T877A Mutant Androgen Receptor Research Articles

Resveratrol, piceatannol and analogs inhibit activation of both wild-type and T877A mutant androgen receptor

Prostate cancer growth and progression are mainly dependent on androgens and many current prostate cancer treatment options target the synthesis or function of androgens. We have previously reported that resveratrol and synthetic analogs of resveratrol with a higher bioavailability inhibit the synthesis of androgens in human adrenocortical H295R cells. Now we have studied the antiandrogenic properties of resveratrol, piceatannol and analogs in two different prostate cell lines; LNCaP and RWPE. LNCaP carry a T877A mutation in the androgen receptor while RWPE has a wild-type androgen receptor. We found that resveratrol, piceatannol and all studied analogs were able to inhibit a dihydrotestosterone-induced activation of the androgen receptor, showing that they act as antiandrogens. In LNCaP cells, all studied compounds were able to statistically significantly decrease the androgenic signaling in concentrations ≥1μM and the synthetic analogs trimethylresveratrol (RSVTM) and tetramethylpiceatannol (PICTM) were the most potent compounds. RWPE cells were not as responsive to the studied compounds as the LNCaP cells. A statistically significant decrease in the androgenic signaling was observed at concentrations ≤5μM for most compounds and RSVTM was found to be the most potent compound. Further, we studied the effects of resveratrol, piceatannol and analogs on the levels of prostate-specific antigen (PSA) in LNCaP cells and found that all studied compounds decreased the level of PSA and that the synthetic analogs diacetylresveratrol (RSVDA), triacetylresveratrol (RSVTA) and RSVTM were the most potent compounds, decreasing the PSA level by approx. 50% at concentrations ≥10μM. In a cell-free receptor binding assay we were unable to show binding of resveratrol or analogs to the ligand binding domain of the androgen receptor, indicating that the observed effects are mediated via other mechanisms than direct ligand competition. We conclude that the resveratrol, piceatannol and analogs are highly interesting for chemoprevention of prostate cancer, since they have a high potency both as inhibitors of androgen synthesis and androgen receptor activation.

Preliminary investigations into triazole derived androgen receptor antagonists

A range of 1,4-substituted-1,2,3-N-phenyltriazoles were synthesized and evaluated as non-steroidal androgen receptor (AR) antagonists. The motivation for this study was to replace the N-phenyl amide portion of small molecule antiandrogens with a 1,2,3-triazole and determine effects, if any, on biological activity. The synthetic methodology presented herein is robust, high yielding and extremely rapid. Using this methodology a series of 17 N-aryl triazoles were synthesized from commercially available starting materials in less than 3h. After preliminary biological screening at 20 and 40μM, the most promising three compounds were found to display IC50 values of 40–50μM against androgen dependent (LNCaP) cells and serve as a starting point for further structure–activity investigations. All compounds in this work were the focus of an in silico study to dock the compounds into the human androgen receptor ligand binding domain (hARLBD) and compare their predicted binding affinity with known antiandrogens. A comparison of receptor–ligand interactions for the wild type and T877A mutant AR revealed two novel polar interactions. One with Q738 of the wild type site and the second with the mutated A877 residue.

The direct inhibitory effect of dutasteride or finasteride on androgen receptor activity is cell line specific

Finasteride and dutasteride were developed originally as 5α-reductase inhibitors to block the conversion of testosterone to dihydrotestosterone (DHT). These drugs may possess off-target effects on the androgen receptor (AR) due to their structural similarity to DHT. A total of four human prostate cancer cell models were examined: LNCaP (T877A mutant AR), 22Rv1 (H874Y mutant AR), LAPC4 (wild-type AR), and VCaP (wild-type AR). Cells were cultured in 10% charcoal-stripped fetal bovine serum, either with or without DHT added to the medium. AR activity was evaluated using the ARE-luciferase assay or the expression of AR regulated genes. Dutasteride was more potent than finasteride in interfering with DHT-stimulated AR signaling. Disruption of AR function was accompanied by decreased cell growth. Cells that rely on DHT for protection against death were particularly vulnerable to dutasteride. Different prostate cancer cell models exhibited different sensitivities to dutasteride and finasteride. LNCaP was most sensitive, LAPC4 and VCaP were intermediate, while 22Rv1 was least sensitive. Regardless of the AR genotype, if AR was transfected into drug-sensitive cells, AR was inhibited by drug treatment; and if AR was transfected into drug-resistant cells, AR was not inhibited. The direct inhibitory effect of dutasteride or finasteride on AR signaling is cell line specific. Mutations in the ligand binding domain of AR do not appear to play a significant role in influencing the AR antagonistic effect of these drugs. Subcellular constituent is an important factor in determining the drug effect on AR function.

