Identification of an anabolic selective androgen receptor modulator that actively induces death of androgen-independent prostate cancer cells.

Prostate cancer (PCa) initially responds to inhibition of androgen receptor (AR) signaling, but inevitably progresses to hormone ablation-resistant disease. Much effort is focused on optimizing this androgen deprivation strategy by improving hormone depletion and AR antagonism. However we found that bicalutamide, a clinically used antiandrogen, actually resembles a selective AR modulator (SARM), as it partially regulates 24% of endogenously 5α-dihydrotestosterone (DHT)-responsive genes in AR(+) MDA-MB-453 breast cancer cells. These data suggested that passive blocking of all AR functions is not required for PCa therapy. Hence, we adopted an active strategy that calls for the development of novel SARMs, which induce a unique gene expression profile that is intolerable to PCa cells. Therefore, we screened 3000 SARMs for the ability to arrest the androgen-independent growth of AR(+) 22Rv1 and LNCaP PCa cells but not AR(-) PC3 or DU145 cells. We identified only one such compound; the 4-aza-steroid, MK-4541, a potent and selective SARM. MK-4541 induces caspase-3 activity and cell death in both androgen-independent, AR(+) PCa cell lines but spares AR(-) cells or AR(+) non-PCa cells. This activity correlates with its promoter context- and cell-type dependent transcriptional effects. In rats, MK-4541 inhibits the trophic effects of DHT on the prostate, but not the levator ani muscle, and triggers an anabolic response in the periosteal compartment of bone. Therefore, MK-4541 has the potential to effectively manage prostatic hypertrophic diseases owing to its antitumor SARM-like mechanism, while simultaneously maintaining the anabolic benefits of natural androgens.

Systems analysis of eleven rodent disease models reveals an inflammatome signature and key drivers.

Common inflammatome gene signatures as well as disease-specific signatures were identified by analyzing 12 expression profiling data sets derived from 9 different tissues isolated from 11 rodent inflammatory disease models. The inflammatome signature significantly overlaps with known drug targets and co-expressed gene modules linked to metabolic disorders and cancer. A large proportion of genes in this signature are tightly connected in tissue-specific Bayesian networks (BNs) built from multiple independent mouse and human cohorts. Both the inflammatome signature and the corresponding consensus BNs are highly enriched for immune response-related genes supported as causal for adiposity, adipokine, diabetes, aortic lesion, bone, muscle, and cholesterol traits, suggesting the causal nature of the inflammatome for a variety of diseases. Integration of this inflammatome signature with the BNs uncovered 151 key drivers that appeared to be more biologically important than the non-drivers in terms of their impact on disease phenotypes. The identification of this inflammatome signature, its network architecture, and key drivers not only highlights the shared etiology but also pinpoints potential targets for intervention of various common diseases.

Identification and characterization of lipopolysaccharide binding protein (LBP) as an estrogen receptor α specific serum biomarker.

Estrogen Receptor α (ERα) and Estrogen Receptor ß (ERß) are steroid nuclear receptors that transduce estrogen signaling to control diverse physiological processes linked to reproduction, bone remodeling, behavior, immune response and endocrine-related diseases. In order to differentiate between ERα and ERß mediated effects in vivo, ER subtype selective biomarkers are essential. We utilized ERα knockout (AERKO) and ERß knockout (BERKO) mouse liver RNA and genome wide profiling to identify novel ERα selective serum biomarker candidates. Results from the gene array experiments were validated using real-time RT-PCR and subsequent ELISA's to demonstrate changes in serum proteins. Here we present data that Lipopolysacharide Binding Protein (LBP) is a novel liver-derived ERα selective biomarker that can be measured in serum.

Characterization of a novel small molecule subtype specific estrogen-related receptor alpha antagonist in MCF-7 breast cancer cells.

BACKGROUND: The orphan nuclear receptor estrogen-related receptor alpha (ERRalpha) is a member of the nuclear receptor superfamily. It was identified through a search for genes encoding proteins related to estrogen receptor alpha (ERalpha). An endogenous ligand has not been found. Novel ERRalpha antagonists that are highly specific for binding to the ligand binding domain (LBD) of ERRalpha have been recently reported. Research suggests that ERRalpha may be a novel drug target to treat breast cancer and/or metabolic disorders and this has led to an effort to characterize the mechanisms of action of N-[(2Z)-3-(4,5-dihydro-1,3-thiazol-2-yl)-1,3-thiazolidin-2-yl idene]-5H dibenzo[a,d][7]annulen-5-amine, a novel ERRalpha specific antagonist. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate this ERRalpha ligand inhibits ERRalpha transcriptional activity in MCF-7 cells by luciferase assay but does not affect mRNA levels measured by real-time RT-PCR. Also, ERalpha (ESR1) mRNA levels were not affected upon treatment with the ERRalpha antagonist, but other ERRalpha (ESRRA) target genes such as pS2 (TFF1), osteopontin (SPP1), and aromatase (CYP19A1) mRNA levels decreased. In vitro, the ERRalpha antagonist prevents the constitutive interaction between ERRalpha and nuclear receptor coactivators. Furthermore, we use Western blots to demonstrate ERRalpha protein degradation via the ubiquitin proteasome pathway is increased by the ERRalpha-subtype specific antagonist. We demonstrate by chromatin immunoprecipitation (ChIP) that the interaction between ACADM, ESRRA, and TFF1 endogenous gene promoters and ERRalpha protein is decreased when cells are treated with the ligand. Knocking-down ERRalpha (shRNA) led to similar genomic effects seen when MCF-7 cells were treated with our ERRalpha antagonist. CONCLUSIONS/SIGNIFICANCE: We report the mechanism of action of a novel ERRalpha specific antagonist that inhibits transcriptional activity of ERRalpha, disrupts the constitutive interaction between ERRalpha and nuclear coactivators, and induces proteasome-dependent ERRalpha protein degradation. Additionally, we confirmed that knocking-down ERRalpha lead to similar genomic effects demonstrated in vitro when treated with the ERRalpha specific antagonist.

