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Recurrent, metastatic prostate cancer continues to be a leading cause of cancer-death in men. The androgen receptor (AR) is important in the development of the prostate by regulating transcription, cellular proliferation, and apoptosis. The GR is expressed in almost every cell in the body and regulates either directly or indirectly genes controlling a wide variety of processes including the development, metabolism, and immune response of the organism.
In the absence of hormone, the glucocorticoid receptor (GR) resides in the cytosol complexed with a variety of proteins including heat shock protein 90 (hsp90), the heat shock protein 70 (hsp70) and the protein FKBP52 (FK506-binding protein 52).[4] The endogenous glucocortiod hormone cortisol diffuses through the cell membrane into the cytoplasm and binds to the glucocorticoid receptor (GR) resulitng in release of the heat shock proteins.
A direct mechanism of action involves homodimerization of the receptor, translocation via active transport into the nucleus, and binding to specific DNA responsive elements activating gene transcription. In the absence of activated GR, other transcription factors such as NF-?B or AP-1 themselves are able to transactivate target genes.
Steroid hormones exert a wide variety of effects on growth, development, and differentiation, including important regulatory and behavioral functions within the reproductive, central nervous system, and adrenal axis. STRUCTURE OF THE STEROID HORMONE RECEPTOR PROTEIN In order to understand how steroid hormone receptors regulate gene function, it is important to know the structure of the receptor proteins as well as the identity and cellular function of the genes that they regulate.
The androgen receptor (AR) is a modular, ligand-inducible transcription factor that regulates the expression of genes that can drive the progression of this disease, and as a consequence, this receptor is a key therapeutic target for controlling prostate cancer. IntroductionProstate cancer (PCa) is predicted to be the leading cause of cancer-related death in men over the next decade [1]. AR undergoes posttranslational modifications that alter its transcription activity, translocation to the nucleus and stability. IntroductionThe androgen receptor (AR) is a member of the steroid hormone receptor family; other family members consist of the estrogen, progesterone, mineralocorticoid, and glucocorticoid receptors [1].
These hormones act through binding to specific intracellular receptor proteins that function as both signal transducers and transcription factors to modulate expression of target genes.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 Molecular cloning has revealed 48 steroid hormone and nuclear receptor genes in humans (Table 1). Members of the steroid receptor superfamily share direct amino acid homology and a common structure (Fig.
In its early stages and when localized to the prostate, this cancer can usually be cured by surgery or radiation therapy. The posttranslational modifications that regulate these events are of utmost importance to understand the functional role of AR and its activity.
AR plays a vital role in the development of the prostate as well as benign prostate hyperplasia and prostate cancer by regulating cellular proliferation [2–5], survival [6], apoptosis [7] and secretion [2]. The pharmacology and classification of the nuclear receptor superfamily: glucocorticoid, mineralocorticoid, progesterone, and androgen receptors".
Unfortunately, with the inevitable progression of the cancer to castration resistance, many of these drugs become ineffective. However, for advanced, metastatic or recurrent disease, alternative systemic treatments are required. The majority of these modifications occur in the activation function-1 (AF1) region of the AR, which contains the transcriptional activation unit 1 (TAU1) and 5 (TAU5). AR is a 919-amino-acid protein encoded from a ~180 kb gene that is located at chromosome Xq11-12. However, there are numerous other regulatory sites on this protein that have not been exploited therapeutically.
In this regard, the androgen receptor (AR), a ligand-inducible transcription factor, is considered to be central for PCa development, growth and metastasis [2,3].The AR belongs to the steroid hormone receptor subfamily of the nuclear receptor superfamily. Identification of the modifications that occur to these regions may increase our understanding of AR activation in prostate cancer and the role of AR in the progression from androgen-dependent to castration-resistant prostate cancer (CRPC). The regulation of AR activity involves a cascade of complex interactions with numerous chaperones, co-factors and co-regulatory proteins, leading ultimately to direct binding of AR dimers to specific DNA androgen response elements within the promoter and enhancers of androgen-regulated genes. The human AR is coded by a gene located on the chromosome at Xq11-12 and is composed of 919 amino acids.
Most of the posttranslational modifications identified to date have been determined using the full-length AR in androgen dependent cells. The largest, comprising of over half of the receptor, is the N-terminal domain (NTD) [9–11], which is highly unstructured and contains one of the two activation function (AF1) motifs. Variability between members of the steroid hormone receptor family is due primarily to differences in the length and amino acid sequence of the amino (N)-terminal domain. As part of the family of nuclear receptors, the AR is organized into modular structural and functional domains with specialized roles in facilitating their inter-molecular interactions.