Interactions of Abiraterone, Eplerenone, and Prednisolone with Wild-Type and Mutant Androgen Receptor: A Rationale for Increasing Abiraterone Exposure or Combining with MDV3100—Letter

We read with interest the report from Richards and colleagues (1) on the potential interactions of abiraterone, eplerenone, and prednisolone with wild-type and mutant androgen receptor (AR). Abiraterone is a CYP17A1 inhibitor that blocks androgen biosynthesis in the testes, adrenals, and castration-resistant prostate cancer (CRPC) tumors, producing androgen ablation with significant clinical benefit in patients with metastatic CRPC. The current report (1) and other studies (2) suggesting that abiraterone downregulates AR expression and produces antiandrogen activity beyond androgen depletion merit further investigation.The suppression of adrenal steroid biosynthesis via abiraterone inhibition of CYP17A1 reduces circulating cortisol, with compensatory release of adrenocorticotropic hormone (3). This induces increased adrenal secretion of mineralocorticoids that can contribute to hypokalemia and hypertension. These manifestations of mineralocorticoid excess are managed by coadministration of low-dose prednisone (a prodrug of prednisolone) in patients treated with abiraterone acetate.Richards and colleagues reported that prednisolone activated the AR in cells transfected with T877A-mutant AR with an EC50 of 25.1 μmol/L. This suggested that coadministration of prednisolone with abiraterone acetate could contribute to disease progression. However, the EC50 is much higher than the plasma concentrations of prednisolone (4–305 nmol/L) measured in 15 patients with CRPC receiving abiraterone acetate plus prednisolone. Furthermore, the authors extrapolated the reported prednisolone plasma concentrations to a prior publication on the in vitro androgen agonist activity of glucocorticoids at nanomolar concentrations in a cell line bearing an AR ligand-binding domain double mutation (T877A plus L701H) to ascribe clinical significance (4). We feel that this comparison is inappropriate in relation to the current findings and the interpretation of the findings related to abiraterone acetate therapy. The prevalence of this double mutation is unknown without established clinical relevance.The authors also reported findings using spironolactone and eplerenone, which produced androgen agonist activity in vitro in cells transfected with wild-type (spironolactone only) or mutant AR at concentrations relevant to clinical activity. The report suggested that this could contribute to disease progression if these drugs were used in patients with prostate cancer. However, neither agent is proposed for use with abiraterone acetate to ameliorate symptoms of mineralocorticoid excess.Abiraterone acetate was studied in conjunction with low-dose prednisone/prednisolone in registration trials leading to approval (5); only prednisone/prednisolone, not spironolactone or eplerenone, are specified in approved labeling. Given the high concentrations of prednisolone investigated in the reported in vitro studies, the clinical significance of the findings is not obvious.D. End is employed as Director of Johnson and Johnson/Janssen and has ownership interest (including patents) in Johnson and Johnson. A. Molina is the Vice President of Johnson and Johnson and has ownership interest (including patents) in the stock options of Johnson and Johnson. No potential conflicts of interest were disclosed by the other authors.

Intratumoral De Novo Steroid Synthesis Activates Androgen Receptor in Castration-Resistant Prostate Cancer and Is Upregulated by Treatment with CYP17A1 Inhibitors

