Anca Azoitei

Stilbene induced inhibition of androgen receptor dimerization: implications for AR and ARΔLBD-signalling in human prostate cancer cells.

Background Advanced castration resistant prostate cancer (CRPC) is often characterized by an increase of C-terminally truncated, constitutively active androgen receptor (AR) variants. Due to the absence of a ligand binding domain located in the AR-C-terminus, these receptor variants (also termed ARΔLBD) are unable to respond to all classical forms of endocrine treatments like surgical/chemical castration and/or application of anti-androgens. Methodology In this study we tested the effects of the naturally occurring stilbene resveratrol (RSV) and (E)-4-(2, 6-Difluorostyryl)-N, N-dimethylaniline, a fluorinated dialkylaminostilbene (FIDAS) on AR- and ARΔLBD in prostate cancer cells. The ability of the compounds to modulate transcriptional activity of AR and the ARΔLBD-variant Q640X was shown by reporter gene assays. Expression of endogenous AR and ARΔLBD mRNA and protein levels were determined by qRT-PCR and Western Blot. Nuclear translocation of AR-molecules was analyzed by fluorescence microscopy. AR and ARΔLBD/Q640X homo-/heterodimer formation was assessed by mammalian two hybrid assays. Biological activity of both compounds in vivo was demonstrated using a chick chorioallantoic membrane xenograft assay. Results The stilbenes RSV and FIDAS were able to significantly diminish AR and Q640X-signalling. Successful inhibition of the Q640X suggests that RSV and FIDAS are not interfering with the AR-ligand binding domain like all currently available anti-hormonal drugs. Repression of AR and Q640X-signalling by RSV and FIDAS in prostate cancer cells was caused by an inhibition of the AR and/or Q640X-dimerization. Although systemic bioavailability of both stilbenes is very low, both compounds were also able to downregulate tumor growth and AR-signalling in vivo. Conclusion RSV and FIDAS are able to inhibit the dimerization of AR and ARΔLBD molecules suggesting that stilbenes might serve as lead compounds for a novel generation of AR-inhibitors.

Androgen receptor aberrations in the era of abiraterone and enzalutamide

Prostate cancer is the most prevalent non-skin cancer and the second leading cause of cancer death in men of the western world. As growth and differentiation of prostate cancer largely depend on androgens, inhibition of the androgen/androgen receptor signaling axis is the main treatment for locally advanced and/or metastatic tumors. Although first-line androgen deprivation therapies like chemical/surgical castration and/or administration of anti-androgens are able to control the disease for several years, prostate cancer almost invariably recurs as castration-resistant prostate cancer. This stage of the disease is characterized by a sustained AR-signaling despite castrate levels of circulating androgens. Various molecular mechanisms were shown to induce castration resistance. This review will discuss the most recent and relevant experimental findings on AR-signaling in castration-resistant prostate cancer in order to provide a comprehensive interpretation of the clinical behavior of this tumor entity following treatments with abiraterone, enzalutamide, ARN-509 or taxanes.

DNA Affects Ligand Binding of the Ecdysone Receptor of Drosophila melanogaster

The heterodimers of all three ecdysone receptor (EcR) isoforms with Ultraspiracle (Usp), the invertebrate orthologue of RXR, bind Ponasterone A with the same affinity in the absence of DNA. Ligand binding is stimulated by ecdysone response elements (EcREs) to different degrees depending on the receptor isoform, the heterodimerization partner, and the type of EcRE. Ligand binding to heterodimers with wtUsp is enhanced 5-fold with hsp27, Pal-1 and DR-1. In the presence of DNA substantial differences in ligand binding were observed, when the AB-domain of wtUsp is replaced by the N-terminus of VP16, which is routinely used for the determination of transcriptional activity to overcome the inhibitory action of the AB-domain of Usp. Enhanced dimerization in the presence of hormone response elements increases mainly the number of binding sites resulting in improved ligand binding, which is observed even if the C-domain of Usp is deleted. RXR, which can partially replace Usp function, confers high affinity ligand binding only in the presence of an EcRE.

