Out of the 4,946 gained FOXA1 sites, 2007 (40.6%) overlap with the gained AR-binding sites (Figure?5E). tumor growth inhibition. These findings suggest that ligand-activated AR may function as a non-canonical inhibitor of ER and that AR agonists may offer a safe and effective treatment for ER-positive breast cancer. were down-regulated by enobosarm, other ER-target genes such as and were not inhibited by enobosarm. These results provide evidence that enobosarm functions in breast cancer by at least partially inhibiting the ER-signaling pathway to reduce cancer growth. The genes enriched for the AR pathway were fed into TCGA database to determine the consequence of altering the AR pathway by an AR agonist. AR pathway genes correlated with a significant increase in survival of breast cancer patients (hazard ratio of 0.64 and log rank P of 1 1.1? 10?8) (Figure?2D). To ensure that enobosarm is not an ER antagonist and the effects are mediated through AR, an ER competitive ligand binding assay (Figure?S4A) and an ER transactivation assay (Figure?S4B) were performed. Both results indicate that enobosarm has no direct interaction with ER, which is in concordance with earlier published Fisetin (Fustel) results (Yin et?al., 2003). Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) Analysis Demonstrates that Enobosarm Reprograms ER and AR Cistromes To determine if the effect of enobosarm on ER function is due to any direct effect on ER binding to DNA, ChIP-sequencing for ER was performed in the tumor samples obtained from animals shown in Figure?1D. ER binding to 1 1,148 regions (q?< 0.05) on the DNA was reprogrammed by enobosarm, with 572 regions statistically enriched with ER and 576 regions depleted of ER (Figure?3A), whereas Principal component analysis (PCA) (Figure?3B) and unsupervised hierarchical clustering (Figure?3D) show the distinct distribution of individual samples, an indication that enobosarm modified the DNA-binding pattern of ER in HCI-13. The motifs that were enriched by the ER represent Fisetin (Fustel) androgen response element (ARE) and FOXA1 response elements (FOXA1RE), whereas the regions that were depleted of ER represent estrogen response element (ERE) and FOXA1RE (Figure?3A right). Although the DNA regions depleted of ER by enobosarm favor the inhibition of the ER-target gene expression pattern, the enrichment of ER at AREs is surprising and has not been previously reported. Figure?S5 shows representative Rabbit polyclonal to YSA1H regions enriched by and depleted of ER. Figure?S6A shows the heatmap of individual tumor specimens. Variability between individual samples can be attributed to the inherent variability between xenograft specimens. Repeating the studies in a cell line model under controlled conditions might provide a robust redistribution outcome. Open in a separate window Figure?3 ChIP-sequencing Shows Reprogramming of ER Binding after Enobosarm Treatment (A) Chromatin immunoprecipitation (ChIP) assay was performed for ER in tumors treated with vehicle (n?= 4) or 10?mg/kg/day enobosarm (n?= 3) (tumors from animals shown in Figure?1D). Next-generation sequencing was performed to determine the genome-wide binding of ER to the DNA. Heatmap of significantly different peaks (q?< 0.05) Fisetin (Fustel) is shown as average of the individual tumor Fisetin (Fustel) samples. The top enriched motifs are shown to the right of the heatmap. (B) Principal Component Analysis (PCA) plot of vehicle- and enobosarm-treated samples that corresponds to ER-ChIP peaks is shown. (C) Pie charts showing the distribution of ER enrichment in enobosarm-treated HCI-13 samples. (D) Unsupervised hierarchical clustering. (E) ChIP assay was performed with ER antibody in HCI-13 specimens treated with vehicle or enobosarm and, real-time PCR was performed with the primers and TaqMan probe to the specified regions. AR, androgen receptor; ER, estrogen receptor; ChIP, chromatin immunoprecipitation; ARE, androgen response elements; ERE, estrogen response element; FOXA1RE, Forkhead box A1 response element. Between 50% and 60% of the ER-enriched and depleted sites were mapped to distal regulatory regions, whereas only around 2%C3% of the sites were mapped to promoter regions (Figure?3C). Interestingly, although the intron and exon binding percentage match with previous reports, the proportion of ER bound to promoters and distal regulatory elements are distinct from that observed in response to estrogens or with a constitutively active ER (Jeselsohn et?al., 2018). Other studies have indicated that the ER cistrome comprises about 30%C40% distal regulatory regions and 7%C22% proximal promoter regions, whereas AR-regulated ER cistrome in this study comprises of 50%C60% and 2%C3% of these regions, respectively. ER binding to pS2 ERE,.