Targeting cuproptosis by zinc pyrithione in triple-negative breast cancer

Summary Triple-negative breast cancer (TNBC) poses a considerable challenge due to its aggressive nature. Notably, metal ion-induced cell death, such as ferroptosis, has garnered significant attention and demonstrated potential implications for cancer. Recently, cuproptosis, a potent cell death pathway reliant on copper, has been identified. However, whether cuproptosis can be targeted for cancer treatment remains uncertain. Here, we screened the US Food and Drug Administration (FDA)-approved drug library and identified zinc pyrithione (ZnPT) as a compound that significantly inhibited TNBC progression. RNA sequencing revealed that ZnPT disrupted copper homeostasis. Furthermore, ZnPT facilitated the oligomerization of dihydrolipoamide S-acetyltransferase, a landmark molecule of cuproptosis. Clinically, high expression levels of cuproptosis-related proteins were significantly correlated with poor prognosis in TNBC patients. Collectively, these findings indicate that ZnPT can induce cell death by targeting and disrupting copper homeostasis, providing a potential experimental foundation for exploring cuproptosis as a target in drug discovery for TNBC patients.


Highlights ZnPT induces cuproptosis by disrupting copper homeostasis and DLAT oligomerization
ZnPT induces cuproptosis in vivo and potentially contributes to chemosensitivity of TNBC ZnPT inhibits progression and stemness of TNBC INTRODUCTION Among tumors in females, breast cancer has the highest incidence and second-highest lethality rate. 1 It accounts for 10%-20% of total breast cancer cases and exhibits a higher prevalence in younger women. 2 Compared to other breast cancer subtypes, triple-negative breast cancer (TNBC) is associated with high recurrence rates, high incidence of distant metastases, and poor overall survival (OS). 3Despite advancements in cancer therapy, effective TNBC treatment remains a formidable challenge due to its high recurrence and metastatic nature.Thus, there remains a pressing need to explore and develop promising and sensitive therapeutic regimens for TNBC.
Cell death has long been observed in cancers and linked to cancer therapy, with radiation and chemotherapy designed to induce malignant cell death.Understanding the mechanisms of cell death has facilitated the development of drugs that directly activate the cell death machinery and led to improved outcomes in cancer patients.Distinct from accidental cell death (ACD), regulated cell death (RCD), also known as programmed cell death (PCD), 4 refers to the autonomous and orderly death of cells orchestrated by various biomacromolecules. 4Notable types of RCD include apoptosis, necroptosis, pyroptosis, and ferroptosis.Pharmacologically targeting RCD with small molecule compounds has emerged as a promising therapeutic approach, 5 with rapid advancements in TNBC therapy. 6Recently, a novel form of RCD induced by copper ions was discovered, demonstrating cytotoxicity when intracellular copper ion levels surpass the threshold required for maintaining homeostatic mechanisms. 7Notably, researchers observed that copper ions directly bind to lipoylated components of the tricarboxylic acid (TCA) cycle, leading to the oligomerization of lipoylated proteins and loss of iron-sulfur cluster proteins, thus triggering proteotoxic stress and ultimately cell death. 7This cuproptosis pathway is marked by an abundance of lipoylated proteins, such as dihydrolipoamide branched chain transacylase E2 (DBT), glycine cleavage system protein H (GCSH), dihydrolipoamide S-succinyltransferase (DLST), and dihydrolipoamide S-acetyltransferase (DLAT). 8The lipoylation of these proteins is governed by various enzymes, including lipolytransferase 1 (LIPT1), lipoyl synthase (LIAS), and dihydrolipoamide dehydrogenase (DLD), 9 as well as ferredoxin 1 (FDX1), which functions as a primary upstream regulator of this lipoylation process. 7The presence of lipoylation-related enzymes and lipoylated proteins is highly correlated with multiple human tumors.Consequently, copper ionophores may offer therapeutic potential for various cancers with these metabolic traits, although specific compounds for cuproptosis are yet to be identified.1][12][13] Currently, ZnPT holds US Food and Drug Administration (FDA) approval as a topical antimicrobial agent for the treatment of psoriasis and ultraviolet (UV) radiation-induced epidermal hyperplasia. 14ZnPT exhibits zinc ionophore properties, which can lead to elevated intracellular zinc levels in yeast, 15,16 resulting in mismetallation and cellular stress. 17ZnPT can also act as a copper ionophore, inducing intracellular copper influx and iron-sulfur protein inactivation, 18 thereby inhibiting fungal growth. 19][27][28][29] In this study, we observed a significant enrichment of metal ion transport and binding in TNBC patients, alongside elevated expression of cuproptosis-related genes.Among the various antineoplastic agents tested, ZnPT emerged as one of the most promising candidates by facilitating the oligomerization of DLAT, a hallmark feature of cuproptosis.Collectively, our results demonstrated that TNBC patients exhibited heightened sensitivity to cuproptosis and ZnPT.Notably, ZnPT was capable of inducing cuproptosis and significantly inhibiting breast cancer progression.

