Identication of A New Kind of Phenazine Compounds Attenuating The Stemness of Breast Cancer Cells Through Triggering Ferroptosis

Background: Breast cancer stem cells (BCSCs) are positively correlated with metastasis, chemoresistance and recurrence of breast cancer. Methods: The stemness were analyzed by qPCR and western blot, ow cytometry, cell spheroid formation assay, and tumor xenograft model. The cell viability was performed by MTT. The transcriptome analysis was used to evaluate the inuence of these three compounds. The migration was analyzed by wound-healing assay, transwell invasion analysis, and metastasis model. The iron concentration was analyzed by uorescence microscopy analysis. The lipid peroxidation and ROS level were analyzed by ow cytometry. The results were presented as mean ± SD, the analysis was Student’s t-test by using GraphPad Prism 5 software. P-values less than 0.05 were considered to be statistically signicant. (*p < 0.05, **p < 0.01, ***p < 0.001). Results: Here, we tried to screen out small-molecule compounds targeting BCSCs from the phenazine library established by us before. We focused on the compounds without affecting cell viability and screened out three potential compounds (CPUL119, CPUL129, CPUL149) that can signicantly attenuate the stemness of breast cancer cells, as evident by the decrease of stemness marker expression, CD44+/CD24- subpopulation, mammary spheroid-formation ability and tumor-initiating capacity. Additionally, these compounds suppressed the metastatic ability of breast cancer cells in vitro and in vivo. Combined with the transcriptome-sequencing analysis in breast cancer cells with or without the treatment of these three compounds, respectively, it was found that ferroptosis was shown on the top of the most upregulated pathways by CPUL119, CPUL129 and CPUL149. Mechanistically, we found that CPUL119, CPUL129 and CPUL149 could trigger ferroptosis by accumulating and sequestering iron in lysosomes through interacting with iron, and by regulating the expression of proteins (IRP2, TfR1, ferritin) engaged in modulating IgG (H+L), HRP conjugate; Goat Anti-Mouse, IgG (H+L), HRP conjugate, proteintech, Cat# SA00001-2, Cat# SA00001-1) and protein signals were detected with High-sig ECL Western Blotting Substrate (Tanon, Cat# 180-5001) on The used (Proteintech,15910-1-AP),

in the process of targeting CSCs, such as Glasdegib targeting leukemia stem cells (LSCs) [6] and ELZONRIS targeting CD123 that is highly expressed in LSCs have already been approved by the USA Food and Drug Administration (FDA) [7] . However, there are still no drugs targeting BCSCs approved for clinical application in breast cancer treatment. Thus, nding new compounds or through drug repositioning to nd compounds targeting BCSCs is becoming more and more important.
Recently, the iron metabolism has been found to be dysregulated in BCSCs [8] . The iron level is increased in the BCSCs, which causes the BCSCs to be more sensitive to iron chelation [9] . The existence of iron could directly change the cell differentiation, and the loss of transferring iron to lysosome could increase the stemness of cancer cells [10] . Iron also could produce OH . through inducing Fenton reaction which was the strongest oxygen-free radical in the reactive oxygen species (ROS), and the OH . can attack lipid to increase lipid peroxidation, which is one critical contributor of ferroptosis [5] . Additionally, the iron homostasis is modulated by different iron regulatatory proteins, such as iron-responsive element binding protein 2 (IRP2), transferrin receptor 1 (TfR1) and ferritin. IRP2 could bind to the iron regulator element (IRE) of mRNA 3'UTRs (untranslation region) or 5'UTRs of the different iron regulatory genes to stabilize or degrade the mRNAs [11] . TfR1 and transferrin degradation in lysosomes have been shown to be increased in BCSCs, while ferroportin (FPN) is decreased [12] . The decrease of transferrin leads to a suppression of ROS and ferroptosis [13] . Ferritin is the container to store iron and superoxide radicals could trigger the Fe 2+ release from the ferritin [14] . The use of lysosome disruptor, siramesine, could increase the iron in breast cancer cells resulting in the increase of ROS and ferroptosis [13] . Besides, the induction of ferroptosis is accompanied by the degradation of ferritin [15] .
It was found that the decrease of iron concentration in lysosomes could maintain the stemness of cancer cells [16] . The decrease of iron output-related gene and the increase of iron input-related gene represent a better prognosis in breast cancer [17] . Recently, iron nanoparticles that could target CSCs through ferroptosis and apoptosis were approved by the FDA, which bring up a new strategy to treat cancer by applying iron [18] . These results suggest that the iron increase in BCSCs promotes the activity of irondependent proteins and avoids the damage caused by iron overload through reducing the iron reserve in the lysosomes, thus reaching an "adjusted iron homeostasis" in line with the CSC metabolism level.
Consistently, it has been shown that salinomycin and its drivatives could target BCSCs through sequestering iron in lysosomes [19][20][21] . Notably, targeting iron in lysosomes has been con rmed to killing CSCs in other tumors, such as nasopharyngeal carcinoma [22] and colorectal cancer [23] . These results amplify that sequestering iron in lysosomes is a promosing method in targeting BCSCs.
In the present study, we reported three phenazine compounds (CPUL119, CPUL129, CPUL149) that can signi cantly attenuate the stemness of breast cancer cells through sequestering iron in lysosomes by interacting with and sequestering iron in lysosomes and thus triggering ferroptosis. This study indicates that CPUL119, CPUL129 and CPUL149 maybe the potential drugs for breast cancer treatment through attenuating the stemness of breast cancer cells.
All the cell culture contamination was at 37 °C under a humidi ed atmosphere with 5% CO2. The process of this experiment was referred to our previous study [24] .