Similar to other nuclear receptors, the AR is organized into three distinct domains: An N-terminal domain (NTD), followed by a DNA binding (DBD) and a C-terminal ligand binding domain (LBD) [4,5]. Further investigations into the role of posttranslational modifications in androgen-independent activation of full-length AR and constitutively active splicing variants are warranted, findings from which may provide new therapeutic options for CRPC.
Within AF1 there are two transcriptional activation unit (TAU) regions: TAU1 (residues 101–360) and TAU5 (residues 370–494) [12]. These regions of the AR present attractive, yet largely unexploited, drug target sites for reducing or eliminating androgen signaling in prostate cancers. Within this family of receptors, the AR shares most of the sequence similarity with progesterone, glucocorticoid and estrogen receptors [6,7]. The second functional region in the AR is the DNA binding domain (DBD), which contains two zinc fingers. Superfamily of steroid nuclear receptors: Positive and negative regulators of gene expression.
The design of small molecule inhibitors targeting these specific AR domains is only now being realized and is the culmination of decades of work, including crystallographic and biochemistry approaches to map the shape and accessibility of the AR surfaces and cavities. Steroidal ligands, principally testosterone and dihydrotestosterone (DHT), bind to a ligand binding pocket on the LBD, called the androgen binding site (ABS), to initiate an activation cascade that results in the transcriptional activation of genes that promote PCa cell viability and growth (for review, see [8]).
The first zinc finger interacts with the half-site of the androgen-response element (ARE) [13–15], and the second facilitates dimerization [13,15]. Here, we review the structure of the AR protein and describe recent advancements in inhibiting its activity with small molecules specifically designed to target areas distinct from the receptor’s androgen binding site. Accordingly, treatment of advanced prostate cancers usually involves some form of surgical or chemical castration to lower the level of circulating androgens and, thereby, to prevent AR transcriptional activity [9]. A short flexible peptide sequence called the hinge region connects the DBD to the ligand binding domain (LBD), wherein the second transcriptional activation function (AF2) resides [16–18].In the absence of androgens, AR is localized primarily in the cytoplasm and remains in an inactive state and interacts with heat shock proteins (HSP90, HSP70, HSP56, and HSP27) [23,24], which prevents it from entering the nucleus [25–27]. It is anticipated that these new classes of anti-AR drugs will provide an additional arsenal to treat castration-resistant prostate cancer. In addition, to maximize androgen blockade, PCa patients are often treated with drugs called anti-androgens, which compete with naturally occurring androgens for the receptor’s androgen-binding site. Upon binding of androgens to the LBD, AR undergoes a conformational change, which releases bound HSPs; AR dimerizes and is rapidly transported into the nucleus [23,28]. Unfortunately, most of these cancers eventually progress to a castration-resistant state, where they no longer respond to androgen deprivation or anti-androgen treatments.In an attempt to overcome resistance to conventional anti-androgens, computational modeling and high throughput screening techniques have been used to identify small molecules that specifically target functional surface sites of the AR. Several groups have employed this approach to systematically and iteratively optimize small molecules for high-affinity binding to the AR and its effective inhibition [10–15]. Coactivators and chromatin remodeling complexes are recruited to facilitate transcription of AR target genes [29]. The development of such inhibitors has been made possible by investigating the three-dimensional structure of the AR and its co-factors by means of X-ray crystallography, site-directed mutagenesis and biophysical measurements probing the AR’s conformational dynamics and interaction with ligands.It is important to note that rational, structure-based drug design has been used extensively to develop inhibitors for a number of other nuclear receptors, including estrogen- and progesterone receptors (ER and PR, respectively), and such efforts have resulted in new anti-cancer drugs (for review, see [16]). A well-known gene regulated by AR is prostate specific antigen (PSA), which currently is used as a biomarker for prostate cancer (PCa). These successes have motivated a rational drug discovery approach, guided by protein structure and biochemical experiments, to study protein-ligand interactions of the AR and to develop new types of anti-AR therapies. Besides PSA, AR regulates many other genes that are involved in regulation of proliferation and apoptosis.The role that androgens play in PCa was first described by Huggins and Hodges in 1941 [30], who noted that upon depletion of androgens, prostate tumors shrink. Since then, androgen depravation therapy has been the mainstay of treatment for advanced PCa.
2 Schematic representation of the common structural and functional domains of the steroid hormone receptors. Unfortunately, PCa usually reoccurs within 18–36 months and becomes a lesion termed as castration-resistant prostate cancer (CRPC) [31–33]. AR expression is often elevated in CRPC [8,34,35], and is believed to be either hypersensitive to androgens [36–38], constitutively active [39], or activated by non-canonical pathways [40]. Moreover, androgens can be synthesized by PCa cells and activate AR in an intracrine fashion [41]. These variants are clinically relevant as they are expressed in PCa cell lines, xenografts, and human tumors [45,48,49]. AR-variants (AR-Vs) are not dependent upon androgens for activation as they lack the LBD (Figure 2) [20,50].
