Elbasvir Triggers Ferroptosis in Esophageal Squamous Cell Carcinoma Through NCOA4-Mediated Ferritinophagy

Elbasvir Triggers Ferroptosis in Esophageal Squamous Cell Carcinoma Through NCOA4-Mediated Ferritinophagy | 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 Elbasvir Triggers Ferroptosis in Esophageal Squamous Cell Carcinoma Through NCOA4-Mediated Ferritinophagy Maoju Tang, Feng Gong, Miyuan Yang, Shuang He, Jiao Cheng, Zhiheng Yang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7262383/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Feb, 2026 Read the published version in Medical Oncology → Version 1 posted 9 You are reading this latest preprint version Abstract Objective: Esophageal squamous cell carcinoma (ESCC) remains treatment-resistant; we explored Elbasvir, an NS5A inhibitor, as a ferroptosis inducer. Methods: Cell viability was assessed by CCK-8 assays. Apoptosis and cell cycle were analyzed via flow cytometry, and key markers via Western blotting. In vivo efficacy was evaluated using BALB/c nude mouse xenografts. Proteomic analysis was conducted by mass spectrometry. Ferroptosis induction was verified via TEM, JC-1, FerroOrange, DCFH-DA, MDA assays, and Western blotting of NCOA4, Ferritin, and FTH1. Binding to NCOA4 was confirmed by surface plasmon resonance (SPR) and drug affinity responsive target stability (DARTS) assays. Results: Elbasvir (40 μM, 48 h) suppressed KYSE150/TE1 viability, induced apoptosis/G0/G1 arrest, and inhibited xenograft growth without toxicity. Proteomics identified ferroptosis as the top pathway. SPR/DARTS confirmed NCOA4 binding. NCOA4 knockdown reduced ferroptosis; overexpression enhanced it. Elbasvir triggered NCOA4-mediated ferritinophagy, FTH1 degradation, iron accumulation, and lipid peroxidation. Discussion: Elbasvir targets NCOA4-FTH1 to induce ferroptosis, offering a repurposing strategy for ESCC. Its safety profile supports clinical translation, with potential applications in iron metabolism-dependent cancers. Elbasvir Esophageal squamous cell carcinoma Ferroptosis NCOA4 FTH1 Targeted therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Esophageal carcinoma (EC) presents as a substantial worldwide disease burden, representing the seventh leading cause of cancer-associated death globally [ 1 ] . EC distribution shows marked geographical disparity, with China alone contributing approximately 50% of worldwide cases [ 2 , 3 ] . Recent data from GLOBOCAN 2022 reveal staggering epidemiological figures, with over 510,000 incident cases and nearly 450,000 deaths annually, underscoring the urgent need for improved therapeutic strategies [ 4 , 5 ] . Histopathologically, EC consists of two main variants - squamous cell carcinoma (ESCC) and adenocarcinoma (EAC) - characterized by distinct geographic prevalence and etiological divergence [ 6 , 7 ] . The Asian predominance of ESCC contrasts with Western EAC prevalence, with tobacco and alcohol use accounting for significant attributable risk in these populations [ 8 – 10 ] . Despite significant advances in molecularly targeted therapies that have revolutionized oncology treatment paradigms, the clinical management of ESCC continues to face substantial challenges. While tislelizumab has emerged as the first dual EU/US-approved immunotherapy for ESCC [ 11 – 13 ] , the broader therapeutic arsenal remains limited, with acquired resistance posing a persistent clinical hurdle [ 14 , 15 ] . These limitations have catalyzed growing interest in alternative drug development strategies, particularly drug repurposing (DRP), which offers distinct advantages over de novo drug development including reduced costs, abbreviated timelines, and established safety profiles [ 16 – 18 ] . The COVID-19 pandemic powerfully demonstrated the utility of this approach, where rapid therapeutic screening identified multiple repurposed candidates [ 19 – 22 ] . Notable successes in oncology DRP include disulfiram [ 23 ] , originally an alcohol-dependence treatment now under investigation as an adjuvant for malignant gliomas [ 24 , 25 ] , and chloroquine, an antimalarial repurposed for cancer therapy through autophagy modulation - including our group's previous demonstration of its anti-EC activity via this pathway [ 26 , 27 ] . Building on this paradigm, we identified Elbasvir, an FDA-approved hepatitis C virus (HCV) NS5A inhibitor, as a promising novel candidate with potent anti-ESCC activity. Our results demonstrate that Elbasvir exerts its antitumor effects predominantly through the activation of ferroptosis. These findings not only highlight the expanded therapeutic potential of this clinically approved agent but also offer new insights into ferroptosis induction as a viable strategy for the treatment of ESCC. 2. Materials and Methods 2.1. Materials and reagents Elbasvir (TargetMol, China), CCK-8 kit (Beyotime Biotechnology, China), Apoptosis kit (BD, USA), cell cycle detection kit (KeyGEN, China), NCOA4 antibody (Abcam, UK), Ferritin antibody (HUABIO, China), FTH1 antibody (Abcam, UK), Streptavidin protease pronase (Roche, Switzerland), Mitochondrial Membrane Potential Assay Kit (Biosharp, China), Fe 2+ Assay Kit (Elabscience, China), Reactive Oxygen Demand Assay Kit (Biosharp, China), Lipid Peroxidation Kit (Biosharp, China), siRNA sequence of NCAO4 gene (Ribobio, China), Overexpression of NCAO4 Plasmid (Sino Biological, China) 2.2. CCK-8 cell viability assay Log-phase KYSE150 and TE1 cells were trypsinized, pelleted, and resuspended to 3000 cells/well, then seeded in 96-well plates (200 µL/well) with triplicate wells per group. To prevent water evaporation, 200 µL of PBS was added in a circle around the edge of the 96-well plate. The cells were incubated in a 37℃, 5% CO 2 incubator until the next day for drug treatment. The experimental group was treated with 40 µM Elbasvir and the control group was treated with an equal volume of DMSO. Cell viability was assessed at 0, 24, 48, and 72 h post-treatment using CCK-8 assay. Following medium removal, 100 µL of freshly prepared CCK-8 working solution (1:9 reagent:serum-free medium) was added per well. After 2 h incubation, absorbance was measured at 450 nm using a microplate reader to determine viability. 2.3. Apoptosis assay Cells were plated in 6-well plates (1×10 5 /well) and cultured for 24 h before treatment with DMSO or 40 µM Elbasvir (48 h). Following trypsinization, cells were washed twice with ice-cold PBS and resuspended in binding buffer (1×10 6 cells/mL). Aliquots (100 µL containing 1×10 5 cells) were stained with 5 µL each of FITC-Annexin V and PI (15 min, RT, dark). After adding 400 µL binding buffer, samples were analyzed by flow cytometry within 1 h. 2.4. Cell cycle assay Following 24 h culture in 6-well plates (1×10 5 cells/well), cells were treated with DMSO or 40 µM Elbasvir for 48 h. After treatment, cells were trypsinized, fixed in 75% ethanol (-20℃, overnight), and washed with PBS. Cell cycle analysis was performed using PI/RNase staining (30 min, RT, dark) with subsequent flow cytometry detection (excitation 488 nm). 2.5. Western Blot The cell sediment was collected, lysed, and centrifuged to obtain the protein supernatant, and the protein concentration was determined by the BCA method. The protein concentration was determined by the BCA method. 40 µg of protein was denatured and added to the SDS-PAGE gel according to the experimental group. Electrophoresis was performed at 80 V for 30 min followed by 120 V for 1 h (adjusted based on target protein size). Proteins were then transferred to PVDF membranes using wet transfer at 250 mA constant current (duration optimized by protein molecular weight). Membranes were blocked with 5% skim milk (2 h, RT), incubated with primary antibody (4℃, overnight), washed 3×10 min with PBST, and probed with species-matched secondary antibody (1 h, RT) followed by additional PBST washes (3×10 min), and then developed with ECL chemiluminescent reagent, and the Sydney number was captured by an imaging system for the analysis of the results. 2.6. Construction and treatment of tumor-bearing nude mouse model Four-week-old male BALB/c nude mice (18–20 g, GemPharmatech, China) were housed under standard conditions (25°C, 55% humidity, 12-h light/dark cycle) at North Sichuan Medical College's Animal Center, with protocols approved by the Institutional Animal Ethics Committee (No.2023033). KYSE150 cells (1×10⁶ in 100 µL) were subcutaneously injected into each mouse. When tumors reached ~ 100 mm³, mice were randomized into two groups receiving either 70 mg/kg Elbasvir (i.p., every other day) or DMSO control. Tumor volume was monitored by caliper measurements (length×width²/2). After 17 days, mice were sacrificed for tissue collection (lungs, liver, kidneys, heart, tumors), which were fixed in 4% PFA for paraffin embedding and HE staining. 2.7. HE staining For histological analysis, the paraffin-embedded tissue slices underwent dewaxing in xylene and subsequent rehydration via a sequence of graduated ethanol solutions, each for 10 min. The sections were rinsed with PBS, subjected to hematoxylin staining for 3 min, and differentiated with 1% hydrochloric acid-alcohol solution for 10–20 s. Following another PBS rinse, the sections were stained with eosin for 2 min. Dehydration was performed through an ascending alcohol gradient, tissue sections were cleared in xylene. Lastly, the specimens were mounted with neutral gum, air-dried, and observed under microscopic examination. 2.8. Proteomic analysis Samples were processed with reaction buffer (1% SDC/100 mM Tris-HCl pH 8.5/10 mM TCEP/40 mM CAA) at 60℃ for 1 h to simultaneously achieve protein denaturation, reduction, and alkylation. After dilution with ultrapure water, tryptic digestion was performed (enzyme:protein = 1:50, 37℃, overnight). The reaction was quenched with TFA, followed by centrifugation (16,000 × g) and desalting using a homemade SDB column. For TMT labeling, aliquots were labeled, pooled, and desalted (Sep-Pak C18). The combined samples were fractionated by high-pH reverse-phase chromatography into 15 fractions, vacuum-dried, and stored at -80℃ prior to LC-MS/MS analysis using a Q Exactive Plus coupled to an EASY-nLC 1200 system. 2.9. Mitochondrial morphological observation Cells from 60 mm dishes were pelleted (1,000 rpm, 5 min) and fixed with 3% glutaraldehyde (4℃, 5 min). After transferring to 1.5 mL tubes and centrifugation (12,000 rpm, 10 min), pellets were post-fixed with 1% osmium tetroxide. Dehydration was performed using an acetone gradient (30%→100%, with three 100% changes), followed by stepwise Epon-812 infiltration (3:1→1:3 resin:acetone). Ultrathin sections (60–90 nm) were collected on copper grids, stained with uranyl acetate (10–15 min) and lead citrate (1–2 min), and imaged using a JEOL JEM-1400FLASH TEM. 2.10. Mitochondrial membrane potential analysis The JC-1 working solution was prepared by diluting JC-1 (200×) 1:160 in ultrapure water and mixing with JC-1 Staining Buffer (5×) at a 1:4 ratio. For positive controls, cells were treated with 10 µM CCCP (1:1000 dilution from 10 mM stock) for 20 min. After PBS washing, cells were incubated with 1 mL working solution per well (37°C, 20 min), followed by two washes with ice-cold JC-1 Staining Buffer (1×). Cells were resuspended in 2 mL culture medium and immediately analyzed by fluorescence or confocal microscopy. 