Carroll, Jason S.

Abstract 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, we have found that estrogen receptor beta (ERβ) protein is expressed in approximately 20% of TN breast tumors. We have demonstrated that ligand-mediated activation of ERβ with estradiol (E2) or ERβ-selective agonists decreases tumor cell proliferation, invasion, and migration in vitro, as well as primary tumor growth and metastatic spread in vivo. As such, we aimed to elucidate the mechanisms by which ERβ elicits its anti-cancer effects in TNBC. Methods: RNAseq and ChIPseq were used to identify ERβ and NFκB regulomes and cistromes in TNBC cells. The global effects of ERβ on H3K27Ac and H3K27Me3 were also assessed via ChIPseq. The influence of ERβ on NFκB signaling was determined using co-immunoprecipitation, luciferase assays transcriptional readouts and ChIPseq. The involvement of EZH2 in mediating ERβ’s suppression of NFκB signaling and cancer cell phenotypes was determined using pharmacological approaches. Results: Pathway analysis of ERβ-regulated genes identified NFκB and inflammatory signaling as being highly suppressed in response to E2 treatment. Numerous NFκB target genes were among the most down-regulated genes by ERβ. ChIPseq revealed that ERβ primarily associated with estrogen response elements (EREs), but was also highly enriched at NFκB binding sites following E2 treatment. However, ERβ had no impact on TNFα-mediated NFκB phosphorylation or nuclear localization, but 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 373 genes to be significantly regulated by TNFα in TNBC cells, of which 107 were blocked in the presence of E2+TNFα. Interestingly, E2+TNFα treatment induced novel NFκB binding sites compared to TNFα alone as identified by ChIPseq for NFκB. 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). These modifications were shown to occur through the recruitment of the histone methyltransferase, EZH2, to enhancer elements of these NFκB target genes. Drug-mediated blockade of EZH2 activity reversed suppression of NFκB target gene expression by ERβ. Finally, knockdown of NFκB expression inhibited the anti-cancer effects of ERβ while expression of a constitutively active form of NFκB enhanced ERβ’s inhibitory effects. Conclusions: Our data suggest that ERβ elicits 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 efficacy of estradiol for the treatment of metastatic ERβ+TNBC and to further evaluate the cross-talk between ERβ and NFκB signaling. Citation Format: Kirsten GM Aspros, Adam W Nelson, Zhenqing Ye, Zhifu Sun, Igor Chernukhin, Jason S Carroll, James N Ingle, Matthew P Goetz, John R Hawse. Estrogen receptor beta repurposes EZH2 to inhibit oncogenic NFκB signaling in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-05-02.

Significance Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer and patients exhibit high rates of recurrence and mortality in part due to lack of treatment options beyond standard-of-care chemotherapy regimens. In the subset of TNBCs that express estrogen receptor beta (ERβ), ligand-mediated activation of ERβ elicits potent anticancer effects. We report here the elucidation of the ERβ cistrome and transcriptome in TNBC and identify a mechanism whereby ERβ induces cystatin gene expression resulting in inhibition of canonical TGFβ signaling and a blockade of metastatic phenotypes. These findings suggest that ERβ-targeted therapies represent a treatment option for the subset of women with ERβ-expressing TNBC.