Metformin reduces basal subpopulation and attenuates mammary epithelial cell stemness in FVB/N mice

Metformin shows promise in breast cancer prevention, but its underlying mechanisms remain unclear. This study investigated the impact of metformin on the repopulation dynamics of mammary epithelial cells (MECs) and the signaling pathways in non-tumorigenic FVB/N mice. This study aimed to enhance our understanding of the role of metformin in reducing the susceptibility of MECs in premalignant tissues to oncogenic factors. In this study, female mice were administered 200 mg/kg/day of metformin via intraperitoneal (i.p.) injection from 8 to 18 weeks of age. After this treatment period, morphogenesis, flow cytometry, analyses of MEC stemness, and RNA sequencing were performed. The study findings indicated that metformin treatment in adult mice reduced mammary gland proliferation, as demonstrated by decreased Ki67+ cells and lateral bud formation. Additionally, metformin significantly reduced both basal and mammary repopulating unit subpopulations, indicating an impact on mammary epithelial cell repopulation. Mammosphere, colony-forming cell, and 3D culture assays revealed that metformin adversely affected mammary epithelial cell stemness. Furthermore, metformin downregulated signaling in key pathways including AMPK/mTOR, MAPK/Erk, PI3K/Akt, and ER, which contribute to its inhibitory effects on mammary proliferation and stemness. Transcriptome analysis with RNA sequencing indicated that metformin induced significant downregulation of genes involved in multiple critical pathways. KEGG-based pathway analysis indicated that genes in PI3K/Akt, focal adhesion, ECM-receptor, small cell lung cancer and immune-modulation pathways were among the top groups of differentially regulated genes. In summary, our research demonstrates that metformin inhibits MEC proliferation and stemness, accompanied by the downregulation of intrinsic signaling. These insights suggest that the regulatory effects of metformin on premalignant mammary tissues could potentially delay or prevent the onset of breast cancer, offering a promising avenue for developing new preventive strategies.