MTT
Cell viability was measured by MTT (Solarbio, Cat# M8180-250 mg). MCF-7, MDA-MB-231 and MCF-7-Adr cells were seeded into 96-well plates at a density of 3000 -5000 cells/well. After 24 h, different concentrations of CPUL119, CPUL129 and CPUL 149 were added. 48 h later, 20 μl 5 mg/ml MTT was added into the medium for a 4 h culturing. Then the medium was removed and added 150 μl DMSO into each well. After shocking for 10 min, the absorbance at 490 nm was analyzed on a Biorad iMark.

GSH determination
The analysis of GSH was performed by Micro Reduced Glutathione (GSH) Assay Kit (Solarbio, Cat# BC1175) following the manufacturer's recommendation.

Analysis of iron content
The content of iron in the cell was measured by Iron Colorimetric Assay Kit (Applygen, Cat# E1042-100) according to the standard protocol. Brie y, MCF-7 and MDA-MB-231 cells were seeded into 24-well plates.
Wound-Healing assay MCF-7 and MDA-MB-231 cells were seeded into 6-well plates and cultured in the complete medium until growing up to 90%. Then, a wound was produced on the cell surface, and washed it with PBS twice to remove the cell fragments. Cells were cultured in the medium with low serum concentration(≤2%) contained compounds or solvent. After 24 h and 48 h, the wounds were observed and taken with a microscope. Migration rate = (D t -D 0 ) / D 0 , the D t represents the distance between the wound at different times.

Invasion assay
Cell invasion ability was assayed by transwell invasion analysis. Finally, the Millicell Hanging cell culture inserts (Merck, Cat# MCEP24H48) coated with Matrigel matrix (Corning, Cat# 356234) were prepared in the 24-well plates. The 2 × 10 5 MCF-7 or MDA-MB-231 cells with 200 μl serum-free medium with different compounds or solvent were added into the upper chamber, and the lower chamber was lled with medium containing 20% serum. After 48 h, the cells were xed with 70% ethanol for 20 min at room temperature. Then, the invaded cells were stained with crystal violet staining solution (Beyotime, Cat# C0121-100 ml), and observed under a microscope. After then, the chambers were washed with 33% glacial acetic and the absorbance at 570 nm was analyzed on a Biorad iMark, which represents the invaded cell number.

Tumorigenesis in vivo
The BALB/c-nude mice (Gempharmatech, Cat# D000521) were female, 3-4 weeks, and were cultured in standard pathogen-free conditions. Each group had six mice. All the experiments were obtained the approval of the Ethics Committee for Animal Experimentation of China Pharmaceutical University. In the tumor-limiting dilution assay, 1×10 6 , 1×10 5 and 1×10 4 MCF-7 and MDA-MB-231 cells pre-treated with compounds (4.5 μM CPUL119, 1.8 μM CPUL129 and 0.9 μM CPUL149) for 48 h were implanted in the inguinal mammary gland of mice orthotopically. After 10 days, the mice were sacri ced, and the amounts of tumors in each group were counted. And the stem cell frequencies were analyzed by ELDA (http://bioinf.wehi.edu.au/software/elda/).

