Estrogen receptor beta suppresses the androgen receptor oncogenic effects in triple-negative breast cancer

Abstract

Background:

Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer associated with poor prognosis and limited treatment options. The androgen receptor (AR) has emerged as a potential therapeutic target for luminal androgen receptor (LAR) TNBC. However, multiple studies have claimed that anti-androgen therapy for AR-positive TNBC only has limited clinical benefits. This study aimed to investigate the role of AR in TNBC and its detailed mechanism.

Methods:

Immunohistochemistry and TNBC tissue sections were applied to investigate AR and nectin cell adhesion molecule 4 (NECTIN4) expression in TNBC tissues. Then, in vitro and in vivo assays were used to explore the function of AR and estrogen receptor beta (ERβ) in TNBC. Chromatin immunoprecipitation sequencing (ChIP-seq), co-immunoprecipitation (co-IP), molecular docking method, and luciferase reporter assay were performed to identify key molecules that affect the function of AR.

Results:

Based on the TNBC tissue array analysis, we revealed that ERβ and AR were positive in 21.92% (32/146) and 24.66% (36/146) of 146 TNBC samples, respectively, and about 13.70% (20/146) of TNBC patients were ERβ positive and AR positive. We further demonstrated the pro-tumoral effects of AR on TNBC cells, however, the oncogenic biology was significantly suppressed when ERβ transfection in LAR TNBC cell lines but not in AR-negative TNBC. Mechanistically, we identified that NECTIN4 promoter –42 bp to –28 bp was an AR response element, and that ERβ interacted with AR thus impeding the AR-mediated NECTIN4 transcription which promoted epithelial–mesenchymal transition in tumor progression.

Conclusions:

This study suggests that ERβ functions as a suppressor mediating the effect of AR in TNBC prognosis and cell proliferation. Therefore, our current research facilitates a better understanding of the role and mechanisms of AR in TNBC carcinogenesis.

Introduction

Triple-negative breast cancer (TNBC) is characterized as lacking in estrogen receptor alpha (ERα), progesterone receptor (PR) expression, and human epithelial growth factor receptor 2 (HER2) amplification.^[1] Although great progress has been made in the diagnosis and treatment of breast cancer, TNBC is still recognized as a targetless breast cancer subtype with high aggressiveness, easy metastases, early recurrence, and drug resistance.^[1] To improve the clinical prognosis of patients with TNBC, novel therapeutic targets and the underlying mechanisms are urgently needed to be investigated.

Previously, Jiang et al^[2] published the genomic and transcriptomic landscape of TNBC, classifying TNBC into four transcriptome-based subtypes: luminal androgen receptor (LAR), immunomodulatory (IM), basal-like immune-suppressed (BLIS), and mesenchymal-like (MES). According to the four TNBC subtypes, the LAR subtype is characterized as androgen receptor (AR) expression, which makes up over 20% of all TNBC samples.^[2] Whether AR expression could be a potential therapeutic target has not been fully understood.^[3,4,5] A meta-analysis reported that AR expression was correlated with better clinical outcomes in breast cancer irrespective of ERα expression.^[6] However, a more recent clinical trial updated this conclusion based on a cohort study of 4147 female patients with invasive breast cancer, and had further demonstrated that AR expression was associated with better prognosis in ERα-positive breast cancers but was associated with worse prognosis in ERα-negative breast cancers including TNBC.^[7] The apparent contradiction implies the dual effects of AR expression in TNBC, and the paradoxical effects of AR across breast cancer with different ERα status indicated the potential molecular mechanisms between the two receptors. Different from ERα, estrogen receptor beta (ERβ) is expressed in 20–30% of TNBC and is elevated as a target for treating TNBC.^[1,8,9] ERβ has been studied for over twenty years since its discovery, yet its role in AR-positive TNBC has been elusive.^[10] Some studies showed a correlation between ERβ positivity and better survival in breast cancer patients;^[11,12] while other researchers demonstrated that ERβ activation inhibited TNBC cell invasion.^[12] Moreover, the mutual relationships between ERβ and AR have not been clearly investigated. In prostate cancer, ERβ has been reported to oppose AR and become a treating target.^[13] Research on AR-positive TNBC reported that ERβ expression increased the sensitivity to the potent AR inhibitor enzalutamide, inhibited the oncogenic role of AR through the phosphatidylinositide 3-kinases (PI3K)/protein kinase B (AKT) signaling pathway, and impeded AR from forming homodimers.^[14] However, whether and how ERβ influences the effect of AR in the LAR subtype of TNBC is still unclear.

In this research, we aimed to verify the promoting effect of AR activation on proliferation, migration, invasion, and metastasis of TNBC cells. On the other hand, we attempted to further investigate the mechanism by which the oncogenic role of AR is suppressed by the molecular interaction between ERβ and AR in the nucleus of TNBC cells, which interferes with the biological function of AR as a transcription factor. To further elucidate the critical role of ERβ-AR interaction in TNBC progression, downstream target genes mediated by AR will be screened, confirming that ERβ suppresses the oncogenic role of AR.

Methods

Clinical samples

Breast cancer tissue sections containing HBreD075Bc01 (75 cases) and HBreD090Bc01 (90 cases) were provided by Outdo Biotech (Shanghai, China). Histological parameters were scored by the criteria of the World Health Organization. Pathologic staging was determined by the current International Union Against Cancer tumor-lymph-node-metastasis classification. The ethical approvals are shown in Supplementary Materials, http://links.lww.com/CM9/B871.

