P4-02-03: Biological Functions of Estrogen Receptor-beta and Its Variants in Breast Cancer.
Authors: JR Hawse, X Wu, M Cicek, M Subramaniam, V Negron, WL Lingle, MP Goetz, TC Spelsberg, JN Ingle
Journal: Cancer Research (2011)

Abstract Background The role of estrogen receptor alpha (ERα) in breast cancer has been studied extensively; yet, much less is known about full-length ERβ (ERβ1) and even less about its 4 variant forms (ERβ2-5). We have recently implicated a role for ERβ1 in sensitizing ERα expressing breast cancer cells to anti-estrogens. However, the ability of ERβ2-5 to modulate ERα and ERβ1 activity, and their association with cancer development, progression, and response to estradiol (E2) and anti-estrogens are not well understood. Here, we provide evidence that the presence of ERβ variants may be of diagnostic and clinical relevance for breast cancer patients and describe the development and characterization of a novel, highly specific monoclonal antibody (MC10) that is able to detect their expression in tumor biopsies. Methods: Transient transfection and luciferase assays were used to determine the transcriptional activity of ERβ2-5 in response to E2 and anti-estrogens alone or in combination with ERα and ERβ1. A novel monoclonal antibody targeting all ERβ variants (MC10) was developed and characterized. The sub-cellular localization of ERβ2-5 was determined via confocal microscopy. Finally, the MC10 antibody was used to assess ERβ positivity in breast tumors and was compared to that of another monoclonal antibody which only detects ERβ1. Results: Unlike ERβ1, ERβ2-5 do not activate an estrogen response element (ERE) in response to E2 and instead, slightly repress the activity of this reporter construct. Expression of ERβ2-5 does not significantly alter the transcriptional activity of ERβ1 following E2 treatment. However, ERβ2, 3 and 5, but not ERβ4, significantly enhance the E2-induced transcriptional activity of ERα. Interestingly, expression of ERβ3, 4 and 5, but not ERβ2, enhance the ability of anti-estrogens to block ERα mediated transcriptional activity. Confocal microscopy revealed that ERβ1 and 2 are almost exclusively localized to the cell nucleus. However, ERβ3-5 exhibit significant cytoplasmic and peri-nuclear localization. Immunohistochemistry of breast cancer biopsies using the MC10 antibody revealed multiple staining patterns including tumors which exhibit primarily nuclear staining and others primarily cytoplasmic, both in the presence and absence of ERα. These results are in contrast to the almost exclusive nuclear staining obtained on the same tumors with an ERβ1-specific antibody. Conclusions: ERβ variants exhibit variable sub-cellular localization patterns and can influence the function of ERα, both in response to E2 and anti-estrogens. Therefore, the differential expression of ERβ variants and their cellular localization may influence breast cancer progression and/or therapeutic responses. The use of ERβ antibodies which do not detect all ERβ variants, or the use of a single ERβ antibody which does not discriminate between ERβ1 and its variants, is unlikely to reveal the complete biological significance of total ERβ expression in breast cancer and may in part explain the conflicting studies which have been reported for ERβ in the field. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-02-03.