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, 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.
Results
We have confirmed the specificity of two independent ERβ monoclonal antibodies, one that detects total (i.e., full length plus splice variants 2–5, which do not include the ligand binding domain) ERβ protein (PPZ0506) and one that detects only the full-length form, which includes the ligand binding domain, of ERβ (PPG5/10). Using these two antibodies, we demonstrate that ERβ is highly expressed in normal human breast tissue as well as in 20–30% of invasive breast cancers. Further, these two antibodies exhibited similar staining patterns across multiple different tissues and were highly concordant with regard to determining ERβ positivity in breast cancers.
Conclusions
ERβ protein was shown to be abundant in the majority of normal breast epithelial cells and is present in 20–30% of breast cancers. Use of these two antibodies, along with their standardized IHC protocols, provide a reference for future studies aimed at determining the utility of ERβ as a prognostic and/or predictive biomarker in various tissues of benign or malignant states.
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.
Estrogen signaling is critical for the development and maintenance of healthy bone, and age-related decline in estrogen levels contributes to the development of post-menopausal osteoporosis. Most ...bones consist of a dense cortical shell and an internal mesh-like network of trabecular bone that respond differently to internal and external cues such as hormonal signaling. To date, no study has assessed the transcriptomic differences that occur specifically in cortical and trabecular bone compartments in response to hormonal changes. To investigate this, we employed a mouse model of post-menopausal osteoporosis (ovariectomy, OVX) and estrogen replacement therapy (ERT). mRNA and miR sequencing revealed distinct transcriptomic profiles between cortical and trabecular bone in the setting of OVX and ERT. Seven miRs were identified as likely contributors to the observed estrogen-mediated mRNA expression changes. Of these, four miRs were prioritized for further study and decreased predicted target gene expression in bone cells, enhanced the expression of osteoblast differentiation markers, and altered the mineralization capacity of primary osteoblasts. As such, candidate miRs and miR mimics may have therapeutic relevance for bone loss resulting from estrogen depletion without the unwanted side effects of hormone replacement therapy and therefore represent novel therapeutic approaches to combat diseases of bone loss.
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Hawse and colleagues employed a mouse model of post-menopausal osteoporosis and estrogen replacement therapy to identify miRs likely to mediate bone-beneficial effects of estrogen on the skeleton. Our studies highlight key differences in estrogen response between cortical and trabecular bone and identify miRs capable of enhancing osteoblast differentiation and matrix mineralization.
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.
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