Cell lines and culture

BT-549 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (22400089, Gibco, Carlsbad, USA) with 10 μg/mL insulin (12585014, Gibco); Hs578t cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (11995065, Gibco) with 10 μg/mL insulin (12585014, Gibco); MDA-MB-468 cells were cultured in DMEM (11995065, Gibco). BT-549, Hs578t, and MDA-MB-468 cell lines were purchased from the American Type Culture Collection (ATCC, USA). BT-549 and Hs578t are LAR TNBC cells, while MDA-MB-468 is AR-negative TNBC cells.^[15] All cells were supplemented with 10% fetal bovine serum (10270106, Gibco) and 1% penicillin/streptomycin (15140122, Gibco) in a 37℃ incubator with 5% CO₂. AR agonist dihydrotestosterone (DHT) (S4757) and inhibitor enzalutamide (Enza) (MDV3100) were obtained from Selleckchem (Houston, TX, USA). Dimethyl sulfoxide (DMSO) was used to dissolve these inhibitors and agonists, and they were stored at –20℃. Cells were treated with DHT at 100 nmol/L for 24 h or Enza at 20 μmol/L for 24 h. The same concentration of DMSO was used as the negative control treatment.

Lentiviral transduction

The BT-549 and Hs578t cells were transfected with pSLenti-EF1-Luc2-F2A-Puro-CMV-ESR2-3xFLAG-WPRE and pSLenti-EF1-Luc2-P2A-BSR-CMV-NECTIN4-HA-WPRE lentivirus. MDA-MB-468 cell line was transfected with pSLenti-EF1-Luc2-F2A-Puro-CMV-ESR2-3xFLAG-WPRE. Lentivirus was packaged by ObiO Technology (Shanghai, China) according to the manufacturer’s instructions and added to a final multiplicity of infection (MOI) of 10. Stable cell lines were obtained by selection with puromycin and blasticidin.

Immunohistochemistry (IHC)

IHC of breast tumor tissue samples was performed, as previously described,^[16] using antibodies against AR (#06-680, Millipore, Billerica, USA, 1:200), NECTIN4 (ab155692, Abcam, Cambridge, UK, 1:100), and ERβ (#14-9336-82, Invitrogen, Carlsbad, USA, 1:50). The two times scoring method based on the positive rate and staining intensity of tumor cells was used: the proportion of positive tumor cells <5% was 0, 5%–25% was 1, 26%–50% was 2, 50%–75% was 3, and >75% was 4. Dyeing intensity: 0 point for non-coloring; 1 point for light yellow; 2 points for dark yellow or brownish yellow; 3 points for tan particles. Multiply the two for its final result, and the expression was judged by the medians. For AR staining, 0–6 is AR low; >6 is AR high. For ERβ staining, 0–10 is ERβ low; more than 10 is ERβ high. For NECTIN4 staining, 0–6.25 is NECTIN4 low; >6.25 is NECTIN4 high.

Immunofluorescence staining for colocalization study

BT-549 and Hs578t cells were seeded and cultured in 12-well plates for 24 h. Then, the cells were fixed with 4% paraformaldehyde for 15 min, washed three times with phosphate buffered saline (PBS) for 5 min, and permeabilized with 0.1% Triton X-100 for 10 min. After blocking for 1 h in 3% bovine serum albumin (BSA) at 37℃, the cells were incubated overnight at 4℃ with primary antibodies anti-AR (ab108341, Abcam), anti-ERβ (#14-9336-82, Invitrogen), anti-E-cadherin (ab40772, Abcam), and anti-N-cadherin (ab76011, Abcam). Subsequently, the secondary antibodies were added for 1 h at 37℃ followed by counterstaining with 4′,6-diamidino-2′-phenylindole (DAPI). The cells were then observed and photographed under the confocal microscope.

Co-immunoprecipitation (co-IP) assays

To investigate AR (#06-680, Millipore, 1:50) and ERβ (#14-9336-82, Invitrogen, 1:50) protein interactions, co-IP was performed in BT-549 and Hs578t cell lines. Cells were treated with DHT at 100 nmol/L for 24 h. Cells were lysed in co-IP buffer (20 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 1% Triton X-100, and 1 mmol/L EDTA) containing protease inhibitors (Roche Applied Science, Mannheim, Germany) on ice for 30 min. Then, the cells were centrifuged, and the supernatant was collected, followed by incubation with primary antibodies and GammaBind Plus Sepharose (#17088601; GE Healthcare, Logan, USA) with gentle rocking overnight at 4℃. The next day, the mixture was pelleted, washed six times with cold 1× co-IP buffer, and then analyzed by western blotting.

Public database analysis

The mRNA data of TNBC patients were downloaded through The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/). We conducted a comparative analysis of four hub gene expressions among lymph node-positive and -negative groups via the Wilcoxon rank-sum test. We divided patients into high- and low-expression groups based on the median value and performed survival analysis of four hub genes through the “survival” R package (R Foundation for Statistical Computing, 2020, https://www.bioconductor.org). Bioinformatics were applied to predict the molecular docking of AR and ERβ. Firstly, we predicted and optimally constructed the protein three dimensional (3D) structures of AR and ERβ using the I-TASSER tool, and then we performed AR protein–ERβ protein molecular docking simulations through the online ZDOCK server (http://zdock.umassmed.edu/) for AR protein and ERβ protein molecular docking simulation.