Identification of ZnPT as a potential inhibitor of TNBC cells based on library screening
Traditionally, the development of new drugs is characterized by lengthy processes and substantial investments. 30Hence, drug repurposing for TNBC treatment is a promising strategy. 31To identify potential therapeutic agents for TNBC, we conducted cell proliferation assays using an FDA-approved drug library containing 638 small molecule compounds (Table S1) against the MDA-MB-231 TNBC cell line (Figures S1A-S1O).The well-recognized inhibitory chemicals for tumor growth include puromycin, validating our screening approach (Figures 1A and 1B).Candidates selected on the basis of their inhibitory effect include napabucasin, ZnPT, Penfluridol, etc. (Figures 1A and 1B).In addition to puromycin, a well-characterized drug, we further validated the remaining three drugs by examining the cell viability.Napabucasin demonstrated a half maximal inhibitory concentration (IC50) of %1.2 mM in the four TNBC cell lines (Figures S2A-S2D), while ZnPT showed inhibitory effects at 1.4 mM (Figures 1C-1F).Penfluridol displayed variable inhibitory effects at concentrations ranging from 1.5 mM to 5.0 mM (Figures S2E-S2H).Napabucasin, a drug specifically developed to target STAT3 in cancer, is underpinned by relatively comprehensive phenotypic and mechanistic studies. 32In contrast, oncological research on ZnPT is not well established.The robust inhibitory effects of ZnPT at 1.6 mM were further confirmed through cell proliferation assays in MDA-MB-231, HCC1806, MDA-MB-468, and HCC1937 cells (Figures 1G, 1H, and S2I-S2J).Based on these results, we selected ZnPT for further investigation.
To further explore the mechanism underlying the inhibitory effects of ZnPT on TNBC proliferation, RNA sequencing (RNA-seq) was performed in ZnPT-treated MDA-MB-231 and HCC1806 cells (Figures S3A-S3D).In the HCC1806 cells, Gene Ontology (GO) 33 biological process enrichment analysis revealed the downregulation of chromosome segregation and nuclear division (Figures S3E-S3F), both crucial processes for eukaryotic cell mitosis, which is dependent on spindle organization. 34This was further validated by GO cellular component enrichment analysis, showing downregulation of the spindle (Figures S3I-S3J), which is essential for the separation of condensed chromosomes during meiosis, a process highly dependent on interactions between microtubules and chromosomal kinetochores. 34GO molecular function enrichment analysis similarly verified the downregulation of microtubule binding (Figures S3M-S3N).The cyclin-dependent kinase (CDK)-RB-E2F axis represents a core transcriptional mechanism essential for cell cycle progression.Axis alterations in this pathway occur in nearly all cancers and result in elevated oncogenic E2F activity leading to uncontrolled proliferation. 35Based on gene set enrichment analysis (GSEA) of the RNA-seq data, hallmark_E2F_targets showed significant downregulation in the ZnPT-treated TNBC cell lines (Figures S3Q-S3R).Additionally, we observed significant downregulation in cell migration and the extracellular matrix (ECM) in MDA-MB-231 cells (Figures S3G-S3H, S3K-S3L, and S3O-S3P).Thus, these findings suggest that ZnPT is a promising compound to inhibit TNBC cell proliferation.