2.11. Ferrous ion (Fe 2+ ) concentration detection All reagents were equilibrated to room temperature prior to use. Iron standards were prepared at specified concentrations for calibration. Treated cells (1×10 6 ) were lysed with 200 µL Reagent I on ice for 10 min, followed by centrifugation (15,000 × g, 10 min) to collect supernatant. For analysis, 80 µL aliquots of standards or test samples were loaded in duplicate onto a microplate, with control wells receiving additional 80 µL of Reagents II and III. After 10 min incubation at 37°C, absorbance was measured at 593 nm. 2.12. Reactive oxygen species (ROS) level detection The in situ loading probe was selected according to the instructions for adherent cells, DCFH-DA probe was diluted with serum-free culture medium at 1:1000 to a final concentration of 10 µM. Cell culture medium was removed, and 1 mL of diluted DCFH-DA was added to each well of a six-well plate. Incubation was performed at 37ºC for 20 min. Following triple washing with serum-free medium to remove extracellular probe, cells were analyzed by flow cytometry (excitation 488 nm/emission 525 nm). Positive controls received 20–30 min stimulation to induce ROS elevation prior to detection. Data was processed for statistical analysis. 2.13. Malonaldehyde (MDA) content detection Cell lysates (0.1 mL per 1×10 6 cells) were centrifuged (12,000 × g, 10 min, 4°C) and supernatants collected for protein quantification (BCA assay). A 0.37% TBA solution was prepared by dissolving 25 mg TBA in 6.76 mL preparation buffer. The MDA working solution (150 µL TBA dilution buffer + 50 µL TBA storage solution + 3 µL antioxidant per sample) and standard curve (1–50 µM) were prepared fresh. Samples (0.1 mL lysate/standards) were mixed with 0.2 mL working solution, heated (100°C, 15 min), cooled, and centrifuged (1,000 × g, 10 min). Supernatant absorbance (200 µL/well) was measured at 532 nm for MDA quantification normalized to protein content. 2.14. RNA interference The sequence of small interfering RNA (siRNA) for NCOA4 gene silencing was designed and provided for synthesis by Reebok Biotech. The sequence is as follows: GACCUUUUUUAUCAGCUUA. 1 × 10 5 KYSE150 and TE1 cells were grown in 6-well plates one night in advance and transfected the next day. The lyophilized powder was transiently dissociated and solubilized by adding DEPC water so that the siRNA concentration was 20 µM and then dispensed. Refer to Lipofectamine 2000 instructions for transfection. To configure the transfection system, remove 3 sterile EP tubes and label the reagent and NC/siRNA tubes. Add 700 µL of serum-free medium to the reagent tube, add 35 µL of Lipofectamine 2000 and mix gently. Add 350 µL of serum-free medium and 17.5 µL of diluted NC/siRNA to the NC/siRNA tubes, mix gently, and allow to stand for 5 min. 350 µL of each of the reagents was then added to the NC/siRNA tubes, mixed gently, and allowed to stand for 15 min. Replace the cell culture medium of the 6-well plate with serum-free medium, and add 200 µL of the allowed cell culture medium to each of the corresponding 6-well plates. Cells in 6-well plates were washed and maintained in serum-free medium prior to treatment. A 200 µL aliquot of the stabilized complex was added dropwise to each well, followed by 6 h incubation at 37℃. The treatment medium was then replaced with complete growth medium for continued culture. 2.15. Plasmid transfection For transformation, 10 µL plasmid was mixed with 50 µL DH5α competent cells, incubated on ice (30 min), heat-shocked (42℃, 90 s), and immediately returned to ice (2 min). After adding 400 µL antibiotic-free LB medium, cells were recovered (37℃, 40 min) before expansion. For plasmid amplification, 100 µL bacterial culture was inoculated into 7 mL LB medium supplemented with kanamycin (1:1000) and incubated overnight (37℃, 220 rpm). Plasmid extraction was performed at visible turbidity, with concentration quantified via Nanodrop 2000. For transfection, cells were seeded in 6-well plates (1×10 5 cells/well) 24 h prior. Two hours before transfection, medium was replaced with fresh complete medium. Plasmid DNA (2 µg) was diluted in 100 µL serum-free medium and combined with 2 µL transfection reagent. After 15 min incubation (RT), the mixture was added dropwise to cells, followed by gentle swirling and incubation (24–48 h). 2.16. Quantitative real-time PCR (qPCR) Total RNA was isolated using Vazyme spin columns, with 1 µg reverse-transcribed to cDNA. qPCR reactions (10 µL total volume) contained 5 µL Taq Pro Universal SYBR Master Mix (2×), 0.4 µL each of forward/reverse primers, 3.2 µL nuclease-free water, and 1 µL cDNA template. Amplification was performed on a LightCycler 480 system (Roche) under the following conditions: 95°C for 30 sec (initial denaturation), followed by 40 cycles of 95°C for 5 sec, 60°C for 30 sec, and 72°C for 20 sec. Data were analyzed using the instrument's proprietary software. The primer sequence of the gene is as follows: NCOA4, the forward primer is 5'-GCCCTACAATGTGAGTGATTGG-3' and the reverse primer is 5'-ACTGGTGCAAGGCTCGTTG-3'; β-actin, the forward primer was 5 '-GCAAGCAGGAGTATGACGAG-3'; The reverse primer is 5 '-CAAATAAAGCCATGCC AATC-3'. 2.17. Prokaryotic expression and purification The NCOA4 coding region was chemically synthesized and ligated into pET28a using BamHI and XhoI restriction enzymes. The empty plasmid cut by the same enzyme and then transformed into receptive cells to screen positive clones and verify by sequencing. The verified recombinant plasmid was transformed into expression strain, and after the transformation by heat shock method, it was coated on the LB plate containing antibiotics. Single colonies were selected and induced by 1.0 mM IPTG (37℃, 4 h) and 0.2 mM IPTG (16℃, 16 h), respectively. Following ultrasonic lysis, protein expression was verified via SDS-PAGE. For large-scale purification, cells were resuspended in PBS, lysed by sonication, and clarified by centrifugation. The supernatant was subjected to Ni-Smart affinity chromatography, with sequential washes (20 mM imidazole) and elution (250 mM imidazole). The purified protein was dialyzed into PBS buffer containing 10% glycerol at 4℃, then concentrated by ultrafiltration tube and determined by SDS-PAGE and Bradford method. 2.18. Surface plasmon resonance (SPR) measurement Place 200 mL of 1×PBS Buffer running buffer, water bottle, and waste bottle in the instrument tray and insert the corresponding inlet tube. Holding the CM5 chip (with the lettered side facing up) in your hand, gently push it into the slot in the direction of the arrow and close the chip compartment door. Channel 2 of the biosensor chip was activated with EDC/NHS (10 µL/min), followed by immobilization of NCOA4 (383-522aa) at 50 µg/mL in sodium acetate buffer (pH 4.0, 10 µL/min, 9 µL coupling volume). Ethanolamine (10 µL/min) was used to block residual active groups. Real-time coupling kinetics were monitored throughout the process. Replace the running buffer with 1 × PBS-P + containing 5% DMSO and insert it into the appropriate inlet tube. The 5% DMSO concentration calibration curve was configured by mixing the 4.5% and 5.8% DMSO master mixes proportionally. The test material was diluted to different concentrations in a 96-well plate and coupled to the target proteins sequentially through the microarray from low to high at a flow rate of 30 µL/min for 150 s. After each concentration point was detected, the microarray was regenerated with a 10 mM glycine hydrochloride (pH 2.0) solution for 5 min, and the operation was repeated until all the concentrations had been detected. Binding kinetics were analyzed using BIAcore T200 systems (GE Healthcare). Sensorgrams were processed with Control Software (v2.0), subtracting reference channel signals. Association and dissociation constants were derived by global fitting to a 1:1 Langmuir model (Evaluation Software v2.0). 2.19. Drug Affinity Responsive Target Stability (DARTS) assay Cells at ~ 80% confluence were harvested from 10 cm dishes, washed twice with PBS, and lysed in ice-cold RIPA buffer (30 min on ice with vortex mixing every 10 min). Lysates were centrifuged (12,000 × g, 10 min, 4°C), and supernatants were aliquoted into input, DMSO control, and Elbasvir treatment groups (400 µM final concentration, 2 h incubation at 25°C). Streptavidin was added at three concentrations (1:1000, 1:5000, 1:10,000) for 30 min (25°C) before reaction termination with PMSF (1:100). Processed samples were subjected to SDS-PAGE and Western blotting for target protein analysis. 2.20. Statistical analysis The experimental data were replicated three times independently, and the data were analyzed using GraphPad Prism 9.5 software and the statistical significance was analyzed by t-test or ANOVA. Differences were considered statistically significant when the P value was < 0.05. 3. Results 3.1. Elbasvir exerts potent antitumor effects in ESCC through growth inhibition and apoptosis induction Elbasvir demonstrates significant antitumor activity in ESCC by suppressing proliferation and inducing apoptosis. To evaluate its therapeutic potential, we first assessed its impact on cell viability using CCK-8 assays in KYSE150 and TE1 cell lines. Elbasvir treatment led to a marked, suppression of ESCC cell proliferation compared to vehicle controls (Fig. 1A). Mechanistic investigations further demonstrated that Elbasvir markedly induced apoptotic cell death, which was quantitatively confirmed by flow cytometric analysis using Annexin V-FITC/PI dual staining (Fig. 1B). Both early and late apoptotic populations were substantially elevated following Elbasvir exposure (Fig. 1C), confirming its pro-apoptotic activity in ESCC cells. Cell cycle profiling demonstrated that Elbasvir treatment induced G0/G1 phase arrest, with a concomitant reduction in G2/M phase populations (Fig. 1D, E). Western blot analysis corroborated these findings, showing upregulation of key apoptotic markers (Fig. 1F) and downregulation of G0/G1 phase regulatory proteins (Fig. 1G). Collectively, these results demonstrate that Elbasvir exerts multifaceted antitumor effects in ESCC by simultaneously inhibiting proliferation, promoting apoptosis while causing a cell cycle blockade at the G0/G1 checkpoint. 