Chromatin immunoprecipitation sequencing (ChIP-seq), chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR), and ChIP-quantitative PCR

ChIP-seq was performed using an anti-AR antibody, as previously described.^[17] Immunoprecipitated deoxyribonucleic acid (DNA) was purified after phenol extraction and genomic libraries were sequenced to 50 bp for an Illumina Hi-Seq 2000 (Illumina, San Diego, USA). ChIP-seq narrow peaks were called against the input using MACS2 software (version 2.0.9, P-value threshold = 1 × e^–5, https://pypi.python.org/pypi/MACS2). All the motifs were searched using the peak summits by extending 250 bp along both ends. Motif discovers using the Hypergeometric Optimization of Motif EnRichment (HOMER) packages (http://homer.salk.edu/homer/). And we used the HOMER’s peaks annotation function to annotate out-called peaks. The sequences of primers were designed for NECTIN4 promoter in chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) analysis. Fifteen primer sets of about 200 bp per primer were synthesized for the NECTIN4 promoter region. According to the data of ChIP-seq, qPCR primers for NECTIN4, DDX5, TUFT1, and CLDN4 were designed to perform ChIP-qPCR. Primers are shown in Supplementary Table 1, http://links.lww.com/CM9/B872. ChIP-PCR primers for NECTIN4 are shown in Supplementary Table 2, http://links.lww.com/CM9/B872.

Luciferase reporter assay

The wild-type or mutated sequences within the predicted binding sites of the promoter region of NECTIN4 were synthesized, inserted into a luciferase reporter plasmid (Outdo Biotech), and transfected into Hs578t cells. After 24 h, the cells were treated with DHT at 100 nmol/L or Enza at 20 μmol/L for 24 h. The luciferase activity was normalized to the Renilla luciferase activity.

Western blotting

Total proteins were extracted from TNBC cells using radioimmunoprecipitation assay buffer (RIPA) lysis buffer containing protease inhibitor cocktail (Roche Applied Science). The lysates were mixed with sodium dodecyl sulphate (SDS) loading buffer, boiled for 8 min, resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to Polyvinylidene Fluoride (PVDF) membranes (Millipore, Bedford, USA). After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies. The primary antibodies used include anti-AR (#06-680, Millipore, 1:1000), anti-ERβ (#14-9336-82, Invitrogen, 1:1000), anti-E-cadherin (ab40772, Abcam, 1:1000), anti-N-cadherin (ab76011, Abcam, 1:5000), anti-NECTIN4 (ab155692, Abcam, 1:1000), and anti-β-actin (ab8226, Abcam, 1:3000).

Wound-healing assay

Cells were seeded in six-well plates and grown to 90% confluence, and then, a wound was scratched in the cell monolayer with a 200 μL sterile pipette tip. Cells were cultured in a serum-free medium after removing floating cells with PBS. We photographed the edge of a scratch at the time 0 h and 24 h. Migration was quantified as a percentage of wound closure.

Cell invasion assay

Transwell chambers (Corning, Tewksbury, MA, USA) were coated with 10 μL of RPMI 1640-or DMEM-diluted Matrigel (BD Biosciences, USA), and 2 × 10⁴ cancer cells cultured in serum-free medium (200 μL) were added to the upper chamber. The medium (800 μL) containing 10% FBS was added to the lower chamber. The cells in the upper side of the chamber were carefully removed with a cotton swab after 24 h incubation. The migrating cells were fixed with 10% methanol for 30 min and then stained with crystal violet for 10 min.

Animal experiments

Animal studies were performed with the approval of the Institutional Animal Care and Use Committee of Nanjing Medical University (No. 2019-SRFA-244). The LAR TNBC cell line Hs578t was used in animal experiments due to its excellent tumorigenic ability.^[18] Female BALB/c nude mice 5-week-old were subcutaneously inoculated or injected by tail vein with 1 × 10⁷ Hs578t cells which were stably transfected with a control vector, ERβ lentivirus and/or NECTIN4 lentivirus. Mice were randomly assigned to receive DHT administration by intraperitoneal injection at the concentration of 0 mg/kg, 5 mg/kg, or 10 mg/kg (n = 5). Mice were monitored for 28 days and were sacrificed at the end of the experiment. Tumors were dissected and weighed. The metastases were monitored using the IVIS^@ Lumina II system (Caliper Life Sciences, Hopkinton, MA, USA) after injecting for 28 days.

Statistical analysis

Statistical analysis in this report was performed by SPSS v26 (SPSS Inc, Chicago, IL, USA). The figures were plotted by GraphPad Prism v7 (https://www.graphpad.com/scientific-software/prism/). Student’s t-test and Fisher’s exact test were used to examine the statistical significance of differences between groups. P-value less than 0.05 was considered as a statistically significant difference.