A comprehensive review of alternatively spliced AR variants was published recently by Dehm and Tindall [51].AR has two activation function (AF) motifs, AF1 in the NTD and AF2 in the LBD. The progesterone and androgen receptors (PR and AR) exist in two distinct forms, A and B, synthesized from the same mRNA by alternate splicing.
However, unlike other nuclear receptors where AF2 has strong transcriptional activity, AF1 is responsible for the majority of AR activity [52–54]. In androgen-dependent PCa cells, the activity of the full-length AR depends on ligand binding, where approximately 50% of ligand-dependent activity is mediated by TAU1 [12]. AF1 is also important for the constitutive transcriptional activity of AR-Vs where the LBD is missing.
Indeed, the constitutive activity of AF1 in AR-Vs is similar to that of the full-length AR activated by androgens [12]. The highly conserved DBD shared by AR, GR, mineralocorticoid receptor (MR), and PR enables them to bind to the same HRE, called the glucocorticoid response element (GRE). When the LBD is truncated from the AR, constitutive activity shifts from the TAU1 region to TAU5 [12]. Within TAU5 of the AF1 region is the core sequence 435WHTLF439 that regulates approximately 50% the androgen-independent activity of AR in CRPC.
3 Schematic diagram of type II zinc finger proteins characteristic of the DNA-binding domain structure of members of the steroid hormone receptor superfamily. Mutation of the core motif WXXLF into AXXLF resulted in a decrease in AR activity by half in C4-2 cells while in androgen-dependent LNCaP cells there was no difference in androgen-dependent transcriptional activity of AR [19]. Zinc fingers are common features of many transcription factors, allowing proteins to bind to DNA. The TAU5-dependent constitutive activity of AR suggests that it is a potential target for treatment of CRPC and therefore is of key interest to understand how the constitutive activation of the AR is regulated.AR undergoes a number of posttranslational modifications that alter its functional activity including transcriptional activity, stability, and cellular localization. Previous reviews by Gioeli and Paschal [55] have identified several of the modifications; specifically, they describe the kinase responsible for phosphorylation of the AR. The CI zinc finger interacts specifically with five base pairs of DNA and determines the DNA sequence recognized by the particular steroid receptor. Similarly, Coffey and Robson reviewed the posttranslational modifications to the AR highlighting acetylation, methylation, SUMOylation, and ubiquitination along with phosphorylation [56]. Lavery and Bevan reviewed the functional consequences of acetylation to the androgen receptor [57], while Clinckemalie et al.
Here we describe the known posttranslational modifications that have been identified to-date. Most of the modifications, identified and reviewed, were discovered in overexpression systems or in androgen dependent cell lines.
N-Terminus Domain (NTD)The multifunctional role of the AR is implemented through its modular domain organization.
Here we summarize the modifications, along with the cell line to give clarity to determine if the modification is present and could result in the activation of the AR in CRPC.


The AR N-terminus domain (NTD) corresponds to the first 558 residues and contains the AF1 functional region. The identification of different modifications between androgen dependent and CRPC may lead to a better understanding of AR reactivation in CRPC.
The NTD is characterized by the presence of two large repeats, termed homopolymers: Poly-glutamine and poly-glycine fragments, averaging 21 and 24 residues, respectively. This region contains the ligand-binding site and dictates hormone binding specificity.30, 31 Greater structural similarity between steroid hormone ligands generally indicates greater amino acid sequence homology in the LBD. The variation in length of the poly-glutamine has been associated with such diseases as X-linked spinal and bulbar muscular atrophy and prostate cancer [62–65]. Furthermore, two transcriptional activation units (TAU) were mapped onto the AR NTD: TAU-1 (residues 101–370) and TAU-5 (residues 360–485) [67,68].
Importantly, the AR-NTD contains an FXXLF motif at residues 23–27 and a WXXLF motif at residues 433–437 that are essential for the interaction with the AR’s LBD. It has been shown that the NTD serves as a binding site for many transcription machinery components, including TFIIF and TFIIH proteins [73–75], co-activators, such as CREB-binding protein (CBP) [76,77], and co-repressors, like SMRT (silencing mediator for retinoic acid and thyroid hormone receptor) [78].
Serine and Threonine Phosphorylation of the ARSerine 16 (S16) phosphorylation is ligand dependent, as phosphorylation at this site increases upon treatment with ligand [60,77,78]. Additionally, the N- and C- terminals of the receptor interact with each other to increase transcriptional activation.74STRUCTURE OF STEROID HORMONE-REGULATED GENES The transcription of DNA to messenger RNA (mRNA) is the most important process regulated by steroid hormones.