3.2. Elbasvir suppresses tumor growth in ESCC xenograft models To evaluate the therapeutic potential of Elbasvir in vivo , we established KYSE150-derived xenograft models. Elbasvir treatment resulted in significant tumor growth suppression compared to vehicle controls, as demonstrated by a significant decrease in both tumor volume and mass (Fig. 2A-C). Importantly, comprehensive histopathological assessment of major organs demonstrated no detectable treatment-related toxicity (Fig. 2D, Supplementary Figure 1), indicating excellent tolerability of Elbasvir at the therapeutic dose. These findings collectively demonstrate that Elbasvir exerts potent antitumor activity against ESCC in vivo while maintaining an advantageous safety profile. 3.3. Elbasvir triggers ferroptosis in ESCC cells Elbasvir exhibits potent anti-tumor effects against ESCC both in vivo and in vitro ; however, its underlying molecular mechanisms remain elusive. To elucidate these mechanisms, we performed proteomic profiling to identify proteins and pathways dysregulated by Elbasvir in ESCC cells. Proteomic analysis identified 642 differentially expressed proteins (427 up-regulated and 215 down-regulated) in Elbasvir-treated cells (Fig. 3A) (Supplementary Table 1). Pathway analysis demonstrated significant enrichment of these proteins in ferroptosis-related processes (Fig. 3B). To elucidate the mechanism underlying Elbasvir's antitumor activity, we first examined mitochondrial ultrastructure by transmission electron microscopy. Elbasvir-treated cells exhibited hallmark morphological features of ferroptosis, including outer membrane rupture, mitochondrial shrinkage, and cristae disintegration (Fig. 4A). Complementary analysis of mitochondrial function revealed significant membrane potential dissipation following Elbasvir treatment (Fig. 4B), confirming functional impairment of mitochondria. These findings collectively demonstrate that Elbasvir triggers characteristic ferroptotic changes at both structural and functional levels. Quantitative analysis of iron metabolism revealed Elbasvir-mediated accumulation of ferrous iron (Fe 2+ ) (Fig. 5A), accompanied by elevated reactive oxygen species and malondialdehyde levels (Fig. 5B-D), consistent with ferroptotic lipid peroxidation. Immunoblot analysis demonstrated significant upregulation of key ferroptosis regulators (NCOA4, Ferritin, and FTH1) in both cellular and xenograft models following Elbasvir treatment (Fig. 5E, Supplementary Figure 2). These findings establish that Elbasvir induces ferroptosis through coordinated iron accumulation, oxidative stress generation, and modulation of iron homeostasis proteins, revealing a novel mechanistic basis for its anti-ESCC activity. 3.4. Elbasvir induces ferroptosis in ESCC through NCOA4-mediated ferritinophagy While our phenotypic and proteomic analyses identified Elbasvir as a potent inducer of ferroptosis in ESCC, its precise molecular target remained unclear. Proteomic profiling of Elbasvir-treated cells revealed significant upregulation of iron metabolism regulators, including NCOA4, SQSTM1, SLC39A1, FTH1, and TNFRSF21 (Supplementary Table 2), with NCOA4 and FTH1 emerging as key mediators of ferroptosis. NCOA4, a selective autophagy receptor, binds Ferritin heavy chain 1 (FTH1) to mediate ferritinophagy, promoting lysosomal degradation of iron-laden Ferritin and subsequent iron release. This process critically regulates cellular iron homeostasis and iron-dependent cell death pathways, including ferroptosis. Consistent with this mechanism, we observed upregulation of NCOA4, Ferritin, and FTH1 at the protein level (Fig. 5E). Bioinformatic and functional analyses implicated NCOA4 as a central player in Elbasvir-induced ferroptosis. Genetic perturbation studies confirmed its pivotal role: NCOA4 knockdown in two ESCC cell lines (Fig. 6A) increased FTH1 protein levels, while NCOA4 overexpression reduced FTH1 (Fig. 6B). NCOA4 depletion enhanced cell viability, whereas its overexpression exacerbated Elbasvir-induced cytotoxicity (Fig. 6C). Intracellular ferrous iron (Fe 2+ ) levels decreased upon NCOA4 knockdown but increased with NCOA4 overexpression (Fig. 6D). Correspondingly, lipid peroxidation (measured by MDA content) was attenuated by NCOA4 silencing but amplified by NCOA4 overexpression (Fig. 6E). These findings demonstrate that NCOA4 drives Elbasvir-triggered ferroptosis by regulating the NCOA4-FTH1 axis, controlling iron release, and promoting oxidative stress. 3.5. Elbasvir directly engages NCOA4 to trigger ferroptosis While NCOA4 is established as a critical regulator of ESCC cell growth, its molecular interaction with Elbasvir remained undefined. For interaction characterization, we successfully cloned, expressed, and purified the functional domain of NCOA4 383-522 (Supplementary Figure 3A-C), followed by binding affinity measurement using surface plasmon resonance (SPR). Elbasvir demonstrated superior binding affinity compared to the known ligand NCOA4-9a (Fig. 7A, B, Supplementary Table 3), with distinct kinetic properties indicating a specific, high-efficiency interaction. Drug affinity responsive target stability (DARTS) assays independently validated this direct binding, showing Elbasvir-mediated stabilization of NCOA4 (Fig. 7C). These biophysical studies establish NCOA4 as a direct molecular target of Elbasvir, revealing a structural mechanism for its ferroptosis-inducing activity in ESCC. 3.6. Elbasvir suppresses ESCC progression by targeting the NCOA4-FTH1 axis to induce ferroptosis Although Elbasvir has been identified as a conjugate of NCOA4, its precise regulatory mechanisms in ESCC ferroptosis have yet to be fully elucidated. To investigate the mechanistic basis, we assessed Elbasvir's involvement in NCOA4 genetic modulation. Notably, Elbasvir co-treatment diminished the pro-survival effect of NCOA4 knockdown, concurrent with marked alterations in ferroptosis-associated FTH1 expression, underscoring NCOA4's pivotal role in Elbasvir-induced ferroptosis (Fig. 8A). Conversely, NCOA4 overexpression significantly suppressed cellular viability while reducing FTH1 expression. This inhibition was further enhanced when co-treated with Elbasvir, and markers associated with iron poisoning showed a corresponding shift (Fig. 8B). Taken together, these findings suggest that Elbasvir triggers iron apoptosis in ESCC by promoting NCOA4-dependent FTH1 degradation, which ultimately leads to tumor cell death. This study reveals the mechanistic basis of Elbasvir's anti-ESCC activity and highlights the therapeutic potential of targeting the NCOA4-FTH1 axis in anti-cancer strategies. 4. Discussion EC remains a formidable global health challenge, ranking among the top causes of cancer-related mortality worldwide [ 28 , 29 ] . Epidemiological studies consistently identify the EC as a significant contributor to premature cancer deaths and reduced life expectancy [ 30 ] . Diagnostic advances, particularly in endoscopic screening, have markedly improved early-stage detection rates [ 31 – 33 ] , while radiotherapy continues to provide clinical benefit for advanced cases [ 34 – 36 ] . The therapeutic paradigm has been transformed by immune checkpoint inhibitors [ 37 – 39 ] , with small molecule inhibitors emerging as particularly promising agents [ 40 – 42 ] . The strategy of drug repurposing - applying approved therapeutics to novel indications - has gained considerable traction in oncology [ 43 , 44 ] . Our investigations identified Elbasvir, an established NS5A inhibitor developed by Merck & Co. for hepatitis C virus (HCV) treatment, as possessing previously unrecognized anti-tumor activity. This highly specific antiviral agent demonstrates picomolar potency against HCV NS5A, effectively suppressing viral replication through multiple mechanisms [ 45 – 49 ] While its clinical efficacy against HCV genotypes 1–4 is well-documented [ 50 ] . Its potential oncological applications remain unexplored [ 51 ] . Elbasvir suppresses ESCC proliferation through dual induction of apoptosis and cell cycle arrest, corroborating known NS5A inhibitor activity, with concordant in vivo validation. The discovery of the effective antitumor activity of Elbasvir against ESCC represents a significant advance in identifying retargeting therapeutic candidates with multimodal mechanisms of action. Beyond conventional cytotoxicity, our findings reveal Elbasvir's novel engagement with ferroptosis - an iron-mediated cell death pathway - wherein it modulates the NCOA4-FTH1 axis to promote ferritinophagy, thereby increasing labile iron pools that drive Fenton chemistry. The Fenton reaction is a biochemical cascade that produces reactive oxygen species and drives deadly lipid peroxidation, ultimately triggering cell ferroptosis [ 52 ] . Our results showed that after Elbasvir treatment, intracellular iron autophagy was enhanced, released Fe 2+ accumulated in the body, downstream ROS levels increased significantly, driving lipid peroxidation, MDA content increased, and cell membrane rupture and death. Ferroptosis is characterized by distinct mitochondrial alterations, including functional impairment, morphological changes, membrane condensation, and cristae reduction or loss. These ultrastructural manifestations of mitochondrial damage represent hallmark features of iron-dependent cell death [ 53 ] . This ferroptotic mechanism represents a distinct cell death pathway in ESCC treatment. Notably, ferroptosis has shown particular therapeutic potential for malignancies exhibiting elevated oxidative stress, including ESCC [ 54 , 55 ] . Targeting ferroptosis has become a new breakthrough in anti-tumor, in which the NCOA4-FTH1 axis mediates iron autophagy and regulates iron metabolism in ferroptosis. Our results show that NCOA4 and FTH1 are significantly activated in Elbasvir-induced ferroptosis responses, and proteomic identification of the NCOA4-FTH1 axis regulation provides key mechanologic insights into Elbasvir activity. NCOA4 serves as a specific ferritinophagy receptor that critically regulates iron homeostasis [ 56 – 58 ] . We demonstrated that Elbasvir can target NCOA4 protein binding while stabilizing NCOA4 protein expression, and that direct binding and subsequent up-regulation of Elbasvir to NCOA4 provides a novel pharmacological strategy to disrupt the balance of the NCOA4-FTH1 axis. Knockdown of NCOA4 inhibits ferroptosis and promotes cell survival by decreasing the content of ferrous and MDA, and the binding FTH1 increases correspondingly. Conversely, Fe²⁺ accumulation and elevated MDA levels potentiated ferroptosis, markedly reducing cell viability while suppressing FTH1 expression. In combination with Elbasvir, the cell growth promoted by NCOA4 knockdown was partially reversed by Elbasvir, and Elbasvir further intensified the inhibitory effect of overexpression of NCOA4 on cell viability. These findings have important therapeutic implications. First, they positioned ferroptosis induction as a viable strategy to combat ESCC, a type of malignancy that is often resistant to apoptosis-based therapies [ 59 ] . Second, the NCOA4-FTH1 axis emerges as a druggable target [ 60 ] , with Elbasvir serving as both a chemical probe and potential therapeutic. Notably, the iron metabolism pathway may represent a metabolic vulnerability in ESCC [ 61 , 62 ] , as many tumors exhibit increased iron requirements to support rapid proliferation [ 63 , 64 ] . Several critical questions emerge from these findings. The selectivity of Elbasvir's NCOA4 binding relative to other ferritinophagy regulators warrants further investigation, as does the potential for synergy with existing ESCC therapies [ 65 ] . Additionally, the tumor microenvironment's role in modulating this ferroptotic response remains unexplored - particularly relevant given the inflammatory nature of ESCC and known interactions between iron metabolism and immune responses [ 66 ] . From a translational perspective, these results suggest that patient stratification based on iron metabolism markers (e.g., Ferritin levels, NCOA4 expression) could optimize Elbasvir's therapeutic application. Furthermore, combining Elbasvir with lipid peroxidation-enhancing agents or system xc- inhibitors may amplify its anti-tumor efficacy while potentially overcoming resistance mechanisms [ 67 ] . 