Metastasis in vivo
The BALB/c-nude mice (Gempharmatech, Cat# D000521) were female, 3-4 weeks, and were cultured in standard pathogen-free conditions. Each group had ve mice. All the experiments were obtained the approval of the Ethics Committee for Animal Experimentation of China Pharmaceutical University. In the metastasis, each mouse was injected with 2×10 6 MDA-MB-231 cells by intravenous. One week later, these mice were injected with different compounds (10 mg/kg 119, 10 mg/kg 129, 5 mg/kg 129) or solvent (23.7% ethanol, 17%PEG400, 59.3% saline) intravenous every two days. After one month later, the mice were sacri ced. And the lungs were made H&E by Servicebio.

Statistical analysis
The results were presented as mean ± SD, the analysis was Student's t-test by using GraphPad Prism 5 software. P-values less than 0.05 were considered to be statistically signi cant. (*p < 0.05, **p < 0.01, ***p < 0.001).

Results
Identi cation of CPUL119, CPUL129 and CPUL149 as novel small-molecules suppressing breast cancer cell stemness We tried to screen out potential compounds that can speci cally attenuate the stemness of breast cancer cells without affecting cell viability from the phenazine-compound library we established before [25] . It was found that nine compounds (W23, W24, W49, W53, CPUL116, CPUL124, CPUL119, CPUL129, CPUL149) had little effect on breast cancer cell viability (Table1), which were chosen for the further studies. As shown in Figure 1A-1I, the compounds (W23, W53, CPUL116, CPUL119, CPUL129, CPUL149) exhibited a certain suppressing effect on the mRNA expression of stemness markers (SOX2, Oct4, Nanog, ALDH1A1) in a concentration-dependent manner. However, only compounds (CPUL119, CPUL129, CPUL149) decreased the protein expression of stemness markers in a concentration-dependent manner without affecting cell viability ( Figure 1J and 1K). The structures of these three compounds were shown in Figure  1L. Furthermore, CPUL119, CPUL129 and CPUL149 had no effect on breast cancer cell viability ( Figure   1M and 1N).
Additionally, we conducted the transcriptome sequencing in breast cancer cells with or without CPUL119, CPUL129, CPUL149 treatment, respectively. As shown in Figure 2A-2C, the transcription of stemnessrelated genes (such as UCA1 [26] , MALAT1 [27] ) was suppressed in cells treated with these three compounds, respectively. Moreover, Gene Set Enrichment Analysis (GSEA) demonstrated that multiple signaling pathways related to stem cell differentiation were enriched in cells treated with these three compounds, respectively (Supplement Figure S1 and S2). To further con rm the effects of CPUL119, CPUL129 and CPUL149 on the stemness of breast cancer cells, we conducted western blot experiments in another two breast cancer cell lines (MDA-MB-231 and MCF-7-Adr) and obtained the consistent results ( Figure 3A and 3B). In addition, the CD44+/CD24-cell-subpopulation with stemness was decreased by these three compounds ( Figure 3C and 3D). Furthermore, CPUL119, CPUL129 and CPUL149 signi cantly suppressed the mammary spheroid-formation ability which is positively correlated with cancer cell stemness, as characterized by the decrease of spheroid size and number ( Figure 3E and 3F). These results suggest that the compounds CPUL119, CPUL129 and CPUL149 can inhibit the stemness of breast cancer cells in vitro. CPUL119, CPUL129 and CPUL149 inhibit the migration, invasion and drug resistance of breast cancer cells As CSCs have regarded as one of a root of tumor metastasis and drug resistance, we continue to investigate the effects of CPUL119, CPUL129 and CPUL149 on the migration, invasion, epithelialmesenchymal transition (EMT) process and drug resistance of breast cancer cells. Firstly, it was found that these three compounds could inhibit the expression of metastasis-related genes and drug resistance based on the transcriptome-sequencing analysis (Figure2A and Supplementary Figure S3). We then performed the wound-healing assay to determine whether these three compounds could in uence the migration ability of breast cancer cells. As shown in the Figure 4A and 4B, cell migration ability was restrained after being treated with these three compounds for 24 h and 48 h. The invasion ability of breast cancer cells was also inhibited by these three compounds for 48 h ( Figure 4C and 4D).
Additionally, it was found that the protein expression of epithelial marker (E-cadherin) was increased, while N-cadherin and MMP-9 expression was decreased by these three compounds ( Figure 5A). Moreover, the effects of these three compounds on drug resistance were further explored. Since paclitaxel (Taxol) and Adriamycin (Adr) are the rst-line drugs for breast cancer, we chose them as the research subjects. As shown in Figure 5B and 5C, compared to the cells that only treated with Taxol or Adr alone, a distinct decrease of IC50 values of Taxol and Adr was observed in cells treated with Taxol or Adr as well as CPUL119, CPUL129 or CPUL149, respectively. Thus, our results demonstrate that CPUL119, CPUL129 and CPUL149 can attenuate the metastasis and drug resistance of breast cancer cells in vitro.

