1 Mechanisms of Androgen Receptor Activation in Advanced

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Mechanisms of androgen receptor activation in advanced prostate cancer:

differential coactivator recruitment and gene expression.

Brooke, G. N.1, Parker, M. G.2 and Bevan, C. L.1*

1Androgen Signalling Laboratory, Department of Oncology and 2Institute of

Reproductive and Developmental Biology, Imperial College London, London W12

0NN, UK

Mechanisms of AR activation in prostate cancer

androgen receptor; prostate cancer; coactivators; antiandrogens; SARMs

* Corresponding author. Tel. 0208 383 3784, Email [email protected].

1 Abstract

Prostate tumour growth depends on androgens; hence treatment includes androgen ablation and antiandrogens. Eventually tumours progress and in approximately 30% of patients this is associated with mutation of the androgen receptor. Several receptor variants associated with advanced disease show promiscuous activation by other hormones and antiandrogens. Such loss of specificity could promote receptor activation hence tumour growth in the absence of conventional ligands, explaining therapy failure. We aimed to elucidate mechanisms by which alternative ligands promote receptor activation. The three most commonly identified variants in tumours

(with amino acid substitutions H874Y, T877A and T877S) and wild-type receptor showed differences in coactivator recruitment dependent upon ligand and the interaction motif utilized. Coexpression and knockdown of coactivators that bind via leucine or phenylalanine motifs, combined with chromatin immunoprecipitation and quantitative PCR, revealed these preferences extend to coactivator recruitment in vivo and affect receptor activity at the transcriptional level, with subsequent effects on target gene regulation. The findings suggest that mutant receptors, activated by alternative ligands, drive growth via different mechanisms to androgen-activated wild-type receptor. Tumours may hence behave differently dependent upon any androgen receptor mutation present and what ligand is driving growth, as distinct subsets of genes may be regulated.

2 Introduction

The growth of prostate tumours, at least initially, is dependent upon androgens, which exert their effects via the androgen receptor (AR). Standard treatment for non-organ confined prostate cancer is androgen blockade, which aims to inhibit the AR pathway using LHRH analogues to suppress testicular androgen secretion, often in conjunction with antiandrogens, which bind to AR but inhibit androgen-dependent transcription

(Goktas & Crawford, 1999). Some antiandrogens can activate the receptor under certain circumstances, these are referred to as partial antiandrogens or Selective

Androgen Receptor Modulators (SARMs) and their tissue-specific effects may be exploited to reduce side-effects of antiandrogen therapy (Segal et al., 2006). While highly successful initially, androgen blockade eventually fails and the tumour recurs in the androgen-depleted environment. This advanced stage of prostate cancer is termed androgen-independent, although the AR signalling pathway often appears to be retained and important in tumour growth. A number of hypotheses have been proposed to explain this therapy failure, including AR amplification and loss of AR specificity caused by mutations of the receptor (Taplin, 2007).

The AR is a member of the nuclear receptor superfamily and shares their common domain structure including two activation functions – AF1 in the N-terminus and AF2 in the C-terminal ligand-binding domain (LBD) (Gelmann, 2002). AF1 appears to be the more important in terms of activity of full-length receptor (McEwan,

2004); although in the context of chromatin it appears the contribution of AF2 may be greater than previously believed (McEwan, 2004; Li et al., 2007). The LBD is composed of 11-12 alpha-helices, the most C-terminal of which (helix 12) moves upon ligand binding to enclose the ligand and form part of a coactivator-binding surface (Wurtz et al., 1996). Coactivators, recruited to activated receptor, alter the

3 topology of chromatin at the promoter by intrinsic or recruited histone acetyltransferase activity, to facilitate access for other transcription factors and/or the preinitiation complex. Many coactivators, including the p160 protein SRC1, interact with the LBD of nuclear receptors via conserved, helical leucine-rich motifs (LxxLL,

L=leucine and x=any amino acid (reviewed in (McKenna et al., 1999)). Recently the

AR LBD has also been demonstrated to interact with similar phenylalanine-rich motifs (Hsu et al., 2003; Dubbink et al., 2004; Hur et al., 2004). However, SRC1 and other coactivators can also interact with and promote activity of AF1 (reviewed in

(McEwan, 2004)).

Mutations of the AR are rare in primary prostate tumors but increase in frequency in advanced disease, suggesting a role in cancer progression(Taplin et al.,

1995; Marcelli et al., 2000). Of those currently described, the majority have been found to cluster in the LBD (Gottlieb et al., 2004). Studies of some of these mutant receptors have demonstrated that such substitutions can alter the specificity of the ligand-binding pocket, allowing non-androgenic ligands to enter the pocket and activate the receptor. The first such mutation to be described was a threonine to alanine substitution at residue 877, which not only responds to androgens but is also activated by oestrogens, progestins and the antiandrogens cyproterone acetate and hydroxyflutamide (Veldscholte et al., 1994). The T877A substitution is the most commonly reported in prostate cancer and lies in helix 11 of the LBD. Helix 11 appears to be a hotspot for somatic mutation since another point mutation causing substitution of the same residue with serine (T877S), and one causing a histidine to tyrosine substitution at nearby residue 874 (H874Y) have also been reported in metastatic prostate tumors (Taplin et al., 1995). Receptors carrying these mutations are activated by hormones including oestradiol and progesterone (Steketee et al.,

