferroptosis inducer erastin triggers ferroptosis by mediating ASM-dependent autophagic degradation of GPX4 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWestern blotting and qPCR indicate that ASM was upregulated in ALDH\u003csup\u003e+\u003c/sup\u003e MDA-MB-231 cells after PS-T treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). The promotion of ASM expression by PS-T was verified using 4T-1 xenograft (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D). Histological examination revealed a positive correlation between PS-T-induced ASM upregulation and autophagic GPX4 degradation in the TNBC stem cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). We performed immunohistochemical analysis of 12 TNBC samples and found a significant increased LC3 abundance, whereas the expression of GPX4 protein decreased in the high-ASM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Furthermore, the ability of PS-T to induce autophagy was significantly inhibited by ASM-IN-1. In addition, ASM-IN-1 treatment reversed the PS-T-induced GPX4 degradation in ALDH\u003csup\u003e+\u003c/sup\u003e MDA-MB-231 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). These results indicate that ASM is required for PS-T-induced GPX4 degradation in TNBC stem cells.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs the most lethal breast cancer subtype, triple-negative breast cancer (TNBC) patients cannot benefit from endocrine therapy and anti-HER-2 targeted therapy, as a result of the lack of hormone receptors. Chemotherapy remains an important systemic therapy for TNBC; however, not all patients benefit from chemotherapy, and many patients often experience recurrence and metastasis within a short period after the completion of chemotherapy, leading to a very poor prognosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Breast cancer stem cells (BCSCs) are a subpopulation of tumor cells with self-renewal abilities and multidirectional differentiation potential. Compared to ordinary tumor cells, BCSCs have stronger chemotherapy resistance, invasion, and metastasis abilities, which may be the seed of recurrence and metastasis after chemotherapy in patients with TNBC [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, finding a new strategy to target and inhibit BCSCs is the focus of the current clinical treatment of TNBC [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStudies have indicated that BCSCs is necessary for chemotherapy resistance, metastasis, and recurrence in TNBC [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. According to the LASSO regression analysis using TCGA data, we found that the abundance of stem cell-related genes was associated with adverse prognosis in patients with TNBC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Our research group is committed to studying the effects and mechanisms of Huaier on the treatment. In a previous clinical study, Huaier granules significantly improved the overall survival rate and delayed disease progression in patients with TNBC [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. To investigate whether PS-T plays an anticancer role by inhibiting BCSCs, we purified polysaccharide of Huaier (PS-T), the main active component of Huaier granules, and performed further experiments [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. ssGSEA enrichment analysis indicated the suppression of the mammary stem cell pathway after PS-T treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Quantitative PCR and flow cytometry analysis of PS-T-treated cells showed that PS-T reduced the level of stem cell markers and the proportion of ALDH\u003csup\u003e+\u003c/sup\u003e and CD44\u003csup\u003ehigh\u003c/sup\u003eCD24\u003csup\u003elow\u003c/sup\u003e cells in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). The ALDH\u003csup\u003e+\u003c/sup\u003e MDA-MB-231 cells were sorted for subsequent experiments. Based on colony formation and mammosphere assays, PS-T suppressed the proliferation and stemness of ALDH\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). Consistently, PS-T significantly limited tumor formation in ALDH\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Taken together, PS-T reduced the stemness characteristics of TNBC stem cells, in vitro and in vivo.\u003c/p\u003e \u003cp\u003eBased on the cell viability experiments and GSEA enrichment analysis, we hypothesized that PS-T inhibits TNBC stem cells via stimulating the ferroptosis pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B, Additional file 1: Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Ferroptosis was reported as a programmed cell death type, triggered by massive ROS accumulation and excessive lipid oxidation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Recent studies have reported that there is a high concentration of iron in cancer stem cells and that iron metabolism is conducive to maintaining the stemness of cancer stem cells [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Promotion of ferroptosis can inhibit the stem cell characteristics of breast, colorectal, and lung cancer [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. As detected by immunofluorescence and colorimetry, PS-T induced intracellular ROS and lipid oxidation in TNBC cells \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-G). The inhibition effect of PS-T on BCSCs was reversed by the addition of ferroptosis inhibitors. