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Endocrinology Vol. 148, No. 9 4334-4343
Copyright © 2007 by The Endocrine Society

A Splicing Variant of the Androgen Receptor Detected in a Metastatic Prostate Cancer Exhibits Exclusively Cytoplasmic Actions

Monika Jagla, Marie Fève, Pascal Kessler, Gaëlle Lapouge, Eva Erdmann, Sebastian Serra, Jean-Pierre Bergerat and Jocelyn Céraline

Faculté de Médecine/Signalisation et Cancer de la Prostate/Equipe d’Accueil 3430 (M.J., M.F., G.L., E.E., J.-P.B., J.C.), Université Strasbourg, and Département d’Hématologie et d’Oncologie (S.S., J.-P.B., J.C.), Hôpital de Hautepierre, Centre Hospitalier Régional Universitaire de Strasbourg, F-67000 Strasbourg, France; and Plate-forme technologique d’Imagerie (P.K.), Institut de Génétique et de Biologie Moléculaire et Cellulaire, F-67404 Illkirch, France

Address all correspondence and requests for reprints to: Dr. Jocelyn Céraline, Université Strasbourg, Faculté de Médecine/Signalisation et Cancer de la Prostate/EA 3430, Médicale A/Centre Hospitalier Régional Universitaire, F-67091 Strasbourg, France. E-mail: jocelyn.ceraline{at}medecine.u-strasbg.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The androgen receptor (AR) is a ligand-activated transcription factor that displays genomic actions characterized by binding to androgen-response elements in the promoter of target genes as well as nongenomic actions that do not require nuclear translocation and DNA binding. In this study, we report exclusive cytoplasmic actions of a splicing variant of the AR detected in a metastatic prostate cancer. This AR variant, named AR23, results from an aberrant splicing of intron 2, wherein the last 69 nucleotides of the intronic sequence are retained, leading to the insertion of 23 amino acids between the two zinc fingers in the DNA-binding domain. We show that the nuclear entry of AR23 upon dihydrotestosterone (DHT) stimulation is impaired. Alternatively, DHT-activated AR23 forms cytoplasmic and perinuclear aggregates that partially colocalize with the endoplasmic reticulum and are devoid of genomic actions. However, in LNCaP cells, this cytoplasmic DHT-activated AR23 remains partially active as evidenced by the activation of transcription from androgen-responsive promoters, the stimulation of NF-{kappa}B transcriptional activity and by the decrease of AP-1 transcriptional activity. Our data reveal novel cytoplasmic actions for this splicing AR variant, suggesting a contribution in prostate cancer progression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING THE LAST decade, increasing data implicate the androgen receptor (AR) in the progression of prostate cancer (PCa) to androgen independence and finally in hormone therapy failure. It is well established that during PCa progression, mutant AR variants with altered activities emerge to support tumor cells growth in an androgen-depleted environment (1). Nevertheless, the exact function of these mutant ARs and their impact on androgen signaling pathways remain to be explored. As a member of the superfamily of nuclear receptors, AR is a ligand-activated transcription factor controlling the expression of specific genes in target tissues (2, 3). In the absence of androgens, AR is localized in the cytoplasm and is associated with heat-shock proteins. Upon stimulation by the agonist 5{alpha}-dihydrotestosterone (DHT), AR dissociates from heat-shock proteins, translocates into the nucleus, and binds to androgen-response elements present in the promoter of AR target genes (4, 5).

These genomic actions of AR are modulated by coregulators, including coactivators and corepressors (6, 7). The coactivator p300 and its functional homolog cAMP response element-binding protein (CREB)-binding protein bridge the transcriptional machinery to AR, whereas members of the p160 family of coactivators modify locally the chromatin structure through their histone acetyltransferase or ATP-dependent chromatin-remodeling activities. On the other side, a number of corepressors that associate with AR have been shown to affect AR transactivation. The silencing mediator of retinoid and thyroid hormone receptors (SMRT)/nuclear hormone receptor corepressor (NCoR) recruits histone deacetylases to transcriptional initiation sites. Other factors such as cyclin D1 or filamin A can interrupt the interaction between AR and its coactivators or the interaction between the N terminus and the C terminus of AR. Corepressors such as calreticulin by interacting with the KVFFKR motif located in AR DNA binding domain (DBD) could inhibit the nuclear translocation of AR (8).

Recent reports suggest that AR can also mediate androgen nongenomic signaling, which does not require AR nuclear translocation and DNA binding (9). AR and other steroid hormone nuclear receptors are able to activate the MAPK/ERK signaling pathway through a mechanism independent of their transcriptional activities (10). Moreover, AR mediates nongenomic activation of phosphatidylinositol 3-OH kinase (PI3K) in androgen-sensitive epithelial cells, which results in the phosphorylation of the downstream AKT/protein kinase B. This activation of the PI3K/AKT pathway by AR acts as an antiapoptotic stimulus (11).

AR mutations are a recurrent event during the progression of PCa on androgen ablation therapy. These mutations are supposed to confer to the AR new functional properties allowing the AR to support PCa cell growth and survival despite the low levels of androgens resulting from the ablation therapy. A previously described yeast-based functional assay (12, 13) is used to screen PCa specimens for AR mutations. In the present study, we report the detection of a splicing AR variant, named AR23, with an insertion of 23 amino acids between the two zinc fingers of the DBD in a hormone-refractory metastatic PCa. We present data demonstrating that this splicing AR variant exhibits exclusively cytoplasmic activities, affecting the activity of transcription factors such as nuclear factor-{kappa}B (NF-{kappa}B) and activator protein-1 (AP-1). The potential role of such a splicing AR variant in PCa progression will be discussed. Also, because this splicing AR variant has been described in two cases of partial androgen insensitivity syndrome (PAIS), its residual functions are worth taking into account (14, 15).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
All steroids, phenol red-free DMEM and RPMI 1640 media, and mycoplasma-screened fetal calf serum (FCS) were from Sigma Aldrich (Saint-Fallavier, France). Primers were from MWG Biotech France SA (Courtaboeuf, France).