5. Conclusion In this study, we identified Elbasvir as a potent anti-ESCC agent through molecular docking and validated its tumor-suppressive effects in vitro and in vivo. Mechanistically, Elbasvir binds directly to NCOA4, stabilizing it and promoting its interaction with FTH1 to activate ferritinophagy, thereby inducing ferroptosis via iron overload, ROS accumulation, and lipid peroxidation (Fig. 9). Our findings unveil the NCOA4-FTH1 axis as Elbasvir’s ferroptotic trigger, offering a novel therapeutic strategy for ESCC. Importantly, Elbasvir’s established safety profile underscores its clinical potential. Beyond ESCC, this work provides a mechanistic framework for targeting iron metabolism in upper GI malignancies and broader ferroptosis-vulnerable cancers. Future studies should prioritize clinical translation while probing the intricate crosstalk between viral protease inhibitors (e.g., Elbasvir) and iron homeostasis pathways to optimize therapeutic exploitation. Declarations Author contributions X.G, X.Z made significant contributions to the conception, design and guidance of the study. M.T, F.G, M.Y jointly completed the experiment implementation, data collection, data analysis, and paper writing. S.H, J.C, Z.Y participated in the experiment. L.X, Q.M, X.G and X.Z made a lot of revisions and revisions to the manuscript. X.G and X.Z finally approved the version to be published. All the authors reviewed the manuscript. Funding Support This study was supported by the Science and Technology Support Program (grant no. 25NSFSC2024), the Medical Research Project of Sichuan (grant no. S23020), the Scientific Research Development Plan Project, Affiliated Hospital of North Sichuan Medical College (grant no. 2024PTZK004 and 2023-2ZD002), and the Scientific Research Development Plan Project, North Sichuan Medical College (grant no. CBY24-QDA27 and CBY22-ZDA03). 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1","display":"","copyAsset":false,"role":"figure","size":1541757,"visible":true,"origin":"","legend":"\u003cp\u003eElbasvir induces growth arrest and apoptosis in esophageal squamous cell carcinoma (ESCC). (A) cell viability in KYSE150 and TE1 cells following Elbasvir treatment (CCK-8 assay). (B) Apoptosis induction assessed dual staining and flow cytometry. (C) Quantitative analysis of early and late apoptotic populations. (D) Cell cycle distribution profiles following Elbasvir exposure. (E) Cell cycle phase quantification demonstrating G0/G1 arrest. (F) Immunoblot analysis of apoptosis-related protein expression. (G) Western blot evaluation of cell cycle regulatory proteins.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7262383/v1/dbfb40064b6fcfed61cb4abf.png"},{"id":92880098,"identity":"d07bca54-f2f2-45d0-996e-30760ccba77f","added_by":"auto","created_at":"2025-10-06 15:26:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3770196,"visible":true,"origin":"","legend":"\u003cp\u003eElbasvir demonstrates potent antitumor activity in ESCC xenograft models. (A) Representative photographs of tumors harvested from control and Elbasvir-treated mice at the experimental endpoint. (B) Tumor growth kinetics following Elbasvir administration. (C) Final tumor weights measured post-excision. (D) Histopathological evaluation of major organs by HE staining. The microscopic scale was: 100 μm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7262383/v1/1713240fa96816ed1b7cf2a5.png"},{"id":92881111,"identity":"f320b877-36dd-406b-876c-77fbb7f21b0c","added_by":"auto","created_at":"2025-10-06 15:34:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":437490,"visible":true,"origin":"","legend":"\u003cp\u003eProteomic analysis of KYSE150 cells treated with Elbasvir. (A) Volcano plot showing differentially expressed proteins (DEPs) in Elbasvir-treated versus control cells (|log2FC| \u0026gt; 1.2, p \u0026lt; 0.05). (B) Top enriched pathways from KEGG analysis of DEPs (ranked by p-value).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7262383/v1/ae8436871039198ab91b996e.png"},{"id":92881114,"identity":"3652d04a-48f6-4704-9b7f-5d9222a8691d","added_by":"auto","created_at":"2025-10-06 15:34:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2999239,"visible":true,"origin":"","legend":"\u003cp\u003eElbasvir induces mitochondrial dysfunction in ESCC cells. (A) TEM micrographs displaying hallmark mitochondrial ultrastructural alterations in KYSE150 and TE1 cells following 48-h treatment with 40 μM Elbasvir (5,000×, 10,000× and 20,000× magnification at a time. The scales are respectively: 2 μm, 1 μm, 500 nm). (B) Confocal microscopy of JC-1 staining revealed Elbasvir-mediated mitochondrial membrane depolarization, evidenced by a shift from red fluorescence (JC-1 aggregates in polarized mitochondria) to green fluorescence (JC-1 monomers in depolarized mitochondria). The microscope scale is: 50 μm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7262383/v1/30060b8f50d2229a1f0e6f14.png"},{"id":92882857,"identity":"5b990a87-f0ce-4817-bf68-e187d3e41319","added_by":"auto","created_at":"2025-10-06 15:50:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":739402,"visible":true,"origin":"","legend":"\u003cp\u003eElbasvir induces ferroptotic signaling in ESCC models. (A) Intracellular Fe\u003csup\u003e2+\u003c/sup\u003eaccumulation following Elbasvir treatment. (B) Malondialdehyde (MDA) levels as a measure of lipid peroxidation. (C) ROS detection by flow cytometry. (D) Quantification of ROS fluorescence intensity. (E) Immunoblot analysis of ferroptosis-related proteins in cellular and xenograft tissues.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7262383/v1/73437a7e636423d0bfcef5aa.png"},{"id":92880102,"identity":"35524fe6-e083-44fd-b3e0-3d70f909fb27","added_by":"auto","created_at":"2025-10-06 15:26:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":500176,"visible":true,"origin":"","legend":"\u003cp\u003eNCOA4 regulates ferroptosis and cell viability in ESCC. (A) NCOA4 mRNA expression following genetic manipulation (qPCR). (B) NCOA4 and FTH1 protein levels by immunoblotting. (C) Cell viability assessment (CCK-8 assay). (D) Intracellular Fe\u003csup\u003e2+\u003c/sup\u003econcentration. (E) Lipid peroxidation (MDA content).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7262383/v1/1c7711593fb2ab9dc3618a66.png"},{"id":92881116,"identity":"e4043d39-4019-495c-af6e-1fc4ea4a96e1","added_by":"auto","created_at":"2025-10-06 15:34:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1304163,"visible":true,"origin":"","legend":"\u003cp\u003eElbasvir directly targets NCOA4 in ESCC. (A) Surface plasmon resonance (SPR) analysis of NCOA4-9a/NCOA4 binding kinetics. (B) SPR characterization of Elbasvir-NCOA4 interaction. (C) Target engagement validation by drug affinity responsive target stability (DARTS) assay.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7262383/v1/317c0ca5df8e2a3c13e2a219.png"},{"id":92882296,"identity":"3b0c2835-917b-46fb-9b7b-c6eba0083f24","added_by":"auto","created_at":"2025-10-06 15:42:18","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1276893,"visible":true,"origin":"","legend":"\u003cp\u003eElbasvir suppresses ESCC progression through the NCOA4-FTH1 axis. (A) Cell viability under combinatorial treatment with Elbasvir and NCOA4 modulation. (B) Immunoblot analysis of NCOA4-FTH1 pathway components.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7262383/v1/8e68a15f3bba5fd553c5e6e2.png"},{"id":103251963,"identity":"de4ef7cd-03d5-4303-be20-a739462ed35a","added_by":"auto","created_at":"2026-02-23 16:12:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13031585,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7262383/v1/1ae2428a-1817-4c40-89ba-9fa22d2d233b.pdf"},{"id":92880108,"identity":"429c3356-4eab-4825-b7a9-045151cbd13d","added_by":"auto","created_at":"2025-10-06 15:26:18","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":128068,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7262383/v1/a4cf02dd873564c79932cc8a.xlsx"},{"id":92880095,"identity":"797f2a9f-e31f-4160-b188-eaca8e75dae5","added_by":"auto","created_at":"2025-10-06 15:26:18","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15623,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7262383/v1/35389271d2d0819a582c01fd.xlsx"},{"id":92881119,"identity":"7dbdc3c8-f5b4-4af0-8770-00a467337520","added_by":"auto","created_at":"2025-10-06 15:34:18","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":6390931,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7262383/v1/5c0114edf3d6b639967d5471.