Metformin enhances alpelisib sensitivity in HER2+ breast cancer by suppressing cancer stemness and oncogenic signaling.

Introduction: Alpelisib, a selective PI3Kα inhibitor that targets PIK3CA mutations, is approved for hormone receptor-positive, HER2-negative breast cancer. However, its therapeutic potential in HER2-positive disease remains underexplored. Hyperglycemia, a frequent adverse event of alpelisib, limits its clinical application. Metformin, an antidiabetic drug with reported anticancer activity, may counteract alpelisib-induced hyperglycemia while enhancing its antitumor efficacy. Methods: We evaluated the effects of alpelisib alone and in combination with metformin in HER2-overexpressing breast cancer cell lines SK-BR-3 and BT-474. Cell viability, colony formation, and tumorsphere assays were performed to assess proliferation and stemness. Flow cytometry was used to analyze ALDH1-positive cell populations and cell-cycle distribution. Synergy was determined using the combination index method. Western blotting examined the effects on signaling pathways and stemness-associated proteins. Results: Alpelisib monotherapy inhibited proliferation, colony formation, and cancer stem cell features in both cell lines. Metformin enhanced these effects, demonstrating strong synergism. Combination treatment induced significant G0/G1 cell-cycle arrest, reduced ALDH1⁺ populations, and decreased tumorsphere formation. Mechanistically, co-treatment broadly suppressed receptor tyrosine kinase-mediated signaling, augmenting inhibition of PI3K/Akt/mTOR, MAPK/ERK, and JAK/STAT pathways, along with downregulation of c-Myc. Stemness-related proteins including β-catenin, Nanog, Sox2, KLF4, and LGR5 were also markedly reduced. Discussion: These findings indicate that metformin synergistically enhances the antitumor activity of alpelisib in HER2-positive breast cancer by inhibiting oncogenic signaling and stemness pathways. Beyond its metabolic benefit in mitigating hyperglycemia, metformin may potentiate PI3K-targeted therapies, supporting further preclinical and clinical evaluation of this combination strategy.