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGPX4 is the key regulator for ferroptosis, and acts by reducing lipid peroxidation in the cell membrane, thereby inhibiting ferroptosis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, PS-T downregulates GPX4 protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C). This reduction further confirmed that PS-T promotes ferroptosis in TNBC stem cells. In addition, our data demonstrated that PS-T downregulated GPX4 levels by inducing autophagy (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, G). When autophagy was pharmacologically inhibited, PS-T no longer promoted ferroptosis or inhibited the stemness of BCSCs in TNBC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH-M). Furthermore, PS-T promotes autophagic GPX4 degradation by upregulating ASM expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), a key enzyme in regulating the sphingomyelin cycle that has a crosstalk with autophagy and GPX4 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, the key PS-T transcription factors that upregulate ASM need further investigation.\u003c/p\u003e \u003cp\u003eThese results suggest that PS-T inhibits BCSCs by promoting the ASM-mediated autophagic degradation of GPX4 and by inducing ferroptosis, ultimately improving the prognosis of patients with TNBC. Exploring and confirming this scientific problem can not only reveal a new mechanism by which PS-T inhibits metastasis and recurrence in TNBC patients, but also provide a theoretical basis and data support for discovering new molecular targets and strategies for TNBC treatment from the perspective of ferroptosis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003ePS-T induces autophagy-dependent GPX4 degradation by upregulating ASM in BCSCs and promotes ferroptosis to inhibit the stemness characteristics, thereby improving the prognosis of patients with TNBC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eWe would like to thank Editage (www.editage.cn) for the English language editing.\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eConceptualization: JJ, HLM and MHW; Methodology: LXZ, ZWW, KFL, ZH and TY; Data analysis: LXZ, ZWW, ZH and PQW; Writing\u0026mdash;original draft preparation: LXZ and ZWW; Writing\u0026mdash;reviewing and editing: JJ, HLM, and MHW. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was funded by the National Natural Science Foundation of China [Grant No. 82474127], the Natural Science Foundation of Chongqing [Grant No. CSTB2023NSCQ-MSX0520], and\u0026nbsp;Military Key Clinical Specialty\u0026nbsp;[Project No. 41561Z23612].\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eAdditional Figures and associated Figure legends are provided in the Supplementary Text and are available online. Raw RNA sequencing data were uploaded to the GEO database (accession number GSE253683).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were approved by the Laboratory Animal Welfare and Ethics Committee of the Army Medical University (SYXK-20170002) and the Ethics Committee of the Southwest Hospital of the Army Medical University (KY2021118).\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLi Y, Zhang H, Merkher Y, Chen L, Liu N, Leonov S, Chen Y: Recent advances in therapeutic strategies for triple-negative breast cancer. 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Cell 2014, 156(1-2):317-331.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Triple-negative breast cancer, Huaier, cancer stem cell, ferroptosis, GPX4, ASM","lastPublishedDoi":"10.21203/rs.3.rs-5706155/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5706155/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBreast 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,\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eLASSO 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 \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e assays and samples from patients with TNBC. Additionally, we confirmed these findings by inhibiting autophagy and ASM expression,\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA high stem-related signature risk score predicts a worse prognosis for TNBC. The proportions of ALDH\u003csup\u003e+\u003c/sup\u003e, CD44\u003csup\u003ehigh\u003c/sup\u003eCD24\u003csup\u003elow\u003c/sup\u003e cells, and cells with stem cell markers decreased proportionally to the dose post-PS-T therapy for TNBC. ALDH\u003csup\u003e+\u003c/sup\u003e 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,\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eTaken 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.\u003c/p\u003e","manuscriptTitle":"Huaier Polysaccharides Targets Cancer Stem Cells in Triple-Negative Breast Cancer via ASM-mediated Autophagy-dependent Ferroptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-30 10:28:18","doi":"10.21203/rs.3.rs-5706155/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4487de0c-2794-45a6-a19a-aefac1a9b5b0","owner":[],"postedDate":"December 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-20T02:08:31+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-30 10:28:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5706155","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5706155","identity":"rs-5706155","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}