Cyclin D1: mechanism and consequence of androgen receptor co-repressor activity.

Androgen receptor regulation is pivotal for prostate growth and development. Activation of the receptor is dictated by association with androgen (ligand) and through interaction with co-activators and co-repressors. We have shown previously that cyclin D1 functions as a co-repressor to inhibit ligand-dependent androgen receptor activation. We demonstrate that cyclin D1 directly binds the N terminus of the androgen receptor and that this interaction is independent of ligand. Furthermore, we show that the interaction occurs in the nucleus and does not require the LXXLL motif of cyclin D1. Although two distinct transactivation domains exist in the N terminus (AF-1 and AF-5), the data shown support the hypothesis that cyclin D1 targets the AF-1 transactivation function. The constitutively active AF-5 domain was refractory to cyclin D1 inhibition. By contrast, cyclin D1 completely abolished androgen receptor activity, even in the presence of potent androgen receptor co-activators. This action of cyclin D1 at least partially required de-acetylase activity. Finally, we show that transient, ectopic expression of cyclin D1 results in reduced cell cycle progression in androgen-dependent LNCaP cells independent of CDK4 association. Collectively, our data support a model wherein cyclin D1 has a mitogenic (CDK4-dependent) function and an anti-mitogenic function (dependent on regulation of the AF-1 domain) that can collectively control the rate of androgen-dependent cellular proliferation. These findings provide insight into the non-cell cycle functions of cyclin D1 and provide the impetus to study its pleiotropic effects in androgen-dependent cells, especially prostatic adenocarcinomas.     

Suppression of androgen receptor signaling in prostate cancer cells by an inhibitory receptor variant

There is increasing evidence that sensitization of the androgen receptor (AR) signaling pathway contributes to the failure of androgen ablation therapy for prostate cancer, and that direct targeting of the AR may be a useful therapeutic approach. To better understand how AR function could be abrogated in prostate cancer cells, we have developed a series of putative dominant-negative variants of the human AR, containing deletions or mutations in activation functions AF-1, AF-5, and/or AF-2. One construct, AR inhibitor (ARi)-410, containing a deletion of AF-1 and part of AF-5 of the AR, had no intrinsic transactivation activity but inhibited wild-type AR (wtAR) in a ligand-dependent manner by at least 95% when transfected at a 4:1 molar ratio. ARi-410 was an equally potent inhibitor of gain-of-function AR variants. Ectopic expression of ARi-410 inhibited the proliferation of AR-positive LNCaP cells, but not AR-negative PC-3 cells. Whereas ARi-410 also marginally inhibited progesterone receptor activity, this was far less pronounced than the effect on AR (50% vs. 95% maximal inhibition, respectively), and there was no inhibition of either vitamin D or estrogen receptor activity. In the presence of ligand, ARi-410 interacted with wtAR, and both receptors translocated into the nucleus. Whereas the amino-carboxy terminal interaction was not necessary for optimal dominant-negative activity, disruption of dimerization through the ligand binding domain reduced the efficacy of ARi-410. In addition, although inhibition of AR function by ARi-410 was not dependent on DNA binding, the DNA binding domain was required for dominant-negative activity. Taken together, our results suggest that interaction between ARi-410 and the endogenous AR in prostate cancer cells, potentially through the DNA binding and ligand binding domains, results in a functionally significant reduction in AR signaling and AR-dependent cell growth.     

Ligand-independent activation of the androgen receptor by interleukin-6 and the role of steroid receptor coactivator-1 in prostate cancer cells

The androgen receptor (AR) can be activated in the absence of androgens by interleukin-6 (IL-6) in human prostate cancer cells. The events involved in ligand-independent activation of the AR are unknown, but have been suggested to involve phosphorylation of the AR itself or a receptor-associated protein. Steroid receptor coactivator-1 (SRC-1) has been shown to interact with the human AR and to modulate ligand-dependent AR transactivation and is regulated by phosphorylation by MAPK. To date, no one has examined the role of SRC-1 in ligand-independent activation of the AR by IL-6 or other signaling pathways known to activate the full-length receptor. This study addressed this and has revealed the following. 1) SRC-1 similarly enhanced ligand-independent activation of the AR by IL-6 to the same magnitude as that obtained via ligand-dependent activation. 2) Androgen and IL-6 stimulated the MAPK pathway. 3) MAPK was required for both ligand-dependent and ligand-independent activation of the AR. 4) Phosphorylation of SRC-1 by MAPK was required for optimal ligand-independent activation of the AR by IL-6. 5) Protein-protein interaction between endogenous AR and SRC-1 was dependent upon treatment of LNCaP cells with IL-6 or R1881. 6) Protein-protein interaction between the AR N-terminal domain and SRC-1 was independent of MAPK. 7) Ligand-independent activation of the AR did not occur by a mechanism of overexpression of either solely wild-type SRC-1 or mutant SRC-1 that mimics its phosphorylated form.     