Clinical report
The patient was diagnosed with a metastatic PCa and was first treated with goserelin and bicalutamide. A clinical response to this complete androgen blockade was obtained for 12 months, and the PCa relapsed thereafter. Different secondary lines of hormone therapy associating successively goserelin with estramustine, cortisol, or etoposide were performed thereafter and gave only short responses. Two years after the first line of hormone therapy and after informed consent, a bone marrow aspirate at a scintigraphic-indicated bone metastasis site was obtained. Microscopic observations from an aliquot of the sample confirmed the presence of metastatic PCa. The sample was assayed for AR mutations with the previously described yeast functional assay (13).

Plasmids
The MMTV-LTR-luciferase reporter plasmid containing glucocorticoid-responsive elements was a gift from Pr. P. Chambon [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France]. The PSA-61-luciferase reporter plasmid containing a 6-kb fragment of the human PSA gene promoter was from Dr. Trapman (Erasmus University, Rotterdam, The Netherlands). Luciferase reporter plasmids for cell proliferation and differentiation (pTA-luc, pTA-AP-1-luc, pTA-CRE-luc, pTA-SRE-luc, and pTA-ISRE-luc) and for protein kinase C and calcium signaling pathways (pTA-NF-{kappa}B-luc and pTA-NFAT-luc), the pEGFP-C3 plasmids, and the pDsRed2-ER plasmid designed for the fluorescent labeling of the endoplasmic reticulum (ER) were purchased from BD Clontech (Ozyme, St. Quentin Yvelines, France). The yeast plasmid pAR23 expressing the mutant AR23 was derived from the yeast functional assay used to screen the tumor sample for AR mutations (13). The pEGFP-ARWT and the pEGFP-AR23 were constructed as follows. The full-length human AR cDNA was amplified from pSV-AR0 with the 5'-primer with XhoI linker, 5'-GCCAAGCTCGAGAGGATGGAAG T-3' and the 3'-primer with BamHI linker, 5'-TAGGGATCCAATGCTTCACTGGG-3'. The amplified XhoI-BamHI AR fragment was inserted into the corresponding cloning site in the pEGFP-C3 vector, yielding the pEGFP-ARWT. Exchanging the EcoRI-BamHI fragment of the pEGFP-ARWT with the mutant counterpart obtained from the yeast expression vector pAR23 generated the pEGFP-AR23 plasmid.

Cell lines and cell cultures
AR-negative COS-1 cells (SV40-transformed African Green Monkey kidney fibroblasts), obtained from Pr. P. Chambon (IGBMC), were cultured in phenol red-free DMEM containing 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. The human androgen-sensitive PCa cell line LNCaP clone FCG (European Collection of Cell Cultures, Salisbury, UK) was cultured in RPMI 1640 medium supplied with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 10 mM HEPES (pH 7.3), and 1 mM sodium pyruvate (Invitrogen, Cergy Pontoise, France).

Luciferase reporter assays
Transient transfections were performed in both cell lines, using JetPEI transfection reagent (PolyPlus Transfection, Illkirch, France) according to the manufacturer’s instructions. To analyze AR-specific transcriptional activities, LNCaP and COS-1 cells were seeded in 12-well plates (8 x 104 cells per well) in complete medium. Twenty-four hours after seeding, cells were transfected with 2 µg reporter plasmid pMMTV-LTR-luc or PSA-61-luc in combination with 1 µg pEGFP-C3, pEGFP-ARWT, or pEGFP-AR23. Twenty-four hours after transfection, 100 nM DHT or vehicle was added. Luciferase activities were assayed 24 h later using the luciferase assay system purchased from Promega (Charbonnieres, France) and a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). To evaluate nongenomic transcription factor activities, LNCaP cells were seeded in 24-well plates (5 x 104 cells) in complete medium and were cotransfected with 0.5 µg of the corresponding pTA-luciferase vector together with 0.5 µg pEGFP-C3, pEGFP-ARWT, or pEGFP-AR23. Cells were allowed to recover 24 h in the presence of 10% FCS medium and then were cultured for an additional 24 h in serum-free medium. Thereafter, cells were treated with 100 nM DHT or vehicle, and luciferase activities were assayed 24 h later. The induction of luciferase activity is indicated in arbitrary units.

Subcellular localization
LNCaP cells (8 x 104 cells) were seeded on Labteck slides (Nalge Nunc Int. Corp., Naperville, IL) in complete medium and transfected 48 h later using the JetPEI transfection reagent (PolyPlus Transfection) and 2 µg pEGFP-C3, pEGFP-ARWT, or pEGFP-AR23. Twenty-four hours after transfection, complete medium was replaced by fresh medium without FCS and containing 100 nM DHT or vehicle. At the indicated time after hormone stimulation, slides were rinsed with PBS and fixed in 2% paraformaldehyde. For the dose-response study, LNCaP cells were seeded in 12-well plates (8 x 104 cells per well) in complete medium and transfected with 0.5, 1, or 2 µg EGFP-AR23 construct. Twenty-four hours after transfection, complete medium was replaced by fresh medium without FCS and containing 100 nM DHT or vehicle. Then, cells were rinsed with PBS 24 h after hormone stimulation and fixed in 2% paraformaldehyde. Nuclei were stained with Hoechst 33258 solution, and slides were mounted for visualization with a confocal microscope (Leica confocal SP2 UV inverted DMIRBE microscope) using the x40 or the x100 oil immersion objective. For cellular fractionation, LNCaP cells were cultured in 100-mm dishes (2 x 106 cells) in phenol red complete medium and were transfected with 15 µg pEGFP-ARWT or pEGFP-AR23 and JetPEI transfection reagent. Twenty-four hours after transfection, cells were treated or not with 100 nM DHT. Then, cellular fractionation was performed at indicated times after DHT stimulation with the ProteoExtract subcellular proteome extraction kit (Calbiochem, Darmstadt, Germany). Protein extracts were thereafter loaded on 7.5% SDS-PAGE and blotted onto nitrocellulose membranes. AR was revealed with mouse IgG2a monoclonal antibody G122-434 (BD Biosciences PharMingen, Le Pont de Claix, France) and peroxidase-conjugated goat antimouse IgG secondary antibodies (BD Biosciences PharMingen) and visualized by chemiluminescence detection (GE Healthcare Life Sciences, Saclay, France). The EGFP tag was detected with rabbit IgG polyclonal anti-GFP (FL) antibody (sc8334; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and peroxidase-conjugated goat-antirabbit IgG (170-6515; Bio-Rad, Marnes-la-Coquette, France) and visualized as for AR.

