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Molecular Biology and Genetics |
Departments of Urology [X-B. S., R. W. d. V. W.] and Biological Chemistry and Cancer Center Basic Science [A-H. M., L. X., H-J. K.], University of California, Davis, School of Medicine, Sacramento, California 95817
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Studies have demonstrated that androgen-independent growth of CaP cells is associated with AR abnormalities. These include amplification and overexpression of the AR gene (3 , 4) and AR mutation (5) . Amplification and overexpression of the AR gene occurs in approximately one-third of hormone-refractory CaPs. This alteration may facilitate the growth of CaP cells in an environment having very low levels of androgens after castration (6) . Many different investigators have studied mutations of the AR gene in CaP (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) . Their studies suggest that AR mutations are rare in untreated early CaPs, ranging in different reports from 0 to 4% (7, 8, 9, 10) . In recurrent and metastatic prostatic tumors, the frequency of mutations reported by different investigators varies considerably. More recent studies have found that AR mutations occur in 5 of 10 (50%) hormone-refractory metastases (16) , in 11 of 25 (44%) advanced CaPs (17) , and in 8 of 30 (27%) microdissected metastatic lesions (10) .
Mutations of the AR gene can alter receptor function. For example, the T877A mutant AR that has been identified in numerous clinical samples and in two CaP cell lines is activated not only by androgens but also by nonandrogenic steroids and hydroxyflutamide, an antiandrogen (12 , 18) . The V715M, V730M, and H874Y AR mutants also respond to nonandrogens and/or antiandrogens (12 , 18) , whereas a recent study has shown that the L701H mutant AR is able to transactivate a reporter gene in the presence of glucocorticoid (19) . In contrast, the C619Y mutant AR from a metastatic CaP has completely lost the ability to transactivate target genes in the presence of androgen (20) . To date, although 56 missense AR mutations have been identified in human CaP,4 only 6 of these have had their transactivational activity determined using multiple ligands (12 , 18, 19, 20, 21) . The functional status of the majority of the mutant ARs found in CaP has not yet been investigated in a systematic way. Because activation of mutant AR by alternative steroids and/or antiandrogens may provide a growth advantage to CaP cells expressing the mutant, determining the function of these mutant ARs is important for understanding their role in the development of hormone-independent CaP.
In the present study, we analyzed the ligand-stimulated transactivational activity of 44 mutant ARs from human CaPs using a colorimetric yeast assay. The ease of genetic manipulations in yeast provides a powerful approach to define critical signal pathways. Because the basic transcriptional machinery and the chromatin remodeling apparatus appear to have been well conserved in yeast (22 , 23) , this system has been fruitfully applied to the investigation of steroid receptor function (24 , 25) . Our goal in this study was to validate this approach and apply it to understand the functional significance of different AR mutations in the development and progression of androgen-independent disease. We found that CaP-derived mutant ARs exhibit diverse transactivational activities including loss-of-function, wt function, gain of function, and promiscuous activity. We also observed that the combination of estradiol and progesterone at physiological concentrations is able to activate certain mutant ARs synergistically that had failed to respond to either ligand used separately. To our knowledge, this is the first systematic and comprehensive analysis of CaP-associated mutant ARs, where their basal transactivational activities are compared and contrasted in single, standardized assays.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Construction of Plasmids.