4 2002). Analysis of the 3-dimensional structure of liganded wild-type AR demonstrated that threonine-877 forms hydrogen bonds with the 17alpha-hydroxyl group of R1881 (a synthetic androgen) (Matias et al., 2000). Substitutions of the smaller alanine or serine are thought to change the size and shape of the pocket thus allowing non-androgenic ligands to enter and to promote an active conformation. In contrast, histidine-874 points away from the ligand pocket and is buried in a cavity between helices 11 and 12, formed when helix 12 relocates after agonist binding

(McDonald et al., 2000). The H874Y substitution is unlikely to cause steric hindrance, but it has been proposed that the more hydrophobic tyrosine side chain could increase the strength of interaction between helix 12 and this crevice

(McDonald et al., 2000), enabling helix 12 to relocate to the active conformation even when a ligand is bound that does not fit optimally. This appears to affect subsequent intra- and inter-receptor interactions since it has been shown that the H874Y mutant has enhanced binding to the p160 coactivators (Duff and McEwan 2005); and more effective binding to the FQNLF motif within AF1 that mediates a ligand dependent

N-/C-terminal interaction, required in some circumstances for maximal AR activity

(He et al., 2000; Askew et al., 2007).

While the contribution of AR mutations to failure of hormone therapy in a subset of patients is generally accepted, and promiscuous activation of several variants has been reported, the mechanisms by which mutant forms are activated and how this influences gene expression is not fully understood. This study aimed to elucidate how alternative ligands promote activation of the H874Y, T877A and

T877S mutant forms of AR, and whether this differs from activation of wild-type AR.

Here we highlight the critical contribution of cofactor binding to aberrant activation.

The data suggest that the wild-type and variant receptors recruit different coactivator

5 complexes dependent upon the ligand bound, and this has consequences for subsequent gene regulation. Hence patterns of androgen-dependent gene transcription, and hence tumour growth, may differ depending on any AR mutation present, with possible implications for disease progression and treatment.

Results

Mutations of AR alter its transactivation profile

Previous studies have established that the substitutions H874Y, T877A and T877S alter activation of AR in response to different ligands. Prior to investigating activation of the mutant receptors in transfected cells, we checked by immunoblotting that all were expressed at similar levels (not shown). We then examined the ability of the receptors to activate an androgen-responsive promoter (TAT-GRE-E1B) in the presence of androgens, SARMs/antiandrogen and other sex steroids (progestins and oestradiol), to build a comprehensive transcriptional profile of these receptors with which to inform subsequent studies on cofactor recruitment. Activation of the reporter was measured across a range of ligand concentrations (Supplementary Figure 1) and the relative activities at maximal concentrations of each ligand are shown (Figure 1).

It was evident that wild-type receptor is not specific for androgens, as it was also activated to varying degrees by the SARMs cyproterone acetate (CPA) and mifepristone (MIF), the progestins medroxyprogesterone acetate (MPA) and progesterone (P4), and oestradiol (E2) (Figure 1 and Supplementary Figure 1).

Interestingly, while the mutant receptors had similar activity to wild-type in the presence of androgens (mibolerone (MIB), dihydrotestosterone (DHT) and androstenedione (ASD)), they exhibited enhanced activity in the presence of most of

6 the non-androgenic ligands tested. One exception was that only ARH874Y showed enhanced activity in the presence of mifepristone, while the other SARMs or

“partial/mixed” antiandrogens (CPA and hydroxyflutamide, OHF) could activate all the mutant receptors to a significantly greater extent than ARWT. The antiandrogen bicalutamide (BIC) was exceptional in that it did not activate any of the receptors, wild-type or mutant. The other non-androgenic ligands, the progestins and oestradiol, showed a similar pattern to the SARMs in activating the mutant receptors to a greater extent than the wild-type. In summary, the mutant receptors were activated to a similar extent as wild-type AR by androgens, but to a greater extent than ARWT by the non-androgenic ligands tested except bicalutamide.

AR variants have different preferences for LxxLL, FxxLY and FxxFF motifs dependent upon ligand bound

Recently it has been demonstrated that AR LBD not only interacts with LxxLL motifs but can also bind phenylalanine-rich motifs, such as FxxLY. We therefore used a 2- hybrid assay to study the effects of mutations and ligands on AR binding to three different interaction motifs - LxxLL, FxxLY or FxxFF. These motifs had previously been identified by phage display and interact with AR LBD in the presence of androgen (Hur et al., 2004).

In the presence of androgen all the ARs were able to interact with the LxxLL,

FxxLY and FxxFF motifs, and interaction was stronger with phenylalanine-rich motifs than the LxxLL motif (Figure 2, compare (b) and (c) to (a)), in accordance with published studies (Hur et al., 2004). In the presence of mibolerone, there was no significant difference in the strength of interaction of ARWT, compared to any of the

7 mutant receptors, with either FxxFF or FxxLY (Figure 2(b) and (c)). However, significantly stronger binding to LxxLL was observed for all the mutants, in particular

ARH874Y, when compared to wild-type receptor. The interaction of ARWT with the

LxxLL motif is not specific for androgen (Figure 2(a)) with similar strength interactions also detected in the presence of cyproterone acetate, medroxyprogesterone acetate and progesterone. In contrast, interaction of ARWT with the phenylalanine-rich motifs appears to be more androgen specific, with strong interaction in the presence of all the androgens and relatively weaker interactions in the presence of oestradiol and progesterone.