Relapse of castration-resistant prostate cancer (CRPC) that occurs after androgen deprivation therapy of primary prostate cancer can be mediated by reactivation of the androgen receptor (AR). One important mechanism mediating this AR reactivation is intratumoral conversion of the weak adrenal androgens DHEA and androstenedione into the AR ligands testosterone and dihydrotestosterone. DHEA and androstenedione are synthesized by the adrenals through the sequential actions of the cytochrome P450 enzymes CYP11A1 and CYP17A1, so that CYP17A1 inhibitors such as abiraterone are effective therapies for CRPC. However, the significance of intratumoral CYP17A1 and de novo androgen synthesis from cholesterol in CRPC, and the mechanisms contributing to CYP17A1 inhibitor resistance/relapse, remain to be determined. We report that AR activity in castration-resistant VCaP tumor xenografts can be restored through CYP17A1-dependent de novo androgen synthesis, and that abiraterone treatment of these xenografts imposes selective pressure for increased intratumoral expression of CYP17A1, thereby generating a mechanism for development of resistance to CYP17A1 inhibitors. Supporting the clinical relevance of this mechanism, we found that intratumoral expression of CYP17A1 was markedly increased in tumor biopsies from CRPC patients after CYP17A1 inhibitor therapy. We further show that CRPC cells expressing a progesterone responsive T877A mutant AR are not CYP17A1 dependent, but that AR activity in these cells is still steroid dependent and mediated by upstream CYP11A1-dependent intraturmoral pregnenolone/progesterone synthesis. Together, our results indicate that CRPCs resistant to CYP17A1 inhibition may remain steroid dependent and therefore responsive to therapies that can further suppress de novo intratumoral steroid synthesis.

The antiandrogenic effect of finasteride against a mutant androgen receptor

Finasteride is known to inhibit Type 2 5α-reductase and thus block the conversion of testosterone to dihydrotestosterone (DHT). The structural similarity of finasteride to DHT raises the possibility that finasteride may also interfere with the function of the androgen receptor (AR). Experiments were carried out to evaluate the antiandrogenic effect of finasteride in LNCaP, C4-2 and VCaP human prostate cancer cells. Finasteride decreased DHT binding to AR, and DHT-stimulated AR activity and cell growth in LNCaP and C4-2 cells, but not in VCaP cells. LNCaP and C4-2 (derived from castration-resistant LNCaP) cells express the T877A mutant AR, while VCaP cells express the wild type AR. When PC-3 cells, which are AR-null, were transfected with either the wild type or the T877A mutant AR, only the mutant AR-expressing cells were sensitive to finasteride inhibition of DHT binding. Peroxiredoxin-1 (Prx1) is a novel endogenous facilitator of AR binding to DHT. In Prx1-rich LNCaP cells, the combination of Prx1 knockdown and finasteride was found to produce a greater inhibitory effect on AR activity and cell growth than either treatment alone. The observation suggests that cells with a low expression of Prx1 are likely to be more responsive to the antiandrogenic effect of finasteride. Additional studies showed that the efficacy of finasteride was comparable to that of bicalutamide (a widely used non-steroidal antiandrogen). The implication of the above findings is discussed in the context of developing strategies to improve the outcome of androgen deprivation therapy.

Abstract LB-420: Intratumoral de novo steroid synthesis mediates prostate cancer resistance to androgen deprivation and CYP17a1 inhibition

Abstract Prostate cancer (PCa) that relapses after androgen deprivation therapy (ADT, medical or surgical castration), termed castration resistant prostate cancer (CRPC), has increased expression of enzymes that convert adrenal dehydroepiandrosterone (DHEA) and androstenedione to testosterone and dihydrotestosterone (DHT). DHEA and androstenedione are synthesized at high levels by the adrenals through the sequential actions of CYP11A1 and CYP17A1; hence CYP17A1 inhibitors such as abiraterone are effective therapies for CRPC. However, mechanisms contributing to CYP17A1 inhibitor relapse remain to be determined. We show that castration resistant VCaP PCa xenografts synthesize androgens de novo at levels that fully restore androgen receptor (AR) activity and respond to abiratrone, and that development of abiraterone resistance is associated with markedly increased intratumoral CYP17A1 expression. Consistent with these results, we find that tumor expression of CYP17A1 is markedly increased in patients after CYP17A1 inhibitor therapy. We further show that CRPC cells expressing a T877A mutant AR are not CYP17A1 dependent, and that AR activity in these cells is instead mediated by upstream CYP11A1 dependent pregnenolone/progesterone synthesis. These findings indicate that CYP17A1 inhibitor resistant tumors are still steroid dependent and may respond to dose escalation, alternative CYP17A1 inhibitors, or agents blocking other steps in steroid synthesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-420. doi:10.1158/1538-7445.AM2011-LB-420