Plasmids were constructed according to standard DNA recombinant methods (26)
. Constructs that included any PCR products were sequenced to verify the correct reading frame and the absence of random mutations. The AR-responsive reporter plasmid pXB11 was constructed by modification of the plasmid pLS210, a gift from Dr. Richard Iggo (ISREC, Epalinges, Switzerland; Ref. 27
). A 0.8-kb ApaI-BamHI fragment containing a triple tandem 26-bp ARE (GRE) consensus oligonucleotide fused upstream to the yeast CYC1 minimal promoter was released from the p2UG plasmid, kindly provided by Dr. Didier Picard (University of Geneva, Geneva, Switzerland; Ref. 28
). This fragment was then inserted into the homologous site of the pLS210 plasmid upstream to the ADE2 reporter gene, yielding the plasmid pXB11. For constructing the wt AR yeast expression plasmid, a 110-bp, PCR-amplified, BamHI- and XmaI-digested AR fragment of the 5'-terminal part was ligated to a 2.7-kb AR fragment that was released using XmaI and SalI endonucleases from the p5HbhAR plasmid, kindly provided by Dr. Elizabeth Wilson (University of North Carolina at Chapel Hill, Chapel Hill, NC), to yield the full-length AR coding region lacking the 5'-terminal untranslated sequences. This resultant AR was then cloned into the yeast expression plasmid pG1 provided by Dr. Picard, yielding the plasmid pXB12. The full-length AR coding region in the pXB12 plasmid was sequenced. It encodes 922 amino acids and contains a repeat length of 25 glutamines and a repeat length of 23 glycines in exon 1. To generate the plasmid pXB15 containing an AR with a deletion of the DBD/LBD region (
DBD/LBD), a KpnI-SalI AR fragment containing codons 502–558 and codons 910–919 plus 92 bp of untranslated sequences were prepared by PCR amplification and endonuclease digestion. This fragment was cloned into the homologous site of pXB12, yielding the plasmid pXB15 that was then linearized by digestion using PshAI.
Yeast Strain.
The Saccharomyces cerevisiae strain yIB12/2 that has a mutated ade2 gene was a gift from Dr. Richard Iggo. To create a reporter strain, the AR-responsive reporter plasmid pXB11 was linearized at the unique ApaI site in URA3 and integrated into the chromosomal URA3 gene, resulting in the yA(G)RE strain with the genotype of MATaade2–1 leu2–3, 112trp1–1his3–11, 15can1–100ura3–1 URA3 3xARE::pCYC1::ADE2. Integration at the URA3 locus was confirmed by PCR amplification of plasmid-based ADE2 sequences from yeast genomic DNA extracts. The yA(G)RE strain was grown on complete YEPD medium supplemented with 200 µg/ml adenine (YPDA+ medium) to avoid selection of spontaneous suppressors of the endogenous mutant ade2 locus.
Site-directed Mutagenesis.
Mutations of the AR gene were prepared by PCR-mediated site-directed mutagenesis, with the exception of the T877A mutation, which was amplified from the cDNA of LNCaP cells. Four primers were requisite for preparing all mutations located in the DBD-hinge-LBD regions: ARsdm-F, 5'-CACTTGTGTCAAAAGCGAAATGGGCCCCTG; ARsdm-R, 5'-CATGACAGACTGTACATCAATAGAGGAA; ARgap1, 5'-TGGATGGATAGCTACTCCGGACCTTACGGGGAC; and AR3UTR, 5'-AAGGCACTGCAGAGGAGTAGTGCAGAGTTATAAC. ARsdm-F and ARgap1 are located at the 3'-terminal part of AR exon 1, and ARsdm-R and AR3UTR are located at the 3'-terminal untranslated region of AR. In addition, two 30-base complementary primers (ARmt-F and ARmt-R) containing specific base changes were synthesized for each target mutation. Mutations were generated by a two-step PCR procedure.Two AR segments were separately amplified from the pXB12 plasmid using the primer pairs ARsdm-F/ARmt-R and ARmt-F/ARsdm-R. The two segments were then purified and fused together by a PCR ligation using the nested primers ARgap1/AR3UTR. The resulting DNA fragment encodes the codons 526–919 of AR plus 92 bp of the untranslated region.
Yeast Transformation and DNA Sequencing.
To obtain the full-length AR coding region, the yA(G)RE yeast cells were cotransformed with 5 µl of PCR products as prepared above and 20 ng of linearized pXB15 plasmid using a lithium acetate procedure (27)
. The transformed yeast was plated on selection plates containing minimal medium minus tryptophan plus adenine (5 µg/ml) and incubated for 3 days at 35°C. Because there are overlaps of 100 bp at the 5'-terminus and 124 bp at the 3'-terminus between the PCR-prepared AR fragment and the linearized plasmid, homologous recombination in yeast resulted in the AR fragment being fused into the plasmid. The AR expression plasmids were extracted from yeast and then transformed into DH5
bacteria to obtain a sufficient amount of plasmid DNA. The AR coding region was sequenced to confirm the specific mutation and the absence of random mutations.
