PFAS is associated with perineural invasion in triple-negative breast cancer with a potential role for Cathepsin D dysregulation: a multi-omics and experimental study.

Per- and polyfluoroalkyl substances (PFAS) are persistent pollutants linked to breast cancer (BC), but their role in perineural invasion (PNI) of triple-negative breast cancer (TNBC) is unclear. Cathepsin D (CTSD), a lysosomal protease, is hypothesized to mediate PFAS-induced PNI, though systematic evidence is lacking. We integrated multi-omics data from TCGA-BRCA, METABRIC, and single-cell RNA-seq datasets. Analyses included differential gene expression, Mendelian randomization, consensus clustering, and machine learning for prognostic modeling. Single-cell analyses were performed using Seurat, Monocle2, and CellChat. GraphBan screened natural CTSD-binding compounds, with binding affinity evaluated by molecular docking and dynamics simulations. Experimental validation included immunohistochemistry, immunofluorescence, Transwell, and Western blot assays. We identified 5 PFAS-associated PNI-related genes (PPGs), with CTSD central to TNBC PNI. PPG-based molecular subtyping revealed a high-risk subgroup exhibiting enhanced epithelial-mesenchymal transition (EMT) activity, proliferation capacity, and significantly poorer overall survival. The PPG-based prognostic model effectively stratified patient outcomes and immunotherapy response. Mendelian randomization confirmed a causal link between genetically predicted CTSD levels and BC risk. Single-cell analysis showed CTSD specifically enriched in myeloid cells; CTSD⁺ myeloid cells displayed immunosuppressive signatures and therapy resistance. CTSD⁺ epithelial cells interacted with cancer-associated fibroblasts via FGF signaling and showed altered metabolism. GraphBan predicted and experiments confirmed Aurantio-obtusin as a high-affinity CTSD inhibitor. Molecular simulations demonstrated stable binding of both PFAS and Aurantio-obtusin to CTSD. Histologically, elevated CTSD expression co-localized with CD68⁺ macrophages in PNI-positive TNBC tissues, while Aurantio-obtusin suppressed CTSD expression and inhibited TNBC cell proliferation and migration. This study suggests that PFAS exposure is associated with PNI and malignant progression in TNBC, potentially involving dysregulation of CTSD. The robust PPG-based prognostic signature and the natural inhibitor Aurantio-obtusin offer novel biomarkers and a potential therapeutic strategy for mitigating PFAS-related cancer risks.