Metformin Enhances Alpelisib Sensitivity in HER2+ Breast Cancer by Suppressing Cancer Stemness and Oncogenic Signaling

Yujie Shi^1,2*Peijia Niu³Qiong Cheng⁴Lexia Chen³Yahan Weng³Xiaohe Yang^5*

• ¹Department of Pathology, People’s Hospital of Zhengzhou University, Zhengzhou, China
• ²Department of Pathology, Fuwai Central China Cardiovascular Hospital, Zhengzhou, China
• ³Fuwai Central China Cardiovascular Hospital, Zhengzhou, China
• ⁴People’s Hospital of Zhengzhou University, Zhengzhou, China
• ⁵North Carolina Central University, Kannapolis, United States

Alpelisib, a PIK3CA-targeting PI3Kα inhibitor, is approved for HR+/HER2− breast cancer, while its efficacy in HER2+ disease remains less studied. Hyperglycemia, a frequent adverse effect, limits alpelisib's clinical use. Metformin, a widely used anti-diabetic agent with reported anti-cancer activity, offers a potential strategy to both counteract alpelisib-induced hyperglycemia and enhance its therapeutic effect. Here, we investigated the therapeutic potential of combining alpelisib with metformin in HER2-overexpressing breast cancer cell lines, SK-BR-3 and BT-474. Alpelisib monotherapy inhibited proliferation, colony formation, and cancer stem cell properties, whereas metformin significantly enhanced these effects, demonstrating strong synergy by combination index analysis. The combination induced significant cell-cycle arrest and reduced stemness, as evidenced by decreased ALDH1+ populations, diminished tumorsphere formation, and inhibition of anchorage-independent growth. Mechanistically, this regimen exerted broad suppression of receptor tyrosine kinase signaling, amplifying inhibition of PI3K/Akt/mTOR effectors (p-Akt, mTOR, S6K), MAPK/ERK and JAK/STAT pathways (p-ERK1/2, p-STAT3), and c-Myc. Importantly, it also targeted stemness-associated networks, downregulating β-catenin, Nanog, Sox2, KLF4, and LGR5, providing a mechanistic rationale for its potent anti-stemness activity. Together, these findings demonstrate that metformin synergistically enhances its anti-tumor efficacy, in addition to its ability to mitigate hyperglycemia, through inhibition of oncogenic signaling and stemness pathways. This dual benefit highlights the translational potential of incorporating metformin into PI3K inhibitor–based regimens to improve therapeutic outcomes, particularly in HER2+ breast cancer, and provides strong rationale for further preclinical and clinical exploration of this combination therapy.