Interaction Proteomics Identifies ERbeta Association with Chromatin Repressive Complexes to Inhibit Cholesterol Biosynthesis and Exert An Oncosuppressive Role in Triple-negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) is characterized by poor response to therapy and low overall patient survival. Recently, Estrogen Receptor beta (ERβ) has been found to be expressed in a fraction of TNBCs where, because of its oncosuppressive actions on the genome, it represents a potential therapeutic target, provided a better understanding of its actions in these tumors becomes available. To this end, the cell lines Hs 578T, MDA-MB-468 and HCC1806, representing the claudin-low, basal-like 1 and 2 TNBC molecular subtypes respectively, were engineered to express ERβ under the control of a Tetracycline-inducible promoter and used to investigate the effects of this transcription factor on gene activity. The antiproliferative effects of ERβ in these cells were confirmed by multiple functional approaches, including transcriptome profiling and global mapping of receptor binding sites in the genome, that revealed direct negative regulation by ERβ of genes, encoding for key components of cellular pathways associated to TNBC aggressiveness representing novel therapeutic targets such as angiogenesis, invasion, metastasis and cholesterol biosynthesis. Supporting these results, interaction proteomics by immunoprecipitation coupled to nano LC-MS/MS mass spectrometry revealed ERβ association with several potential nuclear protein partners, including key components of regulatory complexes known to control chromatin remodeling, transcriptional and post-transcriptional gene regulation and RNA splicing. Among these, ERβ association with the Polycomb Repressor Complexes 1 and 2 (PRC1/2), known for their central role in gene regulation in cancer cells, was confirmed in all three TNBC subtypes investigated, suggesting its occurrence independently from the cellular context. These results demonstrate a significant impact of ERβ in TNBC genome activity mediated by its cooperation with regulatory multiprotein chromatin remodeling complexes, providing novel ground to devise new strategies for the treatment of these diseases based on ligands affecting the activity of this nuclear receptor or some of its protein partners.

Small Non-Coding RNA Profiling Identifies miR-181a-5p as a Mediator of Estrogen Receptor Beta-Induced Inhibition of Cholesterol Biosynthesis in Triple-Negative Breast Cancer

“…In any case, full-length receptor expression was reported in a small fraction (15-20%) of TNBC patients, where its presence was correlated with better survival [13] and response to tamoxifen therapy [13], suggesting its possible use as both a prognostic marker and therapeutic target [14]. In accordance with this data, in our previous study [15] we demonstrated the oncosuppressive role of the full-length ERβ in three TNBC cell lines belonging to different TNBC subtypes.…”

“…Triple-negative breast cancer cell lines HCC1806 (CRL-2335), MDA-MB-468 (HTB-132), and Hs 578T (HTB-126) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in culture as previously described [15]. Lentiviral particles containing viral RNA genome encoding for Tet-On Advanced and Inducible 3xFlag-ERβ were produced using Lenti-X Tet-On Advanced Inducible Expression System components (Takara-Clontech Europe, Göteborg, Sweden) according to the manufacturer's instructions.…”

“…One Tet-On Advanced clone for each cell line was chosen for further transduction using 3xFlag-ERβ-encoding lentiviral particles followed by single clone isolation in the presence of puromycin (Sigma-Aldrich, St Louis, MO, USA). Separate clones were tested for ERβ expression after 24-h induction of transgene expression by 2 µg/mL doxycycline (Sigma-Aldrich) as previously described [15]. For each cell line, one ERβ clone expressing the receptor at a maximum level was chosen for further experiments.…”

“…To verify this hypothesis and investigate the role of ERβ in TNBC, we performed sncRNA sequencing on three previously engineered receptor-expressing cell lines and on 12 ERβ+ and 32 ERβ− TNBC tissue samples where receptor status was assessed by immunohistochemistry [15]. A group of ERβ-regulated sncRNAs was identified both in vitro and in vivo, several of which showed subtype-specific deregulation, while others were independent from the tumor subtype.…”

“…Interestingly, two miRNAs-miR-181a-5p and miR-92a-3p-showed the same response to the receptor in all cell lines and tissues tested. Among them, miR-181a-5p was characterized by high expression and upregulation in TNBC tissues and cell lines and was found to target key components of the cholesterol biosynthesis pathway, previously shown to be inhibited by ERβ in TNBC cells by ERβ-mediated recruitment of transcriptional repressor complexes to regulatory elements of cholesterol biosynthesis genes [15]. Taken together, these findings suggest a dual role of ERβ in epigenetic regulation of gene expression in TNBC: at the transcriptional level via chromatin binding and recruitment of multiple chromatin-modifying complexes to the genome, and post-transcriptionally through sncRNA-mediated regulation of gene expression.…”