The mutant ARs interacted with the LxxLL motif in the presence of the same ligands as wild-type receptor (Figure 2(a)). Interestingly, the receptors with a substitution at residue 877 have stronger binding to the LxxLL motif in the presence of cyproterone acetate and the progestins compared to the interactions induced by androgens. These interactions of mutant receptors with LxxLL were also stronger than those evident for androgen-bound wild-type receptor. Some striking differences were observed in the preference of receptors for different motifs dependent upon ligand. Androgens, progesterone, MPA and cyproterone acetate promoted interaction of all the receptors with the LxxLL motif and hydroxyflutamide did not (Figure 2(a)).

In contrast, neither MPA nor cyproterone acetate promoted interaction with the phenylalanine-rich motifs, while hydroxyflutamide promoted strong interaction of all the mutant, but not the wild-type, receptors with these motifs (Figure 2(b) and (c)).

Interestingly, between the two phenylalanine-rich motifs tested, the preferential interaction with FxxLY>FxxFF, evident in all other situations, was reversed for the receptors carrying substitutions at residue 877 in the presence of hydroxyflutamide.

8 Hence the two SARMs, cyproterone acetetate and hydroxyflutamide, promote a mutually exclusive pattern of interaction of mutant receptors with motifs – CPA

(and MPA) promotes interaction with leucine but not phenylalanine motifs, while

OHF conversely promotes interaction with phenylalanine but not leucine motifs.

Enhancement of AR activity by coactivators is dependent upon interaction motif and ligand

To investigate whether these preferences could affect coactivator recruitment hence transcriptional activity of AR, the wild-type receptor and ART877A were coexpressed with two coactivators: ARA701-401, which interacts with AR LBD mainly via an

FxxLF motif (Hsu et al., 2003); or SRC1, which interacts with AR LBD via LxxLL motifs (Bevan et al., 1999). Activation of an androgen-responsive promoter was measured in the presence of hydroxyflutamide and cyproterone acetate, which showed the most striking differences in the 2-hybrid assay (Figure 3). Neither coactivator was able to enhance the activity of the wild-type receptor in the presence of hydroxyflutamide, which was expected due to the inability of the ligand to activate the receptor in previous transcriptional assays (Figure 1(b)). In the presence of the partial antagonist cyproterone acetate, however, SRC-1 was able to coactivate the receptor to a greater degree than ARA-701-401 (p<0.005). More strikingly, we found that ARA701-401 was a more effective coactivator of ART877A in the presence of hydroxyflutamide (which promoted interaction only with phenylalanine-rich motifs) than in the presence of cyproterone acetate (which preferentially promoted interaction with the LxxLL motif). The converse was true for SRC1, correlating with the results from the mammalian 2-hybrid assays.

Differential recruitment of coactivators to promoters dependent upon ligand

The previous experiments suggested that coactivator recruitment to AR is dependent upon the ligand bound. To investigate recruitment of endogenous coactivators to endogenous promoters, chromatin immunoprecipitation (ChIP) analysis of two androgen-responsive control elements of the Kallikrein 2 (KLK2) gene – the enhancer

(-3842bp) and promoter (-211bp) - was performed in LNCaP cells, which endogenously express ART877A. At both the 1-hour and 2-hour time points, we saw

AR recruited to the enhancer and promoter in the presence of mibolerone and also the two SARMs cyproterone acetate and hydroxyflutamide (Figure 4), with highest recruitment in the presence of the androgen. At the 1-hour time point, ARA70, containing a phenylalanine-rich motif, was found at the response elements after treatment with all three ligands. However at the later time point the coactivator was only present after treatment with hydroxyflutamide and, in the case of the promoter, mibolerone - the ligands that promoted interaction with phenylalanine motifs. In comparison SRC1, whilst always recruited in the presence of androgen, was much more strongly recruited to the promoter in the presence of cyproterone acetate than hydroxyflutamide.

To summarise, the recruitment of these coactivators to the regulatory regions of KLK2 was found to be both ligand- and response element-specific, with hydroxyflutamide promoting recruitment of ARA70 to a greater extent than cyproterone acetate and the inverse for SRC1 recruitment. These preferences were most pronounced at the later (2-hour) time-point. Hence ligand-specific motif preferences evident in the 2-hybrid assay were reflected in in vivo recruitment to

AREs.

Coactivator knockdown demonstrates importance of interaction motif and ligand upon endogenous gene transcription

To test the functional significance of ligand-dependent coactivator recruitment, we used siRNA to knock down ARA70 or SRC1 in LNCaP cells and analysed the effect on regulation of KLK2 in response to different ligands. Knockdown of SRC1 was to the extent of 98% at the RNA and 70% at the protein level, while ARA70 was reduced by 89% at the RNA and 56% at the protein level (not shown). In the presence of scrambled siRNA, KLK2 expression was induced in the order mibolerone

> hydroxyflutamide > cyproterone acetate (Figure 5). Reduced levels of either coactivator resulted in a general reduction in KLK2 expression induced by all of the ligands. SRC1 knockdown had a greater effect in the presence of cyproterone acetate compared to hydroxyflutamide, increasing their difference in induction from 11% to

18%. In contrast, ARA70 knockdown had a greater effect in the presence of hydroxyflutamide, to the extent that the order of induction was reversed and higher

KLK2 expression was found in the presence of cyproterone acetate.