Results

AR exerts oncogenic effects in TNBC

To examine the relationship between AR expression and clinicopathological features of TNBC, IHC staining of AR expression was performed in a cohort of postoperative female patients with TNBC examined as invasive ductal carcinoma (IDC) (n = 159). Patients were grouped into two subgroups based on AR expression [Figure 1A]. IHC staining statistics showed that high AR expression was positively correlated with sentinel lymph node metastasis (χ² = 49.510, P <0.001), higher TNM stages (χ² = 7.937, P = 0.019), and pathological stages (χ² = 6.696, P = 0.035) [Supplementary Table 3, http://links.lww.com/CM9/B872]. This finding based on 159 surgical specimens of TNBC IDC demonstrated that AR expression was correlated with poor clinicopathological status.

Figure 1.

AR promotes proliferation, migration, invasion, and metastases of TNBC. (A) Rrepresentative H&E and IHC staining for AR in paraffin-embedded tissue of patients with TNBC. For AR staining, 0–6 is AR low; 6 is AR high. (B) Western blotting assay of AR expression in BT-549 and Hs578t cells following the indicated treatments. DHT increased the expression of AR in TNBC cell lines. Cells were treated with DHT at 100 nmol/L for 24 h. (C) Colony formation assays were performed to assess the proliferation of BT-549 and Hs578t cells following indicated treatments. DHT promoted the proliferation of TNBC cells and Enza decreased the proliferation of TNBC cells, which were treated with DHT at 100 nmol/L for 24 h or Enza at 20 μmol/L for 24 h. (D) EDU assay was applied to compare the cell proliferation ability of BT-549 and Hs578t cells following indicated treatments. DHT promoted the proliferation of TNBC cells and Enza decreased the proliferation of TNBC cells, which were treated with DHT at 100 nmol/L for 24 h or Enza at 20 μmol/L for 24 h. (E) Wound-healing assay showed increased cell migration after DHT treatment, and decreased cell migration after enzalutamide treatment. Cells were treated with DHT at 100 nmol/L for 24 h or Enza at 20 μmol/L for 24 h. (F) Subcutaneous xenograft experiments showed a dose-dependent increase in tumor weights after DHT treatments at the concentration of 0 mg/kg, 5 mg/kg, or 10 mg/kg (n = 5). (G) In vivo imaging of lateral tail-vein injection murine model showed a dose-dependent increase in lung metastases after DHT treatments at the concentration of 0 mg/kg, 5 mg/kg, or 10 mg/kg (n = 5). ns: No significance, ^*P <0.05, ^†P <0.01, ^‡P <0.005, ^§P <0.001. AR: Androgen receptor; CON: Control; DHT: Dihydrotestosterone; EDU: 5-ethynyl-2-deoxyuridine; Enza: Enzalutamide; H&E: Hematoxylin-eosin staining; IHC: Immunohistochemistry; TNBC: Triple-negative breast cancer.

To test whether there was a precise relationship between AR expression and clinicopathological status, we increased AR expression with or without DHT in TNBC cell lines including BT-549 and Hs578t, respectively [Figure 1B]. Cells were treated with DHT at 100 nmol/L for 24 h. It turned out that AR activation by adding DHT in TNBC cells significantly promoted the abilities of proliferation, migration, and invasion in both tumor cell lines. In contrast, inhibition of AR expression with adding Enza (20 μmol/L for 24 h) in TNBC cells impeded those cells’ biological activities, suggesting that AR could be a pro-tumoral element in TNBC progression [Figure 1C–E]. To further confirm these in vitro results, we performed the in vivo experiments using murine xenograft models to determine the effect of AR expression status on tumor biological behaviors. In situ and tail vein injection in tumor xenograft models showed the promoting effects on tumor growth of AR activation induced by DHT, which is in a dose-dependent manner (DHT concentration:0 mg/kg, 5 mg/kg, and 10 mg/kg) [Figure 1F,G]. Taken together, the above data demonstrated the oncogenic effects of AR on TNBC cells.

ERβ inhibits AR-mediated TNBC progression and function

ERα and ERβ are two subtypes of estrogen receptors with heterogeneous expression and various biological functions in breast cancers.^[19] ERβ expresses in 20–30% of TNBC and is evaluated as a target for precise treatment.^[1,19,20] The LAR TNBC is characterized by the expression of AR and its downstream effects.^[2] To investigate the prognostic value of ERβ to LAR TNBC, we analyzed the outcomes of 253 patients from the TCGA database using the Kaplan–Meier plotter analysis [Figure 2A]. The results showed that LAR patients with high ERβ expression had a significantly prolonged recurrence-free survival (RFS), compared with LAR patients with low ERβ expression, which indicated that ERβ was a potential predictor of good clinicopathological features (n = 253; hazard ratio [HR], 0.53; 95% confidence interval [CI], 0.36–0.78; P <0.05). To determine whether ERβ can impede the key biological functions of AR-positive TNBC, we stably overexpressed ERβ in two AR-positive TNBC cell lines, BT-549 and Hs578t. Colony formation assays showed a decreased proliferation of AR-positive TNBC cell lines after ERβ overexpression [Figure 2B]. In addition, overexpression of ERβ inhibited the other key malignant properties of AR-positive TNBC cells, including the capacities of migration and invasion based on the wound-healing and transwell assays [Figure 2C,D]. These results revealed that ERβ significantly inhibited the AR oncogenic effects in LAR TNBC cell lines.

Figure 2.

