Proteomic Characterization of a 3D HER2+ Breast Cancer Model Reveals the Role of Mitochondrial Complex I in Acquired Resistance to Trastuzumab

HER2‐targeted therapies, such as Trastuzumab (Tz), have significantly improved the clinical outcomes for patients with HER2+ breast cancer (BC). However, treatment resistance remains a major obstacle. To elucidate functional and metabolic changes associated with acquired resistance, we characterized protein profiles of BC Tz‐responder spheroids (RSs) and non‐responder spheroids (nRSs) by a proteomic approach. Three‐dimensional cultures were generated from the HER2+ human mammary adenocarcinoma cell line BT‐474 and a derived resistant cell line. Before and after a 15‐day Tz treatment, samples of each condition were collected and analyzed by liquid chromatography– mass spectrometry. The analysis of differentially expressed proteins exhibited the deregulation of energetic metabolism and mitochondrial pathways. A down‐regulation of carbohydrate metabolism and up‐regulation of mitochondria organization proteins, the tricarboxylic acid cycle, and oxidative phosphorylation, were observed in nRSs. Of note, Complex I‐related proteins were increased in this condition and the inhibition by metformin highlighted that their activity is necessary for nRS survival. Furthermore, a correlation analysis showed that overexpression of Complex I proteins NDUFA10 and NDUFS2 was associated with high clinical risk and worse survival for HER2+ BC patients. In conclusion, the non‐responder phenotype identified here provides a signature of proteins and related pathways that could lead to therapeutic biomarker investigation.