Despite the importance of the NTD for AR function, currently, there is no structural information available for this domain, in part due to intrinsic disordered regions that compromise the stability of the NTD in solution and prevent its crystallization [72]. However, activation of PKA in LNCaP cells by forskolin, failed to increase S16 phosphorylation as determined by 32P-phosphopeptide mapping, suggesting that PKA is not responsible for AR S16 phosphorylation. 4), composed of a structural region in which the DNA encodes the specific amino acids of the protein, and a regulatory region that interacts with various proteins to control the rate of transcription.
Given that forskolin treatment did increase the overall AR phosphorylation level in LNCaP cells [77], PKA may mediate AR phosphorylation at other site(s). Several key elements in the regulatory region of the target gene must be activated before mRNA synthesis can occur. Intriguingly, when the LBD of AR was deleted, it was found that S16 in AR was phosphorylated in the absence of androgens.
Thus, it has been speculated that the S16 could be phosphorylated in androgen-independent PCa [78].
This serine residue lies within the region that interacts with the C-terminus of AR upon dimerization. 4 Schematic diagram of regulatory and structural regions of a steroid hormone responsive gene. Phosphorylation of S16 is androgen dependent in full-length AR, but was phosphorylated in the absence of the LBD, suggesting that this residue may need to be phosphorylated for AR dimerization.Phosphorylation of serine 81 (S81) is mediated by cyclin dependent kinases (CDKs), including CDK1, CDK5, and CDK9. Interestingly, expression of CDK1 increases during the transition from AD to CRPC cells [85].
Hinge RegionThe region between the DBD and the LBD (residues 625–689) is flexible and poorly conserved among NRs.
Cell cycle analysis reveals that there is an increase in AR phosphorylation during mitosis, which coincides with the increased CDK1 activity [64,81]. The signal responsible for nuclear import is encoded by a bipartite nuclear localization signal (NLS: 617-RKCYEAGMTLGARKLKKL-634) formed by two clusters of basic residues belonging to the C-terminus of the DBD and the N-terminus of the hinge region [84–86].
CDK5 also enhances S81 phosphorylation along with its p35 activator and the p25 byproduct of p35 prevents AR phosphorylation at S81 [59,86]. The cellular localization of the AR is controlled by androgen binding, such that the AR is cytoplasmic in its ligand-free state, but upon binding of androgen to the LBD, it undergoes a conformational change, which exposes the NLS and facilitates its interaction with importin-?, which results in translocation of the activated AR to the nucleus [22,87]. AR protein degradation is decreased in LNCaP cells overexpressing CDK5 or p35 compared to CDK5 knockdown cells, suggesting that phosphorylation of S81 induces AR stability. Overexpression of CDK5 in LNCaP cells increased AR nuclear localization while knockdown increased AR cytoplasmic localization [59]. While the pan CDK inhibitor roscovitine blocks the activity of CDK1, CDK5, and CDK9, it inhibits DHT-induced AR phosphorylation at S81.
S81 was not highly phosphorylated in LNCaP cells under androgen-depleted conditions, but upon DHT treatment, there was an increase in phosphorylation [74,81]. In contrast, the AR antagonist bicalutamide was unable to increase the phosphorylation at this site. When circulating levels of steroid hormones exceed the binding capacity of their respective binding proteins, they can then bind nonspecifically, and with low affinity, to albumin, from which they can readily dissociate and enter target cells.207 The unbound and loosely albumin-bound steroids are generally believed to be the most biologically important fractions since the steroid is free to diffuse (or be actively transported) through the capillary wall and lipid plasma membrane bilayer.
These results were confirmed in CWR22Rv1 and LACP-4 cell lines, suggesting that phosphorylation at S81 is regulated by androgens [81]. Moreover, S81 was also phosphorylated when the LBD-truncated AR was overexpressed in 293 cells [78].
Furthermore, one inhibitor of PKC (bisindolylmaleimide) was unable to prevent AR S81 phosphorylation [77]. Thus, besides the CDKs, PKC is a putative kinase potentially responsible for AR S81 phosphorylationAR S81 phosphorylation plays an important role in AR transactivation, cellular localization and stability as well as cell proliferation.
In addition to hsp90, unliganded steroid receptors extracted from animal tissues or mammalian cells showed that GR and PR are complexed with a number of other proteins including hsp70, FKB59, p60, p48 (Hip), and p23.243 These proteins are thought to be required for the assembly and maintenance of ligand-sensitive aporeceptor complexes.