ERβ reverses the oncogenic effect of AR in TNBC. (A) Kaplan–Meier curve showed RFS of LAR patients with low or high ERβ expression in tumor tissues (n = 253, HR 95% CI = 0.53 [0.36–0.78], P = 0.001). (B) Colony formation assays were performed to assess the proliferation of BT-549 and Hs578t cells following the indicated treatments. DHT (100 nmol/L for 24 h) promoted the proliferation of TNBC cells while ERβ overexpression reversed the proliferative effects of AR. (C) Wound-healing assays of TNBC cells following the indicated treatments and ERβ transfection. DHT (100 nmol/L for 24 h) promoted the migration of TNBC cells while ERβ overexpression reversed the oncogenic effects of AR. (D) Transwell assays of TNBC cells following the indicated treatments. DHT (100 nmol/L for 24 h) promoted the invasion of TNBC cells while ERβ overexpression reversed the oncogenic effects of AR. (E) Western blotting assay showed the expression of E-cadherin and N-cadherin in TNBC cells following the indicated treatments. DHT (100 nmol/L for 24 h) increased the expression of N-cadherin and decreased the expression of E-cadherin in TNBC cells while ERβ overexpression reversed the effects. (F) Immunofluorescence showed cellular expression of E-cadherin and N-cadherin in BT-549 and Hs578t cells following the indicated treatments. DHT (100 nmol/L for 24 h) increased the expression of N-cadherin and decreased the expression of E-cadherin in TNBC cells while ERβ overexpression reversed the effects. ns: No significance, ^*P <0.05, ^†P <0.01, ^‡P <0.005, ^§P <0.001. AR: Androgen receptor; CI: Confidence interval; DAPI: 4′,6-diamidino-2-phenylindole; DHT: Dihydrotestosterone; ERβ: Estrogen receptor beta; HR: Hazard ratio; LAR: Luminal androgen receptor; RFS: Recurrence-free survival; TNBC: Triple-negative breast cancer.

To explore the related biological mechanisms underlying the observation that ERβ inhibits AR oncogenic effects on AR-positive TNBC, we dissected the epithelial -mesenchymal transition (EMT) alterations in tumor cells, which were reported to be associated with AR and one of the canonical malignant biology of epithelium-derived carcinogenesis.^[15,21] Compared with the control group, AR activation significantly increased N-cadherin expression and decreased E-cadherin expression, but the increased EMT phenotypic characteristics were notably impeded after ERβ overexpression [Figure 2E]. The immunofluorescence staining also demonstrated that in both AR-positive TNBC cell lines, ERβ overexpression reversed the effects of AR, impeding the EMT phenotype [Figure 2F]. Thus, we proposed that ERβ suppressed the oncogenic role of AR in AR-positive TNBC by inhibiting the AR-mediated EMT signaling pathways. To further determine the effects of ERβ on AR in TNBC, we utilized the AR-negative TNBC cell line MDA-MB-468 for additional experiments.^[15] Androgen agonist DHT failed to activate AR expression in this cell line [Supplementary Figure 1A, http://links.lww.com/CM9/B872]. In addition, the biological functions of AR-negative TNBC cells were barely influenced neither by AR activation nor by ERβ overexpression, including the abilities of proliferation, migration, and invasion [Supplementary Figure 1B–D, http://links.lww.com/CM9/B872]. Meanwhile, the abilities of proliferation, migration, and invasion were decreased after adding androgen inhibitor Enza (20 μmol/L for 24 h) in AR-positive TNBC cells, which could not be rescued by overexpression of ERβ [Supplementary Figure 2, http://links.lww.com/CM9/B872]. Thus, the above findings based on cell lines with different AR expression status indicated that ERβ reversed the protumoral effects of AR in LAR TNBC, but not in AR-negative TNBC.

ERβ interferes with AR-mediated transcription in TNBC

To explore how ERβ reversed the AR oncogenic roles in LAR TNBC, we first performed co-IP assays on TNBC cells to determine whether AR could physically bind with ERβ [Figure 3A]. The results with a co-IP of ERβ and AR suggested a direct interaction of the two proteins. To further probe into the specific molecular mechanism by which ERβ affects AR function in TNBC, we applied bioinformatics to predict the molecular docking of AR and ERβ. AR and ERβ proteins were found to be spatially bound to each other [Figure 3B]. Then, we performed immunofluorescence assays to examine whether the two proteins are co-localized in a TNBC cell. The results clearly showed that AR and ERβ were mostly co-localized in the cell nuclei [Figure 3C]. These results demonstrated that ERβ interacts with AR in TNBC nuclei at the protein level.

Figure 3.

ERβ interferes with AR-mediated transcription. (A) Co-IP assay was performed to investigate the interaction between AR and ERβ in TNBC cells following the indicated treatments. (B) The molecular docking of AR and ERβ predicted by bioinformatics. (C) Colocalization of ERβ and AR in TNBC treated with DHT (100 nmol/L for 24 h) was observed by confocal microscopy. AR: Androgen receptor; Co-IP: Co-immunoprecipitation; DAPI: 4′,6-diamidino-2-phenylindole, DHT: Dihydrotestosterone; ERβ: Estrogen receptor beta; TNBC: Triple-negative breast cancer; ERβ: Estrogen receptor beta; TNBC: Triple-negative breast cancer.

