The co-chaperone p23 promotes prostate cancer motility and metastasis.

Prostate cancer is an androgen receptor (AR)-dependent malignancy at initiation and progression, therefore hormone therapy is the primary line of systemic treatment. Despite initial disease regression, tumours inevitably recur and progress to an advanced castration-resistant state a major feature of which is metastasis to the bone. Up-regulation of AR cofactors and chaperones that overcome low hormone conditions to maintain basal AR activity has been postulated as a mechanism of therapy relapse. p23, an essential component of the apo-AR complex, acts also after ligand binding to increase AR transcriptional activity and target gene expression, partly by increasing chromatin-loaded holo-receptor-complexes. Immunohistochemical studies have demonstrated increased p23 expression in advanced prostate cancer. Here, we further characterise p23 roles in AR signalling and show that it modulates cytosolic AR levels in the absence of hormone, confirming a chaperoning function in the aporeceptor complex and suggesting p23 upregulates AR signalling at multiple stages. Moreover, p23 protein levels significantly increased upon treatment with not only androgen but also clinically relevant anti-androgens. This was in contrast to the HSP90 inhibitor 17-AAG, which did not modulate expression of the cochaperone - important given the HSP90-independent roles we and others have previously described for p23. Further, we demonstrate p23 is implicated in prostate cancer cell motility and in acquisition of invasiveness capacity through the expression of specific genes known to participate in cancer progression. This may drive metastatic processes in vivo since analysis of prostate tumour biopsies revealed that high nuclear p23 significantly correlated with shorter survival times and with development of metastases in patients with lower grade tumours. We propose that increased p23 expression may allow cells to acquire a more aggressive phenotype, contributing to disease progression, and that p23 is a plausible secondary target in combination with HSP90 inhibition as a potential therapy for advanced prostate cancer.

Negative regulation of the androgen receptor gene through a primate-specific androgen response element present in the 5' UTR.

The androgen receptor (AR) is a widely expressed ligand-activated transcription factor which mediates androgen signalling by binding to androgen response elements (AREs) in normal tissue and prostate cancer (PCa). Within tumours, the amount of AR plays a crucial role in determining cell growth, resistance to therapy and progression to fatal castrate recurrent PCa in which prostate cells appear to become independent of androgenic steroids. Despite the pivotal role of the AR in male development and fertility and all stages of PCa development, the mechanisms governing AR expression remain poorly understood. In this work, we describe an active nonconsensus androgen response element (ARE) in the 5' UTR of the human AR gene. The ARE represses transcription upon binding of activated AR, and this downregulation is relieved by disruption of the regulatory element through mutation. Also, multiple species comparison of the genomic region reveals that this ARE is specific to primates, leading to the conclusion that care must be exercised when elucidating the operation of the human AR in PCa based upon rodent promoter studies.

Engineered repressors are potent inhibitors of androgen receptor activity.

Prostate cancer growth is dependent upon the Androgen Receptor (AR) pathway, hence therapies for this disease often target this signalling axis. Such therapies are successful in the majority of patients but invariably fail after a median of 2 years and tumours progress to a castrate resistant stage (CRPC). Much evidence exists to suggest that the AR remains key to CRPC growth and hence remains a valid therapeutic target. Here we describe a novel method to inhibit AR activity, consisting of an interaction motif, that binds to the AR ligand-binding domain, fused to repression domains. These 'engineered repressors' are potent inhibitors of AR activity and prostate cancer cell growth and importantly inhibit the AR under circumstances in which conventional therapies would be predicted to fail, such as AR mutation and altered cofactor levels.

Mini-review: Foldosome regulation of androgen receptor action in prostate cancer.

Steroid hormone receptors play diverse roles in many aspects of human physiology including cell division, apoptosis and homeostasis, tissue differentiation, sexual development and response to stress. These ligand-activated transcription factors require the functional activity of numerous chaperone and chaperone-associated proteins, collectively termed the foldosome, at the crucial step of ligand recognition and binding. Since the initial isolation of foldosome components and pioneering research by Pratt, Toft and colleagues we understand much regarding cytosolic receptor function. The classical view, that the role of foldosome components is restricted to the cytosol, has been modified over recent years by research highlighting additional roles of chaperone proteins in nuclear translocation and target gene expression. Further, dysregulation of chaperone activity and expression has been implicated in various cancers, including breast and prostate cancer. Consequently, the foldosome provides an attractive therapeutic target in steroid hormone receptor-driven malignancies. This review summarises current knowledge of how the foldosome impacts upon androgen receptor signalling, which is the key therapeutic target on prostate cancer, and how foldosome components may be used as biomarkers or therapeutic targets in this disease.

Role of the HSP90-associated cochaperone p23 in enhancing activity of the androgen receptor and significance for prostate cancer.