DNA‐binding properties of Drosophila ecdysone receptor isoforms and their modification by the heterodimerization partner ultraspiracle

Transcriptional activity of ecdysone receptor (EcR) isoforms varies considerably and is modified further by the heterodimerization partner and hormone treatment. To investigate whether differences in DNA binding of receptor complexes are responsible for these variations in transcriptional activity, interaction of Drosophila EcR isoforms, and variants of Ultraspiracle (Usp), the orthologue of RXR, with the ecdysone response elements (EcRE) hsp 27, PAL-1, and DR-1, were determined by electrophoretic mobility shift assays. Receptor proteins were expressed in vertebrate cells (CHO-K1) in order to rule out an influence of endogenous receptor proteins. In the absence of a heterodimerization partner, weak DNA binding of EcR was detected even without hormone with EcR-A and -B1, but not EcR-B2. In the presence of hormone, all three isoforms show increased binding to the hsp 27 EcRE. The heterodimerization partner Usp increased DNA binding considerably. The hormone effect of heterodimers is more pronounced with both EcR-B isoforms compared to EcR-A. Two specific bands were obtained for EcR-A and B1 but only one band is visible with EcR-B2. Deletion of the C-domain of Usp still allows basal DNA binding of the heterodimer, but in contrast to full-length Usp, addition of hormone decreases the intensity of the retarded receptor band of all EcR isoforms and the EcREs hsp27 and DR-1 considerably, whereas interaction with the EcRE PAL-1 is only slightly affected. Synergistic effects on transcriptional activity are associated with the formation of different receptor DNA-complexes observed with 1xhsp27 and 3xhsp27. Comparison of DNA-binding properties of EcR isoforms and EcR/Usp heterodimers revealed that binding of receptor complexes to hsp 27 EcRE is dependent on the AB domain of EcR and the AB-, C-, and D-domains of the heterodimerization partner. Interaction with the hsp 27 EcRE correlates neither with ligand binding nor with transcriptional activity of the various receptor complexes. We, therefore, conclude that the different receptor functions are regulated separately, for example, by interaction with co-modulators or post-transcriptional modifications.

Inhibition of IGF-1R diminishes transcriptional activity of the androgen receptor and its constitutively active, C-terminally truncated counterparts Q640X and AR-V7

AbstractPurposeFailure of endocrine treatment in castration-resistant prostate cancer (CRPC) is often associated with the emergence of C-terminally truncated androgen receptor variants that function as constitutively active transcription factors (i.e., AR∆LBD). The mechanisms involved in the regulation of AR∆LBD signaling are largely unknown. Since the IGF-1 pathway was repeatedly shown to affect AR function, we studied whether an inhibition of IGF-1R could also affect AR∆LBD signaling. MethodsRegulation of androgen receptor (AR) and AR∆LBD signaling was analyzed by reporter gene assays, immunoblotting, ELISA and quantitative RT-PCR.ResultsInhibition of IGF-1R with the small-molecule inhibitor NVP-AEW541 reduced the transcriptional activity of the AR and its truncated counterparts Q640X and AR-V7. As shown in Q640X, the inhibition of transcriptional activity was paralleled by a decreased receptor phosphorylation.ConclusionsInhibition of IGF-1R leads to a down-regulation of AR∆LBD signaling and provides a rationale for CRPC therapies targeting growth factor receptors.

C-terminally truncated constitutively active androgen receptor variants and their biologic and clinical significance in castration-resistant prostate cancer

A mechanism allowing castration resistant prostate cancer cells to escape the effects of conventional anti-hormonal treatments is the synthesis of constitutively active, C-terminally truncated androgen receptor (AR)-variants. Lacking the entire or vast parts of the ligand binding domain, the intended target of traditional endocrine therapies, these AR-variants (termed ARΔLBD) are insensitive to all traditional treatments including second generation compounds like abiraterone, enzalutamide or ARN-509. Although ARΔLBD are predominantly products of alternative splicing, they can also be products of nonsense mutations or proteolytic cleavage. In this review, we will discuss the etiology and function of c-terminally truncated AR-variants and their clinical significance as markers/targets for the treatment of castration resistant prostate cancer.

