Deciphering the Effect of Methyl 4‑Hydroxybenzoate on Breast Cancer by Bioinformatics and Experiments

Methyl 4-hydroxybenzoate (MEP), a widespread environmental contaminant, is suspected to increase breast cancer (BRCA) risk; however, its molecular mechanism remains unclear. Using public health databases, we performed differential expression analysis, network toxicology modeling, and protein-protein interaction mapping to identify candidate biomarkers. Functional interrogation using gene set enrichment analysis, immune infiltration profiling, and molecular docking elucidated the mechanistic roles of these genes in BRCA. Single-cell transcriptomic analysis highlighted the cell populations most relevant to tumor initiation and tracked the biomarker expression patterns within these populations. Subsequently, in vitro assays revealed the direct effects of MEP on malignant behavior. CCNE1, CDK1, E2F1, and EZH2 emerged as core drivers converging on multiple oncogenic pathways. Fifteen immune cell subsets showed markedly altered infiltration patterns in tumors, with macrophages and related populations associated with disease progression. Among the identified biomarkers, CCNE1 exhibited the strongest predicted affinity for MEP (-6.2 kcal mol^-1), highlighting the CCNE1/CDK1 axis as a potential therapeutic target. Fibroblasts were identified as the key cellular context in which these biomarkers are transcriptionally primed during carcinogenesis. Functionally, MEP exposure enhanced malignant behaviors in MCF-7 cells and increased CCNE1, CDK1, E2F1, and EZH2 expression. Collectively, this integrated analysis indicates that MEP may promote BRCA development by acting on four central regulators within fibroblasts, providing mechanistic insights into the environmental origins of BRCA.