Moreover, overexpression of CDK5 in HEK293 cells resulted in an increase in S81 phosphorylation and PSA expression following androgen treatment; whereas in CDK5-knockdown cells no such increase was observed [59]. These data suggest that CDK5-mediated S81 phosphorylation plays a role in androgen-induced transactivation of the AR.It was reported that the S81A mutant remained in the cytoplasm, while a phosphomimicking mutant, S81D, localized in the nucleus in the absence of ligand [64]. In LNCaP cells, there was a decrease in AR nuclear localization when CDK5 was knocked down by siRNAs. 5  Schematic diagram of the mechanism of steroid hormone receptor action in a target cell. The wild-type full length AR consists of eight exons and introns that can be spliced into a plethora of forms, some lacking entire domains (Figure 3). Similarly, when S81A was expressed, lack of S81 phosphorylation prevented nuclear localization of AR [59]. A target cell contains the steroid hormone receptor(s) required to respond to a given steroid hormone. AR splice variants arise primarily through exon skipping and cryptic exon inclusion, where splicing introduces RNA sequences not normally included in the transcript [97].
Although some splice variants, such as AR-45 [98], are found in normal prostate tissue, variants lacking the LBD have been found to be upregulated in tumours (compared to levels in normal prostate cells) [99–102]. Most steroid hormones that enter the cell travel in the bloodstream either in free state or loosely bound to serum albumins. Expression of the S81A mutant in LNCaP cells also resulted in a decrease in cell proliferation compared to wild-type AR [59]. Estrogens and androgens are bound to steroid hormone binding globulin (SHBG) with high affinity.
The finding that AR-S81A was lost after several passages in LAPC4 cells further supports its role of S81 phosphorylation in cell growth and proliferation [75]. Activation of adenylate cyclase by the SHBG receptor or peptide hormones such as LH generate cAMP which activates the protein kinase A (PKA) phosphorylation cascade.
In contrast, knockdown of CDK5 by siRNAs decreased AR half-life, and the proteasome inhibitor MG132 blocked this effect.
Furthermore, expression of CDK5 expression stabilizes wild-type AR but not the S81A mutant [59]. The exception is the glucocorticoid receptor (GR) which is complexed with the hsp90 chaperonin complex in the cytoplasm.
These data suggest that CDK5-mediated phosphorylation of S81 regulates the stability of the AR.Evidence suggests that serine 94 (S94) in AR is constitutively phosphorylated and hormone insensitive [60,74,77,87,88]. Hsp90 and its accompanying proteins are thought to stabilize the receptor until hormone-binding occurs. It has been shown that the transcriptional activity of AR was not altered when both S94 and S81 were mutated to alanine (A) although the effect of single residue mutation on AR transcriptional activity was not determined [74,88]. Although receptors for progesterone, estrogens, and androgens have also been found in complexes with the primarily cytosolic hsps, the function of these associations is unclear.
The finding that S94 was phosphorylated when the LBD-truncated mutant of AR was expressed in 293 cells supports the notion that S94 is constitutively phosphorylated [78].Serine 213 (S213) phosphorylation has been studied extensively. In the case of estrogens, progestins, and androgens, the hormones freely enter the nucleus and bind to their cognate receptor.
To date, AKT [61] and PIM-1 [76,89] have been shown to be responsible for S213 phosphorylation. This allows the receptors to form homodimers and bind to specific hormone response elements (HREs, see Table 2 for specific sequences) in the DNA in the regulatory region of the target gene. Activation of AKT by PI3K increases S213 phosphorylation and the PI3K inhibitor LY294002 suppresses phosphorylation [61].
Treatment of LNCaP, 22Rv1 and LAPC4 cells with the inhibitor isosilybin B suppressed PSA expression [90]. Once bound to the HRE, the liganded steroid hormone receptor induces a DNA bend and, in the presence of agonist ligand, recruits coactivator proteins. In addition to altering the transcriptional activity of AR, isosilybin B was shown to prevent R1881 (a synthetic androgen)-induced nuclear localization of AR in LNCaP cells and inhibit cell growth. The HRE-bound receptor also interacts with other DNA-bound transcription factors, as shown for the interaction of Sp1 with ER. Certain coactivator proteins have histone acetyltransferase (HAT) activity that acetylates lysine residues in histones H3 and H4. The decrease in AR protein was due to the recruitment of the E3 ubiquitination (Ub) ligase Mdm2. Adapted from Naar AM, Beaurang PA, Robinson KM, Oliner JD, Avizonis D, Scheek S, Zwicker J, Kadonaga JT, Tjian R. As demonstrated in both androgen-dependent (LNCaP and LAPC4) and -independent cells (22Rv1) [90], Mdm2 formed a protein complex with both AKT and AR as evident in co-immunoprecipitation (co-IP) assays and this complex was lost when AKT was inactive.Another kinase that regulates the phosphorylation of S213 is PIM-1 [76,89]. Chromatin, TAFs, and a novel multiprotein coactivator are required for synergistic activation by Sp1 and SREBP-1a in vitro. PIM-1 has two distinct isoforms, L (long) and S (short), both of which are able to phosphorylate S213 as determined in LNCaP and COS cells. Even though both PIM isoforms are able to phosphorylate S213, it appears that each has a distinct role in regulation of AR. Ligand-independent recruitment of steroid receptor coactivators to estrogen receptor by cyclin D.