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eElbasvir Triggers Ferroptosis in Esophageal Squamous Cell Carcinoma Through NCOA4-Mediated Ferritinophagy\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEsophageal carcinoma (EC) presents as a substantial worldwide disease burden, representing the seventh leading cause of cancer-associated death globally\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. EC distribution shows marked geographical disparity, with China alone contributing approximately 50% of worldwide cases\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Recent data from GLOBOCAN 2022 reveal staggering epidemiological figures, with over 510,000 incident cases and nearly 450,000 deaths annually, underscoring the urgent need for improved therapeutic strategies\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Histopathologically, EC consists of two main variants - squamous cell carcinoma (ESCC) and adenocarcinoma (EAC) - characterized by distinct geographic prevalence and etiological divergence\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. The Asian predominance of ESCC contrasts with Western EAC prevalence, with tobacco and alcohol use accounting for significant attributable risk in these populations\u003csup\u003e[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDespite significant advances in molecularly targeted therapies that have revolutionized oncology treatment paradigms, the clinical management of ESCC continues to face substantial challenges. While tislelizumab has emerged as the first dual EU/US-approved immunotherapy for ESCC\u003csup\u003e[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, the broader therapeutic arsenal remains limited, with acquired resistance posing a persistent clinical hurdle\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. These limitations have catalyzed growing interest in alternative drug development strategies, particularly drug repurposing (DRP), which offers distinct advantages over de novo drug development including reduced costs, abbreviated timelines, and established safety profiles\u003csup\u003e[\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. The COVID-19 pandemic powerfully demonstrated the utility of this approach, where rapid therapeutic screening identified multiple repurposed candidates\u003csup\u003e[\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Notable successes in oncology DRP include disulfiram\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, originally an alcohol-dependence treatment now under investigation as an adjuvant for malignant gliomas\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, and chloroquine, an antimalarial repurposed for cancer therapy through autophagy modulation - including our group's previous demonstration of its anti-EC activity via this pathway\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Building on this paradigm, we identified Elbasvir, an FDA-approved hepatitis C virus (HCV) NS5A inhibitor, as a promising novel candidate with potent anti-ESCC activity. Our results demonstrate that Elbasvir exerts its antitumor effects predominantly through the activation of ferroptosis. These findings not only highlight the expanded therapeutic potential of this clinically approved agent but also offer new insights into ferroptosis induction as a viable strategy for the treatment of ESCC.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials and reagents\u003c/h2\u003e\u003cp\u003eElbasvir (TargetMol, China), CCK-8 kit (Beyotime Biotechnology, China), Apoptosis kit (BD, USA), cell cycle detection kit (KeyGEN, China), NCOA4 antibody (Abcam, UK), Ferritin antibody (HUABIO, China), FTH1 antibody (Abcam, UK), Streptavidin protease pronase (Roche, Switzerland), Mitochondrial Membrane Potential Assay Kit (Biosharp, China), Fe\u003csup\u003e2+\u003c/sup\u003e Assay Kit (Elabscience, China), Reactive Oxygen Demand Assay Kit (Biosharp, China), Lipid Peroxidation Kit (Biosharp, China), siRNA sequence of \u003cem\u003eNCAO4\u003c/em\u003e gene (Ribobio, China), Overexpression of \u003cem\u003eNCAO4\u003c/em\u003e Plasmid (Sino Biological, China)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. CCK-8 cell viability assay\u003c/h2\u003e\u003cp\u003eLog-phase KYSE150 and TE1 cells were trypsinized, pelleted, and resuspended to 3000 cells/well, then seeded in 96-well plates (200 \u0026micro;L/well) with triplicate wells per group. To prevent water evaporation, 200 \u0026micro;L of PBS was added in a circle around the edge of the 96-well plate. The cells were incubated in a 37℃, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator until the next day for drug treatment. The experimental group was treated with 40 \u0026micro;M Elbasvir and the control group was treated with an equal volume of DMSO. Cell viability was assessed at 0, 24, 48, and 72 h post-treatment using CCK-8 assay. Following medium removal, 100 \u0026micro;L of freshly prepared CCK-8 working solution (1:9 reagent:serum-free medium) was added per well. After 2 h incubation, absorbance was measured at 450 nm using a microplate reader to determine viability.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Apoptosis assay\u003c/h2\u003e\u003cp\u003eCells were plated in 6-well plates (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e/well) and cultured for 24 h before treatment with DMSO or 40 \u0026micro;M Elbasvir (48 h). Following trypsinization, cells were washed twice with ice-cold PBS and resuspended in binding buffer (1\u0026times;10\u003csup\u003e6\u003c/sup\u003ecells/mL). Aliquots (100 \u0026micro;L containing 1\u0026times;10\u003csup\u003e5\u003c/sup\u003ecells) were stained with 5 \u0026micro;L each of FITC-Annexin V and PI (15 min, RT, dark). After adding 400 \u0026micro;L binding buffer, samples were analyzed by flow cytometry within 1 h.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Cell cycle assay\u003c/h2\u003e\u003cp\u003eFollowing 24 h culture in 6-well plates (1\u0026times;10\u003csup\u003e5\u003c/sup\u003ecells/well), cells were treated with DMSO or 40 \u0026micro;M Elbasvir for 48 h. After treatment, cells were trypsinized, fixed in 75% ethanol (-20℃, overnight), and washed with PBS. Cell cycle analysis was performed using PI/RNase staining (30 min, RT, dark) with subsequent flow cytometry detection (excitation 488 nm).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Western Blot\u003c/h2\u003e\u003cp\u003eThe cell sediment was collected, lysed, and centrifuged to obtain the protein supernatant, and the protein concentration was determined by the BCA method. The protein concentration was determined by the BCA method. 40 \u0026micro;g of protein was denatured and added to the SDS-PAGE gel according to the experimental group. Electrophoresis was performed at 80 V for 30 min followed by 120 V for 1 h (adjusted based on target protein size). Proteins were then transferred to PVDF membranes using wet transfer at 250 mA constant current (duration optimized by protein molecular weight). Membranes were blocked with 5% skim milk (2 h, RT), incubated with primary antibody (4℃, overnight), washed 3\u0026times;10 min with PBST, and probed with species-matched secondary antibody (1 h, RT) followed by additional PBST washes (3\u0026times;10 min), and then developed with ECL chemiluminescent reagent, and the Sydney number was captured by an imaging system for the analysis of the results.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Construction and treatment of tumor-bearing nude mouse model\u003c/h2\u003e\u003cp\u003e Four-week-old male BALB/c nude mice (18\u0026ndash;20 g, GemPharmatech, China) were housed under standard conditions (25\u0026deg;C, 55% humidity, 12-h light/dark cycle) at North Sichuan Medical College's Animal Center, with protocols approved by the Institutional Animal Ethics Committee (No.2023033). KYSE150 cells (1\u0026times;10⁶ in 100 \u0026micro;L) were subcutaneously injected into each mouse. When tumors reached\u0026thinsp;~\u0026thinsp;100 mm\u0026sup3;, mice were randomized into two groups receiving either 70 mg/kg Elbasvir (i.p., every other day) or DMSO control. Tumor volume was monitored by caliper measurements (length\u0026times;width\u0026sup2;/2). After 17 days, mice were sacrificed for tissue collection (lungs, liver, kidneys, heart, tumors), which were fixed in 4% PFA for paraffin embedding and HE staining.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. HE staining\u003c/h2\u003e\u003cp\u003eFor histological analysis, the paraffin-embedded tissue slices underwent dewaxing in xylene and subsequent rehydration \u003cem\u003evia\u003c/em\u003e a sequence of graduated ethanol solutions, each for 10 min. The sections were rinsed with PBS, subjected to hematoxylin staining for 3 min, and differentiated with 1% hydrochloric acid-alcohol solution for 10\u0026ndash;20 s. Following another PBS rinse, the sections were stained with eosin for 2 min. Dehydration was performed through an ascending alcohol gradient, tissue sections were cleared in xylene. Lastly, the specimens were mounted with neutral gum, air-dried, and observed under microscopic examination.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Proteomic analysis\u003c/h2\u003e\u003cp\u003eSamples were processed with reaction buffer (1% SDC/100 mM Tris-HCl pH 8.5/10 mM TCEP/40 mM CAA) at 60℃ for 1 h to simultaneously achieve protein denaturation, reduction, and alkylation. After dilution with ultrapure water, tryptic digestion was performed (enzyme:protein\u0026thinsp;=\u0026thinsp;1:50, 37℃, overnight). The reaction was quenched with TFA, followed by centrifugation (16,000 \u0026times; g) and desalting using a homemade SDB column. For TMT labeling, aliquots were labeled, pooled, and desalted (Sep-Pak C18). The combined samples were fractionated by high-pH reverse-phase chromatography into 15 fractions, vacuum-dried, and stored at -80℃ prior to LC-MS/MS analysis using a Q Exactive Plus coupled to an EASY-nLC 1200 system.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Mitochondrial morphological observation\u003c/h2\u003e\u003cp\u003eCells from 60 mm dishes were pelleted (1,000 rpm, 5 min) and fixed with 3% glutaraldehyde (4℃, 5 min). After transferring to 1.5 mL tubes and centrifugation (12,000 rpm, 10 min), pellets were post-fixed with 1% osmium tetroxide. Dehydration was performed using an acetone gradient (30%\u0026rarr;100%, with three 100% changes), followed by stepwise Epon-812 infiltration (3:1\u0026rarr;1:3 resin:acetone). Ultrathin sections (60\u0026ndash;90 nm) were collected on copper grids, stained with uranyl acetate (10\u0026ndash;15 min) and lead citrate (1\u0026ndash;2 min), and imaged using a JEOL JEM-1400FLASH TEM.