AR exhibits differential regulation of target genes dependent upon ligand bound.

Previous experiments suggested that coactivator recruitment to AR is dependent on both ARE/promoter and ligand. We next investigated whether this has functional consequences with regard to gene regulation, i.e. whether different endogenous AR- regulated genes exhibit altered expression patterns in the presence of different ligands. The LNCaP cell line was used, since ART877A responds to a variety of ligands, to compare the expression of KLK2, Differentiation Related Gene-1 (DRG-1),

11 and Cyclin Dependent Kinases 2 and 4 (CDK2 and CDK4) (Figure 6). Both KLK2 and CDK2 were found to be upregulated by all of the ligands tested, but had the highest levels of transcription in the presence of mibolerone. Interestingly, CDK4 was found to be only upregulated by the antiandrogens. In contrast, the regulation of

DRG1 is much more androgen specific, with only modest activation in response to hydroxyflutamide compared to that evident for mibolerone.

Discussion

The ability of ligand binding to nuclear receptors to promote recruitment of coactivators is well established (McKenna et al., 1999), but recruitment may also be affected by promoter context (Rogatsky et al., 2001) and by mutations that influence the function of the receptor (Duff & McEwan, 2005). In this study of AR variants we have demonstrated explicitly that each of these aspects impinges on coactivator recruitment, ultimately resulting in altered expression of endogenous hormone- responsive genes. To illustrate this we used AR variants that have been found to be associated with advanced prostate cancer, carrying somatic mutations in helix 11 of the ligand-binding domain (LBD). These have been postulated to contribute to the progression of prostate cancer to the “androgen independent” stage, since they allow receptor activation, thus presumably cell division, in response to alternative ligands.

We investigated the mechanisms of the previously described gain-of-function effect, using ligands including the commonly used therapeutic antiandrogens (bicalutamide, cyproterone acetate, hydroxyflutamide), an antiandrogen/antiprogestin (mifepristone), naturally occurring hormones likely to be present in patients (dihydrotestosterone, androstenedione, oestradiol, progesterone), and strong synthetic steroids (mibolerone, medroxyprogesterone acetate).

12 The wild-type receptor was transcriptionally active not only in the presence of androgens, but also substantially so (>20% maximal activity) in the presence of cyproterone acetate, oestradiol, mifepristone, medroxyprogesterone acetate and progesterone, confirming previous studies showing that high concentrations of some of these can activate AR (Berrevoets et al., 1993; Taplin et al., 1995; Miyamoto et al.,

1998; Kemppainen et al., 1999; Steketee et al., 2002). We confirmed that variants

ARH874Y, ART877A and ART877S exhibited increased activity in the presence of a range of non-androgenic ligands. These included antiandrogens, the only exception being bicalutamide.

The recruitment of coactivators to steroid receptors is often mediated by

LxxLL-like or leucine motifs. However, the AR is idiosyncratic in that it appears to interact preferentially with phenylalanine-rich motifs, found in several AR coactivators including ARA70, FHL2 and gelsolin (Hsu et al., 2003; Dubbink et al.,

2004; Hur et al., 2004; van de Wijngaart et al., 2006). We therefore next looked at whether the mutant receptors showed altered relative affinity for different interaction motifs in the presence of alternative ligands. It was notable that the mutant receptors all interacted more strongly than ARWT with an LxxLL motif in the presence of androgen, as previously shown for ARH874Y (Duff & McEwan, 2005; Askew et al.,

2007). Indeed the mutants were able to interact with this motif more strongly even in the presence of progestins and cyproterone acetate than did wild-type receptor in the presence of a potent androgen. Striking differences were found in the interactions between the ARs and either a leucine motif or a phenylalanine motif. In the presence of medroxyprogesterone acetate and cyproterone acetate, for example, all the mutant receptors interacted with the LxxLL motif whereas no interaction was detected between any receptor and the FxxFF or FxxLY motifs. In the presence of

13 hydroxyflutamide the reverse was found for mutant receptors, with interaction only evident between these and the phenylalanine motifs. A similar trend was reported for

ART877A by Ozers et al. who showed, using fluorescence resonance energy transfer, that cyproterone acetate antagonized its interaction with an FxxLF-containing peptide, whilst promoting its interaction with an LxxLL-containing peptide (Ozers et al.,

2007).

Comparison of the structure of AR LBD in complex with and without LxxLL and FxxLF motifs revealed that the interaction site for such motifs is an L-shaped hydrophobic cleft, comprised of three distinct subsites that bind the hydrophobic groups found in cognate peptides (Estebanez-Perpina et al., 2005). Conserved charged residues at either end of the cleft, Lys720 and Glu897, form a ‘charge clamp’. The main chain atoms at either end of the FxxLF motif form electrostatic interactions with both charge clamp residues. In contrast, the LxxLL motif forms hydrogen bonds with only one - Lys720 (Hur et al., 2004). Previous studies using computer modeling of AR LBD in complex with androgen or SARMs, that were complete agonists in transcriptional assays, showed that these ligands had distinct effects on the size and shape of the coactivator cleft and the distance between the charge clamp residues (Kazmin et al., 2006). Kazmin et al. proposed that ligand- dependent interactions identified by peptide phage display could be explained by the different conformations formed. Similarly, we hypothesize that the ligand- and mutation-dependent differences in motif preference identified here are as a result of distinct receptor conformations, and the alternative bonds made between the leucine- or phenylalanine-rich motifs and the coactivator groove (i.e. interaction with one or both of the charge clamp residues).

