Modulation of Mitochondrial ERβ Expression Inhibits Triple-Negative Breast Cancer Tumor Progression by Activating Mitochondrial Function

BACKGROUND/AIMS: Breast cancer is a clinically and molecularly heterogeneous disease. Patients with triple-negative breast cancer (TNBC) have poorer outcomes than those with other breast cancer subtypes due to lack of effective molecular targets for therapy. The present study aimed to the identification of estrogen receptor (ER)β as a novel mitochondrial target in TNBC cells, together with underlying mechanisms. METHODS: Expression of ERβ in clinical breast samples were examined by qRT-PCR, immunohistochemistry and immunoblotting. Subcellular distribution and binding of ERβ-Grp75 was determined by confocal microscopic analysis, co-immunoprecipitation experiments, and limited-detergent extraction of subcellular organelles. The effect of mitocondrial ERβ(mitoERβ) overexpression on cell proliferation and cell cycle distribution were assessed CCK-8 assays and FACS. Mitochondrial ROS, membrane potential, and Ca²⁺ level were measured using the specific fluorescent probes Mito-Sox, TMRE, and Rhod-2AM. The tumorigenic effect of mitoERβ overexpression was assessed using an anchorage-independent growth assay, sphere formation and a mouse orthotopic xenograft model. RESULTS: ERβ expression was lower in tumor tissue than in adjacent normal tissue of patients with breast cancer, and low levels of mitochondrial ERβ (mitoERβ) also were associated with increased tumor recurrence after surgery. Overexpression of mitoERβ inhibited the proliferation of TNBC cells and tumor masses in an animal model. Moreover, overexpression of mitoERβ increased ATP production in TNBC cells and normal breast MCF10A cells, with the latter completely reversed by mitoERβ knockdown in MCF10A cells. Grp75 was found to positively regulate ERβ translocation into mitochondria via a direct interaction. Coimmunoprecipitation and subcellular fractionation experiments revealed that ERβ-Grp75 complex is stable in mitochondria. CONCLUSION: These results suggest that the up-regulation of mitoERβ in TNBC cells ensures proper mitochondrial transcription, activating the OXPHOS system to produce ATP. Studying the effects of mitoERβ on mitochondrial activity and specific mitochondrial gene expression in breast cancer might help predict tumor recurrence, inform clinical decision-making, and identify novel drug targets in the treatment of TNBC.