Estrogen Receptor Beta Suppresses the Androgen Receptor Oncogenic Effects in Triple-Negative Breast Cancer
Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the absence of estrogen receptor alpha (ERα), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) amplification. This subtype is associated with poor prognosis, early recurrence, and limited treatment options. Among the various molecular targets explored for TNBC, the androgen receptor (AR) has emerged as a potential therapeutic target, particularly in the luminal androgen receptor (LAR) subtype, which constitutes over 20% of TNBC cases. However, the clinical benefits of anti-androgen therapy in AR-positive TNBC have been limited, suggesting a need for a deeper understanding of the role and mechanisms of AR in TNBC progression.
This study investigates the oncogenic effects of AR in TNBC and explores the role of estrogen receptor beta (ERβ) in suppressing AR-mediated tumor progression. The research employs a combination of immunohistochemistry, in vitro and in vivo assays, chromatin immunoprecipitation sequencing (ChIP-seq), co-immunoprecipitation (co-IP), molecular docking, and luciferase reporter assays to elucidate the molecular mechanisms underlying the interaction between AR and ERβ in TNBC.
AR Exerts Oncogenic Effects in TNBC
The study begins by examining the relationship between AR expression and clinicopathological features in TNBC. Immunohistochemistry (IHC) staining of AR expression was performed on 159 TNBC tissue samples, revealing that high AR expression was positively correlated with sentinel lymph node metastasis, higher TNM stages, and pathological stages. These findings suggest that AR expression is associated with poor clinicopathological status in TNBC.
To further validate the oncogenic role of AR, the researchers conducted in vitro experiments using TNBC cell lines, including BT-549 and Hs578t, which are LAR TNBC cells, and MDA-MB-468, an AR-negative TNBC cell line. Treatment with the AR agonist dihydrotestosterone (DHT) significantly promoted the proliferation, migration, and invasion of AR-positive TNBC cells, while the AR inhibitor enzalutamide (Enza) impeded these biological activities. These results were corroborated by in vivo experiments using murine xenograft models, where AR activation induced by DHT promoted tumor growth in a dose-dependent manner.
ERβ Inhibits AR-Mediated TNBC Progression and Function
The study then shifts focus to ERβ, which is expressed in 20–30% of TNBC and has been evaluated as a target for precise treatment. Analysis of 253 TNBC patients from The Cancer Genome Atlas (TCGA) database revealed that high ERβ expression was associated with prolonged recurrence-free survival (RFS) in LAR TNBC patients, indicating a potential prognostic value for ERβ.
To determine whether ERβ can inhibit the oncogenic effects of AR, the researchers overexpressed ERβ in AR-positive TNBC cell lines. Colony formation assays, wound-healing assays, and transwell assays demonstrated that ERβ overexpression significantly inhibited the proliferation, migration, and invasion of AR-positive TNBC cells. Furthermore, ERβ overexpression reversed the epithelial-mesenchymal transition (EMT) phenotype induced by AR activation, as evidenced by increased E-cadherin expression and decreased N-cadherin expression.
ERβ Interferes with AR-Mediated Transcription in TNBC
The molecular mechanisms underlying the inhibitory effects of ERβ on AR were explored through co-immunoprecipitation (co-IP) assays and bioinformatics analysis. The results indicated a direct physical interaction between AR and ERβ, with the two proteins co-localized in the nuclei of TNBC cells. Bioinformatics prediction of molecular docking further supported the spatial binding of AR and ERβ.
ChIP-seq analysis was performed to identify AR-mediated genes whose transcription was inhibited by ERβ. The study identified 130 genes, including EMT-related genes such as NECTIN4, TUFT1, DDX5, and CLDN4. Among these, NECTIN4 was found to be the most significantly influenced by ERβ, with its expression negatively correlated with clinical prognosis in TNBC patients.
ERβ Selectively Inhibits AR-Mediated NECTIN4 Transcription in TNBC
The researchers then focused on NECTIN4, a transmembrane molecule that promotes EMT and tumor progression. ChIP-PCR and dual-luciferase reporter assays confirmed that AR binds to the NECTIN4 promoter at a specific AR response element (ARE) located 42 bp to 28 bp upstream of the transcription start site (TSS). ERβ overexpression significantly decreased the mRNA expression of NECTIN4, demonstrating the potent inhibition of ERβ on AR-mediated transcription of the NECTIN4 promoter.
IHC staining of TNBC tissue samples revealed that high NECTIN4 expression was positively correlated with lymph node metastasis, further supporting its role in TNBC progression. The study also highlighted the potential of NECTIN4-targeted therapies, such as enfortumab vedotin, which has been approved for the treatment of locally advanced and metastatic urothelial carcinoma.
NECTIN4 is the Pivotal Component Involved in the Regulation of ERβ in LAR TNBC
To confirm the role of NECTIN4 as the downstream effector of ERβ-AR interaction, the researchers conducted rescue experiments in AR-positive TNBC cells. Overexpression of NECTIN4 reversed the inhibitory effects of ERβ on EMT biomarkers, tumor growth, and metastasis. In vivo experiments using murine models demonstrated that ectopic NECTIN4 expression attenuated the effects of ERβ, leading to increased tumor growth and lung metastasis.
Discussion
The study concludes that ERβ functions as a molecular switch that suppresses the oncogenic effects of AR in TNBC. By interacting with AR in the nucleus, ERβ prevents AR from binding to the NECTIN4 promoter, thereby inhibiting EMT and tumor progression. These findings provide a solid basis for a new therapeutic strategy targeting AR-positive TNBC, combining anti-androgen or ERβ activation therapy with NECTIN4 inhibition.
The research highlights the potential of NECTIN4-targeted therapies, such as enfortumab vedotin, for the treatment of AR-positive TNBC. Given the high expression of NECTIN4 in AR-positive TNBC and its role in promoting tumor progression, targeting NECTIN4 could offer a novel approach to treating this aggressive subtype of breast cancer.
Conclusion
This study reveals that ERβ inhibits the oncogenic effects of AR in TNBC by interfering with AR-mediated transcription of NECTIN4. The findings suggest that ERβ activation combined with NECTIN4 inhibition could be a promising therapeutic strategy for LAR TNBC. The research underscores the importance of understanding the complex interactions between hormone receptors in breast cancer and provides new insights into the development of targeted therapies for TNBC.
doi.org/10.1097/CM9.0000000000002930
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