Overexpression of PIM-1S resulted in a decrease in AR half-life, while expression of PIM-1L was unable to alter the half-life of the protein. Phosphorylation of S213 by PIM-1S resulted in the recruitment of Mdm2 as detected by co-IP, while PIM-1L was unable to be co-immunoprecipitated with Mdm2.
However, during this period of the cell cycle there is a decrease in the amount of activated AKT protein. This suggests that a second kinase is responsible for maintaining the phosphorylation of AR during the G2 and M phases of the cell cycle. It can be speculated that PIM-1 may maintain the phosphorylated state of S213 upon translocation into the nucleus and promote the degradation of AR upon entry into the cell cycle in proliferative cells.AR is also phosphorylated at serine 256 (S256) [60,77,91].
Casein kinase II is the putative kinase responsible for this phosphorylation based upon the consensus sequence defined at this site [77].
Binding of the hormone to the receptor may be only one of several factors that activates or transforms the receptor, enabling it to bind as a dimer to specific hormone response elements located adjacent to or sometimes at a distance from the transcription start site of the regulated gene.


The function of this residue phosphorylation has yet to be determined.Two residues in close proximity of each other, threonine 280 and serine 291, are phosphorylated by Aurora A (AurA) in both the absence and presence of androgens. Treatment of LNCaP cells with R1881 had a minimal enhancement of luciferase activity in cells expressed either the active or inactive AurA. This data suggests that T280 and S291 are not critical for androgen induction of AR’s transcriptional activity, but that they may be important in androgen-independent AR activation. These two residues may also work in concert with other phosphorylated residues to enhance AR activity. Phosphorylation of this residue was identified through mutational analysis of residues in the TAU1 region, and decreased AR phosphorylation was observed upon mutation of S308 to a nonphosphorylatable alanine residue [60].
DNA bending appears to be important in cellular processes mediated by multiprotein complexes, including transcription in both prokaryotes,279 and eukaryotes.280, 281, 282, 283 DNA bending is thought to facilitate interactions between components of the transcription complex bound to different sites and to promote DNA looping to allow single proteins to contact multiple DNA elements. Targeting the N-Terminal DomainThe identification of small molecules capable of binding to the AR-NTD has proven to be an elusive goal, given that no structural information is available for this domain. However, since both ligand-dependent and -independent transcriptional activity of the AR is attributed to its N-terminal Tau1 and Tau5 regions (see section 3.1), the NTD remains a very attractive drug target for treating both early stage PCa and CRPC. When bound to the specific HRE on the DNA, the hormone-receptor complex interacts with basal transcription factors and with other proteins to stabilize basal transcription factor binding and promote the assembly of the transcription initiation complex.
This led to the discovery of sintokamides, small peptides with varying degrees of chlorination, which were isolated from the marine sponge Dysidea sp. In conjunction with mutation of six additional residues (S16, S81, S94, S256, S308, and S650) there was a decrease in AR activity [60]. Mutational analysis of S424 to alanine resulted in the loss of phosphorylation at this site [77]. One such peptide variant, sintokamide A, demonstrated inhibition of the growth of the androgen-dependent LNCaP PCa cell line.
However, no studies have identified a role, or the kinase responsible, for S424 phosphorylation.
In addition, sintokamides showed inhibition of transcriptional activity of the NTD fragment of the AR fused to a Gal4 DBD domain (using a luciferase reporter and Gal4 promoter sequence). Further studies on this residue are of importance as it is the sole residue that has been identified to date to be phosphorylated within the TAU5 region, which plays a crucial role in androgen-independent activation of the AR.Serine 515 (S515) undergoes phosphorylation mediated by CDK7 and MAP kinase [70,71]. Since the Gal4-NTD construct has no ligand binding domain, it was suggested that sintokamides are also effective in supressing the AR activity under androgen-independent conditions. The kinase CDK7 is part of the TFIIH transcription complex that regulates the two ubiquitination E3 ligases carboxyl-terminus of Hsc70-interacting protein (CHIP) and mouse homologue of double minute 2 protein (Mdm2). These proteins have also been termed receptor interacting proteins (RIPs) and RAPs (receptor associated proteins); however, not all RIPs are coactivators.
A more recent high-throughput screen of extracts from the marine sponge Niphates digitalis has yielded additional candidate compounds that also antagonize AR transcriptional activity [121]. A mutant S515A that inhibited phosphorylation was found to co-IP with CHIP but was unable to bind to Mdm2.
