DHEA analogues with modifications at positions C3 or C17 were synthesized and evaluated for neuroprotective activity against the neural-crest-derived PC12 cell model of serum deprivation-induced apoptosis. The most potent compounds were the spiro-epoxy derivatives 17beta-spiro[5-androstene-17,2'-oxiran]-3beta-ol (20), (20S)-3beta,21-dihydroxy-17beta,20-epoxy-5-pregnene (23), and (20R)-3beta,21-dihydroxy-17alpha,20-epoxy-5-pregnene (27) with IC(50) values of 0.19 +/- 0.01, 99.0 +/- 4.6, and 6.4 +/- 0.3 nM, respectively. Analogues 20, 23, and 27, up to the micromolar range of concentrations, were unable to activate estrogen receptor alpha and beta (ERalpha and ERbeta) or to interfere with ER-dependent gene expression significantly. In addition, they were unable to stimulate the growth of Ishikawa, MCF-7, and LNCaP cells. Our results suggest that the spiro-epoxyneurosteroid derivatives 20, 23, and 27 may prove to be lead molecules for the synthesis of novel neuroprotective agents.

Aromatase inhibitors are the major first-line treatment of estrogen receptor-positive breast cancer, but resistance to treatment is common. To date, no biomarkers have been validated clinically to guide subsequent therapy in these patients. In this study, we mapped the genome-wide chromatin-binding profiles of estrogen receptor a (ERa), along with the epigenetic modifications H3K4me3 and H3K27me3, that are responsible for determining gene transcription (n ¼ 12). Differential binding patterns of ERa, H3K4me3, and H3K27me3 were enriched between patients with good or poor outcomes after aromatase inhibition. ERa and H3K27me3 patterns were validated in an additional independent set of breast cancer cases (n ¼ 10). We coupled these patterns to array-based proximal gene expression and progression-free survival data derived from a further independent cohort of 72 aromatase inhibitor-treated patients. Through this approach, we determined that the ERa and H3K27me3 profiles predicted the treatment outcomes for first-line aromatase inhibitors. In contrast, the H3K4me3 pattern identified was not similarly informative. The classification potential of these genes was only partially preserved in a cohort of 101 patients who received first-line tamoxifen treatment, suggesting some treatment selectivity in patient classification. Cancer Res; 73(22); 6632-41. Ó2013 AACR.

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Fractionation of the neutral extract of Onobrychis ebenoides (Leguminosae) yielded a new isoflavone, named ebenosin (1), in addition to the known ones, afrormosin (2), formononetin (3) and daidzein (4). Although the relative binding affinities of 1 - 4 for estrogen receptor alpha (ERalpha) were nearly comparable and matched those of 1-3 for ERbeta, that of 4 for the latter receptor was significantly higher than any of the other. Compounds 1 - 4 induced cell proliferation and gene expression in breast and endometrial cancer cells in an ER-dependent manner. Nonetheless, the rank order of induction potencies ( 4 > 3 >or= 2 >or= 1) matched better that of affinities for ERbeta ( 4 > 3 >or= 2 >or= 1) rather than ERalpha ( 4 >or= 3 >or= 2 >or= 1). While the antiestrogen ICI 182,780 could inhibit the induction of proliferation of ER-positive breast cancer cells by 1-4, it could not prevent 1 from exhibiting significant ER-independent cytotoxicity at 10 microM. By contrast, 1 was much less cytotoxic and only weakly estrogenic for ER-positive endometrial adenocarcinoma cells. In conclusion, our data suggest that the C-8 isoprenyl substituent of 1 renders it cytotoxic and/or estrogenic in a cell-dependent manner.