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Mitochondrial membrane potential analysis\u003c/h2\u003e\u003cp\u003eThe JC-1 working solution was prepared by diluting JC-1 (200\u0026times;) 1:160 in ultrapure water and mixing with JC-1 Staining Buffer (5\u0026times;) at a 1:4 ratio. For positive controls, cells were treated with 10 \u0026micro;M CCCP (1:1000 dilution from 10 mM stock) for 20 min. After PBS washing, cells were incubated with 1 mL working solution per well (37\u0026deg;C, 20 min), followed by two washes with ice-cold JC-1 Staining Buffer (1\u0026times;). Cells were resuspended in 2 mL culture medium and immediately analyzed by fluorescence or confocal microscopy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11. Ferrous ion (Fe\u003csup\u003e2+\u003c/sup\u003e) concentration detection\u003c/h2\u003e\u003cp\u003eAll reagents were equilibrated to room temperature prior to use. Iron standards were prepared at specified concentrations for calibration. Treated cells (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e) were lysed with 200 \u0026micro;L Reagent I on ice for 10 min, followed by centrifugation (15,000 \u0026times; g, 10 min) to collect supernatant. For analysis, 80 \u0026micro;L aliquots of standards or test samples were loaded in duplicate onto a microplate, with control wells receiving additional 80 \u0026micro;L of Reagents II and III. After 10 min incubation at 37\u0026deg;C, absorbance was measured at 593 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12. Reactive oxygen species (ROS) level detection\u003c/h2\u003e\u003cp\u003eThe in situ loading probe was selected according to the instructions for adherent cells, DCFH-DA probe was diluted with serum-free culture medium at 1:1000 to a final concentration of 10 \u0026micro;M. Cell culture medium was removed, and 1 mL of diluted DCFH-DA was added to each well of a six-well plate. Incubation was performed at 37\u0026ordm;C for 20 min. Following triple washing with serum-free medium to remove extracellular probe, cells were analyzed by flow cytometry (excitation 488 nm/emission 525 nm). Positive controls received 20\u0026ndash;30 min stimulation to induce ROS elevation prior to detection. Data was processed for statistical analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13. Malonaldehyde (MDA) content detection\u003c/h2\u003e\u003cp\u003eCell lysates (0.1 mL per 1\u0026times;10\u003csup\u003e6\u003c/sup\u003ecells) were centrifuged (12,000 \u0026times; g, 10 min, 4\u0026deg;C) and supernatants collected for protein quantification (BCA assay). A 0.37% TBA solution was prepared by dissolving 25 mg TBA in 6.76 mL preparation buffer. The MDA working solution (150 \u0026micro;L TBA dilution buffer\u0026thinsp;+\u0026thinsp;50 \u0026micro;L TBA storage solution\u0026thinsp;+\u0026thinsp;3 \u0026micro;L antioxidant per sample) and standard curve (1\u0026ndash;50 \u0026micro;M) were prepared fresh. Samples (0.1 mL lysate/standards) were mixed with 0.2 mL working solution, heated (100\u0026deg;C, 15 min), cooled, and centrifuged (1,000 \u0026times; g, 10 min). Supernatant absorbance (200 \u0026micro;L/well) was measured at 532 nm for MDA quantification normalized to protein content.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14. RNA interference\u003c/h2\u003e\u003cp\u003eThe sequence of small interfering RNA (siRNA) for NCOA4 gene silencing was designed and provided for synthesis by Reebok Biotech. The sequence is as follows: GACCUUUUUUAUCAGCUUA. 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e KYSE150 and TE1 cells were grown in 6-well plates one night in advance and transfected the next day. The lyophilized powder was transiently dissociated and solubilized by adding DEPC water so that the siRNA concentration was 20 \u0026micro;M and then dispensed. Refer to Lipofectamine 2000 instructions for transfection. To configure the transfection system, remove 3 sterile EP tubes and label the reagent and NC/siRNA tubes. Add 700 \u0026micro;L of serum-free medium to the reagent tube, add 35 \u0026micro;L of Lipofectamine 2000 and mix gently. Add 350 \u0026micro;L of serum-free medium and 17.5 \u0026micro;L of diluted NC/siRNA to the NC/siRNA tubes, mix gently, and allow to stand for 5 min. 350 \u0026micro;L of each of the reagents was then added to the NC/siRNA tubes, mixed gently, and allowed to stand for 15 min. Replace the cell culture medium of the 6-well plate with serum-free medium, and add 200 \u0026micro;L of the allowed cell culture medium to each of the corresponding 6-well plates. Cells in 6-well plates were washed and maintained in serum-free medium prior to treatment. A 200 \u0026micro;L aliquot of the stabilized complex was added dropwise to each well, followed by 6 h incubation at 37℃. The treatment medium was then replaced with complete growth medium for continued culture.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.15. Plasmid transfection\u003c/h2\u003e\u003cp\u003eFor transformation, 10 \u0026micro;L plasmid was mixed with 50 \u0026micro;L DH5α competent cells, incubated on ice (30 min), heat-shocked (42℃, 90 s), and immediately returned to ice (2 min). After adding 400 \u0026micro;L antibiotic-free LB medium, cells were recovered (37℃, 40 min) before expansion. For plasmid amplification, 100 \u0026micro;L bacterial culture was inoculated into 7 mL LB medium supplemented with kanamycin (1:1000) and incubated overnight (37℃, 220 rpm). Plasmid extraction was performed at visible turbidity, with concentration quantified via Nanodrop 2000. For transfection, cells were seeded in 6-well plates (1\u0026times;10\u003csup\u003e5\u003c/sup\u003ecells/well) 24 h prior. Two hours before transfection, medium was replaced with fresh complete medium. Plasmid DNA (2 \u0026micro;g) was diluted in 100 \u0026micro;L serum-free medium and combined with 2 \u0026micro;L transfection reagent. After 15 min incubation (RT), the mixture was added dropwise to cells, followed by gentle swirling and incubation (24\u0026ndash;48 h).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.16. Quantitative real-time PCR (qPCR)\u003c/h2\u003e\u003cp\u003eTotal RNA was isolated using Vazyme spin columns, with 1 \u0026micro;g reverse-transcribed to cDNA. qPCR reactions (10 \u0026micro;L total volume) contained 5 \u0026micro;L Taq Pro Universal SYBR Master Mix (2\u0026times;), 0.4 \u0026micro;L each of forward/reverse primers, 3.2 \u0026micro;L nuclease-free water, and 1 \u0026micro;L cDNA template. Amplification was performed on a LightCycler 480 system (Roche) under the following conditions: 95\u0026deg;C for 30 sec (initial denaturation), followed by 40 cycles of 95\u0026deg;C for 5 sec, 60\u0026deg;C for 30 sec, and 72\u0026deg;C for 20 sec. Data were analyzed using the instrument's proprietary software. The primer sequence of the gene is as follows: NCOA4, the forward primer is 5'-GCCCTACAATGTGAGTGATTGG-3' and the reverse primer is 5'-ACTGGTGCAAGGCTCGTTG-3'; β-actin, the forward primer was 5 '-GCAAGCAGGAGTATGACGAG-3'; The reverse primer is 5 '-CAAATAAAGCCATGCC AATC-3'.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e2.17. Prokaryotic expression and purification\u003c/h2\u003e\u003cp\u003eThe NCOA4 coding region was chemically synthesized and ligated into pET28a using \u003cem\u003eBamHI\u003c/em\u003e and \u003cem\u003eXhoI\u003c/em\u003e restriction enzymes. The empty plasmid cut by the same enzyme and then transformed into receptive cells to screen positive clones and verify by sequencing. The verified recombinant plasmid was transformed into expression strain, and after the transformation by heat shock method, it was coated on the LB plate containing antibiotics. Single colonies were selected and induced by 1.0 mM IPTG (37℃, 4 h) and 0.2 mM IPTG (16℃, 16 h), respectively. Following ultrasonic lysis, protein expression was verified via SDS-PAGE. For large-scale purification, cells were resuspended in PBS, lysed by sonication, and clarified by centrifugation. The supernatant was subjected to Ni-Smart affinity chromatography, with sequential washes (20 mM imidazole) and elution (250 mM imidazole). The purified protein was dialyzed into PBS buffer containing 10% glycerol at 4℃, then concentrated by ultrafiltration tube and determined by SDS-PAGE and Bradford method.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e2.18. Surface plasmon resonance (SPR) measurement\u003c/h2\u003e\u003cp\u003ePlace 200 mL of 1\u0026times;PBS Buffer running buffer, water bottle, and waste bottle in the instrument tray and insert the corresponding inlet tube. Holding the CM5 chip (with the lettered side facing up) in your hand, gently push it into the slot in the direction of the arrow and close the chip compartment door. Channel 2 of the biosensor chip was activated with EDC/NHS (10 \u0026micro;L/min), followed by immobilization of NCOA4 (383-522aa) at 50 \u0026micro;g/mL in sodium acetate buffer (pH 4.0, 10 \u0026micro;L/min, 9 \u0026micro;L coupling volume). Ethanolamine (10 \u0026micro;L/min) was used to block residual active groups. Real-time coupling kinetics were monitored throughout the process. Replace the running buffer with 1\u003cem\u003e\u0026times;\u003c/em\u003e PBS-P\u0026thinsp;+\u0026thinsp;containing 5% DMSO and insert it into the appropriate inlet tube. The 5% DMSO concentration calibration curve was configured by mixing the 4.5% and 5.8% DMSO master mixes proportionally. The test material was diluted to different concentrations in a 96-well plate and coupled to the target proteins sequentially through the microarray from low to high at a flow rate of 30 \u0026micro;L/min for 150 s. After each concentration point was detected, the microarray was regenerated with a 10 mM glycine hydrochloride (pH 2.0) solution for 5 min, and the operation was repeated until all the concentrations had been detected. Binding kinetics were analyzed using BIAcore T200 systems (GE Healthcare). Sensorgrams were processed with Control Software (v2.0), subtracting reference channel signals. Association and dissociation constants were derived by global fitting to a 1:1 Langmuir model (Evaluation Software v2.0).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e2.19. Drug Affinity Responsive Target Stability (DARTS) assay\u003c/h2\u003e\u003cp\u003eCells at ~\u0026thinsp;80% confluence were harvested from 10 cm dishes, washed twice with PBS, and lysed in ice-cold RIPA buffer (30 min on ice with vortex mixing every 10 min). Lysates were centrifuged (12,000 \u0026times; g, 10 min, 4\u0026deg;C), and supernatants were aliquoted into input, DMSO control, and Elbasvir treatment groups (400 \u0026micro;M final concentration, 2 h incubation at 25\u0026deg;C). Streptavidin was added at three concentrations (1:1000, 1:5000, 1:10,000) for 30 min (25\u0026deg;C) before reaction termination with PMSF (1:100). Processed samples were subjected to SDS-PAGE and Western blotting for target protein analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e2.20. Statistical analysis\u003c/h2\u003e\u003cp\u003eThe experimental data were replicated three times independently, and the data were analyzed using GraphPad Prism 9.5 software and the statistical significance was analyzed by t-test or ANOVA. Differences were considered statistically significant when the P value was \u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. Elbasvir exerts potent antitumor effects in ESCC through growth inhibition and apoptosis induction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eElbasvir demonstrates significant antitumor activity in ESCC by suppressing proliferation and inducing apoptosis. To evaluate its therapeutic potential, we first assessed its impact on cell viability using CCK-8 assays in KYSE150 and TE1 cell lines. Elbasvir treatment led to a marked, suppression of ESCC cell proliferation compared to vehicle controls (Fig. 1A). Mechanistic investigations further demonstrated that Elbasvir markedly induced apoptotic cell death, which was quantitatively confirmed by flow cytometric analysis using Annexin V-FITC/PI dual staining (Fig. 1B). Both early and late apoptotic populations were substantially elevated following Elbasvir exposure (Fig. 1C), confirming its pro-apoptotic activity in ESCC cells. Cell cycle profiling demonstrated that Elbasvir treatment induced G0/G1 phase arrest, with a concomitant reduction in G2/M phase populations (Fig. 1D, E). Western blot analysis corroborated these findings, showing upregulation of key apoptotic markers (Fig. 1F) and downregulation of G0/G1 phase regulatory proteins (Fig. 1G). Collectively, these results demonstrate that Elbasvir exerts multifaceted antitumor effects in ESCC by simultaneously inhibiting proliferation, promoting apoptosis while causing a cell cycle blockade at the G0/G1 checkpoint.