Site-specific CpG hypermethylation contributes to breast cancer progression by GLP-1R down-regulation

Breast cancer (BC) is the most prevalent malignancy in women. Metabolic alterations contribute to BC progression and therapy resistance. Despite improvement in the survival rate, its incidence continues to rise. Metabolic syndrome supports BC onset and progression, affecting incidence, prognosis and treatment response efficacy. Recently, the repositioning of antidiabetic drugs for cancer treatment has been investigated with encouraging outcome on cancer prevention especially upon glucagon-like peptide-1 receptor agonists (GLP-1RAs). Therefore, BC patients treated with GLP-1RAs exhibited significantly improved all-cause overall survival compared with non-users. Despite the clinical relevance of GLP-1R targeting, its involvement in BC is barely investigated. GLP-1R was investigated in 1) public BC patient transcriptomic or DNA methylation datasets; 2) two independent BC cohorts at diagnosis; 3) BC cell lines and 4) an orthotopic triple-negative BC (TNBC) mouse model. Integrated analyses, validated by immunohistochemistry, revealed a progressive GLP-1R decline from normal ductal epithelium to invasive BC, inversely associated with tumor aggressiveness. Hypermethylation of the cg15041550 CpG site within the GLP-1R regulatory region was identified as a silencing event linked to the normal-to-invasive transition. Methylated DNA immunoprecipitation and pharmacological modulation confirmed the epigenetic sensitivity of GLP-1R expression. GLP-1R restoration reduced proliferation in vitro and metastatic spread in vivo, while phospho- proteomics suggested that GLP-1R loss activates CDK1 and mTOR signaling. In TNBC patients, low GLP-1R expression and high cg15041550 methylation were associated with metastasis, reduced pathological complete response, and poorer disease- free survival. These findings identify cg15041550 methylation as an epigenetic switch in BC progression and support GLP-1R downregulation as a prognostic biomarker and potential therapeutic target in invasive BC.