CPUL119, CPUL129 and CPUL149 inhibit the stemness and metastasis of breast cancer cells in vivo
We then evaluated the tumor-initiating ability of MCF-7 and MDA-MB-231 cells pre-treated with or without CPUL119, CPUL129 or CPUL149, respectively. As shown in Figure 6A-6F, the tumor-initiating ability was decreased by the pre-treatment of CPUL119, CPUL129 or CPUL149, which was characterized by the decreased tumor-formation rate and con dence intervals for 1/ (stem cell frequency). Notably, CPUL129 just impaired the tumor-initiating ability of MCF-7 cells in the high concentration of injected cell number, this result suggested CPUL129 may not inhibit the stemness of breast cancer cells in vivo, at least in the lower cell concentrations. Additionally, we constructed a lung metastatic model through injecting MDA-MB-231 cells by tail intravenous in nude mice. After one week, these nude mice were treated with CPUL119, CPUL129 and CPUL149 by tail intravenous every three days, respectively. One month later, these mice were sacri ced ( Figure 7A). As shown in Figure 7B and 7C, through H&E staining analysis, mice treated with these three compounds had smaller and fewer lung metastatic nodules compared with control mice which were treated with the solvent. Importantly, the groups treated with these three compounds exhibited a healthier phenotype by detecting mice weight ( Figure 7D). Consequently, these results demonstrate that CPUL119, CPUL129 and CPUL149 can inhibit the stemness and metastasis of breast cancer cells in vivo.