Furthermore, it has been proposed that one such compound, niphatenone B alkynyl ether, may covalently bind to the AF1 region of the NTD [121].As an alternative approach, a decoy peptide comprising the entire AR-NTD sequence (from amino acid 1 to 538) has been shown to inhibit the transcriptional activity of the full length AR (detected with a PSA reporter construct) [122]. Both CHIP and Mdm2 appear to be equally associated with AR and both E3 ligases are able to promote AR polyubiquitination while monoubiquitination was only identified when CHIP was present [70]. The mechanism of action of the AR1–538 peptide remains unclear, but it likely competes with known AR cofactors for NTD binding. A phosphomimetic mutant S515E appears to increase the transcriptional activity of AR as measured in a PSA luciferase assay.
It has also been shown that AR1–538 peptide is effective in both ligand-dependent and -independent conditions.
The S515 phosphorylated AR or the phosphomimetic mutant S515E had a shorter half-life presumably due to the recruitment of Mdm2 to promote AR for protein degradation since Mdm2 had a lower affinity for binding to the S515A mutant.
AR protein degradation could be rescued using the MG132 proteasome inhibitor demonstrating that AR protein loss was a result of degradation [70]. This molecule, being a close analog of bisphenol A diglycidic ether, could block transcription of a PSA reporter construct in LNCaP cells.
Epidermal growth factor (EGF) treatment induces S515 phosphorylation in COS cells expressing truncated AR?507-660. These proteins have been identified in yeast and mammalian two hybrid screening, during purification, in immunoprecipitation assays, and by cross-linking studies.
In addition, EPI-001 showed similar activity in 22rv1 cells, which contain both full length AR and the AR-V7 splice variant (Figure 3).
Using a PSA reporter construct, EPI-001 was shown to also inhibit the transcriptional activity of a truncated form of the AR lacking the LBD domain (AR1–653). Together, these results suggest that EPI-001 has the capability to directly target the NTD of the AR splice variants. As demonstrated in CWR-R1 cells derived from the CRPC cell line CWR22, expression of the phosphorylation-resistant alanine mutation at S515 resulted in decreased AR transcriptional activity as measured by PSA-Enh-Luc assay [71].Although serine 578 (S578) lies within a PKC consensus phosphorylation motif, the kinase responsible for phosphorylation at this site is still undetermined.
Importantly, the activity of progesterone receptors and glucocorticoid receptors was not affected by EPI-001, which illustrates its specificity for the AR. Treatment of CWR-R1 and Ishikawa cells with EGF resulted in an increase in AR transcriptional activity, which coincided with an increase in phosphorylation of S578. However, in the absence of the AR-NTD crystal structure, it is unclear how EPI-001 binds to the AR. Both FLAG-AR-(amino acids 507–660)-S578D (phosphomimetic) and FLAG-AR-(amino acids 507–660)-S578A (phosphorylation-resistant) were transfected into COS cells to investigate cellular localization of AR. Nevertheless, intrinsic fluorescence of tyrosine and tryptophan residues within an AF1 peptide could be modulated by this compound, suggesting direct binding to the NTD. The phosphomimetic mutant was equally distributed between the cytoplasm and the nucleus, while the alanine mutant exclusively resided in the nucleus [71]. Whether EPI-001 binds to the AF1 region within the context of the full length AR remains to be determined. These findings suggest that S578 or other residues in this region (507–660) play an important role in regulation of AR cellular localization.Phosphorylation of serine 650 (S650) occurs both in the presence and absence of androgens [77] and regulates AR localization and transcriptional activity [73,74,77,87].
Some support for this interaction was shown by co-immunoprecipitation experiments with the AR, where EPI-001 caused a modest (21%) reduction of the pull-down of CBP, a co-factor known to bind to the AR AF1.
The inhibitory effect of EPI-001 in vivo was validated by showing a volume reduction in CRPC tumour xenografts in mice.
Currently, EPI-001 is the best characterized compound that appears to target and inhibit the activity of the AR-NTD and, therefore, is a good candidate drug for treating CRPC. S650 is present within the CK II kinase phosphorylation consensus motif; but the CK II inhibitor DRB was unable to prevent the phosphorylation of S650 in LNCaP cells [73].