Immunoblotting
Protein extracts were prepared from transfected LNCaP cells, loaded on a 7.5% SDS-PAGE gel, and transferred onto nitrocellulose membranes by electroblotting. Blots were probed with the mouse IgG2a monoclonal antibody G122-434 against human AR (BD Biosciences PharMingen) or after stripping, with the mouse IgG1{kappa} monoclonal antibody C4 against ß-actin (Chemicon International, Hampshire, UK). Blots were incubated with peroxidase-conjugated secondary antibodies (goat antimouse IgG; BD Biosciences PharMingen) and detection was carried out with the chemiluminescence Western blotting kit (GE Healthcare). Signals were quantified using the Gel Doc 2000 imaging system and the QuantityOne software (Bio-Rad) and normalized with the signal corresponding to ß-actin.

Colocalization studies
LNCaP cells (8 x 104 cells) were seeded on Labtek slides (Nalge Nunc Int. Corp., Naperville, IL) in complete medium and transfected 48 h later using the JetPEI transfection reagent and 2 µg pEGFP-C3, pEGFP-ARWT, or pEGFP-AR23. ER colocalization experiments were done by cotransfecting cells with 1 µg of one of the pEGFP plasmids in combination with 1 µg pDsRed2-ER vector. Twenty-four hours after hormone stimulation, slides were rinsed with PBS and fixed in 2% paraformaldehyde. Nuclei were stained using Hoechst solution (1 µg/ml), and slides were mounted for visualization with a confocal microscope (Leica confocal SP2 UV inverted DMIRBE microscope) using the x100 oil immersion objective.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A new AR variant detected in a hormone-refractory metastatic PCa results from an aberrant exon/ intron splicing
A new AR variant with an insertion of 69 nucleotides in position 2882 (GenBank NM_000044) was detected in a patient with a hormone-refractory metastatic PCa by using a previously described yeast functional assay (13). The 69-nucleotide insertion was corresponding to the 3'-part of intron 2 of the human AR gene (GenBank M27425), suggesting the persistence of an intronic sequence in the mature AR transcript. The insertion occurred at the junction of exon 2 and exon 3, which encode, respectively, for the first and the second zinc finger. The normal splicing of the intron 2 in AR pre-mRNA implies the 5'-splice donor site AG/GU in position 99376 and the 3'-splice acceptor site AG/GG in position 141977 (GenBank NC_000023) (Fig. 1AGo). For this AR variant, an alternative cryptic 3'-splice acceptor site AG/AA present in position 141908 (GenBank NC_000023) was used during the maturation process. The analysis of this new AR variant cDNA sequence did not reveal any mutation in the classical donor or acceptor splice sites. As a result of this aberrant splicing, the last 69 nucleotides of intron 2 persist in AR transcripts (Fig. 1BGo). These supplementary nucleotides do not lead to a premature termination codon but give rise to a mutant AR, named AR23 p.[Glu588_Gly589insGlulleProGluGluArgAspSerGlyAsnSerLeuSerGlyLeuSerThrLeuValPheValLeuPro], containing an insertion of 23 amino acids in position 588 between the two zinc fingers of AR23 DBD (Fig. 1CGo).


Figure 1
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  		FIG. 1. The 69-nucleotide insertion in AR23 results from an exon/intron splicing error. A, Representation of the region between exon 2 and exon 3 of the human AR gene (GenBank NC_000023) indicating the normal splicing of intron 2. Positions corresponding to the normal 5'-splice donor (99376) and 3'-splice acceptor (141977) sites are represented. B, Representation of the cryptic splice acceptor site at the 3'-end of intron 2 (position 141908) was used to generate a transcript with an additional 69 nucleotides upstream of exon 3. C, Schematic representation of the human AR DBD showing the two zinc fingers, the P- and D-boxes, the nuclear export signal (NES), and the 23 amino acids inserted in position 588 (GenBank NM_000044). HR, Hinge region; LBD, ligand binding domain; AF-2, activation function-2.

 
The 23-amino-acid insertion affects the AR intracellular trafficking
To check whether this insertion could influence the nuclear translocation of AR23, we further analyzed the cellular localization of this mutant AR after DHT stimulation in the LNCaP cell line. Cells were transfected with pEGFP-C3, pEGFP-ARWT, or pEGFP-AR23 expression vector and were treated with 100 nM DHT or vehicle. The localization of fluorescent protein was examined by confocal microscopy 24 h after hormone treatment. The intracellular distribution of the EGFP-C3 alone (control) was not affected by the hormone treatment (Fig. 2AGo, a and b). As expected, the EGFP-ARWT was translocated in the nucleus after DHT stimulation and presented a cytosolic pattern in vehicle-treated cells (Fig. 2AGo, c and d). On the contrary, the DHT-activated AR23 was unable to enter in the nucleus but formed rather cytosolic and perinuclear aggregates (Fig. 2AGo, e and f, and inset 2A, f). The formation of these aggregates was DHT dependent, because in vehicle-treated cells, AR23 presented a similar expression pattern to that of wild-type AR. Similar results were obtained in PC-3 cells, an AR-negative PCa cell line, and in COS-1 cells, an AR-negative and nonprostatic cell line (data not shown). This particular intracellular distribution of DHT-activated AR23 was not due to the EGFP tag because aggregates were also evidenced by AR23 immunoassaying in COS-1 cells (data not shown). Furthermore, a dose-response study using increasing amounts of EGFP-AR23 construct was conducted to check whether the aggregate formation was not due to an overexpression of the mutant AR. The formation of AR23 aggregates was independent of the quantity of AR23 expression plasmid used (Fig. 2BGo), suggesting that aggregates did not result from an overexpression of AR23.