Analysis of Yeast-expressed AR Protein.
The expressed AR protein was extracted from yeast following the procedure described by Doesburg et al. (29)
and analyzed by Western blotting using the anti-AR antibody AR441 (Neomarker, Fremont, CA).
Functional Scoring of AR.
Single yeast colonies expressing specific mutant AR were grown overnight in 3 ml of YPDA+ medium. Approximately 1 µl of yeast culture was inoculated onto selection plates containing different concentrations of one of the ligands studied and grown at 35°C for 3 days. On the basis of the colors of the yeast colonies, the transactivational activity of AR was assigned to one of five categories: white indicates complete activity (scored as ++++); white-pink indicates moderate activity (+++); pink indicates partial activity (++); reddish-pink indicates weak activity (+); and red indicates lack of activity (-).
Transient Transfection Assay.
DU145 cells (5x104 cells/well) were plated in 24-well plates for 24 h in RPMI 1640 containing 10% charcoal-stripped FBS (HyClone, Logan, UT). The cells were transfected with wt or mutant AR expression plasmids, the pGL3B-PSA6 firefly luciferase reporter plasmid kindly provided by Dr. Chawnshang Chang (University of Rochester, Rochester, NY), and the pRL-SV40 Renilla luciferase plasmid (Promega Corp., Madison, WI) using the Fugene 6 transfection reagent (Roche, Indianapolis, IN). The next day, the cells were fed with fresh medium containing 1% charcoal-stripped FBS plus different ligands at the indicated concentrations. Cells were harvested 24 h after hormone addition, and the levels of luciferase were measured using a dual luciferase assay (Promega) in an EG & G Berthold LB96V MicrolumatPlus microplate luminometer (Perkin-Elmer-Wallac, Inc., Gaithersburg, MD).
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RESULTS |
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We tested whether the transactivational activity of AR in yeast is appropriately dependent on the presence of ARE sequences, intact AR protein, and androgen. The yA(G)RE yeast cells were transformed with one of three types of plasmids: the pXB12 plasmid that expresses wt AR; the DBD/LBD-deleted pXB15 plasmid; or the PG-1 plasmid that lacks the AR gene. In addition, yeast cells lacking the ARE-promoter were transformed with the pXB12 plasmid. The transformed yeast cells were grown on selection plates in the presence or absence of 10-8 M DHT. Only the yA(G)RE yeast cells containing ARE-reporter and expressing intact AR protein yielded white yeast colonies in the presence of DHT. The remaining yeast cultures gave red yeast colonies (data not shown). These results indicate that the combination of an ARE-reporter, an intact AR, and androgen are required for the AR functional assay in yeast.
Response of wt AR to Androgens, Nonandrogens, and Antiandrogens.
The yA(G)RE cells were transformed with the pXB12 plasmid and grown on selection plates containing various concentrations of the nine ligands studied. Transactivational activity was assessed based on the yeast scores as described in "Materials and Methods." The results were well reproducible in two independent experiments. The dose responses of wt AR to these ligands are given in Table 1
. These data can be summarized as follows: (a) the wt AR exhibited different sensitivities to the three androgens with the activity of DHT > testosterone > R1881; (b) the adrenal androgen precursor DHEA activated wt AR in a dose-dependent fashion, with slight activation occurring at 10-7 M and complete activation at 10-4 M; (c) similar to the previous study (18)
, wt AR could be activated in yeast with 10-8 M progesterone or estradiol, concentrations that are approximately 10- and 100-fold greater than male physiological levels, respectively; (d) wt AR failed to respond to up to 10-4 M flutamide, bicalutamide, or hydrocortisone. Thus, neither glucocorticoid nor antiandrogens affect the function of wt AR through the ARE (GRE) consensus sequences used in the yeast system.
