Androgen receptor (AR) is playing an important role in the progression of a subset of TNBC. We evaluated the impact of ERβ expression along with anti-AR drugs in AR-positive TNBC.
We used MDA-MB 453 human cell line, representative for AR+/ERβ- molecular profile. pEGFP-C1-ERβ plasmid was transfected into MDA-MB 453 cells. Cell proliferation, metastatic potential and apoptosis were examined using MTT assay, scratch assay and Annexin V-FITC assay, respectively. Protein levels of PI3K/AKT molecules were assessed using Western blot. All assays were also conducted in the presence of anti-androgens; bicalutamide and enzalutamide. The localization of AR and ERβ was detected by immunofluorescence. In order to test if a physical association (ERβ/AR) occurs, proximity ligation assay (PLA), which enables the visualization of interacting proteins in fixed cells and tissues, was performed.
MDA- MD 453/ERβ cells exhibited reduced cell proliferation (19%±0.06), lower metastatic potential (50%±2.4) and increased late apoptosis (12%) compared to MDA-MB 453 mock cells. ERβ suppressed PI3K/AKT pathway through PTEN and inhibited the activation and nuclear translocation of AR. Also, ERβ significantly impeded AR from forming homodimers, reversing the aggravating role of AR. It was also shown that enzalutamide was superior to bicalutamide regarding cell proliferation, metastatic potential and stimulated apoptosis. In addition, using PLA assay, we demonstrated a strong physical interaction between ERβ and AR in MDA- MD 453/ERβ cells. The administration of enzalutamide enhanced the formation of ERβ/AR heterodimers reducing further proliferation (54%±0.003) and metastatic ability (81%±4.5) and inducing late apoptosis (21%). Lastly, employing PLA assay in TBNC human paraffin embedded tissues, we found a strong interaction between ERβ and AR, recapitulating the
Our results suggest that ERβ has oncosuppressive potential in AR-positive TBNC development and provide mechanistic insights regarding its’ predictive role for the efficacy of anti-AR agents in this TNBC group.
Michalis V. Karamouzis
Astellas Pharma Europe Ltd.
All authors have declared no conflicts of interest.