4-Hydroxyestradiol Promotes Breast Carcinogenesis via Androgen Receptor: Evidence from Urinary Metabolite Profiling and Functional Studies

Objective Breast cancer (BC) is a major global health threat with increasing incidence, and estrogen metabolites are implicated in its pathogenesis. To investigate alterations in urinary estrogen metabolites in BC patients and elucidate the oncogenic role and molecular mechanism of the metabolite 4-hydroxyestradiol (4-OH-E2) in BC progression. Methods A total of 126 treatment-naive BC patients and 103 healthy women were recruited to detect urinary estrogen metabolites using gas chromatography-tandem mass spectrometry (GC-MS/MS). MCF10A cells were treated with estradiol (E2) or 4-OH-E2 for 8 weeks (designated MCF10A-E and MCF10A-H, respectively), followed by assessments of cell morphology, migration (Transwell, scratch assay), invasion (Transwell), colony formation, and cell cycle (flow cytometry). Tumorigenicity assays were performed in BALB/c nude mice. Potential targets of 4-OH-E2 were predicted via SwissTargetPrediction and validated using GEPIA database. Loss-of-function experiments (AR silencing) were conducted to explore the role of androgen receptor (AR) in BC cells, with qRT-PCR, Western blot, and functional assays (proliferation, migration, invasion) used for verification. Results BC patients demonstrate dysregulated estrogen metabolism, marked by significantly elevated urinary 4-OH-E2. Chronic 4-OH-E2 exposure drives malignant transformation in mammary epithelial cells, enhancing tumorigenic phenotypes in vitro and in vivo. AR is a critical mediator of 4-OH-E2’s oncogenic effects, and its loss-of-function accelerates BC progression. Conclusion 4-OH-E2 is significantly elevated in BC patients and promotes breast carcinogenesis by enhancing cell malignant phenotypes and tumorigenicity, with AR as a critical target. These findings highlight 4-OH-E2 and AR as potential targets for BC prevention and treatment.