ZnPT induces cuproptosis by disrupting intracellular copper homeostasis and DLAT oligomerization
To explore the mechanism underlying the inhibitory effects of ZnPT on proliferation, we conducted in-depth analysis of the transcriptomic data.Based on GO enrichment analysis, we observed a significant upregulation in detoxification and stress response to copper ions (Figures 1I-1L).The GSEA results validated notable changes in copper homeostasis and response to metal ions (Figures 1M-1P and S2S-S2T).In addition, using inductively coupled plasma mass spectrometry (ICP-MS), we confirmed higher levels of intracellular copper and zinc in the TNBC cell lines following ZnPT treatment (Figures 1Q-1T).Previous research has indicated that dysregulation of copper ions  impairs [4Fe-4S] cluster maturation by displacing iron bound to cysteine (Cys) residues with high binding affinity. 36Our GO enrichment analysis identified dysregulated expression of genes related to the iron-sulfur protein NADH and NAD(P)H dehydrogenase (Figures S2K-S2L).
Copper exposure can induce protein aggregation, leading to protein misfolding. 37Similarly, our results showed significant upregulation in misfolded protein binding (Figures S2M-S2N).Additionally, we observed a significant upregulation in autophagosome and phagophore assembly sites (Figures S2O-S2R), consistent with previous studies showing increased autophagy levels in response to copper-carrying molecules. 38Recent research reported on a novel form of copper-dependent cell death, known as cuproptosis, which uniquely involves the binding of copper to lipoylation enzymes in the TCA cycle, leading to protein aggregation, proteotoxic stress, and ultimately cell death. 7e confirmed these findings through immunofluorescence, observing an increase in DLAT foci with increasing concentrations of ZnPT in TNBC cell lines (Figures 2A-2D and S4A-S4D).We further validated the occurrence of ZnPT-mediated cuproptosis based on analysis of ZnPT-treated HCC1806 and MDA-MB-231 cells using native-polyacrylamide-gel electrophoresis (PAGE) and western blotting assays.Notably, our results showed oligomerization of lipoylated DLAT upon ZnPT treatment in HCC1806 and MDA-MB-231 cells (Figures S4N- S4O).In addition to oligomerization of DLAT, another marker of cuproptosis is the loss of F-S cluster proteins.Consistent with previous findings, treatment with ZnPT significantly downregulated F-S cluster proteins ACO2 and SDHB (Figures 2E, 2F, and S4E-S4F).
Cell death entails complex signaling cascades and well-defined molecular effector mechanisms involving proteins and lipids.Previous studies have shown that ZnPT can induce lysosome-dependent apoptotic cell death. 39However, our results revealed that ZnPT-induced cell death did not involve the cleavage or activation of caspase3 activity (Figure 2K and S4K), a hallmark of apoptosis. 40Flow cytometry assays further supported these findings, demonstrating that ZnPT induced cell death through an apoptosis-independent pattern, as evidenced by Annexin V/PI staining (Figures 2G-2J and S4G-S4J), a sensitive indicator of apoptosis. 41These results provide robust evidence for the role of ZnPT in inducing cuproptosis.

