GPER as an actionable target in endocrine resistant breast cancer

Background: The G protein-coupled estrogen receptor (GPER) mediates rapid non-genomic estrogen signaling and has been implicated in breast cancer progression and resistance to endocrine therapy. Notably, tamoxifen functions as a GPER agonist, potentially promoting tumor growth despite antagonizing estrogen receptor alpha (ERalpha). Resistance to endocrine therapy therefore remains a major clinical challenge, underscoring the critical need for therapeutic strategies that effectively target both ERalpha and GPER. This study aimed to define the clinical and functional significance of GPER across breast cancer subtypes, define its clinical subcellular localization and functional significance and explore its association with the tumor microbiome and identify pharmacologic approaches to target it. Methods: GPER expression was assessed in breast cancer subtypes using immunohistochemistry, Western blotting, and RT-qPCR. GPER function and signaling were evaluated using CRISPR/Cas9 knockdown and overexpression models, intracellular calcium assays, and pharmacologic inhibition with Ormeloxifene, while tumor-associated microbiota was profiled by 16S rRNA sequencing to assess associations with tumor aggressiveness and molecular subtype. Results: GPER was broadly expressed across all breast cancer subtypes, with significantly higher expression in metastatic tumors, highlighting its relevance as a therapeutic target. Genetic suppression of GPER reduced proliferation, migration, and clonogenic survival in both breast cancer cells and organoid models. We report for the first time that the clinically approved contraceptive ormeloxifene functions as a dual ERalpha-GPER inhibitor, suppressing estrogen- and GPER-mediated signaling by blocking G1-induced calcium mobilization, reducing GPER protein levels, inhibiting tumor growth across hormone receptor-positive, HER2-enriched, and triple-negative subtypes, and reversing tamoxifen resistance. This was supported by molecular docking analyses demonstrating direct binding to both receptors. Microbiome profiling using 16S rRNA sequencing further identified subtype-specific bacterial signatures associated with aggressive disease features. Combination studies with CDK and PARP inhibitors revealed enhanced therapeutic responses in the context of GPER-targeted therapy. Statistical analyses included multigroup comparisons, diversity metrics, and correlation analyses to define molecular, functional, and microbial determinants of aggressive breast cancer phenotypes. Conclusions: This study establishes GPER as a key mediator of endocrine resistance and identifies ormeloxifene as a clinically translatable dual ERalpha-GPER inhibitor, providing a translatable strategy to overcome endocrine resistance in breast cancer. Dual targeting of ERalpha and GPER represents a distinct therapeutic approach, while integration of tumor microbiome signatures offers additional opportunities for precision oncology.