Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2‑positive breast cancer

Despite advances in HER2‑targeted therapy for HER2‑positive breast cancer, resistance to trastuzumab and tumor recurrence remain major barriers to durable outcomes. The present study evaluated the therapeutic potential of ebastine, a second‑generation H1‑antihistamine, as a repurposing candidate to overcome trastuzumab resistance by targeting HER2 signaling and cancer stem cell (CSC)‑associated phenotypes in HER2‑positive breast cancer cells. Molecular docking studies revealed that ebastine bound to the ATP‑binding site of the HER2 tyrosine kinase domain, thereby suppressing the phosphorylation of HER2, p95HER2 and HER3, as assessed by immunoblotting. Immunoprecipitation assay further demonstrated that this binding disrupted HER2/HER3 and HER2/EGFR heterodimerization, leading to reduced downstream AKT activation. Ebastine significantly decreased aldehyde dehydrogenase (ALDH)1 activity, decreased the CD44^high/CD24^low CSC‑like population, as assessed by flow cytometry, and inhibited mammosphere formation. In a trastuzumab‑resistant xenograft model, ebastine markedly suppressed tumor growth, decreased the Ki‑67 proliferation index and angiogenesis and induced apoptosis. These effects were accompanied by decreased expression of HER2, HER3, ALDH1, CD44, and vimentin in tumor tissues, as determined by immunohistochemistry. Furthermore, serum biochemical analyses revealed no significant hepatotoxicity or nephrotoxicity, indicating a favorable in vivo safety profile. These findings demonstrated that ebastine effectively disrupts key pathways involved in CSC‑like traits and HER2 activity, even under trastuzumab‑resistant conditions. Its multifaceted inhibitory effects support the repositioning of ebastine as a promising therapeutic strategy for treating refractory HER2‑positive breast cancer.