Stimulation of LNCaP cells with an upstream activator of PKC, phorbol-12-myristate-13-acetate, resulted in a phosphorylation event at S650, whereas use of one PKC inhibitor (bisindolylmaleimide) was unable to prevent the phosphorylation event [77]. These observations suggest that the bisindolylmaleimide-insensitive isoforms of PKC (?, ?, and ?) may be responsible for S650 phosphorylation. However, inhibition of either p38 (SB203580) or JNK1 (SP600125) significantly decreased the amount of S650 phosphorylation. Additionally, when MKK4 or MKK6 knocked down by siRNAs, there was an increase in the expression of PSA mRNA in the absence or presence of DHT [73]. A second function identified for S650 is the regulation of shuttling of the AR between the nucleus and the cytoplasm. Interestingly, treatment of LNCaP cells with the PKA activator (forskolin) resulted in increased phosphorylation of S650 in the absence of R1881, with maximal phosphorylation observed at 2 h post-treatment [77]. These data suggest that S650 is potentially regulated in a cAMP dependent mechanism.Phosphorylation of serine 791 (S791) is similar to S213 in that it is dependent on AKT. Phosphomimetic mutations in both S213 and S791 resulted in an impaired translocation of AR to the nucleus in COS1 cells [62]. This suggests that phosphorylation of AR at S791 plays a role in the ubiquitination of AR and its degradation; this residue is the only modified residue within 50 amino acids of the ubiquitinated lysine 845 and 847 [62].
The phosphorylation of T850 is cycle-cycle dependent with increase phosphorylation during the G2 and M phase, which coincides with increased PIM-1 (S or L) expression. AR mutation at T850 resulted in lower AR protein levels during the M phase, these data suggest that phosphorylation at T850 stabilizes AR during the M phase. Upon overexpression of PIM-1L, there was no decrease in AR half-life while the overexpression of the 1S did.
Future OutlookUnderstanding the three-dimensional structure and function of AR domains has played a major role in the development of inhibitors against the receptor’s transcriptional activity. Ubiquitination of AR by RNF6 does not degrade AR, but increases its transcriptional activity. Whereas most small molecules have been designed to compete with DHT for the androgen binding site in the AR-LBD, additional distinct, yet functionally significant pockets in other regions of the protein, including the BF3 and AF2 surface sites, as well as DBD and NTD domains, should also be targeted to deal with CRPC.While AR-NTD inhibitors show promise in blocking AR transcriptional activity and treating CRPC tumours driven by AR variants, more detailed information on the structure of the AR-NTD is necessary in order to understand the molecular mechanism of action of small molecule inhibitors, such as EPI-001, as well as peptide molecules, which appear to interact with the AF1 region. Knockdown of RNF6 by shRNA in CWR-R1 and LNCaP cells had reduced AR transcriptional activity measured by ARR2-Luc assay. At the very least, site-directed mutagenesis of amino-acid residues in the AF1 region should be performed when testing NTD inhibitors to provide evidence that they indeed interact directly at the expected site on the AR.The AR-DBD may allow for a more rational approach to drug design, given that its crystal structure is already known [83]. Residues that were ubiquitinated were identified by MS as K845 and K847, which are two highly conserved residues in AR among species. In particular, exposed regions at the DBD dimerization (D-box) and DNA binding (P-box) interfaces (Figure 2A) could potentially be targeted by small molecule inhibitors. This will likely require accurate in silico modelling coupled with high throughput screening of small molecules, as well as assessment of their inhibitory effect on AR transcriptional activity using reporter assays. Knockdown of RNF6 resulted in retarded proliferation in C4-2B and CWR-R1 cells suggesting that it plays a role in proliferation most likely due to the expression levels of AR.
Mutagenesis of key amino-acids in the D-box and P-box weakens the dimerization and transcription factor activity of the AR [24] and clearly shows the importance of these surface exposed regions.
Interestingly, the interaction between AR and RNF6 was enhanced upon androgen stimulation, suggesting that low levels of androgen are needed for AR phosphorylation at T850 and its protective activity [76].
Phosphatases That Affect the ARThe reversible process of phosphorylation of the AR has not been extensively studied. The conformational change that results from androgen binding to AR, allows for the binding of PP2A to the C-terminus of AR [60].
PP2A was only loaded onto the AR when cells expressed the SV40 small t antigen, which resulted in the dephosphorylation of AR at S81, S94, S258, S308, and S424 [60]. Inhibition of PP1 resulted in an increase phosphorylation of S650 and increased nuclear localization. Modest phosphorylation of AR at S256 and S424 was observed when PP1 was inhibited, suggesting that PP1 plays a critical role in regulating the phosphorylated state of AR [91]. PP1 overexpression resulted in increased AR protein levels and enhanced transcriptional activity.
The increased level of protein was a result of the inhibition of the proteasome-mediated AR degradation, suggesting that specific AR phosphorylation is required for AR ubiquitination and degradation.
In LNCaP and CWR22Rv1 cells that were treated with the pan phosphatase inhibitor okadaic acid, there was a decrease in endogenous AR protein, and the loss of protein could be rescued by treatment with MG132.



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