Figure 2
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  		FIG. 2. The 23-amino-acid insertion affects the AR intracellular trafficking. LNCaP cells were transfected as described in Materials and Methods with the indicated pEGFP construct, and the intracellular localization of the EGFP, EGFP-ARWT, or EGFP-AR23 was followed by confocal microscopy. A, Localization of EGFP as a control (a and b) and EGFP-ARWT (c and d) or EGFP-AR23 (e and f) fusion proteins, 24 h after DHT treatment. Vertical panels represent vehicle-treated (a, c, and e) or 100 nM DHT-treated (b, d, and f) cells. AR23 was unable to enter in the nucleus upon DHT stimulation but formed rather cytoplasmic and perinuclear aggregates (compare d and f, and see inset f). B, Dose-response study indicating that the formation of aggregates was not due to an overexpression of AR23. LNCaP cells were transfected with increasing amounts of pEGFP-AR23 construct, 0.5 µg (a and b), 1 µg (c and d), and 2 µg (e and f) and were treated (b, d, and f) or not (a, c, and e) with 100 nM DHT. Images were collected with a Leica SP2 (UV) inversed DMIRBE confocal microscope, using a x40 objective. Plasmid dose influences only the percentage of transfected cells and not the formation of aggregates. C, Kinetics experiments revealing the formation of aggregates 40 min after DHT treatment (compare e and f). Confocal microscopy was used to realize a time-course experiment to evaluate the formation of aggregates by AR23 upon 100 nM DHT stimulation. Results for pEGFP-ARWT (a, c, and e) or pEGFP-AR23 (b, d, and f) are shown. Scale bar, 12.7 µM. Confocal images were collected with a Leica SP2 (UV) inversed DMIRBE confocal microscope, using a x100 oil immersion objective. D, Intracellular movement dynamics of EGFP-ARwt and EGFP-AR23 were studied by cellular fractionation of LNCaP cells and Western blot analysis. The wild-type AR exhibited a cytoplasmic expression pattern (F1) in the absence of DHT. Within 40 min after DHT addition, the wild-type AR was redistributed in membrane and nuclear fractions (F2 and F3), whereas the mutant AR23 was not detected in the nuclear fraction (F3) but remained in the cytosolic and membrane fractions (F1 and F2).

 
Next, a time-course experiment was performed in LNCaP cells to follow the intracellular trafficking of AR23 before the formation of aggregates. Although the DHT-activated wild-type AR was completely translocated into the nucleus 20 min after stimulation (Fig. 2CGo, a and c), the AR23 still exhibited a diffuse cytoplasmic expression pattern (Fig. 2CGo, b and d). The perinuclear aggregates formed by AR23 was noticeable 40 min after DHT stimulation (Fig. 2CGo, f). By this time, AR23 depicted any nuclear distribution (Fig. 2CGo, f). These data indicate that the 23-amino-acid insertion between the two zinc fingers impairs AR23 nuclear entry after hormone stimulation and suggest also that perinuclear aggregates are unlikely due to a nuclear export process of the AR23.

These data were supported by subcellular fractionation followed by Western blot analysis performed in transfected LNCaP cells at different times after DHT stimulation. In these experiments, intracellular movement dynamics of EGFP-ARWT and EGFP-AR23 could be followed. As expected, the evolution of the signal corresponding to the wild-type AR in the cytosolic (F1), membrane (F2), and nuclear (F3) fractions was reflecting normal dynamics of intracellular movement of AR in LNCaP cells after DHT stimulation (Fig. 2DGo). Besides, the intranuclear trafficking of the AR23 seemed to be altered. Indeed, the AR23 was never retrieved in the nuclear fraction (F3) after DHT stimulation, whatever the time of observation, but seemed rather to accumulate in the cytosolic and in the membrane fractions (Fig. 2DGo).

Altogether, these results suggest that the insertion of 23 amino acids between the two zinc fingers in the DBD leads to the cytoplasmic retention of AR after DHT treatment and that this DHT-activated AR23 forms aggregates in the cytoplasm and in a perinuclear membrane compartment.

AR23 localizes partially in ER after DHT stimulation
Because the insertion of the following 23 amino acids p.[Glu588_Gly589insGluIleProGluGluArgAspSerGlyAsnSerLeuSerGlyLeuSerThrLeuValPheValLeuPro] between the two zinc fingers of the AR DBD seems to retain the AR in a perinuclear membrane compartment upon DHT stimulation, we next screened this peptide sequence for any similarity with known targeting signal in databases. Sequence alignments and standard dynamic programming-based alignments methods (BLAST over SWISS-PROT) were used to identify any functional motif or membrane-targeted signal within the 23-amino-acid insertion. A canonical casein kinase II (CK-II) phosphorylation site was present within the 23-amino-acid insertion. Potential relatedness (59% of identity) was found with the cytoplasmic domain of the Vpu protein of HIV-1. This cytoplasmic domain is known to be responsible for the transport of Vpu to the rough ER/Golgi cellular compartment. Similarly, identities were also observed with a short peptide within the Rho-GTPase activating protein (88% of identity) and the protocadherin {gamma}-C3 precursor (77% of identity). These two proteins are also known to interact with the Golgi/rough ER, thus suggesting that AR23 could carry an ER interaction domain.