AR has been reported to be a transcription factor and could increase the expression of certain genes by binding to the AR response elements (AREs) in the promoters. To detect the possible mechanisms by which ERβ can regulate AR-mediated transcription, ChIP-seq analysis was performed in two groups of Hs578t cells, one control group and the other with ERβ overexpression [Supplementary Figure 3A, http://links.lww.com/CM9/B872]. ChIP-seq results showed the differentiated peaks bound by AR in each group. In total, we identified 1082 AR-mediated genes whose transcription was inhibited by ERβ (Gene set #1). For further filtering, we turned to the JASPAR database (https://jaspar.genereg.net/). According to the JASPAR database, 4995 genes were reported to be mediated by AR as a transcription factor (Gene set #2). Next, we took the intersection of Gene set #1 and Gene set #2 and narrowed it down to 130 genes whose transcription was impeded by ERβ expression in AR-positive TNBC [Supplementary Figure 3B, http://links.lww.com/CM9/B872]. The ChIP-seq and filtering analysis suggested that ERβ can impede AR from binding to the promoters of these 130 genes as a transcription factor. Supplementary Figure 3C, http://links.lww.com/CM9/B872 illustrates the top enriched AR-binding motifs. Among the 130 AR-mediated genes, we identified EMT-related genes including NECTIN4, TUFT1, DDX5, and CLDN4. ERβ hindered AR from binding to the promoter regions which was defined as 3000 bp upstream of the transcription start site (TSS) [Supplementary Figure 3D, http://links.lww.com/CM9/B872].

ERβ selectively inhibits AR-mediated NECTIN4 transcription in TNBC

To validate whether the expression of the four AR-mediated genes was inhibited by ERβ, we performed ChIP-qPCR in two AR-positive TNBC cell lines (BT-549 and Hs578t). At the transcription level, ERβ inhibited the expression of all four AR-mediated genes, and TUFT1 and NECTIN4 were the most significantly influenced in BT-549 and Hs578t cells [Figure 4A]. Then, we validated four AR-mediated genes whose expression was notably decreased by the AR–ERβ interaction.

Figure 4.

ERβ inhibits AR-mediated NECTIN4 transcription. (A) ChIP-qPCR was performed to assess the binding of AR with the promoter regions of representative EMT-related genes (NECTIN4, TUFT1, DDX5, CLDN4) in TNBC cells. (B) NECTIN4 was positively correlated with lymph node metastasis of TNBC patients from the TCGA database (n = 162, P = 0.028). (C) NECTIN4 was associated with poor OS of TNBC patients from the TCGA database (n = 162, P = 0.036). (D) ChIP-PCR was performed to assess the enrichment of AR on the NECTIN4 promoter in TNBC cells following the indicated treatments. By dividing the 3000 bp promoter region into 15 segments of 200 bp each, ChIP-PCR showed that ARE of NECTIN4 was located in 200 bp upstream of TSS (P15). (E) Dual-luciferase assays demonstrated the ARE in NECTIN4 promoter. ns: No significance, ^*P <0.05, ^†P <0.01, ^‡P <0.005, ^§P <0.001). AR: Androgen receptor; ARE: AR response element; CON: Control; EMT: Epithelial–mesenchymal transition; Mut: Mutation; NECTIN4: Nectin cell adhesion molecule 4; OS: Overall survival; TCGA: The Cancer Genome Atlas; TSS: Transcription start site; TNBC: Triple-negative breast cancer; WT: Wild type.

To determine whether these four AR-mediated genes were correlated with poor clinicopathological features, we analyzed the TNBC samples of the TCGA cohort (n = 162). Bioinformatics analysis showed that high expression of NECTIN4 was associated with lymph node metastasis (P = 0.028) and poor overall survival (OS) (P = 0.036) [Figure 4B,C], while the other three genes (TUFT1, DDX5, and CLDN4) did not meet the criteria [Supplementary Figure 4, http://links.lww.com/CM9/B872]. These results suggested that AR-mediated NECTIN4 transcription was hindered by ERβ, and NECTIN4 expression was negatively correlated with clinical prognosis.

Subsequently, we identified the ARE in the NECTIN4 promoter region using ChIP-PCR assay [Figure 4D]. By dividing the 3000 bp promoter region into 15 segments of 200 bp each, ChIP-PCR showed that ARE of NECTIN4 was located at 200 bp upstream of TSS in both AR-positive TNBC cell lines. Notably, ERβ significantly decreased the mRNA expression of this promoter segment, demonstrating the potent inhibition of ERβ to AR-mediated transcription of the NECTIN4 promoter. Therefore, we identified that AR bound to NECTIN4 promoter, and ARE located in –42 bp to –28 bp (GACGCACCTGTTCTG). For further consolidation, we performed dual-luciferase reporter assays in AR-positive TNBC cells. After transfecting Hs578t cells with plasmids of wild-type and mutated NECTIN4 promoters, we verified that GACGCACCTGTTCTG was the ARE of the NECTIN4 promoter [Figure 4E].