14 Having found that various ligands promote distinct interactions with motifs, the logical conclusion was that this would extend to differential coactivator recruitment. In support of this, enhancement of AR activity by SRC1 (interacts with the LBD via LxxLL motifs) or ARA701-401 (interacts via a phenylalanine-rich motif) varied dependent upon the ligand bound. Our hypothesis is that these coactivator- specific effects are due to their differential recruitment to the LBD. To determine the contribution of different coactivators to activation of endogenous gene expression by various ligands, we first investigated their occupancy of the endogenous KLK2 enhancer and promoter in LNCaP cells, i.e. in the presence of endogenous ART877A.

The first point of note was that mibolerone promoted the strongest interaction of AR with these regulatory sequences, and cyproterone acetate and hydroxyflutamide an equal, weaker interaction, correlating with levels of mRNA expression induced by these ligands. Next, in agreement with our interaction and reporter assays, we saw

ARA70 recruited in the presence of hydroxyflutamide and, in the case of the promoter, mibolerone, conditions under which it was able to coactivate AR activity and interact with ART877A via a phenylalanine-rich motif. Conversely cyproterone acetate treatment (promoted LxxLL interaction and coactivation by SRC1) resulted in promoter occupancy by the LxxLL-containing coactivator SRC1. Interestingly, some differences between cofactor recruitment to the enhancer and promoter were evident.

In particular, at the 2-hour time point ARA70 was recruited to the enhancer only in the presence of hydroxyflutamide, whereas it was additionally recruited to the promoter in the presence of mibolerone. One possible explanation for this is that cofactors are known to cycle on and off promoters with varying periodicity (Metivier et al., 2003), and we hypothesise that response element context and/or ligand may affect said periodicity. Overall, however, ARE occupancy by endogenous coactivators

15 showed similar preferences in the presence of alternative ligands to those proposed by our in vitro interaction studies. That this can alter gene expression in vivo was shown by the fact that reducing levels of ARA70 and SRC1 had opposite effects on AR activation by hydroxyflutamide versus cyproterone acetate.

These findings have clinical and diagnostic implications. Both SRC1 and

ARA70 are expressed in human prostate cancer cell lines and in normal prostate, in the luminal epithelial cells that also express AR (Gregory et al., 2001; Linja et al.,

2001; Hu et al., 2004; Powell et al., 2004; Agoulnik et al., 2005). There is both in vitro and in vivo evidence that SRC1 is required for optimal androgen signalling and growth in the prostate, since knockdown of SRC1 attenuated growth and androgen- dependent gene expression in androgen-sensitive prostate cancer cell lines (Agoulnik et al., 2005), and SRC1-null mice exhibit attenuation of androgen-stimulated prostatic growth (Xu et al., 1998). Although we saw that knockdown of ARA70 had more effect on KLK2 expression than SRC1 knockdown, implying that ARA70 may be more important in AR signalling in vivo, the relatively weak effect of SRC1 knockdown could be due to functional redundancy between it and the highly related p160 coactivators, TIF2 and AIB1. This possibility is supported by study of the

SRC1-null mouse, where increased levels of TIF2 mRNA were found in hormone- responsive tissues and hypothesised to compensate for loss of SRC1, resulting in only relatively minor hormone resistance being evident in the animals (Xu et al., 1998).

Alterations in cofactor levels is often cited as a potential mechanism for progression of prostate tumours to the hormone-refractory stage, but studies investigating differences in cofactor levels between tumour and normal or benign tissue are often inconclusive or even contradictory (reviewed in (Chmelar et al.,

2007)). At the protein level, several studies have shown a link between increased

16 SRC1 expression and prostate cancer progression, with increased expression in hormone-refractory tumours (Gregory et al., 2001; Maki et al., 2006) or linked to tumour stage/grade (Agoulnik et al., 2005). At the mRNA level, however, decreased or unaltered SRC1 levels in malignant versus benign prostate have been reported

(Fujimoto et al., 2001; Linja et al., 2004; Maki et al., 2006). Studies on expression of

ARA70 in prostate cancer are equally contradictory with the coactivator being found to be both up-regulated at the protein level (Hu et al., 2004) and down-regulated at the

RNA level (Li et al., 2002) in tumours versus benign tissue, whereas others have found no change in mRNA levels (Mestayer et al., 2003). In light of the current study, a possible explanation of the conflicting results of such studies could be differences in hormonal treatment regimes in the patients. Potentially, treatments could cause selection for tumour cells expressing certain cofactors – for example tumours from patients treated with hydroxyflutamide may be more prone to overexpress coactivators that interact with AR via phenylalanine-rich motifs, such as ARA70.

Of course another factor in gene expression is the promoter context. We investigated expression of genes previously identified as androgen-regulated, involved in prostate differentiation (KLK2 and DRG1) and in cell cycle progression

(CDK2 and CDK4). Similar to previous reports, KLK2 and CDK2 were found to be upregulated in response to androgen (Lu et al., 1997; Gregory et al., 1998; Nelson et al., 2002). In addition, we found both genes were also upregulated in response to the

SARMs, cyproterone acetate and hydroxyflutamide. Evidence for regulation of CDK4 by the androgen receptor pathway is contradictory since Lu et al. found up-regulation in response to androgen, whereas Gregory et al. found no effect (Lu et al., 1997;

Gregory et al., 1998). We also found no androgen-dependent regulation of CDK4.