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) were obtained from advanced CaPs and 9 from earlier stages of this disease. The reported frequencies and the locations of these mutations in the AR are presented in Table 2
. It is noteworthy that the T877A mutation has been found in 2 cell lines and 16 clinical samples, indicating that this is a hot spot in CaP. Of these mutations, 6 are located in the DBD, 4 in the hinge region, and 34 in the LBD. In addition, we prepared the nonfunctional C784Y AR identified in a patient with complete androgen insensitivity syndrome (30)
and two mutant ARs with double mutations (G724D/P801T and L574P/Q902R) that exhibited loss-of-function in our preliminary yeast studies. These three mutants were used as negative controls.
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, demonstrating dose-dependent responses of these mutants to DHT, estradiol, or progesterone in concentrations from 10-10 to 10-7 M. The three negative controls failed to respond to the seven ligands tested (data not shown). Table 2
gives the functional scores of these 44 mutants in response to no ligand, to four steroid hormones used at physiological concentrations, and to hydrocortisone and antiandrogens used at the chosen concentrations.
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. For 37 of the 44 AR mutants, different extents of transactivational activity were obtained in response to DHT used at the physiological level (10-9 M). Of these 37 mutants, three (7%) were completely activated (++++) only by DHT, showing wt function, whereas 14 (32%) were partially activated (+ to +++) by DHT, suggesting partial function. The remaining 20 mutants (45%) exhibited activity that was greater than wt AR in the presence of 10-7 M DHEA (Table 2)
, suggesting gain of function.
Of the 20 gain-of-function mutants, 5 (K580R, L701H, V715M, H874Y, and T877A) also responded to estradiol, progesterone, hydrocortisone, flutamide, or bicalutamide in the concentrations given in Table 2
indicating promiscuous activity. The extent of promiscuous response to the five nonandrogen ligands varied. V715M and H874Y responded only to estradiol and/or progesterone, whereas T877A was also activated by flutamide. The K580R and L701H mutants were activated to different extents by all five nonandrogen ligands. The response of the promiscuous mutant ARs to nonandrogens was dose dependent. As shown in Fig. 1a
, an increased number of mutant ARs were activated by estradiol or progesterone at doses higher than male physiological levels. In addition, the T877A AR responded to 10-4 M hydrocortisone, and the V715M, G750S, and H874Y ARs responded to 10-4 M flutamide (data not shown).
Protein Expression Levels of Loss-of-Function and Gain-of-Function Mutant ARs.
All mutant AR genes were expressed from the same promoter in our yeast system. To investigate whether reduction or loss of protein expression occurs in loss-of-function AR mutations and increased protein expression in gain-of-function AR mutations, Western blot analysis was performed on AR protein extracted from each of the transformed yeast strains that were adjusted to the same growth density. Fig. 2
shows that the expression levels of AR protein were similar in both loss-of-function and gain-of-function mutant AR transformants, indicating that the functional difference in response to ligands is not attributable to the changes in AR expression levels.
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and Fig. 1a
). However, the combination of both ligands weakly or moderately activated an additional 7 mutants: S647N, Q670R, R726L, V730M, G750S, D890N, and A896T (Fig. 1b)
. These results suggest that some mutant ARs respond synergistically to the two ligands at physiological concentrations.
Comparison between the Yeast Assay and a Mammalian Cell Assay.