Prostate tumor growth initially depends on androgens, which act via the androgen receptor (AR). Despite androgen ablation therapy, tumors eventually progress to a castrate-resistant stage in which the AR remains active. The mechanisms are poorly understood but it may be that changes in levels or activity of AR coregulators affect trafficking and activation of the receptor. A key stage in AR signaling occurs in the cytoplasm, where unliganded receptor is associated with the heat shock protein (HSP)90 foldosome complex. p23, a key component of this complex, is best characterized as a cochaperone for HSP90 but also has HSP90-independent activity and has been reported as having differential effects on the activity of different steroid receptors. Here we report that p23 increases activity of the AR, and this appears to involve steps both in the cytoplasm (increasing ligand-binding capacity, possibly via direct interaction with AR) and the nucleus (enhancing AR occupancy at target promoters). We show, for the first time, that AR and p23 can interact, perhaps directly, when HSP90 is not present in the same complex. The effects of p23 on AR activity are at least partly HSP90 independent because a mutant form of p23, unable to bind HSP90, nevertheless increases AR activity. In human prostate tumors, nuclear p23 was higher in malignant prostate cells compared with benign/normal cells, supporting the utility of p23 as a therapeutic target in prostate cancer.

Repression of androgen receptor activity by HEYL, a third member of the Hairy/Enhancer-of-split-related family of Notch effectors.

The Hairy/Enhancer-of-split-related with YRPW-like motif (HEY) family of proteins are transcriptional repressors and downstream effectors of Notch signaling. We previously reported that HEY1 and HEY2 selectively repress androgen receptor (AR) signaling in mammalian cell lines and have shown that in human tissue HEY1 is excluded from the nuclei in prostate cancer but not benign prostatic hyperplasia. We have now characterized a third member of this family, HEYL, which is a more potent repressor of AR activity. HEYL interacted with and repressed AR activation function-1 domain and competitively inhibited SRC1e activation of AR transcriptional activity. Using a cell line inducibly expressing exogenous HEYL, we showed that HEYL represses endogenous AR-regulated genes and reduces androgen-dependent prostate cancer cell growth. Using a trans-repression assay, we identified both trichostatin-sensitive and -insensitive domains within HEYL; however, analysis of endogenous AR target genes suggested that HEYL represses AR activity through histone deacetylase I/II-independent mechanisms. Immunohistochemical analyses of tissue indicated that, in a fashion similar to that previously reported for HEY1, HEYL is excluded from the nuclei in prostate cancer but not adjacent benign tissue. This suggests that nuclear exclusion of HEY proteins may be an important step in the progression of prostate cancer.

Androgen receptor signalling in prostate cancer: the functional consequences of acetylation.

The androgen receptor (AR) is a ligand activated transcription factor and member of the steroid hormone receptor (SHR) subfamily of nuclear receptors. In the early stages of prostate carcinogenesis, tumour growth is dependent on androgens, and AR directly mediates these effects by modulating gene expression. During transcriptional regulation, the AR recruits numerous cofactors with acetylation-modifying enzymatic activity, the best studied include p300/CBP and the p160/SRC family of coactivators. It is known that recruitment of histone acetyltransferases (HATs) and histone deacetylases (HDACs) is key in fine-tuning responses to androgens and is thus likely to play a role in prostate cancer progression. Further, these proteins can also modify the AR itself. The functional consequences of AR acetylation, the role of modifying enzymes in relation to AR transcriptional response, and prostate cancer will be discussed.

Binding affinity and kinetic analysis of nuclear receptor/co-regulator interactions using surface plasmon resonance.

Knowledge of the kinetics of protein-protein interactions has become important in defining nuclear receptor function. Such knowledge allows characterization of interactions that occur with high affinity and/or selectivity. Surface plasmon resonance is a useful and sensitive tool for studying protein-protein interactions. This technique involves immobilization of a "ligand" to the surface of a sensor chip and subsequently passing over multiple concentrations of "analyte" to generate binding curves. Interaction between the receptor and target protein is monitored by the density at the sensor chip surface and allows calculation of the association and dissociation stages (and therefore affinity) of interactions to be assessed in real-time. Using software packages, these kinetic parameters can be quantified. Importantly, the levels of recombinant protein required are much less than that needed for other affinity techniques such as isothermal titration calorimetry and anisotropy fluorescence spectroscopy.

Control of patterns of corneal innervation by Pax6.