To determine whether the DHT-activated AR23 colocalized with the ER, LNCaP cells were cotransfected with a plasmid encoding the fluorescent protein DsRed2 tagged with the KDEL peptide, an ER localization motif, in combination with the indicated EGFP-AR fusion construct. A similar distribution pattern could be observed between AR23 aggregates and the ER marker. Indeed, the cytoplasmic and perinuclear aggregates formed by the AR23 partially colocalized with the small dots formed by the DsRed2-ER fusion protein in the cytoplasm and in nuclear invaginations (Fig. 3Go, A–D). Such a codistribution pattern was not observed with the EGFP-ARwt construct or the control (Fig. 3Go, E–L). Together, all these data suggest that the DHT-activated AR23 was partially retained in the ER.


Figure 3
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  		FIG. 3. AR23 localizes partially in ER after DHT stimulation. Confocal microscopy shows partial AR23 colocalization with ER. LNCaP cells were transiently transfected with pEGFP-AR23 (A–D), pEGFP-ARWT (E–H), or pEGFP-C3 as a control (I–L), in combination with pDsRed2-ER expression plasmid in complete medium. Vertical panels represent separate channels, DAPI, EGFP, DsRed2-ER, and merged images. The EGFP-AR23 partially colocalized with ER marker in the cytoplasm and in nuclear invaginations (A–D). This codistribution pattern was not observed with the EGFP-ARwt construct or the control (E–L). Scale bar, 12.7 µM. Confocal images were collected with a Leica SP2 (UV) inversed DMIRBE confocal microscope, using a x100 oil immersion objective.

 
The AR23 loses its genotropic actions but remains partially active in prostate cancer cells
Due to the particular intracellular distribution of the AR23 upon DHT stimulation, classical genomic activities of the AR are expected to be completely abolished. To verify this possibility, AR23 transcriptional activities were analyzed by Luciferase reporter assays performed in COS-1 cells. Cells were cotransfected with the PSA-61-luc or the MMTV-LTR-luc promoter constructs in combination with the indicated pEGFP plasmid. Both PSA-61 and the MMTV-LTR promoters are known to be AR responsive. Basal levels of MMTV-LTR and PSA promoters activities were obtained in the presence of pEGFP-C3-negative control plasmid (Fig. 4Go, A and B). As expected, a strong and ligand-dependent up-regulation of these two promoters was observed in the presence of the wild-type AR upon DHT stimulation. Indeed, an 8-fold increase of transcriptional activities was obtained from the two promoters in the presence of DHT-activated wild-type AR. Besides, upon DHT stimulation, AR23 failed to induce transcription from these two promoters (Fig. 4Go, A and B). These data could not be explained by a lower expression of AR23 in transfected COS-1 cells compared with wild-type AR, because Western blot analyses showed that the two ARs depicted identical expression levels in transfected cells (Fig. 4CGo). These data are in agreement with the cytosolic retention of DHT-activated AR23.


Figure 4
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  		FIG. 4. The AR23 loses its genomic actions but remains partially active in PCa cells. Transcriptional activities of wild-type AR or of the mutant AR23 were measured in COS-1 (A and B) or in LNCaP (D and E) cells. Cells were transfected with pEGFP-C3 as a control or pEGFP-ARWT or pEGFP-AR23 plasmids in combination with pPSA61-Luc (A and C) or the pMMTV-Luc (B and E) luciferase reporter vectors. Cells were treated with DHT (+) or vehicle (–) for 48 h. Cells extracts were subsequently assayed for luciferase activity. In the AR-negative COS-1 cells, the wild-type AR exhibited genotropic actions from the two androgen-responsive promoters, whereas the AR23 was unable to directly activate transcription from these two promoters. C, Western blot analysis representing the expression level of EGFP-ARWT and EGFP-AR23 in COS-1 cells. A similar expression pattern could be observed for the two receptors. D and E, Transcriptional activities of the wild-type AR or AR23 from PSA61 (D) and MMTV-LTR (E) were measured in androgen-sensitive LNCaP cell line. Both the wild-type AR and AR23 enhance transcription of the two androgen-responsive promoters in a DHT-dependent manner. Values are given in arbitrary units (AU). Data represent the mean assays performed in triplicate ± SEM. F, Western blot analysis representing the evaluation of expression level of the endogenous AR in LNCaP cells transfected with the EGFP-ARWT or the EGFP-AR23. The histogram represents the ratio of quantified signals corresponding to the transfected and endogenous ARs normalized with the signal corresponding to ß-actin. The expression level of the endogenous AR is 1.5-fold increased in the presence of the DHT-activated AR23 than in the presence of the wild-type AR.

 
To confirm these results, AR23 transcriptional activities were investigated in a PCa cell line. The AR-positive LNCaP cells were cotransfected with the PSA-61-luc or the MMTV-LTR-luc promoter constructs in combination with the pEGFP-ARWT, the pEGFP-AR23, or the pEGFP-C3 expression plasmid as control. In the presence of the pEGFP-C3 control plasmid, transcriptional activities measured in LNCaP cells referred to the endogenous AR. As expected, a weak and DHT-dependant up-regulation of the two promoters by the endogenous AR could be observed (Fig. 4Go, D and E). In the presence of the wild-type AR, transcriptional activities from the two promoters were further increased after DHT treatment. In these conditions, the transcriptional activity observed in the presence of the wild-type AR was three times (PSA-61) and one and a half times (MMTV-LTR) higher than that observed in the presence of the control plasmid alone and results from the combined transcriptional activities of the endogenous and the wild-type ARs. Unexpectedly, a DHT-dependent augmentation of transcriptional activities from the two promoters, reaching a similar magnitude to that observed in the presence of the wild-type AR, was obtained with the AR23 (Fig. 4Go, D and E), despite the impaired nuclear localization of this AR variant (Fig. 2AGo, inset f). These data revealed unexpected actions of the AR23 in a PCa cell line, despite its exclusively cytoplasmic localization. As a consequence of the cytoplasmic retention of the DHT-activated AR23, the up-regulation of the two androgen-responsive promoters in the presence of this receptor should involve a second intermediate factor, which could be the endogenous AR present in LNCaP cells.