To explain the potential of NECTIN4 targeted therapy in AR-positive TNBC, we examined NECTIN4 expression in TNBC samples. We performed IHC staining of the same TNBC cohorts investigated previously for AR expression (n = 151) [Figure 5A]. IHC staining and the following analysis suggested that high NECTIN4 expression was positively correlated with lymph node metastases (P = 0.004) [Figure 5B]. The pathological examination of TNBC surgical specimens verified the clinical value of NECTIN4 in AR-positive TNBC. Moreover, antibody–drug conjugates (ADC) targeting NECTIN4, such as enfortumab vedotin, had been approved in locally advanced and metastatic urothelial carcinoma.^[22] The availability of ADC targeting NECTIN4 enhanced the value of this research, suggesting the possibility of applying it in treating advanced LAR TNBC. Collectively, we identified the pivotal mechanisms based on the observation that ERβ switched off AR oncogenic effects in TNBC. Through ERβ–AR interaction, ERβ prevented AR from binding to the ARE located in the NECTIN4 promoter and inhibited EMT in tumor progression [Figure 5C].

Figure 5.

NECTIN4 expression in TNBC and schematic diagram. (A) Immunohistochemical staining of NECTIN4 from TNBC tissues with different AR status. (B) Correlation between NECTIN4 expression and clinicopathological features of patients with TNBC. (C) The schematic diagram showed that ERβ attenuated the AR-mediated transcription of NECTIN4. ^*T stage of 2 sample in NECTIN4 low and high expression group were missed, respectively; ^†Nodal status of 22 samples in NECTIN4 low and 26 samples in NECTIN4 high expression group were missed, respectively. AR: Androgen receptor; ERβ: Estrogen receptor beta; IHC: Immunohistochemistry; NECTIN4: Nectin cell adhesion molecule 4; TNBC: Triple-negative breast cancer; TNBC: Triple-negative breast cancer.

To better evaluate the clinical application of our findings, we jointly examined the IHC staining of AR, ERβ, and NECTIN4 in a TNBC cohort [Supplementary Figure 5A, http://links.lww.com/CM9/B872]. In this TNBC cohort, 13.70% (20/146) of surgically excised specimens were double-expression of AR and ERβ, 24.66% (36/146) were AR-high and ERβ-low, 21.92% (32/146) were AR-low and ERβ-high, and 39.73% (58/146) were AR-low and ERβ-low [Supplementary Figure 5B, http://links.lww.com/CM9/B872]. In addition, the predominant NECTIN4 expression in AR-high TNBC samples indicated potential prospects of a combination strategy of endocrine therapy plus NECTIN4 inhibition [Supplementary Figure 5C, http://links.lww.com/CM9/B872]. Given that NECTIN4 had served as a novel target in treating urothelial cancer, together with the substantial percentage of NECTIN4 expression, our current study may provide a pre-clinical basis for the attempt to target NECTIN4 in treating AR-positive TNBC.

NECTIN4 is the pivotal component involved in the regulation of ERβ in LAR TNBC

To confirm the effects of NECTIN4 in AR-positive TNBC and to determine whether NECTIN4 was the downstream effector of ERβ-AR interaction, we designed the rescue experiment. AR-positive TNBC cells were transfected with NECTIN4 or/and ERβ. With increased NECTIN4 expression, we also found that EMT biomarkers were recovered [Figure 6A]. Afterward, we performed an in situ breast cancer model using the transfected Hs578t cells, suggesting that ectopic NECTIN4 expression attenuated the effects caused by ERβ [Figure 6B]. To explore the effects of NECTIN4 in TNBC metastasis, tail-vein injection models were used. Bioluminescence imaging tracked the lung metastasis of murine models, indicating the increasing metastases of NECTIN4-overexpressing TNBC cells [Figure 6C,D]. It is clearly shown that ectopic ERβ expression reversed the oncogenic effects of AR in vivo, while for TNBC cells transfected with both ERβ and NECTIN4, the oncogenic activities of AR recovered and cells harvested increased abilities of EMT, proliferation, and metastasis. Therefore, we concluded that NECTIN4 was not only the key downstream effector of ERβ-AR interaction but also a promising target against AR-positive TNBC.

Figure 6.

NECTIN4 overexpression eliminates the effect of ERβ. (A) Western blotting assay showed the expression of AR, NECTIN4, N-cadherin, and E-cadherin in BT-549 and Hs578t cells following the indicated treatments. (B) Subcutaneous xenograft experiments showed tumor sizes and weights after the indicated treatments (n = 5). (C) Bioluminescence imaging showed lung metastases after lateral tail vein injection of Hs578t cells in murine models (n = 5). (D) H&E staining of lung specimens derived from the murine model of lung metastasis. ns: No significance, ^*P <0.05, ^†P <0.01, ^‡P <0.005, ^§P <0.001. AR: Androgen receptor; ERβ: Estrogen receptor beta; H&E: Hematoxylin-eosin staining; NECTIN4: Nectin cell adhesion molecule 4.

Discussion

The effects and networks of hormone receptors are critical in the progression of breast cancer, making endocrine therapy one of the most widely used strategies against breast cancer, which is the most common malignancy in the world.^[23] For TNBC, though characterized as ERα, PR, and HER2 negative, the expression of AR is positive in 12%–46% of TNBC clinical reports.^[7,24] A systemic review indicated that AR was associated with prolonged OS and disease-free survival (DFS) in AR-positive breast cancers, irrespective of ER expression^[6] while a more recent study including 4147 female patients with invasive breast cancer reported different perspective.^[7] For ERα-positive breast cancers, AR expression was associated with improved prognosis, while for ERα-negative breast cancers, AR expression was related to worse prognosis in the first 5–10 years postdiagnosis,^[7] confirming the dual effects of AR. Simultaneously, a follow-up study of 72.7 months published the different associations of AR and clinical prognosis according to ER status.^[25] Additionally, pre-clinical and clinical trials demonstrated the therapeutic effects of AR inhibition and pro-tumoral effects of AR activation in AR-positive TNBC,^[3,4] confirming the oncogenic potential of AR in TNBC, and the reversible or dual effects of this protein. Due to the remarkable heterogeneity of TNBC and the absence of an explicit cut-off value for AR positivity,^[26] the mechanisms underlying the different roles of AR expression in TNBC remain elusive.

