Targeting ABCB6 induces ferroptosis in osteosarcoma cells via HIF1A/GPX4 pathway

Osteosarcoma (OS) is a malignant bone tumor that predominantly affects adolescents. Due to its early metastatic tendency and chemoresistance, long-term survival rates in patients have not shown significant improvement over time, highlighting the urgent need for novel molecular targets and therapeutic strategies. Ferroptosis, a form of programmed cell death driven by iron-dependent lipid peroxidation, offers a promising avenue for overcoming drug-resistant tumors. ATP-binding cassette subfamily B member 6 (ABCB6) belongs to the ATP-binding cassette (ABC) transporter superfamily and is mainly localised to the outer mitochondrial membrane, where it participates in haem synthesis and intracellular iron transport. Recent studies have indicated that ABCB6 is aberrantly expressed in various malignancies and regulates tumor progression, yet its role and mechanism in OS remain unexplored. The present study aims to systematically characterise the expression pattern, clinical prognostic significance, and biological functions of ABCB6 in OS, and to dissect the molecular mechanism by which ABCB6 governs ferroptosis in OS cells, thereby providing a theoretical foundation for ABCB6-targeted therapy.

Human OS cell lines (HOS and 143B) and human bone marrow mesenchymal stem cells (BMSCs) were used as research subjects. Stable ABCB6-knockdown OS cell lines were established via lentivirus-mediated RNA interference, and rescue experiments were performed using lentiviral overexpression of hypoxia-inducible factor 1-alpha (HIF1A). Cell proliferation was assessed by cell counting kit-8 (CCK-8) and colony formation assays; migration was evaluated by Transwell and scratch wound-healing assays; apoptosis was detected by flow cytometry. Ferroptosis-related phenotypic assays were employed to determine the impact of ABCB6 expression on ferroptosis. Transcriptome sequencing was conducted to analyse differentially expressed genes and enriched pathways; chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were applied to verify the direct binding of HIF1A to the promoter of glutathione peroxidase 4 (GPX4) and its transcriptional regulation. Iron chelator and proteasome inhibitor interventions were used to validate the regulation of HIF1A protein stability by Fe²⁺. In vivo, ABCB6-knockdown and control 143B cells were injected subcutaneously into nude mice to establish a xenograft tumor model, and tumor growth was monitored.

Both messenger RNA (mRNA) and protein levels of ABCB6 were significantly higher in OS cell lines than in BMSCs (both P<0.05), and analysis of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET)-OS cohort revealed that patients with high ABCB6 expression had markedly worse overall survival than those with low expression [hazard ratio (HR)=2.94, 95% confidence interval (CI) 1.36 to 6.36, P<0.05]. ABCB6 knockdown significantly suppressed OS cell proliferation and colony formation, reduced migratory capacity, and increased the apoptosis rate, accompanied by downregulation of N-cadherin and upregulation of E-cadherin and cleaved caspase-3 (all P<0.05). ABCB6 silencing markedly elevated the proportion of Liperfluo-positive cells and dihydroethidium (DHE) oxidation levels, increased intracellular Fe²⁺ concentration, decreased the glutathione (GSH)/oxidized glutathione (GSSG) ratio, reduced mitochondrial membrane potential, and induced typical ferroptotic mitochondrial morphological changes (shrinkage and cristae reduction) as observed by transmission electron microscope (all P<0.05); these oxidative alterations were reversed by the ferroptosis inhibitors. ABCB6 knockdown downregulated GPX4 at both protein and mRNA levels, whereas solute carrier family 7 member 11 (SLC7A11) expression remained unchanged (P>0.05). Transcriptome sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed significant enrichment of the HIF-1 signalling pathway in the control group (P<0.001); ABCB6 knockdown decreased HIF1A protein expression, and treatment with an iron chelator or a proteasome inhibitor reversed this degradation through Fe²⁺ chelation or inhibition of the ubiquitin-proteasome pathway, respectively (all P<0.05). Chromatin immunoprecipitation followed by quantitative polymerase chain reaction (ChIP-qPCR) and dual-luciferase reporter assays confirmed that HIF1A directly binds to the GPX4 promoter and activates its transcription. Overexpression of HIF1A in ABCB6-knockdown cells restored GPX4 expression and significantly rescued the proliferation inhibition, migration impairment, and apoptosis increase caused by ABCB6 silencing, while also restoring mitochondrial membrane potential (all P<0.05). In the xenograft model, tumors from the ABCB6-knockdown group exhibited significantly reduced volume and weight compared with the control group (all P<0.05); immunohistochemistry confirmed downregulation of HIF1A and GPX4, reduced Ki-67 and N-cadherin positivity, and increased cleaved caspase-3 and E-cadherin positivity in the knockdown group (all P<0.05).

This study systematically uncovers, for the first time, the oncogenic role and prognostic value of ABCB6 in OS, and delineates the complete "ABCB6/Fe²⁺/HIF1A/GPX4" axis that governs ferroptosis in OS cells. These findings not only provide a novel biomarker for molecular subtyping and prognostic evaluation of OS, but also establish a theoretical basis for therapeutic strategies that target the ABCB6/HIF1A/GPX4 axis to induce ferroptosis. Future investigations may explore the upstream regulators of ABCB6 and its potential utility in reversing chemoresistance in OS.