Abstract 2104: Mechanisms for the inhibition of estrogen receptors by estrogen related receptor beta and oxysterols
Authors: Starkey Nicholas J.E. , Yuan Lu , Li Yufei , Lubahn Dennis B. 
Published: May 2015

Abstract
We have previously shown that Estrogen Related Receptor Beta - Short Form (ERRβ-SF) inhibits the transcriptional activity of the Estrogen Receptors Alpha (ERα) and Beta (ERβ). Our lab and others have also shown the importance of these receptors in prostate cancer (PCa) as well as other ER positive, hormonally responsive, cancers such as breast and uterine. We have recently found, via RNA-Seq anlyses, that the overexpression of ERRβ-SF in the DU-145 PCa cell line changes the expression of many oxysterol metabolizing enzymes. These include, for example, CYP27A1 increased 69% and CYP46A1 increased 300% (although with a very low read number). These enzymes are responsible for the production of 27-hydroxycholesterol (27-OHC) and 24(S)-hydroxycholesterol (24(S)-OHC), respectively. Interestingly, the enzymes that catabolize these cholesterol metabolites were: CYP7A1 - not expressed, CYP7B1 - not expressed, and CYP39A1 which decreased by 63%. We hypothesized, that this represents two potential mechanisms for the inhibition of ER activity by ERRβ-SF. Oxysterol concentrations are increased when ERRβ-SF is overexpressed, this will either: 1) inhibit ERs through the known mechanism of directly binding to and inhibiting ERs, as has been previously shown for 27-OHC, or 2) inhibit by a novel mechanism in which oxysterols bind to ERRβ-SF and increase its ability to bind to and inhibit ER activities. To test these hypotheses, we used a transcriptional assay with an Estrogen Response Element driven Luciferase (ERE-Luc) reporter gene. ERs were expressed independently, or with ERRβ-SF, and treated with various oxysterols in the Ishikawa uterine cancer cell line. Using this assay, we have found that 27-OHC inhibits ER activity with a 4 fold difference between ERα and ERβ (IC50s = 4µM and 1µM, respectively). 24(S)-OHC inhibits ERβ only, with no effect on ERα at concentrations as high as 10µM. Interestingly, 10µM 24(S)-OHC increased the growth of Ishikawa cells &gt;20% within 24hrs treatment under all conditions tested. Unfortunately, due to the overlap in ERE binding by the ERR and ERs, it was difficult to test the proposed novel mechanism using the ERE-Luc assay. To address this we are creating a new FRET based assay to observe the effects of the ligands on the ERR-ER interaction. We are further confirming the endogenous concentrations of the 24(S)-OHC and 27-OHC metabolizing enzymes via qRT-PCR. In conclusion, we have identified a potential molecular mechanism via oxysterols for the inhibitory action of ERRβ-SF on the ERs. This may explain the role of cholesterol metabolites in prostate cancer progression.
Citation Format: Nicholas J.E. Starkey, Lu Yuan, Yufei Li, Dennis B. Lubahn. Mechanisms for the inhibition of estrogen receptors by estrogen related receptor beta and oxysterols. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2104. doi:10.1158/1538-7445.AM2014-2104