Next, we wondered whether the AR23 could influence the level of expression of the endogenous AR. A Western blot analysis was performed on protein extracts prepared from LNCaP cells transfected with pEGFP-ARWT or pEGFP-AR23 expression plasmid. Cells were treated with 100 nM DHT or left untreated. As expected, the intensity of the signal corresponding to endogenous AR significantly increased after DHT treatment. Moreover, the intensity of the signal corresponding to the endogenous AR was 1.5-fold higher in the presence of DHT-activated AR23 than in the presence of the wild-type AR (Fig. 4FGo). These data indicate a potential effect of the AR23 on the level of expression or on the stability of the endogenous AR in transfected LNCaP cells.

The cytoplasmic AR23 affects transcriptional activities of NF-{kappa}B and AP-1
Transcription factors involved in cell proliferation and differentiation like CREB, E2F, signal transducer and activator of transcription (STAT1/STAT2), serum response factor (SRF), AP-1, and those implicated in protein kinase C and calcium signaling pathways such as NF-{kappa}B, nuclear factor-activated T cells (NFAT), could be regulated from the cytoplasm. Thus, to investigate whether the cytoplasmic AR23 is able to influence the activity of these transcription factors, we performed different luciferase gene reporter assays, in which the luciferase gene was placed under the tight control of a minimal promoter containing a TATA box (pTA-luc) alone or linked to known transcription factor- binding sites. Thus, LNCaP cells were transiently cotransfected with one of the EGFP-AR constructs together with the indicated luciferase reporter plasmid, and luciferase activities were measured 24 h after DHT treatment. Luciferase activities measured reflected the level of activity of the corresponding transcription factor studied. By cotransfecting LNCaP cells with an AR expression plasmid, our intention was to determine the impact of the expressed AR on the activity of the transcription factors present in this cell line (Fig. 5AGo).


Figure 5
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  		FIG. 5. Effects of wild-type AR and AR23 on AP-1 and NF-{kappa}B transcriptional activities. A, Schematic representation of the study conducted to determine the impact of AR23 on the activities of different transcription factors (TF) CREB, E2F, STAT1/STAT2, SRF, AP-1, NF-{kappa}B, and NFAT in LNCaP transfected cells. If the expressed AR influences the activity of a TF, the luciferase gene expression from the promoter construct containing the binding site for this TF will be affected. B–E, Histograms representing luciferase activities obtained, respectively, from pTA-Luc (B), pTA-SRE-Luc (C), pTA-NF-{kappa}B-Luc (D), and pTA-AP-1-Luc (E) in LNCaP cells cotransfected with the pEGFP-C3 as control or the pEGFP-AR expression plasmid and with the indicated reporter plasmid. Cells were thereafter treated with 100 nM DHT (+) or left untreated (–). Luciferase activities were recorded 24 h after DHT stimulation. The cytoplasmic DHT-activated AR23 influences AP-1 and NF-{kappa}B transcriptional activities. Values are indicated in arbitrary units (AU). Data represent the mean of assays performed in triplicate ± SEM.

 
Transcriptional activities from the pTA-luc construct were extremely low, whatever the pEGFP construct used. These activities correspond to the negative control and indicate that further regulatory elements are necessary to conduct transcription from this minimal promoter (Fig. 5BGo). In our experimental conditions, the use of regulatory elements corresponding to CRE-, E2F-, ISRE-, and NFAT-luciferase constructs did not enhance any further this basal transcriptional activity (data not shown).

On the contrary, transcriptional activity from the SRE construct was enhanced in the presence of the DHT-activated wild-type AR (Fig. 5CGo). Besides, only a basal transcriptional activity, similar to that obtained in the presence of the endogenous AR, was observed in AR23-transfected cells. These data indicate that contrary to the wild-type AR, the AR23 variant was unable to stimulate activities of the SRF transcription factor.

A weak NF-{kappa}B transcriptional activity was observed when LNCaP cells were cotransfected with pEGFP-C3 and pTA-NF-{kappa}B-luc (Fig. 5DGo). This basal activity was enhanced by DHT treatment and could result from the activation of the endogenous AR in LNCaP cells. The NF-{kappa}B transcriptional activity was further increased in the presence of EGFP-ARWT and amplified again by DHT treatment. Similarly, NF-{kappa}B transcriptional activity was also induced in the presence of DHT-stimulated AR23.

Also, AR23 seemed to have an effect on AP-1 activities. Indeed, an unexpected decrease of AP-1 transcriptional activity was obtained in the presence of AR23 (Fig. 5EGo). In this experiment, basal AP-1 transcriptional activities were observed in LNCaP cells cotransfected with pEFGP-C3 and pTA-AP-1-luc construct. These basal AP-1 activities were not affected by hormone treatment. A moderate increase of AP-1 activity (1.9-fold) was observed in LNCaP cells in the presence of DHT-activated EGFP-ARWT compared with the vehicle-treated cells. However, in the presence of AR23, AP-1 activity was below the basal level measured in LNCaP cells transfected with the control plasmid (Fig. 5EGo).

Together, our findings indicate that AR23 could affect transcriptional activities of AP-1 and NF-{kappa}B transcription factors in LNCaP cells.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well established that during PCa progression, mutant ARs with distinguished transcriptional activities emerge to support tumor cell growth in an androgen-depleted environment (16). In this study, we described new functional properties of AR23, an AR splicing variant detected in a hormone-refractory metastatic PCa.