We compared the yeast assay to a mammalian cell assay. Four representative ARs were selected: the wt AR, one loss-of-function mutant (S865P), and two promiscuous AR mutants (H874Y and T877A). For the mammalian cell assay, the coding regions of these ARs were cloned into pcDNA3.1+ plasmids (Invitrogen) and then used to transfect DU145 CaP cells, which lack expression of the AR. The results from luciferase activity assay in DU145 cells were compared with those from transactivational activity assay in yeast, in the presence of each of five ligands in different concentrations. As shown in Fig. 3
, the results are generally in agreement between the colorimetric yeast assay and the quantitative luciferase assay in DU145 cells using four of five ligands tested: DHT, estradiol, progesterone, and DHEA. Although both H874Y and T877A were activated in yeast in the presence of 10-5 M and/or 10-4 M flutamide, this agreement was not observed in DU145 cells because high doses of flutamide were found to be toxic to DU145 cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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also avoids much of the difficulties of interassay comparisons. In addition, the large size of the AR gene makes the detection of AR mutations from a large number of CaP samples difficult to perform. The approach developed here would permit mutations in the DBD/LBD region of the AR to be rapidly assembled into plasmids. This would allow high through-put screening for AR mutations from clinical CaP specimens. Because this is the first report to investigate a large number of mutant ARs, a major concern is whether the yeast system permits us to analyze AR function correctly. The results obtained provide a very positive answer: (a) we found that AR-transformed yeast cells require the presence of both an ARE-promoter and androgen for transactivation; (b) we compared the yeast assay and a DU145-based luciferase reporter assay, and the results from both systems correlated well; (c) our yeast findings are consistent with published information for the six previously tested mutants (12 , 18, 19, 20) ; and (d) the C784Y AR mutant found in the complete androgen insensitivity syndrome, which is inactive in mammalian cells, also failed to respond to DHT in yeast. Taken together, these results show that AR activity determined in yeast is closely comparable with that in mammalian cells.
Because of the expression of various coregulators of steroid receptors in different cell types under different culture conditions, it is sometimes difficult to compare the reported AR activities in the literature. In this regard, yeast provides a valuable system to define the basal transactivational activity of wt and mutant ARs. Because yeast does not have steroid receptors nor most of the coregulators found in mammalian cells, it provides a readout of the intrinsic activity of steroid receptors, without the complication of coregulators that are present to various extents in mammalian cells. The results of yeast assays, when compared with those obtained in mammalian cells, could help delineate the contributions of these coregulators to hormonal responsiveness. If there is a discrepancy for any mutant AR in the two systems, this affords a chance to introduce candidate coregulator(s) into yeast cells to see whether they play a crucial role in the final outcome of AR activity. In a previous study, introduction of the p160 TIF2 coactivator into yeast enhanced the ligand-dependent transactivational activity of the thyroid hormone receptor (31) . Because two members of the p160 coactivator family have been found to be overexpressed in CaP (32) , experiments are in progress to ascertain the effect of these coactivators on the activity of mutant ARs in yeast.
Our findings in these 44 AR mutants may provide valuable information for understanding the role of mutant ARs in the progression of CaP. For example, 17 mutant ARs responded only to DHT. It is predictable that CaP cells containing this type of mutant ARs may be responsive to antiandrogen treatment. It should, however, be noted that other factors, such as AR amplification (4) as well as coactivators (32) , may also contribute to resistance to this treatment. The significance of the seven loss-of-function mutant ARs in CaP is uncertain. It seems probable that CaP cells expressing a function-abolished AR might confer growth or survival advantages in tumor cells if they have already developed a growth loop that is independent of the androgen-AR pathway. This is consistent with the seemingly paradoxical finding that when the AR is reintroduced back into the PC3 cell line which lacks AR expression, androgen treatment suppresses cell growth and in some cases, induces apoptosis (33) . In addition, some variants of the LNCaP cell line that are no longer dependent on androgen for growth have been found to undergo growth arrest upon androgen treatment (34) . Because androgen functions as both a growth and a differentiation factor for prostate development, it is probable that androgen-induced genes are involved in both growth stimulation and growth arrest. The final outcome of AR action thus would depend on the particular cellular environment.
Twenty (45%) AR mutants were activated with 1 x 10-7 M DHEA, which is close to the normal concentration in prostate tissue (18 , 35 , 36) . Because yeast does not convert DHEA into estradiol (37) , the possibility of conversion into androgens probably does not exist in the yeast system. Thus, our results suggest that DHEA is able to exert a direct action on some mutant ARs. The significance of these gain-of-function mutants is that, after androgen ablation, DHEA may still be able to stimulate the growth of such CaP cells. This is supported by a study of mutant AR in CaP cells where antiandrogens failed to block the transactivational activity of mutant AR that is stimulated by androstenedione that has been converted from DHEA (38) .
