Identification of ferroptosis-related biomarker in breast cancer and analysis of its role in breast cancer progression

Background Ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation, has emerged as a crucial biological process in suppressing tumor growth. Accumulating evidence suggests that the dysregulation of ferroptosis is closely linked to the progression and therapeutic resistance of breast cancer (BC). However, the specific mechanisms driving ferroptosis in BC progression remain to be fully elucidated. Objectives This study aims to elucidate the mechanisms underlying BC progression. Methods Proteomic data from the CPTAC database (PDC000120) were analyzed to identify differentially expressed proteins (DEPs). Ferroptosis-related genes were obtained from the GeneCard database, and an intersection with DEPs was performed to obtain common targets. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to characterize these targets. Weighted gene co-expression network analysis (WGCNA) was employed to identify clinical modules, and their intersection with the common targets yielded core targets. Furthermore, machine learning algorithms were utilized to screen for hub genes. The tumor immune microenvironment was evaluated using the CIBERSORT algorithm. Finally, in vitro experiments, including Western blotting, EdU assays, flow cytometry, and biochemical measurements, were performed to validate the function of the selected gene. Results A total of 76 ferroptosis-related DEPs were identified. WGCNA identified the “red” and “turquoise” modules as the most clinically significant, and 39 core targets were obtained by intersecting module genes with the DEPs. Through machine learning algorithms, BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) and lysine-specific demethylase 5C (KDM5C) were identified as candidate hub genes. Immune infiltration analysis revealed that BUB1B and KDM5C expression was significantly correlated with various immune cells, including NK cells and macrophages. Pan-cancer analysis indicated that BUB1B exhibited more extensive differential expression compared to KDM5C and was upregulated in BC tissues and cell lines. Functional experiments demonstrated that silencing BUB1B significantly inhibited BC cell proliferation, promoted apoptosis, and induced ferroptosis, evidenced by increased Fe 2+ , malondialdehyde, and lipid reactive oxygen species levels, as well as decreased glutathione levels. Further assays revealed that BUB1B knockdown inhibited GPX4 and promoted ACSL4 protein expression in BC cells. Conclusion This study systematically identified BUB1B as a key ferroptosis-related gene associated with BC progression. BUB1B promoted tumor malignancy by regulating cell proliferation, apoptosis, and ferroptosis. These findings suggest that BUB1B holds promise as a potential therapeutic target for BC.