ZnPT induces cuproptosis in vivo and shows potential in chemosensitivity for TNBC patients
To investigate the role of ZnPT in regulating cuproptosis in vivo, we conducted experiments in nude mice subcutaneously transplanted with HCC1806 cells.Once tumors reached a size (at least 0.1 3 0.1 cm), intraperitoneal injections of 5 mg/kg ZnPT were administered once every four days.Remarkably, the tumors treated with ZnPT exhibited regression, while the control tumors continued to grow (Figures 2L-2M), with all mice showing a partial response to treatment after 24 days (Figure 2N).Furthermore, immunohistochemical staining for Ki67 confirmed the inhibitory effects of ZnPT treatment on proliferation (Figures S4L-S4M).Notably, DLAT oligomerization was observed upon ZnPT treatment (Figures 2O-2P), validating that the observed cuproptosis was mediated by ZnPT.While ZnPT has clinical approval for topical use, its potential side effects when administered as an intraperitoneal injection in mice remain unclear.However, at the doses applied in our experiments, we observed no marked changes in body weight, immune system, or liver and renal function in the mice (Figures S4P-S4W), suggesting it may be a potentially safe approach for tumor treatment.
To further assess the clinical benefit of ZnPT in TNBC patients, we analyzed transcriptomic data of TNBC patients obtained from The Cancer Genome Atlas (TCGA) database.Results revealed that proteins within the cuproptosis pathway, specifically those involved in lipoylation such as DLAT and GCSH, were highly expressed in TNBC patients (Figures 3A and S5A).Additionally, upstream regulators of lipoylation, namely FDX1 and DLD, also showed high expression levels in TNBC patients (Figures 3A and S5A).Analysis of OS confirmed that patients with high expression of cuproptosis-related genes at the mRNA and protein levels exhibited poorer outcome (Figures 3B, 3C, and S5B-S5C).Furthermore, GO enrichment analysis indicated that metal ion transmembrane transporter activity and the ion channel complex were significantly upregulated in patients with high expression of cuproptosis-related genes (Figures 3D, 3E, and S5D-S5H).Moreover, zinc ion binding, metal ion binding, and metal ion transmembrane transporter activity were enriched in breast cancer patients (Figure 3G).Overall, this evidence strongly suggests that TNBC patients exhibit chemosensitivity to the cuproptosis pathway under ZnPT treatment.

ZnPT inhibits TNBC self-renewal and stemness markers
3][44][45] Similarly, our data showed a significant correlation between cuproptosis-related genes and breast  3G, and S5I-S5L), suggesting that ZnPT may also target these cells.CSCs are known for their high drug efflux properties, which contribute to their resistance to anticancer drugs. 46Additionally, CSCs contain abundant antioxidant molecules, such as glutathione (GSH), which play crucial roles in maintaining homeostasis, stemness, proliferation, and survival. 47According to previous studies, mitochondrial GSH can slow copper ion-induced cell death by inhibiting enzymatic lipoylation and oligomerization of DLAT. 48These unique properties of CSCs have paved the way for the discovery of small molecule drugs and advancements in targeting these cells.
Previous studies have shown that ALDH, a marker found in many CSCs, 49 reduces oxidative stress and increases breast cancer resistance to chemotherapeutic agents. 50In our study, we observed a remarkable reduction in ALDH expression in the TNBC cell lines upon treatment with 2.0 mM ZnPT (Figures 3H-3K and S5N-S5Q).The transmembrane protein CD44, another major BCSC surface marker, has also been implicated in metastasis, recurrence, and chemoresistance. 51CD44 promotes the expression of multidrug resistance genes, and its suppression is associated with enhanced chemosensitivity in cancer cells. 52Our analysis confirmed a significant suppression of CD44 protein levels with increasing concentrations of ZnPT (Figures 3M and S5R).In addition to ALDH and CD44 being universally acknowledged as BCSC markers, the STAT3 transcription factor is also widely recognized to govern BCSC behavior. 53Interestingly, a significant fraction of STAT3 is localized in the mitochondria, 54 where cuproptosis occurs.Indeed, we found that the correlation between cuproptosis-related genes and STAT3 was higher than that with ALDH and CD44 (Figures 3F, 3G, and S5M).Overall, STAT3 and phosphorylated STAT3 were downregulated after ZnPT treatment (Figure 3L and S5R).
To investigate the potential impact of ZnPT on BCSCs, we conducted clonal formation assays, which are commonly used to assess BCSC stemness. 55,56Our results demonstrated that the colony formation ability of the BCSCs was partially inhibited upon 1.0 mM ZnPT treatment and completely repressed upon 2.0 mM ZnPT treatment (Figures 4A, 4B, and S6A-S6B).Tumor spheroids are more resistant to treatment than cells in 2D culture and can recapitulate the drug resistance observed in solid tumors. 57,58The potential anti-CSC agents will be determined by counting the number and the size of formed tumorspheres. 59Results showed that ZnPT (2.0 mM) treatment significantly inhibited the number and size of tumorspheres (Figures 4C-4E and S6C-S6E).Thus, these results suggest that ZnPT plays a critical role in inhibiting BCSC stemness.