Transcripts of this particular AR variant are characterized by a 69-nucleotide insertion between exon 2 and exon 3. The inserted sequence results from an aberrant splicing using a cryptic acceptor site in intron 2 and corresponds to the last 69 nucleotides of intron 2. Actually, according to the AR23 cDNA sequencing, we could not explain this aberrant splicing by the presence of a mutation. Nevertheless, we cannot exclude a mutation relating to the donor or to the acceptor splicing site in the intronic sequence.

AR splicing variants resulting from the use of the same cryptic acceptor site within the intron 2 have already been reported in two cases of PAIS (14, 15). Moreover, other splicing errors at different exon/intron junctions have already been described in patients with androgen insensitivity (17, 18, 19). In addition, naturally occurring or pathological splice variants are common in other members of the nuclear receptor superfamily (20). However, to our knowledge, the present study is the first report of an AR splicing variant isolated from PCa.

The 69-nucleotide insertion between the two zinc fingers of the DBD does not lead to a premature termination codon but to the addition of 23 amino acids: p.[Glu588_Gly589insGluIleProGluAspSerGlyAsnSerLeuSerGlyLeuSerThrLeuValPheValLeuPro]. Because this peptide sequence is similar to that detected in two cases of PAIS previously described, we can predict that the AR23 displays also normal ligand-binding capacities (14, 15).

This particular AR variant being detected in both PAIS, wherever the AR conserves a residual activity, and in PCa suggests that the AR23 retains some function. In this regard, little is known about how the inserted peptide affects AR activities.

Importantly, the 23-amino-acid insertion occurs in a key region of the DBD, between the carboxyl-terminal part of the first zinc finger, which contains the P-box responsible for specific DNA binding and the nuclear export signal, and the second zinc finger, which contains the dimerization domain and a nuclear localization signal. We demonstrated that the addition of 23 amino acids in this key region of the DBD influences the subcellular compartmentalization of the AR by impairing its translocation from the cytoplasm to the nucleus after ligand binding. The DHT-activated AR23 forms rather cytoplasmic and perinuclear aggregates that are unrelated to an overexpression of the mutant AR. These cytoplasmic aggregates should not be confused with the androgen-induced nuclear aggregation of ARs that contain an expanded glutamine tract in spinal and bulbar muscular atrophy (21). In our study, the AR23 is unable to be translocated into the nucleus. In the case of the wild-type AR, androgen binding induces a conformational change that reveals the bipartite nuclear-localization signal that overlaps the DBD and hinge region (22). Once exposed, the nuclear-localization signal can be recognized by import receptors such as importin-{alpha} and importin-ß, which mediate translocation of the AR through the nuclear-pore complex (23). The conformation adopted by the 23 amino acids may impair these interactions with importins and affects the normal nucleo-cytoplasmic shuttling of the receptor. Consequently, the DHT-activated AR23, which cannot be translocated into the nucleus, should aggregate in the cytoplasm or within a particular organelle. Another possibility could be the presence of an additional targeting signal within the amino acid insertion, which targets the mutant AR to a special subcellular compartment.

By the use of subcellular fractionation, we determined that the DHT-activated AR23 was predominantly in the membrane fraction, and confocal microscopy analyses indicate a partial colocalization with the ER. This is in agreement with the homology between the 23-amino-acid insertion and short peptides from proteins known to interact with the ER such as the Vpu of HIV-1 (24), the Rho-GTPase activating protein, and the protocadherin {gamma}-C3 precursor (25, 26). Indeed, within protocadherin {gamma}-C3, six amino acids (EIPEER) of the 23 were identical to a part of the cadherin repeat domain 1, a calcium-binding motif (26). Thus, the 23-amino-acid insertion could contain a signal motif for targeting intracellular organelles such as the ER. Nevertheless, the determination of the exact localization of the DHT-activated AR23 requires additional investigations. In the light of these preliminary results, we wonder what could be the functional consequences of such a localization of an AR variant.

The ER is an important and dynamic organelle that represents the storage compartment for the majority of intracellular calcium (27). Calcium has been shown to be a critical factor for regulating cell proliferation, cell adhesion, and cell migration (28, 29, 30). It remains to be determined whether or not the mutant AR could interfere with these calcium-mediated signaling pathways. Moreover, it has been reported that membrane microdomains commonly called lipid rafts are implicated in several signal-transduction mechanisms by serving as platforms for assembling signaling complexes. It has also been demonstrated that wild-type AR is able to form complexes with other signaling complexes in rafts that were isolated from LNCaP cells (31, 32). Besides, the 23-amino-acid insertion tail, which is enriched in hydrophobic amino acids such as the LVFVL motif, could stabilize the anchoring of the AR23 in those lipid rafts (Fig. 1CGo).

Thus, it seems possible that the AR23 is at a crucial position to trigger signals from the cytoplasm. This observation is confirmed by data obtained from functional analyses performed in LNCaP cells. We demonstrated that the coexpression of the AR23 in this cell line, which expresses an endogenous AR, leads to an increase of transcriptional activities from the MMTV-LTR and the human PSA gene promoters, two AR-responsive promoters, after DHT stimulation. These activities could not be attributed to a direct action of the AR23, because this mutant AR could not be translocated into the nucleus after DHT binding. Furthermore, the AR23 has no effect on these two promoters in AR-negative COS-1 cells. The presence of the endogenous AR in LNCaP cells seems to be a prerequisite for AR23 activities on androgen-responsive promoters. Thus, the cytoplasmic DHT-activated AR23 was still functional in AR-positive PCa cells, and could trigger a signal to the nucleus to enhance transcription from androgen-responsive promoters. Nevertheless, this model supposes the coexpression of AR23 together with a normal AR in PCa cells.

