Abstract P5-05-03: Estrogen receptor beta elicits anti-cancer effects in triple negative breast cancer through suppression of NFκB signaling
Authors: Aspros K , Nelson A , Ye Z , Sun Z , Chernukhin I , Carroll J , Ingle J , Goetz M , Hawse J 
Published: May 2019

Background: 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. Using well-validated antibodies, Estrogen Receptor Beta (ERβ) protein has been shown to be expressed in approximately 25% of TNBCs and is associated with improved patient outcomes. Using multiple ERβ +/- TNBC cell lines and PDX models, we have demonstrated that ligand-mediated activation of ERβ by estradiol (E2) decreases cell proliferation, invasion, and migration in vitro, as well as primary tumor growth and metastatic spread in vivo.

Methods: To determine the mechanisms by which ERβ elicits these anti-cancer effects, we elucidated the ERβ transcriptome and cistrome via Microarray and ChIPseq, respectively, in TNBC cells stably expressing ERβ in a doxycycline-inducible manner. We also performed gene expression and luciferase assays to assess the impact of ERβ on NFκB signaling, followed by ChIP-PCR and ChIPseq to assess how ERβ modifies chromatin architecture near NFκB target genes.

Results: Pathway analysis of ERβ-regulated genes identified NFκB signaling as one of the most suppressed pathways in response to E2 treatment. Indeed, numerous NFκB target genes were among the most down-regulated genes following E2 treatment but only in the presence of ERβ expression. Chromatin Immunoprecipitation followed by sequencing (ChIPseq) revealed that ERβ primarily associated with estrogen response elements (EREs), but was also enriched around NFκB binding sites following E2 treatment. In fact, 12% of all ERβ binding sites were enriched for NFκB response elements and ERβ was shown to physically associate with NFκB protein. Using an NFκB reporter construct and qPCR, ERβ was shown to block TNFα-mediated induction of NFκB signaling and NFκB target gene expression. Globally, RNAseq identified 200 genes to be significantly regulated by TNFα in TNBC cells, of which 81 were significantly altered in the presence of E2+TNFα. ChIPseq 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). These modifications are also associated with recruitment of the histone methyltransferase, EZH2, to enhancer elements of these NFκB target genes. Drug-mediated blockade of HDAC and EZH2 activity reversed suppression of NFκB target gene expression by ERβ.

Conclusions: Our data suggest that ERβ may elicit its anti-cancer effects in part via formation of a novel co-repressor complex consisting of ERβ, NFκB, and EZH2. These data are in keeping with prior observations of the importance of NFκB signaling as it relates to TNBC cell proliferation and invasion, and that decreased expression of NFκB target genes is associated with improved outcomes in TNBC patients. Currently, a Mayo Breast SPORE prospective study is underway to investigate the role of estradiol in ERβ expressing TNBC and to further evaluate the cross-talk between ERβ and NFκB signaling in TNBC.

Citation Format: Aspros K, Nelson A, Ye Z, Sun Z, Chernukhin I, Carroll J, Ingle J, Goetz M, Hawse J. Estrogen receptor beta elicits anti-cancer effects in triple negative breast cancer through suppression of NFκB signaling [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-03.