CPUL119, CPUL129 and CPUL149 regulates the iron hemostasis and ferroptosis in breast cancer cells
To explore the underlying mechanisms underlying the effects of CPUL119, CPUL129 and CPUL149 on the stemness of breast cancer cells, we re-analyzed the transcriptome-sequencing datasets and found that the ferroptosis (HSA04216) was signi cantly enriched in cells treated with these three compounds, respectively ( Figure 8A-8C). Consistently, GO analysis revealed that some biological process related lysosome function and iron transport were enriched, such as lysosomal lumen, iron-sulfur cluster assembly, protein maturation by iron-sulfur (Supplementary Figure S4-6). KEGG pathway analysis showed that glutathione (GSH) metabolism, which is also involved in ferroptosis, was enriched too (Supplementary Figure S4-6). Firstly, we detected the effects of these three compounds on GSH levels; however, it was found that GSH level was not changed by the treatment of these three compounds, respectively ( Figure 9A). Then, we evaluated iron concentration in breast cancer cells with or without treatment of CPUL119, CPUL129 or CPUL149, respectively and found that the iron concentration was signi cantly increased by these three compounds ( Figure 9B). Furthermore, we examined the effect of these compounds on iron homestasis. Treatment of cells with CPUL119, CPUL129 or CPUL149 induced a response trait of cytoplasmic deletion of iron, which was characterized by the increase of TfR1 and IRP2 along with decreased ferritin expression ( Figure 9C-9F). Besides, an increase of labile iron was observed in cells treated with these three compounds, respectively ( Figure 10A). Besides, the compounds may have a combination with iron in MCF-7 cells according to the Colocalization coe cient Rr ( Figure 10B). There are two factors regulating lipid peroxides during ferroptosis, including GSH and iron concentration [5] . As our previous results showed that CPUL119, CPUL129 or CPUL149 had no effect on GSH level, we wondered whether the increased iron concentration could increase the production of lipid peroxides.
Indeed, the ROS was increased in cells treated with these three compounds, respectively ( Figure 10C and  10D). Besides, the GO analysis is enriched genes-associated with oxidation-reduction process such as Oxidoreductase activity (Supplementary Figure S5). Furthermore, the lipid peroxidation was increased by the treatment of these three compounds (Supplementary Figure S7 A-D).
CPUL119, CPUL129 and CPUL149 interact and sequester iron in lysosomes in breast cancer cells We wondered whether CPUL119, CPUL129 and CPUL149 are located in lysosomes. As shown in Figure  11A and 11B, CPUL119, CPUL129 and CPUL149 were located in lysosomes, while a few of them were also located in mitochondria which plays a critical role in oxidative metabolism [28] , demonstrating that these three compounds mostly accumulate in lysosomal compartment. Additionally, it was found that CPUL119, CPUL129 and CPUL149 interacted with iron and led to the iron aggregation in lysosome, while iron was diffused in the cytosol of untreated-cells ( Figure 11C and 11D). These data indicated that CPUL119, CPUL129 and CPUL149 can interact and induce iron aggregation in lysosome.
CPUL119, CPUL129 and CPUL149 attenuate the stemness of breast cancer cells partially through triggering ferroptosis Finally, we investigated whether CPUL119, CPUL129 and CPUL149 attenuate the stemness of breast cancer cell through triggering ferroptosis. To determine whether other cell death pathways, including necrosis and apoptosis, which are commonly engaged in lysosomal cell death are involved in these three compound-mediated inhibitions on breast cancer cell stemness, apoptosis inhibitor Z-VAD-FMK, necrosis inhibitor Nec-1, ferroptosis inhibitor Fer-1, or ROS scavenger NAC were added in cells with CPUL119, CPUL129 or CPUL149 treatment respectively. As shown in Figure 12A, ferroptosis inhibitor or ROS scavenger NAC, but not Z-VAD-FMK or Nec-1, rescued the suppression of CPUL119, CPUL129 or CPUL149 on stemness marker expression. Additionally, the decreased subpopulation of CD44+/CD24-ratio led by CPUL119, CPUL129 or CPUL149 was partially abrogated by Fer-1 or NAC, respectively ( Figure 12B-12C).
Furthermore, a consistent result was obtained when the spheroid formation ability was evaluated ( Figure  12D and 12E). Besides, we explored whether the effects of CPUL119, CPUL129 or CPUL149 on the stemness of breast cancer cells was indeed dependent on lysosome function or iron. CA-074 (an inhibitor of the lysosomal protease cathepsin B) or iron chelating agent deferoxamine (DFO) was added in cells treated with these three compounds, respectively. As shown in Supplementary Figure S7A-D, the increased lipid peroxides led by these three compounds was attenuated by CA-074, and enhanced by DFO. Consistently, CA-074 rescued the effects of CPUL119, CPUL129 and CPUL149 on the expression of ferritin and stemness markers ( Figure 13A and 13B). These above results link the lysosomal degradation of ferritin to the production of ROS in this organelle through the release of additional soluble redox-active iron, thus attenuating the stemness of breast cancer cell stemness. Ferric ammonium citrate (FAC) was added into breast cancer cells to increase the iron concentration and the results indicated that FAC indeed promoted the expression of stemness markers and ferritin and even reversed the effects of CPUL119, CPUL129 and CPUL149 on the expression of ferritin and stemness markers ( Figure 13C and 13D).
Overall, our results demonstrate that CPUL119, CPUL129 and CPUL149 suppress the stemness of breast cancer cells through sequestering iron in lysosomes and triggered ferroptosis.