Allosteric conversation in the androgen receptor ligand-binding domain surfaces

Androgen receptor (AR) is a major therapeutic target that plays pivotal roles in prostate cancer (PCa) and androgen insensitivity syndromes. We previously proposed that compounds recruited to ligand-binding domain (LBD) surfaces could regulate AR activity in hormone-refractory PCa and discovered several surface modulators of AR function. Surprisingly, the most effective compounds bound preferentially to a surface of unknown function [binding function 3 (BF-3)] instead of the coactivator-binding site [activation function 2 (AF-2)]. Different BF-3 mutations have been identified in PCa or androgen insensitivity syndrome patients, and they can strongly affect AR activity. Further, comparison of AR x-ray structures with and without bound ligands at BF-3 and AF-2 showed structural coupling between both pockets. Here, we combine experimental evidence and molecular dynamic simulations to investigate whether BF-3 mutations affect AR LBD function and dynamics possibly via allosteric conversation between surface sites. Our data indicate that AF-2 conformation is indeed closely coupled to BF-3 and provide mechanistic proof of their structural interconnection. BF-3 mutations may function as allosteric elicitors, probably shifting the AR LBD conformational ensemble toward conformations that alter AF-2 propensity to reorganize into subpockets that accommodate N-terminal domain and coactivator peptides. The induced conformation may result in either increased or decreased AR activity. Activating BF-3 mutations also favor the formation of another pocket (BF-4) in the vicinity of AF-2 and BF-3, which we also previously identified as a hot spot for a small compound. We discuss the possibility that BF-3 may be a protein-docking site that binds to the N-terminal domain and corepressors. AR surface sites are attractive pharmacological targets to develop allosteric modulators that might be alternative lead compounds for drug design.     

Inhibition of Plk1 represses androgen signaling pathway in castration-resistant prostate cancer

Prostate cancer (PCa) is the second leading cause of cancer-related death in males in the United States. Majority of prostate cancers are originally androgen-dependent and sensitive to androgen-deprivation therapy (ADT), however, most of them eventually relapse and progress into incurable castration-resistant prostate cancer (CRPC). Of note, the activity of androgen receptor (AR) is still required in CRPC stage. The mitotic kinase polo-like kinase 1 (Plk1) is significantly elevated in PCa and its expression correlates with tumor grade. In this study, we assess the effects of Plk1 on AR signaling in both androgen-dependent and androgen-independent PCa cells. We demonstrate that the expression level of Plk1 correlated with tumorigenicity and that inhibition of Plk1 caused reduction of AR expression and AR activity. Furthermore, Plk1 inhibitor BI2536 down-regulated SREBP-dependent expression of enzymes involved in androgen biosynthesis. Of interest, Plk1 level was also reduced when AR activity was inhibited by the antagonist MDV3100. Finally, we show that BI2536 treatment significantly inhibited tumor growth in LNCaP CRPC xenografts. Overall, our data support the concept that Plk1 inhibitor such as BI2536 prevents AR signaling pathway and might have therapeutic potential for CRPC patients.     

Cyclin E as a coactivator of the androgen receptor

Androgens play an important role in the growth of prostate cancer, but the molecular mechanism that underlies development of resistance to antiandrogen therapy remains unknown. Cyclin E has now been shown to increase the transactivation activity of the human androgen receptor (AR) in the presence of its ligand dihydrotestosterone. The enhancement of AR activity by cyclin E was resistant to inhibition by the antiandrogen 5-hydroxyflutamide. Cyclin E was shown to bind directly to the COOH terminus portion of the AB domain of the AR, and to enhance its AF-1 transactivation function. These results suggest that cyclin E functions as a coactivator of the AR, and that aberrant expression of cyclin E in tumors may contribute to persistent activation of AR function, even during androgen ablation therapy.