Christine Helsen

Diverse roles of androgen receptor (AR) domains in AR-mediated signaling

Androgens control male sexual development and maintenance of the adult male phenotype. They have very divergent effects on their target organs like the reproductive organs, muscle, bone, brain and skin. This is explained in part by the fact that different cell types respond differently to androgen stimulus, even when all these responses are mediated by the same intracellular androgen receptor. To understand these tissue- and cell-specific readouts of androgens, we have to learn the many different steps in the transcription activation mechanisms of the androgen receptor (NR3C4). Like all nuclear receptors, the steroid receptors have a central DNA-binding domain connected to a ligand-binding domain by a hinge region. In addition, all steroid receptors have a relatively large amino-terminal domain. Despite the overall structural homology with other nuclear receptors, the androgen receptor has several specific characteristics which will be discussed here. This receptor can bind two types of androgen response elements (AREs): one type being similar to the classical GRE/PRE-type elements, the other type being the more divergent and more selective AREs. The hormone-binding domain has low intrinsic transactivation properties, a feature that correlates with the low affinity of this domain for the canonical LxxLL-bearing coactivators. For the androgen receptor, transcriptional activation involves the alternative recruitment of coactivators to different regions in the amino-terminal domain, as well as the hinge region. Finally, a very strong ligand-induced interaction between the amino-terminal domain and the ligand-binding domain of the androgen receptor seems to be involved in many aspects of its function as a transcription factor. This review describes the current knowledge on the structure-function relationships within the domains of the androgen receptor and tries to integrate the involvement of different domains, subdomains and motifs in the functioning of this receptor as a transcription factor with tissue- and cell-specific readouts.

Structural basis for nuclear hormone receptor DNA binding

The gene family of nuclear receptors is characterized by the presence of a typical, well conserved DNA-binding domain. In general, two zinc coordinating modules are folded such that an α-helix is inserted in the major groove of the DNA-helix displaying a sequence similar to one of two hexameric consensus motifs. Both zinc molecules coordinate four cysteines. Although the DNA-binding domains as well as the hormone response elements are very similar, each nuclear receptor will affect transcription of a specific set of target genes. This is in part due to some important receptor-specific variations on the general theme of DNA interaction. For most nuclear receptors, the DNA-binding domain dimerizes on DNA, which explains why most hormone response elements consist of a repeat of two hexamers. The hexamer dimers can be organized either as direct, inverted or everted repeats with spacers of varying lengths. The DNA can be bound by homodimers, heterodimers and for some orphan receptors, as monomer. Another key element for DNA binding by nuclear receptors is the carboxy-terminal extension of the DNA-binding domain extending into the hinge region. This part not only co-determines sequence specificity, but also affects other functions of the receptors like nuclear translocation, intranuclear mobility and transactivation potential. Moreover, allosteric signals passing through towards other receptor domains, explain why to some extent, the DNA elements can also be considered as controlling ligands.

A satellite cell-specific knockout of the androgen receptor reveals myostatin as a direct androgen target in skeletal muscle

Androgens have well‐established anabolic actions on skeletal muscle, although the direct effects of the androgen receptor (AR) in muscle remain unclear. We generated satellite cell‐specific AR‐knockout (satARKO) mice in which the AR is selectively ablated in satellite cells, the muscle precursor cells. Total‐limb maximal grip strength is decreased by 7% in satARKO mice, with soleus muscles containing ~10% more type I fibers and 10% less type IIa fibers than the corresponding control littermates. The weight of the perineal levator ani muscle is markedly reduced (–52%). Thus, muscle AR is involved in fiber‐type distribution and force production of the limb muscles, while it is a major determinant of the perineal muscle mass. Surprisingly, myostatin (Mstn), a strong inhibitor of skeletal muscle growth, is one of the most androgen‐responsive genes (6‐fold reduction in satARKO) through direct transcription activation by the AR. Consequently, muscle hypertrophy in response to androgens is augmented in Mstn‐knockout mice. Our finding that androgens induce Mstn signaling to restrain their own anabolic actions has implications for the treatment of muscle wasting disorders.—Dubois, V., Laurent, M. R., Sinnesael, M., Cielen, N., Helsen, C., Clinckemalie, L., Spans, L., Gayan‐Ramirez, G., Deldicque, L., Hespel, P., Carmeliet, G., Vanderschueren, D., and Claessens, F. A satellite cell‐specific knockout of the androgen receptor reveals myostatin as a direct androgen target in skeletal muscle. FASEB J. 28, 2979–2994 (2014). www.fasebj.org

Emerging mechanisms of enzalutamide resistance in prostate cancer

The majority of prostate cancers are hormone-dependent at diagnosis highlighting the central role of androgen signalling in this disease. Surprisingly, most forms of castration-resistant prostate cancer (CRPC) are still dependent on the androgen receptor (AR) for survival. Therefore, the advent of new AR-targeting drugs, such as enzalutamide, is certainly beneficial for the many patients with metastatic CRPC. Indeed, this compound provides a substantial survival benefit—but it is not curative. This Perspectives article describes the different ways through which cancer cells can become resistant to enzalutamide, such as AR truncation and other mutations, as well as by-pass of the AR dependence of prostate cancer cells through expression of the glucocorticoid receptor. The clinical relevance of these mechanisms and emerging questions concerning new therapeutic regimens in the treatment of metastatic CRPC are being discussed.