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Elbasvir suppresses tumor growth in ESCC xenograft models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the therapeutic potential of Elbasvir \u003cem\u003ein vivo\u003c/em\u003e, we established KYSE150-derived xenograft models. Elbasvir treatment resulted in significant tumor growth suppression compared to vehicle controls, as demonstrated by a significant decrease in both tumor volume and mass (Fig. 2A-C). Importantly, comprehensive histopathological assessment of major organs demonstrated no detectable treatment-related toxicity (Fig. 2D, Supplementary Figure 1), indicating excellent tolerability of Elbasvir at the therapeutic dose. These findings collectively demonstrate that Elbasvir exerts potent antitumor activity against ESCC in vivo while maintaining an advantageous safety profile.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Elbasvir triggers ferroptosis in ESCC cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eElbasvir exhibits potent anti-tumor effects against ESCC both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e; however, its underlying molecular mechanisms remain elusive. To elucidate these mechanisms, we performed proteomic profiling to identify proteins and pathways dysregulated by Elbasvir in ESCC cells. Proteomic analysis identified 642 differentially expressed proteins (427 up-regulated and 215 down-regulated) in Elbasvir-treated cells (Fig. 3A) (Supplementary Table 1). Pathway analysis demonstrated significant enrichment of these proteins in ferroptosis-related processes (Fig. 3B).\u003c/p\u003e\n\u003cp\u003eTo elucidate the mechanism underlying Elbasvir\u0026apos;s antitumor activity, we first examined mitochondrial ultrastructure by transmission electron microscopy. Elbasvir-treated cells exhibited hallmark morphological features of ferroptosis, including outer membrane rupture, mitochondrial shrinkage, and cristae disintegration (Fig. 4A). Complementary analysis of mitochondrial function revealed significant membrane potential dissipation following Elbasvir treatment (Fig. 4B), confirming functional impairment of mitochondria. These findings collectively demonstrate that Elbasvir triggers characteristic ferroptotic changes at both structural and functional levels.\u003c/p\u003e\n\u003cp\u003eQuantitative analysis of iron metabolism revealed Elbasvir-mediated accumulation of ferrous iron (Fe\u003csup\u003e2+\u003c/sup\u003e) (Fig. 5A), accompanied by elevated reactive oxygen species and malondialdehyde levels (Fig. 5B-D), consistent with ferroptotic lipid peroxidation. Immunoblot analysis demonstrated significant upregulation of key ferroptosis regulators (NCOA4, Ferritin, and FTH1) in both cellular and xenograft models following Elbasvir treatment (Fig. 5E, Supplementary Figure 2). These findings establish that Elbasvir induces ferroptosis through coordinated iron accumulation, oxidative stress generation, and modulation of iron homeostasis proteins, revealing a novel mechanistic basis for its anti-ESCC activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Elbasvir induces ferroptosis in ESCC through NCOA4-mediated ferritinophagy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhile our phenotypic and proteomic analyses identified Elbasvir as a potent inducer of ferroptosis in ESCC, its precise molecular target remained unclear. Proteomic profiling of Elbasvir-treated cells revealed significant upregulation of iron metabolism regulators, including NCOA4, SQSTM1, SLC39A1, FTH1, and TNFRSF21 (Supplementary Table 2), with NCOA4 and FTH1 emerging as key mediators of ferroptosis. NCOA4, a selective autophagy receptor, binds Ferritin heavy chain 1 (FTH1) to mediate ferritinophagy, promoting lysosomal degradation of iron-laden Ferritin and subsequent iron release. This process critically regulates cellular iron homeostasis and iron-dependent cell death pathways, including ferroptosis. Consistent with this mechanism, we observed upregulation of NCOA4, Ferritin, and FTH1 at the protein level (Fig. 5E). Bioinformatic and functional analyses implicated NCOA4 as a central player in Elbasvir-induced ferroptosis. Genetic perturbation studies confirmed its pivotal role: \u003cem\u003eNCOA4\u003c/em\u003e knockdown in two ESCC cell lines (Fig. 6A) increased FTH1 protein levels, while \u003cem\u003eNCOA4\u003c/em\u003e overexpression reduced FTH1 (Fig. 6B). \u003cem\u003eNCOA4\u003c/em\u003e depletion enhanced cell viability, whereas its overexpression exacerbated Elbasvir-induced cytotoxicity (Fig. 6C). Intracellular ferrous iron (Fe\u003csup\u003e2+\u003c/sup\u003e) levels decreased upon \u003cem\u003eNCOA4\u003c/em\u003e knockdown but increased with \u003cem\u003eNCOA4\u003c/em\u003e overexpression (Fig. 6D). Correspondingly, lipid peroxidation (measured by MDA content) was attenuated by \u003cem\u003eNCOA4\u0026nbsp;\u003c/em\u003esilencing but amplified by \u003cem\u003eNCOA4\u003c/em\u003e overexpression (Fig. 6E). These findings demonstrate that NCOA4 drives Elbasvir-triggered ferroptosis by regulating the NCOA4-FTH1 axis, controlling iron release, and promoting oxidative stress.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. Elbasvir directly engages NCOA4 to trigger ferroptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhile NCOA4 is established as a critical regulator of ESCC cell growth, its molecular interaction with Elbasvir remained undefined. For interaction characterization, we successfully\u0026nbsp;cloned, expressed, and purified the functional domain of NCOA4\u003csup\u003e383-522\u003c/sup\u003e(Supplementary Figure 3A-C), followed by binding affinity measurement using surface plasmon resonance (SPR). Elbasvir demonstrated superior binding affinity compared to the known ligand NCOA4-9a (Fig. 7A, B, Supplementary Table 3), with distinct kinetic properties indicating a specific, high-efficiency interaction. Drug affinity responsive target stability (DARTS) assays independently validated this direct binding, showing Elbasvir-mediated stabilization of NCOA4 (Fig. 7C). These biophysical studies establish NCOA4 as a direct molecular target of Elbasvir, revealing a structural mechanism for its ferroptosis-inducing activity in ESCC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6. Elbasvir suppresses ESCC progression by targeting the NCOA4-FTH1 axis to induce ferroptosis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlthough Elbasvir has been identified as a conjugate of NCOA4, its precise regulatory mechanisms in ESCC ferroptosis have yet to be fully elucidated. To investigate the mechanistic basis, we assessed Elbasvir\u0026apos;s involvement in NCOA4 genetic modulation. Notably, Elbasvir co-treatment diminished the pro-survival effect of NCOA4 knockdown, concurrent with marked alterations in ferroptosis-associated FTH1 expression, underscoring NCOA4\u0026apos;s pivotal role in Elbasvir-induced ferroptosis (Fig. 8A). Conversely, NCOA4 overexpression significantly suppressed cellular viability while reducing FTH1 expression. This inhibition was further enhanced when co-treated with Elbasvir, and markers associated with iron poisoning showed a corresponding shift (Fig. 8B). Taken together, these findings suggest that Elbasvir triggers iron apoptosis in ESCC by promoting NCOA4-dependent FTH1 degradation, which ultimately leads to tumor cell death. This study reveals the mechanistic basis of Elbasvir\u0026apos;s anti-ESCC activity and highlights the therapeutic potential of targeting the NCOA4-FTH1 axis in anti-cancer strategies. \u0026nbsp;\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eEC remains a formidable global health challenge, ranking among the top causes of cancer-related mortality worldwide\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Epidemiological studies consistently identify the EC as a significant contributor to premature cancer deaths and reduced life expectancy\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Diagnostic advances, particularly in endoscopic screening, have markedly improved early-stage detection rates\u003csup\u003e[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e, while radiotherapy continues to provide clinical benefit for advanced cases\u003csup\u003e[\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. The therapeutic paradigm has been transformed by immune checkpoint inhibitors\u003csup\u003e[\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e, with small molecule inhibitors emerging as particularly promising agents\u003csup\u003e[\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe strategy of drug repurposing - applying approved therapeutics to novel indications - has gained considerable traction in oncology\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Our investigations identified Elbasvir, an established NS5A inhibitor developed by Merck \u0026amp; Co. for hepatitis C virus (HCV) treatment, as possessing previously unrecognized anti-tumor activity. This highly specific antiviral agent demonstrates picomolar potency against HCV NS5A, effectively suppressing viral replication through multiple mechanisms\u003csup\u003e[\u003cspan additionalcitationids=\"CR46 CR47 CR48\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003eWhile its clinical efficacy against HCV genotypes 1\u0026ndash;4 is well-documented\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. Its potential oncological applications remain unexplored\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eElbasvir suppresses ESCC proliferation through dual induction of apoptosis and cell cycle arrest, corroborating known NS5A inhibitor activity, with concordant in vivo validation. The discovery of the effective antitumor activity of Elbasvir against ESCC represents a significant advance in identifying retargeting therapeutic candidates with multimodal mechanisms of action. Beyond conventional cytotoxicity, our findings reveal Elbasvir's novel engagement with ferroptosis - an iron-mediated cell death pathway - wherein it modulates the NCOA4-FTH1 axis to promote ferritinophagy, thereby increasing labile iron pools that drive Fenton chemistry. The Fenton reaction is a biochemical cascade that produces reactive oxygen species and drives deadly lipid peroxidation, ultimately triggering cell ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. Our results showed that after Elbasvir treatment, intracellular iron autophagy was enhanced, released