Interestingly, however, the kinase was found to be significantly (although slightly)

17 up-regulated by the SARMs cyproterone acetate and hydroxyflutamide. We found that DRG1 also showed differential regulation by the ligands, being strongly upregulated by mibolerone and only weakly by hydroxyflutamide. A previous study of

DRG1 regulation in LNCaP cells also showed high specificity for androgens, although in this case SARMs were not studied (Ulrix et al., 1999). This all adds to a growing body of evidence supporting the notion that differential expression of steroid target genes is dependent upon many factors including the promoter, receptor interactions (both intra-receptor and with other proteins) and post-translational modifications (He et al., 2002; Li et al., 2003; Callewaert et al., 2004; Powell et al.,

2004). The implications go beyond differential expression of androgen responsive genes. The AR shares a common response element with the glucocorticoid, mineralocorticoid and progesterone receptors. Since different hormones have distinct effects, it remains unclear how a single response element can mediate distinct physiological effects in response to such ligands. One contributing factor is likely to be that differences in coactivator complexes recruited by different receptors could affect the rate of transcription from a given promoter under control of a given steroid receptor, hence be a mechanism to control receptor specificity (Li et al., 2003), and our data are supportive of this.

Mutations of the AR have been found in 10-40% of cases of advanced, hormone independent prostate cancer (Taplin, 2007). Those that reduce ligand specificity, as presented here, may promote AR activation hence tumour growth in the absence of conventional AR ligands, explaining the failure of androgen blockade therapy. We have shown that receptors activated by non-androgenic ligands appear to behave differently to those activated by androgens, both in their mechanism of activation and their pattern of gene regulation. Genes differentially regulated include

18 those involved in prostate differentiation and in androgen-stimulated proliferation.

This implies that prostate tumours may behave differently dependent upon which (if any) AR mutation is present and what ligand is driving growth, as alternative subsets of genes may be regulated.

Methods

Ligands and antibodies

Ligands were dissolved in EtOH (VWR, Leicestershire, UK) and stored at -20°C.

Androstenedione, cyproterone acetate, dihydrotestosterone, medroxyprogesterone acetate, mifepristone, 17ß-oestradiol and progesterone were from Sigma (Dorset,

UK), mibolerone from PerkinElmer (Buckinghamshire, UK). Bicalutamide and hydroxyflutamide were kind gifts from Astra-Zeneca Pharmaceuticals (Cheshire, UK) and Schering-Plough (Hertfordshire, UK) respectively. Anti-AR (N-20), -SRC1 (M-

341) and -ARA70 (H-300) antibodies were from Santa-Cruz (Santa-Cruz, CA).

Plasmids

The following have been previously described: pSG5-SRC1e (Kalkhoven et al., 1998) and PB-PROM-LUC (Verrijdt et al., 2003). The following were kind gifts: pSVAR from Albert Brinkmann (Rotterdam); TAT-GRE-EIB-LUC from Guido Jenster

(Rotterdam); pKBU-LxxLL (containing SSRGLLWDLLTKDSR), pKBU-FxxFF

(containing SRFADFFRNEGLGSR) and pKBU-FxxLY (containing

SRFEALYLDRVTGLHTDTSR) Gal4 DBD fusion proteins from Benjamin Beuhrer and Maria Sjøberg (KaroBio, Huddinge, Sweden) (Hur et al., 2004).

Mutations encoding H874Y, T877S and T877A were introduced into residues

503-919 of AR inserted into pBluescript (Stratagene, La Jolla, CA), by PCR

19 mutagenesis, and this fragment subcloned into pSVAR using KpnI and BamHI

(Roche, Basel, Switzerland). pSG5-ARA701-401 was created by amplification of amino acids 1-401 of ARA70 from pSG5-ARA70 (Bevan et al., 1999) and insertion into pSG5 (Stratagene) using EcoRI and BamHI. VP16-AR fusion vectors were created by amplifying wild-type or mutant AR from pSVAR and inserting into

BamH1 and XbaI sites of pVP16 (Clontech, Mountain View, CA). All plasmids were verified by sequencing.

Cell Culture

Cell lines were obtained from American Type Culture Collection (Manassas, VA).

COS-1 and HeLa cells were cultured in DMEM and LNCaP in RPMI 1640 as described previously (Gamble et al., 2006).

Transcription Assays

COS-1 cells and HeLa cells at 60% confluence in 24-well plates were incubated for

24 hours in androgen-depleted media. For dose response experiments, HeLa cells were transfected using FuGENE 6 with 100ng AR expression vector, 100ng PDM-

LAC-Z-ß-GAL and 1µg reporter per well. For coexpression assays, COS-1 cells were transfected using the calcium phosphate method (Chen & Okayama, 1987) with

50ng AR expression vector, 200ng or 400ng SRC1 expression vector or pSG5-

ARA701-401, 100ng PDM-LAC-Z-ß-GAL and 1µg luciferase reporter per well. For 2- hybrid assays, COS-1 cells were transfected as above with 100ng pVP16-AR (wild- type or mutant), 100ng pKBU-LxxLL, -FxxLY or -FxxFF, 100ng of PDM-LAC-Z-β-

GAL and 1µg 5-GAL-TATA-LUC. After transfection cells were incubated with ligand for 24 hours before lysis, and luciferase and β-galactosidase activities

20 measured using the LucLite Plus (PerkinElmer, Waltham, MA) and Galacto-light Plus

(Tropix, Bedford, MA) kits respectively.