Nuclear receptor interacting protein (Nrip1), also known as RIP140, is a co-regulator for nuclear receptors that plays an essential role in ovulation by regulating the expression of the epidermal growth factor-like family of growth factors. Although several studies indicate a role for RIP140 in breast cancer, its role in the development of the mammary gland is unclear. By using RIP140-null and RIP140 transgenic mice, we demonstrate that RIP140 is an essential factor for normal mammary gland development and that it functions by mediating oestrogen signalling. RIP140-null mice exhibit minimal ductal elongation with no side-branching, whereas RIP140-overexpressing mice show increased cell proliferation and ductal branching with age. Tissue recombination experiments demonstrate that RIP140 expression is required in both the mammary epithelial and stromal compartments for ductal elongation during puberty and that loss of RIP140 leads to a catastrophic loss of the mammary epithelium, whereas RIP140 overexpression augments the mammary basal cell population and shifts the progenitor/differentiated cell balance within the luminal cell compartment towards the progenitors. For the first time, we present a genome-wide global view of oestrogen receptor-α (ERα) binding events in the developing mammary gland, which unravels 881 ERα binding sites. Unbiased evaluation of several ERα binding sites for RIP140 co-occupancy reveals selectivity and demonstrates that RIP140 acts as a co-regulator with ERα to regulate directly the expression of amphiregulin (Areg), the progesterone receptor (Pgr) and signal transducer and activator of transcription 5a (Stat5a), factors that influence key mitogenic pathways that regulate normal mammary gland development.

Oestrogen receptor α (ERα) is a nuclear receptor that is the driving transcription factor expressed in the majority of breast cancers. Recent studies have demonstrated that the liver receptor homolog-1 (LRH-1), another nuclear receptor, regulates breast cancer cell proliferation and promotes motility and invasion. To determine the mechanisms of LRH-1 action in breast cancer, we performed gene expression microarray analysis following RNA interference for LRH-1. Interestingly, gene ontology (GO) category enrichment analysis of LRH-1–regulated genes identified oestrogen-responsive genes as the most highly enriched GO categories. Remarkably, chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-seq) to identify genomic targets of LRH-1 showed LRH-1 binding at many ERα binding sites. Analysis of select binding sites confirmed regulation of ERα−regulated genes by LRH-1 through binding to oestrogen response elements, as exemplified by the TFF1/pS2 gene. Finally, LRH-1 overexpression stimulated ERα recruitment, while LRH-1 knockdown reduced ERα recruitment to ERα binding sites. Taken together, our findings establish a key role for LRH-1 in the regulation of ERα target genes in breast cancer cells and identify a mechanism in which co-operative binding of LRH-1 and ERα at oestrogen response elements controls the expression of oestrogen-responsive genes.

Five new isoflavones, named 5,4'-dihydroxy-7-methoxy-3'-(3-methylbuten-2-yl)isoflavone (1), 5,2',4'-trihydroxy-7-methoxy-5'-(3-methylbuten-2-yl)isoflavone (2), 5,4'-dihydroxy-7-methoxy-3'-(3-methyl-2-hydroxybuten-3-yl)isoflavone (3), 3'-formyl-5,4'-dihydroxy-7-methoxyisoflavone (4), and 5-hydroxy-3''-hydroxy-2'',2''-dimethyldihydropyrano[5'',6'':3',4']isoflavone (5), as well as six known compounds, wighteone (6), 3'-isoprenylgenistein (7), isolupabigenin (8), alpinumisoflavone (9), erypoegin D (10), and crystacarpin (11), were isolated from Erythrina poeppigiana. The structures of the isolated compounds were elucidated on the basis of chemical and spectroscopic analysis. The affinity of these compounds for the estrogen receptors ERalpha and ERbeta was evaluated using a receptor binding assay. While isoprenyl and dimethylpyrano substituents in ring A reduced the affinity of binding to ERbeta ca. 100-fold compared to genistein, the isoprenyl substituent in ring B was better accommodated, allowing 7 to bind with ca. 10-fold lower affinity than genistein.

Protein kinase A (PKA)-induced estrogen receptor alpha (ERa) phosphorylation at serine residue 305 (ERaS305-P) can induce tamoxifen (TAM) resistance in breast cancer. How this phospho-modification affects ERa specificity and translates into TAM resistance is unclear. Here, we show that S305-P modification of ERa reprograms the receptor, redirecting it to new transcriptional start sites, thus modulating the transcriptome. By altering the chromatin-binding pattern, Ser305 phosphorylation of ERa translates into a 26-gene expression classifier that identifies breast cancer patients with a poor disease outcome after TAM treatment. MYC-target genes and networks were significantly enriched in this gene classifier that includes a number of selective targets for ERaS305-P. The enhanced expression of MYC increased cell proliferation in the presence of TAM. We demonstrate that activation of the PKA signaling pathway alters the transcriptome by redirecting ERa to new transcriptional start sites, resulting in altered transcription and TAM resistance.