ZnPT represses TNBC capability of migration and ECM
Despite significant advancements in cancer diagnosis and treatment, metastasis continues to pose a major challenge in achieving favorable clinical outcomes, given that more than 90% of cancer-related deaths are attributed to metastatic disease. 60Primary tumor metastasis is a complex and multistep process involving local migration and invasion of cancer cells into adjacent tissues at the primary site, followed by dissemination into the circulatory system. 61Once in the circulation, circulating tumor cells extravasate into distant organs and often remain dormant after colonization.However, in certain cases, dormancy is disrupted, leading to the development of lethal macrometastases. 62,63In the sequential steps in metastasis, dysregulation of ECM homeostasis within solid tumors can have a marked effect. 64ased on RNA-seq analysis, our results revealed a significant downregulation in ECM constituents, organization, binding, and cell migration in mesenchymal-like cells MDA-MB-231.In line with these results, we performed a wound healing assay, a classic method for studying directional cell migration in vitro. 65Results showed that cell migration decreased with increasing concentrations of ZnPT (Figures 4F, 4G, and S6F-S6G).We also conducted a transwell-based assay, commonly used to assess cancer cell migration or invasion capacity. 66As anticipated, the ability of the TNBC cells to cross the porous membranes was partially inhibited upon treatment with 1.5 mM ZnPT and completely repressed upon treatment with 2.0 mM ZnPT (Figures 4H-4K and S6H-S6I).
Cell migration relies on the reorganization of microtubules, intermediate filaments, and actin filaments, which form different arrays to support cell propulsion. 67Vimentin, a component of intermediate filaments, promotes directional cell migration by coordinating the dynamics of actin filaments and microtubules. 68,69Thus, we measured the expression of vimentin in different ZnPT-treated TNBC lines and found that ZnPT reduced the protein levels of vimentin in a dose-dependent manner (Figures 4L and S6J).Furthermore, fibronectin, a versatile adhesive-like glycoprotein, plays a crucial role in regulating the function and structure of the interstitial ECM as well as cell attachment and migration. 70Consequently, we examined the levels of the fibronectin protein and found that they were significantly inhibited with increasing ZnPT concentrations (Figures 4L and S6J).In summary, our findings indicate that ZnPT suppresses wound healing and cell migration in TNBC cell lines, highlighting its potential role in regulating metastasis in TNBC patients.

TNBC signaling pathways are disrupted by ZnPT treatment
To investigate the intracellular signaling pathways affected by ZnPT, we conducted Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis 71 using previously obtained transcriptomic data.Our analysis demonstrated significant enrichment in 1.9 2.5 2.0 1.7 0.9 0.4 downregulated genes within the PI3K-AKT signaling pathway (Figures 5C and 5D).The PI3K-AKT signaling pathway is frequently activated in various types of solid tumors, playing a dominant role in crucial aspects of carcinoma, such as cell proliferation, stemness, migration, and chemoresistance. 72,73Our results demonstrated that AKT3 gene expression was significantly downregulated in the PI3K-AKT signaling pathway (Figures 5A and 5B).To further explore whether ZnPT regulates AKT and its phosphorylation, we conducted western blot analysis of ZnPT-treated cell extracts.Results indicated inactivation of AKT phosphorylation and a reduction in total AKT protein levels (Figures 5I-5L).Carcinogenic activation of the PI3K-AKT signaling pathway can occur in TNBC due to the overexpression of upstream regulators, such as EGFR. 74Consistently, we also observed a reduction in EGFR protein levels upon ZnPT treatment (Figures 5I-5L).
6][77] Correspondingly, KEGG pathway enrichment analysis demonstrated that the mitogen-activated protein kinase (MAPK) signaling pathway was markedly upregulated under ZnPT treatment (Figures 5E-5H).Additionally, we observed a significant increase in the activation of phosphorylated ERK with increasing ZnPT concentrations (Figures 5I-5L).The novel copper complex HYF127c/Cu was previously shown to activate the MAPK signaling pathway by inducing reactive oxygen species (ROS), ultimately promoting autophagy. 78Consistently, KEGG analysis showed a significant upregulation in mitophagy, a form of autophagy that selectively degrades mitochondria (Figures 5E-5H).These findings suggest that ZnPT-induced enhancement of intracellular copper ions can lead to the oligomerization of mitochondrial proteins, such as DLAT, ultimately triggering proteotoxic stress and mitochondrial dysregulation.In conclusion, our results indicated that ZnPT downregulates AKT expression, thereby inhibiting the PI3K-AKT signaling pathway, while concurrently enhancing the MAPK signaling pathway and mitophagy by elevating intracellular copper ion levels.