Previous studies have demonstrated promiscuous activity in the L701H, V715M, H874Y, and T877A ARs (12 , 18, 19, 20, 21) , and to this list we have now added the K580R AR. All these promiscuous ARs were detected in patients with advanced CaPs. A recent study found that patients with CaPs expressing the T877A AR progressed to androgen independence after antiandrogen treatment (39) ; it is probable, therefore, that the other four promiscuous mutant ARs also contribute to disease progression.
Although only five mutant ARs responded to estradiol and progesterone, we observed that the number and the extent of response of activated mutant ARs was significantly increased when ligand concentrations were increased. Although the increased concentrations are unlikely to be of physiological significance to CaP growth, we hypothesized that a combination of these two steroids at physiological concentrations might activate other mutant ARs besides these five. Our results have confirmed this. This synergistic stimulation might contribute to resistance to antiandrogen treatment in CaP cells containing such AR mutations, because combined androgen blockade does not completely inhibit the production of steroid hormones (40)
In conclusion, CaP-derived mutant ARs can be classified as having loss of function, wt function, partial function, gain of function, or promiscuous activity. These mutations may contribute to the progression of CaP, resulting in resistance to treatment via different pathways. Recent studies suggest that mutant ARs enhance binding of adrenal androgens and increase the recruitment of coactivators after androgen deprivation (32) . To further investigate the function of these CaP-derived mutant ARs and to better understand their roles in CaP, a careful evaluation of the effect of coactivators on AR should be carried out. The present work provides a framework for future studies in this direction.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NCI Grant CA77662-NCI. 
2 To whom requests for reprints should be addressed, at Department of Urology, University of California, Davis, School of Medicine, 4860 Y Street, Suite 3500, Sacramento, CA 95817. Phone: (916) 734-2824; Fax: (916) 734-8094; E-mail: rwdeverewhite{at}ucdavis.edu
3 The abbreviations used are: CaP, prostate cancer; wt, wild-type; AR, androgen receptor; DHT, dihydrotestosterone; DHEA, dehydroepiandrosterone; DBD, DNA-binding domain; LBD, ligand-binding domain; R1881, methyltrienolone.
4 Internet address: http://www.mcgill.ca/androgendb (updated 3/28/2001).
Received 9/25/01. Accepted 12/27/01.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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J. L. Mohler, C. W. Gregory, O. H. Ford III, D. Kim, C. M. Weaver, P. Petrusz, E. M. Wilson, and F. S. French The Androgen Axis in Recurrent Prostate Cancer Clin. Cancer Res., January 15, 2004; 10(2): 440 - 448. [Abstract] [Full Text] [PDF]
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 W. G. Nelson, A. M. De Marzo, and W. B. Isaacs Prostate Cancer N. Engl. J. Med., July 24, 2003; 349(4): 366 - 381. [Full Text] [PDF]
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 N. Poujol, S. Lumbroso, B. Terouanne, J.-M. A. Lobaccaro, W. Bourguet, and C. Sultan Pathophysiology of Androgen Insensitivity Syndromes: Molecular and Structural Approaches of Natural and Engineered Androgen Receptor Mutations at Amino Acid 743 J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5793 - 5800. [Abstract] [Full Text] [PDF]
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 E. P. Gelmann Molecular Biology of the Androgen Receptor J. Clin. Oncol., July 1, 2002; 20(13): 3001 - 3015. [Abstract] [Full Text] [PDF]
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Y. B. Wetherill, C. E. Petre, K. R. Monk, A. Puga, and K. E. Knudsen The Xenoestrogen Bisphenol A Induces Inappropriate Androgen Receptor Activation and Mitogenesis in Prostatic Adenocarcinoma Cells Mol. Cancer Ther., May 1, 2002; 1(7): 515 - 524. [Abstract] [Full Text] [PDF]
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