Purpose: Significant controversy exists regarding the expression patterns of estrogen receptor beta (ERβ) in normal and diseased breast tissue. To address this issue, we have validated two ERβ antibodies, have optimized the IHC protocols for both antibodies and now report the expression patterns of ERβ in normal and malignant breast tissues.Methods: ERβ antibody specificity was determined using western blot and IHC analysis. ERβ protein expression patterns were assessed via IHC in normal breast tissue and invasive breast carcinoma. Further, we report the detailed protocol of the ERβ IHC assay developed in our CAP/ CLIA certified laboratory to provide a standardized method for future studies.

Triple Negative Breast Cancer (TNBC) accounts for 15–20% of all breast cancer cases, yet is responsible for a disproportionately high percentage of breast cancer mortalities. Thus, there is an urgent need to identify novel biomarkers and therapeutic targets based on the molecular events driving TNBC pathobiology. Estrogen receptor beta (ERβ) is known to elicit anti-cancer effects in TNBC, however its mechanisms of action remain elusive. Here, we report the expression profiles of ERβ and its association with clinicopathological features and patient outcomes in the largest cohort of TNBC to date. In this cohort, ERβ was expressed in approximately 18% of TNBCs, and expression of ERβ was associated with favorable clinicopathological features, but correlated with different overall survival outcomes according to menopausal status. Mechanistically, ERβ formed a co-repressor complex involving enhancer of zeste homologue 2/polycomb repressive complex 2 (EZH2/PRC2) that functioned to suppress oncogenic NFκB/RELA (p65) activity. Importantly, p65 was shown to be required for formation of this complex and for ERβ-mediated suppression of TNBC. Our findings indicate that ERβ+ tumors exhibit different characteristics compared to ERβ− tumors and demonstrate that ERβ functions as a molecular switch for EZH2, repurposing it for tumor suppressive activities and repression of oncogenic p65 signaling.

Triple Negative Breast Cancer (TNBC) affects approximately 15-20% of BC patients, yet accounts for a disproportionately higher rate of BC morbidity and mortality, in part due to lack of targeted therapies. We have shown that estrogen receptor beta (ERβ) is expressed in approximately 20% of TN breast tumors and that ligand-mediated activation of ERβ with estradiol (E2) or ERβ-selective agonists decreases tumor cell proliferation, invasion and migration in vitro and in vivo. Therefore, we aimed to elucidate the mechanisms by which ERβ elicits its anti-cancer effects in TNBC. RNAseq analysis of ERβ-expressing MDA-MB-231 cells demonstrated that ERβ significantly downregulates NFκB signaling in the presence of E2. ChIPseq for ERβ in these cells revealed that ERβ primarily associated with estrogen response elements, but 12% of all ERβ binding sites were located at NFκB consensus motifs. Using an NFκB reporter construct and qPCR, ERβ was shown to block TNFα-mediated induction of NFκB signaling and NFκB target gene expression. RNAseq analysis of MDA-MB-231-ERβ cells treated with TNFα or E2+TNFα revealed substantial global inhibition of TNFα regulated genes in the presence of E2. ChIPseq for NFκB demonstrated that ERβ significantly alters NFκB’s cistrome whereby it can both diminish NFκB binding and redistribute NFκB throughout the genome. ChIPseq also demonstrated that ligand-mediated activation of ERβ significantly diminished an activating histone mark (H3K27Ac) at many of these NFκB target genes while enhancing a repressive mark (H3K27Me3). The addition of H3K27Me3 at these loci was shown to occur through the recruitment of the histone methyltransferase, EZH2. Drug-mediated blockade of EZH2 activity reversed suppression of NFκB target gene expression by ERβ. Knockdown of NFκB or Mutation of ERβ’s DNA binding domain rendered ERβ incapable of associating with DNA, recruiting EZH2, methylating NFκB target gene loci, repressing NFκB target gene expression and inhibiting proliferation. Interestingly, ERβ was shown to elicit more potent anti-cancer effects in TNBC cells expression a constitutively active form of NFκB. These finding suggest that a primary mechanism by which ERβ functions as a tumor suppressor is through inhibition of NFκB pathway activity. Our studies have also revealed that ERβ functions as a molecular switch for EZH2 and repurposes it for tumor suppressive activities, as EZH2 has previously been reported to enrich NFκB signaling in TNBC. These findings could address the paradox that high EZH2 expression is associated with worse TNBC patient outcomes, while high H3K27Me3 expression is associated with improved patient outcomes. Currently, a Mayo Clinic Breast Cancer SPORE prospective phase II clinical trial is underway to investigate the efficacy of estradiol for the treatment of metastatic ERβ+TNBC and to further evaluate the cross-talk between ERβ, EZH2 and NFκB signaling.

Triple negative breast cancer (TNBC) is an aggressive sub-type of the disease which accounts for a disproportionately high percentage of breast cancer morbidities and mortalities. For these reasons, a better understanding of TNBC biology is required and the development of novel therapeutic approaches are critically needed. Estrogen receptor beta (ERβ) is a reported tumor suppressor that is expressed in approximately 20% of primary TNBC tumors, where it is associated with favorable prognostic features and patient outcomes. Previous studies have shown that ERβ mediates the assembly of co-repressor complexes on DNA to inhibit the expression of multiple growth promoting genes and to suppress the ability of oncogenic transcription factors to drive cancer progression. To further elucidate the molecular mechanisms by which ERβ elicits its anti-cancer effects, we developed MDA-MB-231 cells that inducibly express a mutant form of ERβ incapable of directly binding DNA. We demonstrate that disruption of ERβ’s direct interaction with DNA abolishes its ability to regulate the expression of well characterized immediate response genes and renders it unable to suppress TNBC cell proliferation. Loss of DNA binding also diminishes the ability of ERβ to suppress oncogenic NFκB signaling even though it still physically associates with NFκB and other critical co-factors. These findings enhance our understanding of how ERβ functions in this disease and provide a model system that can be utilized to further investigate the mechanistic processes by which ERβ elicits its anti-cancer effects.