Chromatin Immunoprecipitation

LNCaP cells were grown to approximately 70% and serum-starved for 72hrs. Cells were treated for 1 or 2hrs with ligand (100nM mibolerone, 1µM other ligands) before cross-linking with formaldehyde (Sigma) for 10min at 37°C. ChIP was performed using the Upstate Chromatin Immunoprecipitation kit (Upstate, Charlottesville, VA) following manufacturer’s instructions. DNA was recovered by phenol-chloroform extraction and semi-quantitative PCR performed using primers designed to anneal either side of an ARE in the KLK2 enhancer (For-

5’GTTGAAAGCAGACCTACTCTGGA-3’; Rev-5’-

CTGGACCATCTTTTCAAGCAT-3’) and promoter (For-5'-

GGGAATGCCTCCAGACTGAT-3'; Rev-5'-CTTGCCCTGTTGGCACCTA-3')

(Kang et al., 2004).

Coactivator knockdown

LNCaP cells were grown to approximately 60% confluence in 6 well plates then changed to androgen-depleted media and transfected with (100nM final concentration) pools of scrambled (D-001810-10), ARA70- (L-010321-00) or SRC1- targeting (L-005196-00) siRNA using Dharmafect 4 (Dharmacon, Northumberland,

UK). Cells were incubated for 72hrs with siRNA before addition of ligand and left for a further 24hrs before harvesting.

Real-time quantitative PCR

21 LNCaP cells were grown to 60% confluence in 6 well plates for 72hrs. Cells were treated with ligand (100nM mibolerone, 1µM other ligands) and incubated for a further 24hrs before harvesting. RNA was recovered using an RNAeasy kit with the additional DNAse step (Qiagen, West Sussex, UK) and reverse transcription performed on 2µg of RNA. Expression of KLK2, CDK2, CDK4, DRG1 and GAPDH were measured using real-time quantitative PCR (ABI 7500, Applied Biosystemns,

Foster City, CA) using FAM-labelled oligo sets (Ambion, Warrington, UK). Results were normalized using GAPDH data.

Acknowledgements

We are grateful to Simak Ali and to members of the Androgen Signalling Laboratory for extensive discussion and criticism and for technical help. This study was supported by the Medical Research Council and the Hammersmith Hospitals Trust

Research Committee

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29 Legends to Figures

Figure 1. Transcriptional profile of wild-type and mutant receptors. HeLa cells were transiently transfected with WT or mutant AR vectors, an androgen-responsive luciferase reporter (TAT-GRE-E1B-LUC) and ß-galactosidase expression vector

(PDM-LAC-Z-β-GAL). Cells were treated for 24 hours with MIB and DHT at 100nM and all other ligands at 1µM. Luciferase data were normalized for ß-galactosidase activity and expressed as a percentage of wild-type AR activity in the presence of

100nM MIB. Results are mean ±1SE of at least 3 independent experiments performed in duplicate. T-test for significance of difference to corresponding result for WT receptor: * p<0.01, ** p<0.001. EtOH = ethanol, MIB = Mibolerone, DHT = dihydrotestosterone, ASD = androstenedione, BIC = bicalutamide, CPA = cyproterone acetate, OHF = hydroxyflutamide, MIF = mifepristone, MPA = medroxyprogesterone acetate, P4 = progesterone, E2 = 17ß-oestradiol.

Figure 2. Interactions of the receptors with LxxLL, FxxLY and FxxFF motifs. COS-

1 cells were transfected with Gal4-responsive luciferase reporter (5-GAL-LUC), ß- galactosidase expression vector, plasmid expressing the VP16 activation domain fused to full length wild-type or mutant AR and plasmid expressing the Gal4 DNA- binding domain fused to an (a) LxxLL, (b) FxxLY or (c) FxxFF peptide. Luciferase activity normalized as for Figure 1 and activity expressed as a percentage, with 100% taken as luciferase activity when VP16-ARWT coexpressed with LxxLL peptide in the presence of MIB. Results are mean ± 1SE of 3 independent experiments performed in duplicate. *p<0.05, **p<0.005 (Student’s t-test).

30 Figure 3. Enhancement of transcriptional activity by coactivators is ligand dependent.

COS-1 cells were transfected with plasmids expressing wild-type or T877A mutant

AR, ARA701-401 or SRC-1, androgen-responsive luciferase reporter vector (PB-

PROM-LUC) and β-galactosidase expression vector. Luciferase activity was normalized for β-galactosidase activity and expressed as percentage of wild-type AR activity in the presence of mibolerone and absence of coactivator. Results are mean ±

1SE of at least 3 independent experiments performed in duplicate. * = p<0.05,

**P<0.005 (Student’s t-test).

Figure 4. Differential recruitment of coactivators to endogenous androgen-responsive regulatory sequences is dependent upon agonist. Chromatin immunoprecipitation was performed on LNCaP cell extract as described with anti-AR, anti-ARA70 or anti-

SRC1 antibodies. PCR was performed using primers designed to anneal either side of androgen response elements in the enhancer and promoter sequences respectively of the Kallikrein 2 (KLK2) gene.