Estrogen-related receptor-alpha antagonist inhibits both estrogen receptor-positive and estrogen receptor-negative breast tumor growth in mouse xenografts.

Estrogen-related receptors (ERR) are orphan members of the nuclear receptor superfamily most closely related to estrogen receptors (ER). Although ERalpha is a successful target for treating breast cancer, there remains an unmet medical need especially for estrogen-independent breast cancer. Although estradiol is not an ERR ligand, ER and ERR share many commonalities and overlapping signaling pathways. An endogenous ERR ligand has not been identified; however, novel synthetic ERRalpha subtype-specific antagonists have started to emerge. In particular, we recently identified a novel compound, N-[(2Z)-3-(4,5-dihydro-1,3-thiazol-2-yl)-1,3-thiazolidin-2-yl idene]-5H dibenzo[a,d][7]annulen-5-amine (termed compound A) that acts specifically as an ERRalpha antagonist. Here, we show that compound A inhibited cell proliferation in ERalpha-positive (MCF-7 and T47D) and ERalpha-negative (BT-20 and MDA-MD-231) breast cancer cell lines. Furthermore, we report the differential expression of 83 genes involved in ERRalpha signaling in MCF-7 and BT-20 breast cancer cell lines. We show that compound A slowed tumor growth in MCF-7 and BT-20 mouse xenograft models, and displayed antagonistic effects on the uterus. Furthermore, a subset of genes involved in ERRalpha signaling in vitro were evaluated and confirmed in vivo by studying uterine gene expression profiles from xenograft mice. These results suggest for the first time that inhibition of ERRalpha signaling via a subtype-specific antagonist may be an effective therapeutic strategy for ER-positive and ER-negative breast cancers.

Estrogen receptor ligands. Part 10: Chromanes: old scaffolds for new SERAMs.

The discovery, synthesis, and SAR of chromanes as ER alpha subtype selective ligands are described. X-ray studies revealed that the origin of the ER alpha-selectivity resulted from a C-4 trans methyl substitution to the cis-2,3-diphenyl-chromane platform. Selected compounds from this class demonstrated very potent in vivo antagonism of estradiol in an immature rat uterine weight assay, effectively inhibited ovariectomy-induced bone resorption in a 42 days treatment paradigm, and lowered serum cholesterol levels in ovx'd adult rat models. The best antagonists 8F and 12F also exhibited potent inhibition of MCF-7 cell growth and were shown to be estrogen receptor down-regulators (SERDs).

Immunolocalization of estrogen receptor beta in the mouse brain: comparison with estrogen receptor alpha.

Estrogen receptor alpha (ER alpha) and ER beta are members of the steroid nuclear receptor family that modulate gene transcription in an estrogen-dependent manner. ER mRNA and protein have been detected both peripherally and in the central nervous system, with most data having come from the rat. Here we report the development of an ER beta-selective antibody that cross-reacts with mouse, rat, and human ER beta protein and its use to determine the distribution of ER beta in the murine brain. Further, a previously characterized polyclonal antibody to ER alpha was used to compare the distribution of the two receptors in the first comprehensive description of ER distribution specifically in the mouse brain. ER beta immunoreactivity (ir) was primarily localized to cell nuclei within select regions of the brain, including the olfactory bulb, cerebral cortex, septum, preoptic area, bed nucleus of the stria terminalis, amygdala, paraventricular hypothalamic nucleus, thalamus, ventral tegmental area, substantia nigra, dorsal raphe, locus coeruleus, and cerebellum. Extranuclear-ir was detected in several areas, including fibers of the olfactory bulb, CA3 stratum lucidum, and CA1 stratum radiatum of the hippocampus and cerebellum. Although both receptors were generally expressed in a similar distribution through the brain, nuclear ER alpha-ir was the predominant subtype in the hippocampus, preoptic area, and most of the hypothalamus, whereas it was sparse or absent from the cerebral cortex and cerebellum. Collectively, these findings demonstrate the region-selective expression of ER beta and ER alpha in the adult ovariectomized mouse brain. These data provide an anatomical framework for understanding the mechanisms by which estrogen regulates specific neural systems in the mouse.

Identification and characterization of a functionally distinct form of human estrogen receptor beta.

Estrogen receptors are important for the development and maintenance of many different tissues in the body including the breast, uterus, brain and bone. There are two known genes encoding estrogen receptors, Estrogen Receptor alpha (ER alpha) and Estrogen Receptor beta (ER beta). These receptors are transcription factors with distinct functional domains involved in DNA binding, ligand binding and transcriptional regulation. A novel isoform of human ER beta (ER beta 548) which includes an extended amino terminal domain has been identified. Isoform specific antibodies confirm the presence of this receptor in human tissue. Transactivation analysis with different estrogenic ligands indicates that ER beta 548 is functionally distinct from previously reported forms of ER beta.