Discussion
BCSCs have been considered to be the root of breast cancer progression, thus, targeting BCSCs maybe a promising way to treat breast cancer thoroughly [4,[29][30][31] . In the present work, we aim to screen out smallmolecule compounds targeting BCSCs from the phenazine library established by us before. We nally screened out CPUL119, CPUL129 and CPUL149 as promosing compounds which can speci cally attenuating the stemness of breast cancer cells without affecting cell viability, as evident by the ability to reduce the stemness, invasion and chemoresistance in vitro and in vivo, and CPUL149 showed a better activity among the three compounds.
Targeting the abnormal signaling pathways, such as Wnt, Notch, Hedgehog and autophagy, has been shown to potentially killing CSC as we reviwed in a recent study [5] , however, most of small-molecule compounds are just in the preclinic stage. Targeting iron metabolism using the iron nanoparticles can be used to treat cancer through ferroptosis, which has been approved by FDA [32] . Besides, inducing ferroptosis through sequenstering iron in lysosome has been con rmed to speci cally killing CSCs including BCSCs [10,20,22,23] . In this work, we found CPUL119, CPUL129 and CPUL149 could increase the content of iron in breast cancer cells accompanying with the change of iron-regulatory protein such as IRP2, TfR1 and ferritin. Speci cally, these three compounds can interact, induce iron aggregation in lysosome and thus trigger ferroptosis. We noted that although iron in lysosomes could be further increased after the treatment of these compounds, iron also locates in lysosomes in the normal situation ( Figure 11C and 11D), this indicates an iron-rich environment in lysosomes [33] . Additionally, the decreased stemness resulted by these three compounds was attenuated or even reversed by ferroptosis inhibitor (fer-1), NAC and ferritin degradation inhibitor (CA-074), and the stemness was increased if we used the FAC to increase the iron. Notably, a small number of CPUL119, CPUL129 and CPUl149 were located in mitochondria which plays a critical role in oxidative metabolism, and apoptosis or necrosis inhibitor could also resuced the effects of these three compounds on the stemness of breast cancer cells although without signi cance. The GO analysis enriched necrosis correlated biological processes such as tumor necrosis factor receptor and tumor necrosis factor receptor binding (Supplementary Figure S2A). These results suggest that CPUL119, CPUL129 or CPUL149 might suppress the stemness of breast cancer cells through other signaling pathways, or these three compounds suppress the stemness of breast cancer cells through different signaling pathways as shown in the GO-or KEGG-enrichment analysis.
Furthermore, since cancer cells can be transformed into CSCs under certain conditions [34,35] , the effects of apoptosis or necrosis inhibitor on the stemness of breast cancer cells might be led by the effects on cancer cells. Moreover, we determined the location of these compounds only using lysosome probe and mitochondrion probe, and other organelle probes, such as endoplasmic reticulum or Golgi apparatus, was not used. Therefore, it is still unclear whether CPUL119, CPUL129 and CPUL149 locate in other organelles. Finally, supplementing with FAC enhanced the stemness of breast cancer cells, this is consistent with the previous study indicating that glioblastoma stem-like cells prefer to iron tra cking [36] .
Although the compounds target BCSCs through ferroptosis have been shown to have better research trend and anti-BCSCs ability, the risks should be considered. At rst, the iron metabolism is very vital in hematologic, cardiomyopathy and liver [37][38][39] , thus the iron overload maybe cause the unpredictable effects to this system; however, these three compounds may have no effect on hematopoietic system as a healthier phenotype was observed in mice treated with these three compounds by detecting mice weight ( Figure 7D). Besides, once the ferroptosis happens, abundant oxidative substance can be produced, such as OH·, these oxidative substances maybe attack the normal tissue. Therefore, these reasons may be challenging to make these compounds in the clinic, and more experiments are still needed. Notably, this study demonstrates that these three compounds may act as iron chelators, this speculation should be con rmed by other experiments, such as Nuclear Magnetic Resonance (NMR), and the detailed binding sites should be further explored.
Although these compounds showed better activity target BCSCs in the laboratory, we didn't take any pharmacodynamics, pharmacokinetics and toxicological evaluation. So, these may cause these compounds to face the challenge to be a drug. But these three compounds maybe become lead compounds to further enrich the library of molecules targeting BCSCs because of their better antistemness activity.

Conclusions
This study suggests that CPUL119, CPUL129 and CPUL149 can speci cally inhibit the stemness of breast cancer cells through ferroptosis and may be the potential compounds for breast cancer treatment.