Interaction of the N-terminus of ecdysone receptor isoforms with the ligand-binding domain

Ecdysone receptor (EcR) isoforms exert different biological functions, although they vary only in their N-terminal domain. Despite identical C-termini, which mediate hormone-induced activity, the influence of ligand is isoform specific, which indicates an N/C-interaction. The position of helix 12 with and without hormone varies among isoforms and modifies N/C-interaction determined by fluorescence resonance-energy transfer (FRET), which depends on the salt bridge between helices 4 and 12 of the ligand-binding domain (LBD). Disruption of the salt bridge by mutation of K497 (helix 4) had no effect on basal N/C-interaction, but prevented the hormone-induced increase, which was partially restored by a salt bridge with reversed polarity. The heterodimerization partner Ultraspiracle (Usp) can compensate for the disruption of the salt bridge. Without ligand the AB-domains of EcR-A and EcR-B1, but not EcR-B2, interact with the LBD via K497 and repress transcriptional activity. This intramolecular cross talk between N- and C-terminus along with the position of helix 12 stabilized by K497 regulates transcriptional activity of EcR isoforms.

Functional Analysis of Ecdysteroid Receptor from Drosophila melanogaster “In Vitro”

Ecdysone receptor (EcR) was expressed in vertebrate cells to study its functional properties in the absence of the heterodimerization partner Ultraspiracle (Usp) and to avoid interference with endogenous receptor isoforms. Comparison of different isoforms affords determination of receptor concentration, which was achieved either by determination of ligand binding sites by Scatchard analysis or by quantitative evaluation of specific Western blot signals, but not by normalization on transfection efficiency as determined by cotransfection with a constitutive reporter plasmid. Ligand- and DNA- binding, and transcriptional activity of EcR isoforms and the influence of Ultraspiracle (Usp) were described.

Influence of hormone response elements (HREs) on ecdysteroid receptor concentration

Transcriptional activity of nuclear receptors is the result of transactivation capability and receptor protein concentration. The concentration of ecdysteroid receptor (EcR) constitutively expressed in vertebrate cells varies depending on the isoforms. Besides ligand binding and heterodimerization with ultraspiracle (USP), which stabilizes receptor protein concentration, degradation is regulated by interaction of the receptor complex with different ecdysteroid response elements (EcREs). Coexpression of EcREs significantly reduces ecdysteroid receptor concentration depending on the type of EcRE. Transcriptional activity and interaction with hormone response elements (HREs) as determined by Electrophoretic Mobility Shift Assay (EMSA) are often inversely related to receptor protein concentration. The complex regulation of receptor protein concentration offers an additional opportunity to regulate transcriptional activity in an isoform‐ and target cell‐specific manner and allows the temporal limitation of hormone action.

Influence of helix 12 of Ultraspiracle on Drosophila melanogaster ecdysone receptor function.

Although it has no ligand, helix 12 in the ligand binding domain of Ultraspiracle (USP) is locked in an antagonistic position. To investigate whether this position is of functional importance, we enhanced the flexibility of helix 12 by mutating two amino acids (259, located in L1–3 and F491 in helix 12). Mutated USP reduces the stability of USP and all isoforms of the ecdysone receptor (EcR) and impairs nuclear localization and DNA binding of EcR/USPL259A/F491/A, resulting in lower levels of basal transcriptional activity. Although the affinity of the ligand ponasterone A to EcR/USPL259/F491 is moderately diminished, hormone‐induced stimulation of transcriptional activity is normal. Potentiation of the ecdysone response by juvenile hormone (JH) is selectively increased in mutated heterodimers with EcR‐B1, demonstrating that the antagonistic position impairs functional interaction of the EcR complex with JHIII.