DISCUSSION
Although ZnPT has a long application history as an antifungal agent, its exact cytotoxic mechanisms remain underexplored.However, recent studies have highlighted its promising antitumor capabilities.For instance, Forcina et al. (2017) used two human tumor cell lines to assess the impacts of over 1,800 bioactive compounds on cell death dynamics at the population level, identifying 13 compounds that rapidly triggered cell death, including ZnPT. 79In our study, ZnPT ranked third in potency among the 638 FDA-approved compounds tested against the MDA-MB-231 cell line.Based on detailed analyses of four TNBC cell lines, our results also indicated that ZnPT had significant inhibitory effects on cell proliferation, stemness, migration, and invasion at concentrations as low as 2 mM.Our xenograft experiments on nude mice further demonstrated the excellent therapeutic effects of ZnPT, with no discernible impact on mouse weight or blood, liver, and kidney function.In addition, our data revealed that ZnPT significantly inhibited multiple aberrantly activated signaling pathways in breast cancer, including EGFR, AKT, and STAT3.Taken together, our findings highlight the excellent potential of ZnPT as an effective agent against cancer cells.
Within cells, copper serves a dual function: while it acts as an essential cofactor for numerous proteins, excess copper can also induce stress and promote cell death.The cell permeability of simple copper salts is poor. 802][83] As a known ionophore, pyrithione also aids metal ion transport across cell membranes. 84In this study, direct ICP-MS evidence and indirect bioinformatics evidence indicated an elevated intracellular copper ion concentration with ZnPT treatment.
Cuproptosis primarily occurs within mitochondria, involving processes such as DLAT protein oligomerization and iron-sulfur protein downregulation, which ultimately trigger acute proteotoxic stress, as evidenced by elevated HSP70 protein levels. 7Rudolf and Cervinka (2010) observed the accumulation of zinc in mitochondria and disturbance of mitochondrial membrane potential in cervical tumor Hep-2 cells upon ZnPT treatment. 39 Reeder et al. (2011) demonstrated the inactivation of iron-sulfur proteins in yeast following ZnPT exposure, 18 while Justiniano et al. (2017) confirmed the upregulation of proteotoxic stress markers, including HSP70, in SCC-25 cells upon ZnPT treatment. 25hese findings collectively support the potential of ZnPT at inducing cuproptosis in cancer cells.Consistent with these results, our data also revealed dysregulation of mitochondrial and iron-sulfur proteins, such as NAD(P)H, in TNBC cells.The confocal and native-PAGE analyses of DLAT further validated the oligomerization of DLAT, a hallmark feature of cuproptosis.In conclusion, based on the screening of The mechanism of cuproptosis is distinct from that of apoptosis, which is dependent on caspase3 activation.Notably, our results demonstrated that treatment with ZnPT for 24 h did not lead to the upregulation of cleaved caspase3.Similarly, Forcina et al. (2017) demonstrated that ZnPT-induced cell death is not modulated by BAX and BAK inhibition or treatment with the pan-caspase inhibitor Q-VD-OPh. 79However, other studies have shown that ZnPT can induce apoptosis. 39This discrepancy may be due to the high ZnPT treatment concentration used in the experiments or the concurrent use of zinc and copper ions.Furthermore, as a non-specific copper ionophore, ZnPT may also promote the accumulation of intracellular iron levels.Indeed, our KEGG enrichment analysis of transcriptomic data showed that ferroptosis was significantly upregulated.These findings imply that ZnPT may modulate cell death depending on the presence of different metal ions.Considering the capacity of ZnPT to induce cell death via metal ions and the distinct characteristics of metal ion transport and binding observed in breast cancer, especially in TNBC, targeted therapies and diagnostics involving cuproptosis may be a unique and promising approach for effective TNBC treatment.
In conclusion, our study demonstrated that ZnPT dysregulates copper homeostasis and inhibits cell viability and proliferation in TNBC cells.Remarkably, ZnPT promotes the aggregation of DLAT, a biomarker of cuproptosis, both in vitro and in vivo.Furthermore, ZnPT     Amoebiasis Amoebiasis Amoebiasis Amoebiasis Amoebiasis Amoebiasis Amoebiasis Amoebiasis Amoebiasis Amoebiasis Amoebiasis Amoebiasis Amoebiasis Amoebiasis Amoebiasis Amoebiasis Amoebiasis Small cell lung cancer Small cell lung cancer Small cell lung cancer Small cell lung cancer Small cell lung cancer Small cell lung cancer Small cell lung cancer Small cell lung cancer Small cell lung cancer Small cell lung cancer Small cell lung cancer Small cell lung cancer Small cell lung cancer Small cell lung cancer Small cell lung cancer Small cell lung cancer Small cell lung cancer TNC TNC TNC TNC TNC TNC TNC TNC TNC TNC TNC TNC TNC TNC TNC TNC TNC         suppresses the migration, invasion, and stemness in TNBC cells and inhibits TNBC development by downregulating the EGFR-PI3K-AKT signaling pathway and upregulating the MAPK signaling pathway.Collectively, this study revealed that ZnPT induces cell death by targeting copper homeostasis, providing an experimental basis for exploring cuproptosis as a potential target in drug discovery for TNBC patients.

