Abstract
Members of the nuclear receptor subfamily 2, group C (NR2Cs), despite sharing structural homology with established biomarkers like estrogen receptor (ER), progesterone receptor (PR), and androgen receptor (AR), remain underexplored in breast cancer (BC). This study aims to elucidate the subtype-specific prognostic significance of NR2Cs and investigate the molecular and immunomodulatory mechanisms of NR2C2 in restricting BC progression. We integrated transcriptomic data from TCGA with functional genomics to analyze NR2Cs expression and patient prognosis. Mechanistic landscapes were characterized using pathway enrichment, protein-protein interaction networks, immune infiltration algorithms, and pharmacogenomic drug sensitivity correlation. In vitro validation was performed using cell proliferation, colony formation, 5-Ethynyl-2'-deoxyuridine (EdU), and wound healing assays. In vivo antitumor efficacy and T-cell infiltration levels were evaluated using orthotopic human MDA-MB-231 xenografts in BALB/c nude mice and syngeneic murine EO771 tumors in immunocompetent C57BL/6 mice, supplemented by multiplexed immunohistochemistry. Both NR2C1 and NR2C2 were significantly downregulated in BC tissues compared with normal controls. NR2C2 demonstrated a robust, pan-subtype protective profile, correlating with prolonged survival across major molecular subtypes, particularly in ER-positive cohorts. In silico profiling connected NR2C2 to transforming growth factor-β (TGF-β) pathway inhibition and tumor-immune T-cell infiltration enhancement. In vitro, NR2C2 overexpression significantly suppressed BC cell proliferation, colony formation, DNA synthesis, and migratory capabilities. In vivo, NR2C2 overexpression markedly restricted tumor growth in both models. Mechanistically, this tumor-suppressive effect was characterized by cell-intrinsic growth arrest (decreased Ki-67), induced apoptosis (increased Cleaved Caspase-3), and significant inhibition of the TGF-β pathway (decreased Smad). Concurrently, in the immunocompetent syngeneic model, Nr2c2 overexpression promoted a robust recruitment of infiltrating CD4+ and cytotoxic CD8+ T cells into the tumor microenvironment. Our findings establish NR2C2 as a pivotal tumor suppressor in BC that restrains tumor progression by attenuating TGF-β signaling, thereby simultaneously suppressing cell-intrinsic proliferation and promoting T-cell infiltration into the tumor microenvironment. This study offers a promising therapeutic paradigm bridging orphan nuclear receptor biology with active cancer immunotherapy.