Fe\u003csup\u003e2+\u003c/sup\u003e accumulated in the body, downstream ROS levels increased significantly, driving lipid peroxidation, MDA content increased, and cell membrane rupture and death. Ferroptosis is characterized by distinct mitochondrial alterations, including functional impairment, morphological changes, membrane condensation, and cristae reduction or loss. These ultrastructural manifestations of mitochondrial damage represent hallmark features of iron-dependent cell death\u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. This ferroptotic mechanism represents a distinct cell death pathway in ESCC treatment. Notably, ferroptosis has shown particular therapeutic potential for malignancies exhibiting elevated oxidative stress, including ESCC\u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. Targeting ferroptosis has become a new breakthrough in anti-tumor, in which the NCOA4-FTH1 axis mediates iron autophagy and regulates iron metabolism in ferroptosis. Our results show that NCOA4 and FTH1 are significantly activated in Elbasvir-induced ferroptosis responses, and proteomic identification of the NCOA4-FTH1 axis regulation provides key mechanologic insights into Elbasvir activity. NCOA4 serves as a specific ferritinophagy receptor that critically regulates iron homeostasis\u003csup\u003e[\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e. We demonstrated that Elbasvir can target NCOA4 protein binding while stabilizing NCOA4 protein expression, and that direct binding and subsequent up-regulation of Elbasvir to NCOA4 provides a novel pharmacological strategy to disrupt the balance of the NCOA4-FTH1 axis. Knockdown of NCOA4 inhibits ferroptosis and promotes cell survival by decreasing the content of ferrous and MDA, and the binding FTH1 increases correspondingly. Conversely, Fe\u0026sup2;⁺ accumulation and elevated MDA levels potentiated ferroptosis, markedly reducing cell viability while suppressing FTH1 expression. In combination with Elbasvir, the cell growth promoted by NCOA4 knockdown was partially reversed by Elbasvir, and Elbasvir further intensified the inhibitory effect of overexpression of NCOA4 on cell viability. These findings have important therapeutic implications. First, they positioned ferroptosis induction as a viable strategy to combat ESCC, a type of malignancy that is often resistant to apoptosis-based therapies\u003csup\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/sup\u003e. Second, the NCOA4-FTH1 axis emerges as a druggable target\u003csup\u003e[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/sup\u003e, with Elbasvir serving as both a chemical probe and potential therapeutic. Notably, the iron metabolism pathway may represent a metabolic vulnerability in ESCC\u003csup\u003e[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/sup\u003e, as many tumors exhibit increased iron requirements to support rapid proliferation\u003csup\u003e[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSeveral critical questions emerge from these findings. The selectivity of Elbasvir's NCOA4 binding relative to other ferritinophagy regulators warrants further investigation, as does the potential for synergy with existing ESCC therapies\u003csup\u003e[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]\u003c/sup\u003e. Additionally, the tumor microenvironment's role in modulating this ferroptotic response remains unexplored - particularly relevant given the inflammatory nature of ESCC and known interactions between iron metabolism and immune responses\u003csup\u003e[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]\u003c/sup\u003e. From a translational perspective, these results suggest that patient stratification based on iron metabolism markers (e.g., Ferritin levels, NCOA4 expression) could optimize Elbasvir's therapeutic application. Furthermore, combining Elbasvir with lipid peroxidation-enhancing agents or system xc- inhibitors may amplify its anti-tumor efficacy while potentially overcoming resistance mechanisms\u003csup\u003e[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, we identified Elbasvir as a potent anti-ESCC agent through molecular docking and validated its tumor-suppressive effects in vitro and in vivo. Mechanistically, Elbasvir binds directly to NCOA4, stabilizing it and promoting its interaction with FTH1 to activate ferritinophagy, thereby inducing ferroptosis via iron overload, ROS accumulation, and lipid peroxidation (Fig. 9). Our findings unveil the NCOA4-FTH1 axis as Elbasvir’s ferroptotic trigger, offering a novel therapeutic strategy for ESCC. Importantly, Elbasvir’s established safety profile underscores its clinical potential. Beyond ESCC, this work provides a mechanistic framework for targeting iron metabolism in upper GI malignancies and broader ferroptosis-vulnerable cancers. Future studies should prioritize clinical translation while probing the intricate crosstalk between viral protease inhibitors (e.g., Elbasvir) and iron homeostasis pathways to optimize therapeutic exploitation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.G, X.Z made significant contributions to the conception, design and guidance of the study. M.T, F.G, M.Y jointly completed the experiment implementation, data collection, data analysis, and paper writing. S.H, J.C, Z.Y participated in the experiment. L.X, Q.M, X.G and X.Z made a lot of revisions and revisions to the manuscript. X.G and X.Z finally approved the version to be published. All the authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Support\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Science and Technology Support Program (grant no. 25NSFSC2024), the Medical Research Project of Sichuan (grant no. S23020), the Scientific Research Development Plan Project, Affiliated Hospital of North Sichuan Medical College (grant no. 2024PTZK004 and 2023-2ZD002), and the Scientific Research Development Plan Project, North Sichuan Medical College (grant no. CBY24-QDA27 and CBY22-ZDA03).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the data supporting the conclusions of this study are all included in this article and its supplementary materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Author Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the course of this research, there were no any commercial or financial relationships that could potentially constitute a conflict of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBetancourt-Cuellar S L, Benveniste M, Palacio D P, et al. Esophageal Cancer: Tumor-Node-Metastasis Staging[J]. Radiol Clin North Am, 2021,59(2):219-229.\u003c/li\u003e\n\u003cli\u003eYang H, Wang F, Hallemeier C L, et al. Oesophageal cancer[J]. 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Nat Rev Clin Oncol, 2020,17(6):382.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"medical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"medo","sideBox":"Learn more about [Medical Oncology](https://www.springer.com/journal/12032)","snPcode":"12032","submissionUrl":"https://submission.nature.com/new-submission/12032/3","title":"Medical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Elbasvir, Esophageal squamous cell carcinoma, Ferroptosis, NCOA4, FTH1, Targeted therapy","lastPublishedDoi":"10.21203/rs.3.rs-7262383/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7262383/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eEsophageal squamous cell carcinoma (ESCC) remains treatment-resistant; we explored Elbasvir, an NS5A inhibitor, as a ferroptosis inducer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eCell viability was assessed by CCK-8 assays. Apoptosis and cell cycle were analyzed via flow cytometry, and key markers via Western blotting. In vivo efficacy was evaluated using BALB/c nude mouse xenografts. Proteomic analysis was conducted by mass spectrometry. Ferroptosis induction was verified via TEM, JC-1, FerroOrange, DCFH-DA, MDA assays, and Western blotting of NCOA4, Ferritin, and FTH1. Binding to NCOA4 was confirmed by surface plasmon resonance (SPR) and drug affinity responsive target stability (DARTS) assays.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Elbasvir (40 μM, 48 h) suppressed KYSE150/TE1 viability, induced apoptosis/G0/G1 arrest, and inhibited xenograft growth without toxicity. Proteomics identified ferroptosis as the top pathway. SPR/DARTS confirmed NCOA4 binding. NCOA4 knockdown reduced ferroptosis; overexpression enhanced it. Elbasvir triggered NCOA4-mediated ferritinophagy, FTH1 degradation, iron accumulation, and lipid peroxidation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiscussion: \u003c/strong\u003eElbasvir targets NCOA4-FTH1 to induce ferroptosis, offering a repurposing strategy for ESCC. Its safety profile supports clinical translation, with potential applications in iron metabolism-dependent cancers.\u003c/p\u003e","manuscriptTitle":"Elbasvir Triggers Ferroptosis in Esophageal Squamous Cell Carcinoma Through NCOA4-Mediated Ferritinophagy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-06 15:26:13","doi":"10.21203/rs.3.rs-7262383/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-17T21:48:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-09T05:18:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"49870821748621733885149397842270428165","date":"2025-10-19T04:30:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-09T04:02:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"218573583071650404736452508574678378241","date":"2025-09-24T03:50:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-24T03:34:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-01T13:44:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-01T13:44:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Medical Oncology","date":"2025-07-31T12:38:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"medical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"medo","sideBox":"Learn more about [Medical Oncology](https://www.springer.com/journal/12032)","snPcode":"12032","submissionUrl":"https://submission.nature.com/new-submission/12032/3","title":"Medical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1643bdd2-5044-487c-9e20-234d096c2385","owner":[],"postedDate":"October 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T16:07:48+00:00","versionOfRecord":{"articleIdentity":"rs-7262383","link":"https://doi.org/10.1007/s12032-026-03249-y","journal":{"identity":"medical-oncology","isVorOnly":false,"title":"Medical Oncology"},"publishedOn":"2026-02-21 15:59:49","publishedOnDateReadable":"February 21st, 2026"},"versionCreatedAt":"2025-10-06 15:26:13","video":"","vorDoi":"10.1007/s12032-026-03249-y","vorDoiUrl":"https://doi.org/10.1007/s12032-026-03249-y","workflowStages":[]},"version":"v1","identity":"rs-7262383","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7262383","identity":"rs-7262383","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}