Proteomic Analysis of Mixed Cell Membrane Extracts From Triple-Negative Breast Cancer Cell Lines Revealed Broad Metabolic Reprogramming Associated With Tumor Progression.

Triple-negative breast cancer (TNBC) represents the most aggressive subtype with limited therapeutic options and the poorest clinical outcomes. Using high-resolution nUPLC-QTOF-MS/MS, we performed comprehensive proteomic profiling of TNBC cell lines representing disease progression stages: HCC-1937 (stage IIB), HCC-1599 (stage IIIA), and MDA-MB-231 (stage IV), benchmarked against nontumorigenic MCF-12F cells. From 11,864 initially detected proteins, 5623 high-confidence proteins were retained after quality filtering, spanning a dynamic range of six orders of magnitude. Differential abundance protein analysis identified distinct stage-associated proteomics profiles, finding 381, 490, and 550 differentially abundant proteins (DAPs) in HCC-1937, HCC-1599, and MDA-MB-231, respectively, with no shared DAPs among the three TNBC cell lines. Sparse PLS-DA and k-means clustering analyses showed distinct clustering patterns among TNBC cell lines. STRING network topology analysis uncovered dysregulated hubs in oxidative phosphorylation, mitochondrial electron transport chain complexes, including multiple subunits of Complex I (NDUFB3, NDUFB9, NDUFB10, NDUFB11), Complex III (UQCRC1, UQCRC2, UQCRB, UQCRFS1), and Complex IV (COX4I1, MT-CO2) in HCC-1937 and MDA-MB-231 cells, glycolytic enzymes, and cytoskeletal remodeling proteins. Functional enrichment analysis revealed significant alterations in metabolic pathways, including coordinated upregulation of glycolysis-associated proteins and downregulation of oxidative phosphorylation-related proteins. Cancer hallmark analysis identified enrichment in tissue invasion/metastasis (FDR < 0.001), immune evasion, genomic instability, and inflammatory signaling across all TNBC stages. The mitochondrial ribosomal proteins MRPL24, MRPL49, MRPL58, MRPS18B, and MRPS34 were found downregulated, which likely impairs assembly of functional respiratory chain complexes across multiple TNBC lines, which provides mechanistic insight into the observed OXPHOS suppression. In addition, the coordinated downregulation of PMPCA, the mitochondrial processing peptidase and TOMM22 and TIMM10, which are members of the mitochondrial import machinery, also contributes to the impaired mitochondrial function. These stage-resolved proteomics landscapes provide mechanistic insights into TNBC progression and identify potential therapeutic vulnerabilities and prognostic biomarker candidates for precision oncology. Data are available via ProteomeXchange with identifier PXD071351.