A 629RKLKK633 motif in the hinge region controls the androgen receptor at multiple levels

The androgen receptor protein has specific domains involved in DNA binding, ligand binding, and transactivation, whose activities need to be integrated during transcription activation. The hinge region, more particular a 629RKLKK633 motif, seems to play a crucial role in this process. Indeed, although the motif is not part of the DNA-binding domain, its positive residues are involved in optimal DNA binding and nuclear translocation as shown by mutation analysis. When the mutated ARs are forced into the nucleus, however, the residues seem to play different roles in transactivation. Moreover, we show by FRAP analysis that during activation, the AR is distributed in the nucleus in a mobile and two immobile fractions, and that mutations in the 629RKLKK633 motif affect the distribution of the AR over these three intranuclear fractions. Taken together, the 629RKLKK633 motif is a multifunctional motif that integrates nuclear localization, receptor stability, DNA binding, transactivation potential and intranuclear mobility.

Evidence for DNA-binding domain--ligand-binding domain communications in the androgen receptor.

ABSTRACT DNA binding as well as ligand binding by nuclear receptors has been studied extensively. Both binding functions are attributed to isolated domains of which the structure is known. The crystal structure of a complete receptor in complex with its ligand and DNA-response element, however, has been solved only for the peroxisome proliferator-activated receptor γ (PPARγ)-retinoid X receptor α (RXRα) heterodimer. This structure provided the first indication of direct interactions between the DNA-binding domain (DBD) and ligand-binding domain (LBD). In this study, we investigated whether there is a similar interface between the DNA- and ligand-binding domains for the androgen receptor (AR). Despite the structural differences between the AR- and PPARγ-LBD, a combination of in silico modeling and docking pointed out a putative interface between AR-DBD and AR-LBD. The surfaces were subjected to a point mutation analysis, which was inspired by known AR mutations described in androgen insensitivity syndromes and prostate cancer. Surprisingly, AR-LBD mutations D695N, R710A, F754S, and P766A induced a decrease in DNA binding but left ligand binding unaffected, while the DBD-residing mutations K590A, K592A, and E621A lowered the ligand-binding but not the DNA-binding affinity. We therefore propose that these residues are involved in allosteric communications between the AR-DBD and AR-LBD.

Androgen receptor antagonists for prostate cancer therapy

Androgen deprivation is the mainstay therapy for metastatic prostate cancer (PCa). Another way of suppressing androgen receptor (AR) signaling is via AR antagonists or antiandrogens. Despite being frequently prescribed in clinical practice, there is conflicting evidence concerning the role of AR antagonists in the management of PCa. In the castration-resistant settings of PCa, docetaxel has been the only treatment option for decades. With recent evidence that castration-resistant PCa is far from AR-independent, there has been an increasing interest in developing new AR antagonists. This review gives a concise overview of the clinically available antiandrogens and the experimental AR antagonists that tackle androgen action with a different approach.

The Role of Single Nucleotide Polymorphisms in Predicting Prostate Cancer Risk and Therapeutic Decision Making

Prostate cancer (PCa) is a major health care problem because of its high prevalence, health-related costs, and mortality. Epidemiological studies have suggested an important role of genetics in PCa development. Because of this, an increasing number of single nucleotide polymorphisms (SNPs) had been suggested to be implicated in the development and progression of PCa. While individual SNPs are only moderately associated with PCa risk, in combination, they have a stronger, dose-dependent association, currently explaining 30% of PCa familial risk. This review aims to give a brief overview of studies in which the possible role of genetic variants was investigated in clinical settings. We will highlight the major research questions in the translation of SNP identification into clinical practice.

The discovery of novel human androgen receptor antagonist chemotypes using a combined pharmacophore screening procedure

Unraveling the mechanisms involved in castration‐ and therapy‐resistant prostate cancer has led to a renewed interest in androgen receptor (AR)‐targeted therapeutics. Anti‐androgens that block the activity of the AR therefore remain a valid therapeutic option. However, they must be more effective than, or display a distinct mechanism of action or binding mode from those of bicalutamide and hydroxyflutamide, which are currently in clinical use. For that reason, the second‐generation anti‐androgen MDV3100 was developed. MDV3100, however, shares its 4‐cyano‐3‐(trifluoromethyl)phenyl group with bicalutamide and hydroxyflutamide required for binding to the AR. In this work, we used a combined strategy to find new antagonist structures distinct from the 4‐cyano‐3‐(trifluoromethyl)phenyl group to avoid cross‐resistance for these compounds and to find structures without agonist activity on mutant ARs (AR W741C and AR T877A). We found two novel chemotypes with AR‐antagonistic activity (IC50: 3–6 μM) by virtual screening and confirmed their biological activity in an androgen‐responsive reporter assay. The design of our computational approach was validated by the observation of strongly decreased or absence of agonistic activity on the two mutant ARs. Further structural derivatization to optimize the potency of these compounds can render these chemotypes into very promising, alternative AR antagonists for prostate cancer therapy.

The Genomic Landscape of Prostate Cancer

By the age of 80, approximately 80% of men will manifest some cancerous cells within their prostate, indicating that prostate cancer constitutes a major health burden. While this disease is clinically insignificant in most men, it can become lethal in others. The most challenging task for clinicians is developing a patient-tailored treatment in the knowledge that this disease is highly heterogeneous and that relatively little adequate prognostic tools are available to distinguish aggressive from indolent disease. Next-generation sequencing allows a description of the cancer at an unprecedented level of detail and at different levels, going from whole genome or exome sequencing to transcriptome analysis and methylation-specific immunoprecipitation, followed by sequencing. Integration of all these data is leading to a better understanding of the initiation, progression and metastatic processes of prostate cancer. Ultimately, these insights will result in a better and more personalized treatment of patients suffering from prostate cancer. The present review summarizes current knowledge on copy number changes, gene fusions, single nucleotide mutations and polymorphisms, methylation, microRNAs and long non-coding RNAs obtained from high-throughput studies.