Because these data highlight possible cytoplasmic actions of the AR23, we further investigated whether AR23 could also affect from the cytoplasm the signaling pathway of transcription factors involved in cell proliferation and survival. In this report, we demonstrated that luciferase activity measured from the SRE-luc construct increased in a DHT-dependent manner in the presence of the wild-type AR. This correlates with the observation that the DHT-activated AR can be recruited by SRF to activate SRE-containing promoters (33). On the contrary, luciferase activity was not enhanced from this SRE-luc reporter construct in the presence of AR23, due to the inability of this mutant AR to translocate into the nucleus. These results illustrate one of the consequences of the loss of genomic activities of AR23.

On the contrary, data obtained with the NF-{kappa}B-luc and AP-1-luc constructs showed that despite this lack of genomic actions, the AR23 could affect transcriptional activities of NF-{kappa}B and AP-1. Therefore, we demonstrated that the AR23 was able to increase NF-{kappa}B activity in PCa cells. The NF-{kappa}B family of transcription factors is known to regulate the expression of adhesion molecules that promote cell migration and cell-cell interactions (34). Another function of NF-{kappa}B is to induce expression of genes that contribute to tumor progression such as antiapoptotic genes and genes that regulate cell growth (34, 35). We demonstrated that in transfected LNCaP cells, NF-{kappa}B transcriptional activity was enhanced in the presence of AR23 at a similar level to that observed in the presence of the wild-type AR. These effects could not be attributed to a direct action of the wild-type or mutant AR on the promoter construct used in these assays, because this promoter does not contain androgen-responsive elements but three NF-{kappa}B binding sites juxtaposed to a TATA box to control the expression of the luciferase gene.

Although the relationship between the AR expression and NF-{kappa}B activation is still poorly understood (35), our results suggest that the cytoplasmic mutant AR23 could influence the activity of NF-{kappa}B in PCa cells. Therefore, the DHT-activated AR is also known to activate in a nongenomic way the PI3K/Akt signaling pathway (11), and Akt can activate NF-{kappa}B pathway by phosphorylation of I{kappa}B{alpha} kinases (36, 37). It remains unknown whether this AR nongenomic pathway contributes to NF-{kappa}B activities measured in our experiments in the presence of the wild-type or the mutant AR.

The decrease of AP-1 activity observed in the presence of AR23 is intriguing. AP-1 is a complex transcription factor composed of members of the Jun family associated with related proteins of the Fos or ATF family (38, 39). Interactions between c-Jun and the DBD/hinge region of the wild-type AR have been reported to modulate reciprocally both AP-1 and AR transcriptional activities (40, 41). To explain the loss of AP-1 activity in the presence of AR23, it remains to be known whether these interactions are affected by the 23-amino-acid insertion within the DBD. Nevertheless, as demonstrated by confocal microscopy performed in transfected LNCaP cells, the endogenous c-Jun is not sequestered in AR23 cytoplasmic aggregates, suggesting a different mechanism than c-Jun sequestration to explain the loss of AP-1 activity (data not shown).

The transcription factor AP-1 has been implicated in cell growth, differentiation, and development by mediating gene regulation in response to a various range of physiological and pathological stimuli (39). Also, AP-1 is involved in PCa cell death by enhancing the expression of proapoptotic factors such as TNF-{alpha} and FasL or by repressing the transcription of the antiapoptotic factor cellular FADD-like IL-1ß-converting enzyme (FLICE)-inhibitory protein long isoform (c-FLIP) in PCa (39, 42, 43, 44). These studies together with our data indicating a decrease in AP-1 activity in the presence of AR23 suggest that AR23 may contribute to tumor progression by protecting PCa cells from apoptosis. However, additional studies are required to evaluate consequences of this loss of AP-1 activity on cell proliferation or survival.

These cytoplasmic activities should be distinguished from classical nongenomic activities. Indeed, nongenomic activities are measured within minutes after hormone stimulation and not after several hours as observed with AR23 (9, 10, 11).

Taken together, our data demonstrate that the 23-amino-acid insertion abolishes genomic actions of the AR and promotes novel cytoplasmic actions. These novel functions are worth being taken into account for residual activity of the AR splicing variant in PAIS. Also, our results suggest that an exclusively cytoplasmic AR could play an important role in PCa progression and open up a new field of investigation in nongenomic AR functions.


   Acknowledgments
 
We thank S. Serra (Département d’Oncologie Hôpital Civil, Strasbourg, France) and V. Lindner (Service d’Anatomo-Pathologie, Hôpital de Hautepierre, Strasbourg, France) for clinical sample acquisition. We are grateful to P. Kessler, J. Vonesch, and D. Hentsch from the imagery unit at IGBMC (IGBMC, Illkirch, France) for rendering possible the confocal microscopy studies.


   Footnotes
 
This work is part of a clinical study managed by the Hôpitaux Universitaires de Strasbourg approved by the Ethic Committee, CCPPRB–Alsace1, and supported by the Programe Hospitalier de Recherche Clinique National 2002. These studies were supported by the Faculté de Médecine of the Université Strasbourg and grants from the associations ARECOH (Association pour la recherche clinique en onco-hématologie), ATGC (association alsace contre le cancer), and ARTP (Association pour la recherche sur les tumeurs prostatiques), the Ligue Nationale contre le Cancer, and the Association pour la Recherche sur le Cancer.

Disclosure Statement: The authors have nothing to declare.

First Published Online May 31, 2007

Abbreviations: AP-1, Activator protein-1; AR, androgen receptor; CREB, cAMP response element-binding protein; DHT, 5{alpha}-dihydrotestosterone; DBD, DNA binding domain; ER, endoplasmic reticulum; FCS, fetal calf serum; NFAT, nuclear factor-activated T cells; NF-{kappa}B, nuclear factor-{kappa}B; PAIS, partial androgen insensitivity syndrome; PCa, prostate cancer; PI3K, phosphatidylinositol 3-OH kinase; STAT, signal transducer and activator of transcription.

Received April 5, 2007.

Accepted for publication May 22, 2007.


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 Materials and Methods
 Results
 Discussion
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