Figure 5. Knockdown of coactivators has different effects on gene expression dependent upon agonist. LNCaP cells were transfected with siRNA to knock down

ARA70 or SRC1. Following 72hrs of incubation with siRNA, cells were treated as indicated for 24hrs before harvesting RNA. Reverse transcription was performed and expression of Kallikrein 2 (KLK2) analysed by real-time quantitative PCR. Result is a representative figure of 3 independent experiments and the mean of 8 PCR replicates ± 1SE. ** p<0.005 (Student’s t-test).

Figure 6. Differential expression of target genes dependent upon the agonist bound.

LNCaP cells were grown for 72hrs in stripped media and treated as indicated for

31 24hrs before cells were harvested, RNA extracted and cDNA created using reverse transcription. Expression of Kallikrein 2 (KLK2), cyclin dependent kinases 2 and 4

(CDK2 and CDK4) and Differentiation Related Gene-1 (DRG1) was analysed using real-time quantitative PCR. Result is a representative figure and the mean of 8 PCR replicates ± 1SE. ** p<0.005 (Student’s t-test).

32 Brooke et al Figure 1

180 WT ** 160 H874Y T877A 140 T877S * **** ** ** 120 ** ** * 100 * ** 80 ** %activity ** 60 ** 40 ** ** 20

E2 P4 MIF BIC MIB OHF CPA DHT ASD MPA EtOH Androgens Anti-androgens Other (SARMs) hormones Brooke et al Figure 2

(a) LxxLL WT AR 3000 * * H874Y 2500 T877A T877S 2000 1500 * %activity 1000 ** ** ** ** ** 500 ** * ** ** ** * 0 EtOH MIB DHT ASD BIC CPA OHF MIF MPA P4 E2 (b) FxxLY 12000 ** 10000 ** ** 8000 6000

%activity 4000 ** ** ** 2000 ** 0 EtOH MIB DHT ASD BIC CPA OHF MIF MPA P4 E2 (c) FxxFF 6000 ** 5000

4000 ** ** ** 3000

%activity 2000 ** * 1000 ** ** 0 EtOH MIB DHT ASD BIC CPA OHF MIF MPA P4 E2 Androgens Anti-androgens Other (SARMs) hormones Brooke et al Figure 3

CPA 1400 * OHF 1200 * 1000 800 ** 600 % activity % 400 200 0

ARA701-401 SRC1 ARA701-401 SRC1 WT T877A Brooke et al Figure 4

Enhancer Promoter EtOH EtOH MIB CPA OHF EtOH MIB CPA OHF Input AR

1hr 1hr ARA70 SRC1

Input AR

2hr 2hr ARA70 SRC1 Brooke et al Figure 5

3.5

3 +11% ** 2.5 +18% ** 2 EtOH MIB CPA 1.5 -23% OHF ** 1 Relativeexpression

0.5

0 Scram siRNA ARA70 siRNA SRC1 Brooke et al Figure 6

** EtOH 12 MIB CPA OHF

Relative Expression Relative ** ** ** 2 ** ** ** ** **

0 KLK2 CDK2 CDK4 DRG1 Brooke et al Supplemental Figure 1

a b 200 MBMIB 200 DHTDHT

150 150

100 100 % activity %activity

50 %activity 50

0 0 0 0.01 0.1 1 10 100 0 0.01 0.1 1 10 100 [MIB] nM [DHT] nM c d 200 DHT 200 ASD EE2 2

150 150

100 100 % activity %activity % activity %activity 50 50

0 0 0 0.01 0.1 1 10 100 0 0.1 1 10 100 1000 [ASD] nM [E2] nM e f 200 200 MIFMIF MPMPAA

150 150 WT 100 100 H874Y % activity %activity % activity %activity 50 50 T877A T877S 0 0 0 0.1 1 10 100 1000 0 0.1 1 10 100 1000 [MIF] nM g h [MPA] nM 200 P 200 BIC P4 4 BIC

150 150

100 100 % activity %activity % activity %activity 50 50

0 0 0 0.1 1 10 100 1000 0 0.1 1 10 100 1000 [P4] nM [BIC] nM

i j OHF 200 CPCPAA 200 OH-FOHF

150 150

100 100 % activity %activity % activity %activity

50 50

0 0 0 0.1 1 10 100 1000 0 0.1 1 10 100 1000 [CPA] nM [OHF] nM Supplementary Figure 1. Dose response curves of the wild-type and mutant receptors in the presence of different ligands. HeLa cells were transiently transfected with WT or mutant AR, a luciferase reporter (TAT-GRE-E1B-LUC) and a ß-galactosidase expression vector (PDM-LAC-Z- β-GAL). Luciferase data were normalised for ß-galactosidase activity and expressed as a percentage of wild-type AR activity in the presence of 100nM MB. Results are the mean ±1SE of at least 3 independent experiments performed in duplicate. Ligands tested were A - mibolerone (MIB),

B - dihydrotestosterone (DHT), C - androstenedione (ASD), D - 17ß-estradiol (E2), E - mifepristone (MIF), F - medroxyprogesterone acetate (MPA), G - progesterone (P4), H - bicalutamide (BIC), I - cyproterone acetate (CPA), J - hydroxyflutamide (OHF)..