Limitations of the study
This study has several limitations.First, although we identified that ZnPT targeted cuproptosis to induce DLAT aggregation, we did not demonstrate the condition under which oligomerization occurred or the presence of lipoylated DLAT.Second, our study lacks relevant evidence concerning the interactions between copper ions and DLAT that initiate the lipoylation of DLAT.Third, our research only used the TCGA data and in vitro methods to speculate that TNBC is sensitive to cuproptosis, lacking experimental data in mice and patients.Additionally, our research lacks direct evidence how PI3K-AKT pathway and mitochondrial autophagy contributed to the cuproptosis.Despite these limitations, our research extends our understanding for future clinical trials on cuproptosis as a targeted approach for TNBC.

Cell proliferation assay
Cells were seeded in 96-well plates at a concentration of 2 000 cells/well with complete medium overnight, then treated with serial dilutions of compounds for five days.Cell proliferation was then measured using the MTS assay every day with CellTiter 96 AQueous One Solution Reagent (Promega) at 490 nm.

Immunofluorescence
Cells were seeded on coverslips in six-well plates (50 000 cells/well) with complete medium overnight, then treated with serial dilutions of compounds for two days.Cells were then fixed in 4% paraformaldehyde (PFA) and blocked with 0.1% TritonX-100 in 5% goat serum for 1 h, followed by incubation with primary antibodies against DLAT (12362S, CST, 1:100) overnight at 4 C.After washing three times with PBS, fluorescein-labeled (KPL, 1:200) and Alexa Fluor 555-labeled secondary antibodies (Life Technologies, 1:2 000) were used to treat cells for 1 h.Finally, after washing three times with PBS, the coverslips were mounted with 4',6-diamidino-2-phenylindole (DAPI) (H-1200, Vector Laboratories) and observed via laser-scanning confocal microscopy (Nikon).The fluorescence counting function in ImageJ was then used to count the number of DLAT foci.

Analysis of foci number
Using ImageJ, the images (Select File/Open Samples/image.file) were converted to grayscale (Image/Type/8-bit) and the threshold was set using the automated routine (Process/Binary/Make Binary).After analyzing the Particles (Analyze/Analyze Particles), a particle count summary was shown in another data window.