In this study, we verified the oncogenic effects of AR activation in AR-positive TNBC and demonstrated the therapeutic effects of AR inhibition on disease progression. Via bioinformatics analysis and phenotypic study, we discovered that ERβ predicted a good prognosis in LAR TNBC. ERβ and ERα share the same structural system,^[27] but regulate respectively transcription through the recruitment of different transcriptional co-regulators, which may explain the different roles played in gene activation or repression by ERβ and ERα.^[28] Mechanistically, ERβ inhibited AR as a transcription factor by molecular interaction. Next, we filtered and determined that NECTIN4 was the pivotal suppressed gene by ERβ–AR interaction. Without ERβ, AR bound to the NECTIN4 promoter upstream of the TSS, while with ERβ ectopic expression, this binding was significantly impeded, leading to decreased EMT progression and malignant biology. The tumor-suppressive functions of ERβ made it an attractive target in the seemingly targetless TNBC, and various research aimed to bring forth a selective ERβ agonist in cancer therapy. Previous research demonstrated that LY500307, a selective ERβ agonist, modulated the tumor immunity in the tumor microenvironment of lung metastases, which served as a potential target for immune checkpoint blockade-resistant TNBC.^[29] Since AR has been reported to be a transcription factor in tumor cells,^[30,31] we speculated that ERβ inhibited the oncogenic role of AR by interfering with the AR-mediated transcription. NECTIN4 is a transmembrane molecule of which the expression cannot be detected in normal tissues of adults.^[32] In a comparative study of breast cancer cell lines and tissues, NECTIN4 was reported to be a biomarker of disease progression, and serum NECTIN4 level was associated with tumor metastases.^[33] Interestingly, across breast cancer subtypes, NECTIN4 expression was positively correlated with basal-like properties which is similar to TNBC.^[33] Our research revealed that in TNBC cell lines, NECTIN4 increased N-cadherin expression, decreased E-cadherin expression, and promoted the metastases of TNBC cells via the EMT process, which corresponded with previous research on papillary thyroid carcinoma that NECTIN4 facilitated EMT via the PI3K/Akt pathway.^[34] Since NECTIN4 is almost exclusively expressed in tumor cells, the significant differences between tumor tissues and normal tissues bring about opportunities for NECTIN4-directed ADC such as enfortumab vedotin to benefit TNBC patients.^{[32,35,36,37,38]} To date, enfortumab vedotin has been proven by clinical trials to benefit pretreated patients with locally advanced or metastatic urothelial carcinoma.^[22,39]

This study revealed that ERβ is a molecular switch of the oncogenic AR, and the key mechanism is via the NECTIN4 and EMT process. It represents the establishment of the molecular and functional relationship between the two well-known receptors that is ERβ–AR interaction and NECTIN4 considered as one novel but popular anti-tumoral target. Moreover, this research is potently prevalent in clinical practice: (1) NECTIN4 is highly expressed in AR-positive breast cancers, and its expression was proved to be suppressed by ERβ–AR interaction. (2) NECTIN4 has been confirmed to be oncogenic in various solid tumors. (3) Valid NECTIN4 targeted drugs are available and in development. Therefore, the findings of this research provide a solid basis for a new strategy targeting TNBC, that is anti-androgen or ERβ activation therapy in combination with NECTIN4 targets. With intense research on NECTIN4, new targeting strategies emerging and two clinical trials are in process to investigate the application of NECTIN4 targets in advanced solid tumors including TNBC (NCT05234606 and NCT04561362, https://clinicaltrials.gov/). These two clinical trials are preliminary attempts to expand the indication of NECTIN4 targeted therapy. Simultaneously, our research indicates an even more accurate regimen against AR-positive TNBC, infusing new insights into the treatment of the seemingly targetless TNBC. Although there are novel findings that ERβ regulates AR-medicated transcription of NECTIN4, the current study has several limitations. Firstly, detailed isoforms of ERβ are not included in this report.^[20,40] Furthermore, the downstream regulatory mechanisms of AR-mediated NECTIN4 expression have not been fully investigated, which is our next research focus.

Collectively, our research proposes that ERβ functions as a molecular switch of AR oncogenic effects in TNBC. Moreover, we suggest that ERβ activation combined with NECTIN4 inhibition is a potential therapeutic strategy in LAR TNBC and validates the novel attempt of endocrine therapy in combination with immunotherapy in treating TNBC, which is the most aggressive subtype of breast cancer.

Funding

This work was supported by grants from the Key International Cooperation of the National Natural Science Foundation of China (No. 81920108029), the Key Foundation for Social Development Project of the Jiangsu Province, China (No. BE2021741), and Youth Fund of the National Natural Science Foundation of China (No. 82002783).

Conflicts of interest

None.