Computationally Identified Novel Diphenyl- and Phenylpyridine Androgen Receptor Antagonist Structures

We have identified and profiled a set of androgen receptor (AR) binding compounds representing two nonsteroidal scaffolds from a public chemical database supplied by Asinex with virtual screening procedure incorporating our recently published 3D QSAR model of AR ligands. The diphenyl- and phenylpyridine-based compounds act as antagonists in wild-type AR in CV1 cells and also retain this antagonistic character in CV1 cells expressing T877A mutant receptor. This mutation is frequently associated with prostate cancer. Two of the compounds repress the androgen-dependent cell growth of LNCaP prostate cancer cells expressing the T877A AR mutant. Molecular modeling of the observed in vitro antagonism with induced fit docking suggests that W741 and M895 could be mechanistically involved in the initiation of the antagonism. The results indicate finding of nonsteroidal AR antagonist compounds from a public chemical database with computational methods. Compounds could serve as a novel platform to develop more potent AR antagonists with inhibitory activity in both wild-type and T877A mutant AR.

Antiandrogen effects of mifepristone on coactivator and corepressor interactions with the androgen receptor.

Mifepristone is a potent antagonist of steroid hormone receptors such as glucocorticoid and progesterone receptors. We investigated the potential for mifepristone to act as an antiandrogen and compared it with partial androgen receptor (AR) agonists and antagonists, in particular bicalutamide. Mifepristone was an effective antiandrogen in vitro that inhibited transcription from three androgen-responsive promoters and blocked the agonist R1881 in a dose-dependent manner. Like bicalutamide, mifepristone also antagonized the action of androgen receptor with a (T877A) mutation. Mifepristone competed effectively with R1881 with a relative binding affinity comparable to that of cyproterone acetate, and much higher than that of hydroxyflutamide and bicalutamide in a binding assay. Mifepristone could effectively induce the binding of the herpes simplex viral protein 16/AR fusion protein to the hormone response elements in the murine mammary tumor virus-luciferase reporter. With either wild-type or T877A mutant AR, mifepristone alone was unable to induce any detectable interaction with coactivators transcriptional intermediary factor-2 or beta-catenin but could inhibit the R1881-induced binding of AR to transcriptional intermediary factor-2 and beta-catenin. Similarly, mifepristone could inhibit the R1881-induced N/C-terminal interaction in a dose-dependent manner even though mifepristone alone has no effect on the N/C-terminal interaction of AR. We found that mifepristone could induce a strong interaction between AR and corepressors nuclear receptor corepressor and silencing mediator for retinoid and thyroid hormone receptors in both transactivation and two-hybrid assays to a greater degree than hydroxyflutamide, cyproterone acetate, and bicalutamide. The AR-corepressor interaction was also seen in coimmunoprecipitation assays. Finally, mifepristone at high concentrations induced a low level of prostate-specific antigen expression in LNCaP and antagonized prostate-specific antigen expression induced by R1881. Mifepristone also antagonized R1881 action on the growth of LNCaP prostate cancer cells.

Constitutive activation of the androgen receptor by a point mutation in the hinge region: a new mechanism for androgen-independent growth in prostate cancer.

Androgen receptor (AR) mutations that modify both the ligand binding and the transactivation capacities of the AR represent one of the mechanisms involved in the transition of prostate cancer (PCa) from androgen-dependent to androgen-independent growth. We use a yeast-based functional assay to detect and analyze mutant ARs in PCa. We report the detection of 2 different mutant ARs within the same metastatic tumour sample harvested in a patient with advanced PCa who had escaped androgen deprivation. Concomitantly to the widely described T877A mutant AR, we identified an additional double mutant AR harboring the nonsense mutation Q640Stop just downstream the DNA binding domain together with the T877A point mutation. This type of mutation, which leads to a c-terminal truncated AR, has not been described yet in PCa. Using luciferase reporter assays we demonstrated that this truncated AR exhibited constitutive transactivation properties. In conclusion, our data suggest that mutation-induced constitutive activation of the AR could be a mechanism used by PCa cells to escape androgen deprivation.