PURPOSE: Corneal nerves play essential roles in maintaining the ocular surface through provision of neurotrophic support, but genetic control of corneal innervation is poorly understood. The possibility of a neurotrophic failure in ocular surface disease associated with heterozygosity at the Pax6 locus (aniridia-related keratopathy [ARK]) was investigated. METHODS: Patterns of corneal innervation were studied during development and aging in mice with different Pax6 dosages and in chimeras. Immunohistochemistry and ELISA-based assays were used to determine the molecular basis of defects seen in Pax6 mutants, and wound healing assays were performed. RESULTS: In adults, the Pax6(+/-) epithelium was less densely innervated than the wild-type epithelium, and radial projection of epithelial nerves was disrupted. Neurotrophic support of the corneal epithelium appeared normal. Directed nerve projection correlated with patterns of epithelial cell migration in adult wild-types, but innervation defects observed in Pax6(+/-) mice were not fully corrected in wound healing or chimeric models where directed epithelial migration was restored. CONCLUSIONS: Pax6 dosage nonautonomously controls robust directed radial projection of corneal neurons, and the guidance cues for growth cone guidance are not solely dependent on directed epithelial migration. There is little evidence that ARK represents neurotrophic keratitis.

Structural characterization of the native NH2-terminal transactivation domain of the human androgen receptor: a collapsed disordered conformation underlies structural plasticity and protein-induced folding.

The androgen receptor (AR) mediates the action of the steroid hormones testosterone and dihydrotestosterone. The protein contains two globular alpha-helical domains responsible for binding hormone and DNA. In contrast, the N-terminal domain is less well structurally defined and contains the main determinants for receptor-dependent transactivation, termed AF1. Previously, we have shown this region has the propensity to form alpha-helix structure. Significantly, the binding of specific protein targets or a natural osmolyte resulted in a more protease resistant conformation for the AF1 domain, consistent with an increase in conformational stability. Computational and experimental analyses were used to investigate the conformational properties of the native AF1 domain. This region of the receptor is predicted to contain significant regions of natural disordered structure, when analyzed by amino acid composition, PONDR (Predictor of Natural Disordered Regions), RONN (Regional Order Neural Network), and GlobPlot, but is grouped with ordered proteins on a charge-hydropathy plot. The binding of a hydrophobic fluorescence probe, 8-anilinonaphthalene-1-sulfonic acid (ANS), together with size-exclusion chromatography suggests that native AR-AF1 exists in a collapsed disordered conformation, distinct from extended disordered (random coil) and a stable globular fold. This state has also been described as premolten or molten globule-like. These findings are discussed in terms of the functional importance of the intrinsic plasticity of the AF1 domain.

Functional characterization of the native NH2-terminal transactivation domain of the human androgen receptor: binding kinetics for interactions with TFIIF and SRC-1a.

The androgen receptor (AR) is a ligand-activated transcription factor that mediates the actions of the steroid hormones testosterone and dihydrotestosterone at the level of gene transcription. The main transactivation function is modular in structure, maps to the N-terminal domain (NTD), and is termed AF1. This region of the AR is structurally flexible and functions in multiple protein-protein interactions with coregulatory proteins and components of the general transcription machinery. Using surface plasmon resonance, the binding kinetics for the interaction of AR-AF1 with the large subunit of the general transcription factor TFIIF, termed RAP74, and the coactivator SRC-1a were measured. AR-AF1 interacts with both the NTD and CTD of RAP74 and the CTD of SRC-1a. The dissociation constants ( Kd) for the binding of polypeptides derived from RAP74 are in the submicromolar range, while a peptide from SRC-1a bound with a Kd of 14 microM. Significantly, the individual NTD and CTD of RAP74 interacted with AR-AF1 with distinct binding kinetics, with the NTD exhibiting slower on and off rates. TFIIF is involved in transcription initiation and elongation, and the CTD of RAP74 binds to the RNA polymerase II enzyme, the general transcription factor TFIIB, and a CTD phosphatase, FCP1. We have mutated hydrophobic residues in the RAP74-CTD structure to disrupt secondary structure elements and show that binding of AR-AF1 depends upon helix 3 in the winged-helix domain of the RAP74-CTD polypeptide. Altogether, a model is suggested for AR-AF1-dependent transactivation of receptor-target genes.

Structure and function of steroid receptor AF1 transactivation domains: induction of active conformations.

Steroid hormones are important endocrine signalling molecules controlling reproduction, development, metabolism, salt balance and specialized cellular responses, such as inflammation and immunity. They are lipophilic in character and act by binding to intracellular receptor proteins. These receptors function as ligand-activated transcription factors, switching on or off networks of genes in response to a specific hormone signal. The receptor proteins have a conserved domain organization, comprising a C-terminal LBD (ligand-binding domain), a hinge region, a central DBD (DNA-binding domain) and a highly variable NTD (N-terminal domain). The NTD is structurally flexible and contains surfaces for both activation and repression of gene transcription, and the strength of the transactivation response has been correlated with protein length. Recent evidence supports a structural and functional model for the NTD that involves induced folding, possibly involving alpha-helix structure, in response to protein-protein interactions and structure-stabilizing solutes.