Immunohistochemistry
Tumor tissues were fixed and embedded with paraffin for one week.Paraffin sections were deparaffinized and rehydrated in a series of degraded alcohols.Antigen retrieval was performed using 10 mM citrate buffer in a microwave for 20 min.Sections were incubated in 3% H 2 O 2 for 15 min to inactivate endogenous peroxidase.The samples were blocked with 10% goat serum for 2 h and incubated with primary antibodies against Ki67 (ab15580, Abcam, 1:100) overnight at 4 C.After washing three times with PBS, the samples were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Sigma, 1:200) at room temperature for 1 h.Finally, after again washing three times with PBS, the slides were stained with 3,3-diaminobenzidine (DAB) and countered with hematoxylin.

Flow cytometry
Cells were seeded in 10-cm plates (1 3 10 6 cells) and cultured for 24 h, then treated with serial dilutions of compounds for two days.To examine the ratio of BCSCs, ALDH enzymatic activity was assessed using an ALDEFLUOR kit (1700, Stem Cell Technologies) according to the provided manual.Cell apoptosis was assayed by flow cytometry with a FITC Annexin V apoptosis detection kit I (556547; BD Biosciences) according to the manufacturer's instructions.

Clonal formation assay
Cells were seeded in six-well plates (500 cells/well) and incubated with serial dilutions of compounds at 37 C for 2 weeks.The cells were sequentially fixed with 4% PFA for 10 min and the clones were stained with 0.2% crystal violet.

Figure 2 .
Figure 2. ZnPT promotes oligomerization of DLAT in vitro and in vivo (A and B) Representative images of immunofluorescence in ZnPT-treated TNBC cells, scale bar 5mm.(C and D) Statistical analysis of DLAT foci number in ZnPT-treated TNBC cells.(E and F) Western blotting of ACO2 and SDHB proteins in ZnPT-treated TNBC cells.(G and I) Representative images of apoptosis cell ratios using flow cytometry.(H and J) Statistical analysis of early and late apoptosis cell ratios in ZnPT-treated TNBC cells.(K) Western blotting of cleaved caspase3 proteins in ZnPT-treated TNBC cells.(L) Image of tumor size at growth endpoint, with each experimental group containing three mice (two tumor blocks in left and right mammary glands of each mouse).(M) Statistical analysis of tumor weight at growth endpoint, with each experimental group containing three mice (two tumor blocks in left and right mammary glands of each mouse).(N) Tumor growth curve of nude mice xenografted with HCC1806 cells with or without ZnPT treatment, with each experimental group containing three mice.(O) Representative immunofluorescence images of DLAT in ZnPT-treated nude mice, scale bar 2mm.(P) Statistical analysis of DLAT foci numbers in ZnPT-treated nude mice.Data were presented as means G SD. Two-tailed t test.

Figure 3 .
Figure 3. Analysis of cuproptosis-associated genes in breast cancer patients from TCGA database (A) Transcript expression levels of DLAT and FDX1 in normal breast tissue and different breast cancer subtypes from TCGA.(B) Kaplan-Meier survival analysis showing correlation between OS and DLAT and FDX1 mRNA expression.(C) Kaplan-Meier survival analysis showing correlation between OS and DLAT and FDX1 protein expression.(D and E) GO enrichment analysis of upregulated genes in breast cancer patients with high DLAT and FDX1 expression.(F) GO enrichment analysis of differentially expressed genes in breast cancer tissue compared with normal tissues.(G and H) Analysis of TCGA dataset for expression correlation between DLAT and FDX1 and ALDH1A3, CD44, and STAT3.(I and K) Representative images of ALDH+ cell ratios using flow cytometry.(J and L) Statistical analysis of ALDH+ cell ratios in ZnPT-treated TNBC cells.(M) Western blot analysis of CD44, p-STAT3, and STAT3 proteins in ZnPT-treated TNBC cells.Data were presented as means G SD. Two-tailed t test.