Mutation of the androgen receptor causes oncogenictransformation of the prostateGuangzhou Han*†, Grant Buchanan‡, Michael Ittmann§, Jonathan M. Harris¶, Xiaoqing Yu*†, Francesco J. DeMayo*,Wayne Tilley‡, and Norman M. Greenberg*†�**

Departments of *Molecular and Cellular Biology, §Pathology, and �Urology, Baylor College of Medicine, Houston, TX 77030; ‡University of Adelaide andHanson Institute, Adelaide SA 5000, Australia; and ¶Queensland University of Technology, Brisbane QLD 4001, Australia

Communicated by James P. Allison, Memorial Sloan–Kettering Cancer Center, New York, NY, December 14, 2004 (received for review August 1, 2004)

Recent evidence demonstrates that the androgen receptor (AR) con-tinues to influence prostate cancer growth despite medical therapiesthat reduce circulating androgen ligands to castrate levels and�orblock ligand binding. Whereas the mutation, amplification, overex-pression of AR, or cross-talk between AR and other growth factorpathways may explain the failure of androgen ablation therapies insome cases, there is little evidence supporting a causal role betweenAR and prostate cancer. In this study, we functionally and directlyaddress the role whereby AR contributes to spontaneous cancerprogression by generating transgenic mice expressing (i) AR-WT torecapitulate increased AR levels and ligand sensitivity, (ii) AR-T857Ato represent a promiscuous AR ligand response, and (iii) AR-E231G tomodel altered AR function. Whereas transgenes encoding eitherAR-WT or AR-T857A did not cause prostate cancer when expressed atequivalent levels, expression of AR-E231G, which carries a mutationin the most highly conserved signature motif of the NH2-terminaldomain that also influences interactions with cellular coregulators,caused rapid development of prostatic intraepithelial neoplasia thatprogressed to invasive and metastatic disease in 100% of miceexamined. Taken together, our data now demonstrate the oncogenicpotential of steroid receptors and implicate altered AR function andreceptor coregulator interaction as critical determinants of prostatecancer initiation, invasion, and metastasis.

The work by Huggins and Hodges in the early 1940s demon-strated that prostate cancer, like the gland from where it arises,

is initially dependent on androgens for growth and survival (1).Since then, androgen ablation has been used for the treatment oflocally advanced and metastatic prostate cancer (2). Despite ini-tially favorable responses, androgens are not the prostate cancerAchilles heel, as most patients receiving this therapy ultimatelyprogress to develop hormone ablation therapy-resistant disease (2).Moreover, studies in a autochthonous mouse model have directlydemonstrated that androgen ablation can frequently select for moreaggressive and metastatic disease (3).

Because androgen receptor (AR) is expressed in almost allclinical and murine prostate cancer both before and after androgenablation therapy (4, 5), AR activity likely contributes to all stagesof prostate cancer progression. Global gene expression profilingdemonstrated AR as the only gene consistently up-regulated inemerging therapy-resistant human prostate cancer xenografts (6),and AR gene amplification after androgen ablation therapy hasbeen reported in almost one-third of prostate tumors (7). Further-more, many recurrent prostate cancers overexpress coactivatorsTIF2 and SRC1 that can increase AR activity at physiologicalconcentrations of adrenal androgen (8). As well, IGF-I, KGF, EGF,and Her-2�neu growth factor pathways activate AR in the absenceof androgen in prostate cancer (9–11). Collectively, inappropriateAR activation might provide cancer cells with a growth survivaladvantage after androgen ablation; despite progression in anandrogen-depleted environment, the cancers may still be ARdependent.

Somatic missense mutations in AR have been identified inprimary, recurrent, and metastatic forms of clinical prostate cancerand cell lines (12–14). The prototypical example is AR-T877A, a

missense mutation identified in the LNCaP cell line originallyisolated from the lymph node metastasis of a hormone refractoryprostate cancer patient. Located in the ligand-binding domain(LBD) T877A confers promiscuity to AR allowing activation byprogesterone, estrogen, adrenal androgens, and hydroxyflutamidein addition to androgens (15). Although �60 missense mutations inthe AR have been identified from clinical specimens (16), theirassociation with disease progression and emergence of a hormone-independent phenotype has only recently been appreciated (4).Much like somatic mutations in p53, the spontaneous mutations inAR cluster to discrete hot spots that collectively cover only 8% ofthe receptor coding sequence, implying they define importantstructural and functional regions (17). We have reported thatspontaneous somatic AR mutations identified in prostate tumorsderived from a genetically engineered mouse model also collocateto the same hot spots (17), supporting the conservation of ARstructure–function relationships in man and mouse.

An important finding of our previous analysis in a geneticallyengineered mouse model was that androgen ablation resulted in apredominance of AR gene mutations in the N-terminal domain(NTD) of the receptor compared with untreated animals (5). Inparticular, two mutations were of interest because they resulted inamino acid substitutions (A229T, E231G) in a short AR NTDsignature motif (ARNSM; Fig. 1A and ref. 18). The ARNSM isunique to AR and is the region of the AR-NTD most highlyconserved during evolution (18), suggesting it is critical for NTDstructure and recruitment of transcription factors (19, 20). BothAR-A229T and AR-E231G display increased ligand-independentbasal activity, whereas AR-E231G has increased responsiveness tocoactivators ARA160 and ARA70 (5). Recently, the AR-NTD wasshown to facilitate direct interaction with the Hsp70-interactingprotein (CHIP) that functions as a negative regulator of ARtranscriptional activity (21). Indeed, the AR-A229T andAR-E231G mutations reduced the interaction between CHIP andAR by 16% and 43%, respectively. These data confirm that the ARNTD contains an evolutionarily conserved motif that likely has acritical role in modulating AR action.

Owing to a paucity of appropriate experimental systems, it hasbeen difficult to prove a direct and�or causal relationship betweenAR expression, activation, or function, and the initiation or pro-gression of prostate disease beyond the fact that genes like prostate-specific antigen can be detected in tumors growing independent ofnormal physiologic levels of testicular androgens. Therefore, thecurrent study was undertaken to directly assess the consequences ofprostate-restricted expression of (i) wtAR to mimic increased

Freely available online through the PNAS open access option.

Abbreviations: AR, androgen receptor; LBD, ligand-binding domain; NTD, N-terminaldomain; HA, hemagglutinin; rPB, rat probasin; DP lobe, prostate dorsal lobe; LP lobe,prostate lateral lobe; VP lobe, prostate ventral lobe; AP lobe, prostate anterior lobe; rmsd,rms deviation; H&E, hematoxylin�eosin; ARNSM, AR NTD signature motif.

†Present address: Clinical Research Division, Fred Hutchinson Cancer Research Center,Seattle, WA 98109.

**To whom correspondence should be addressed. E-mail: ngreenberg@fhcrc.org.

© 2005 by The National Academy of Sciences of the USA

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receptor levels and androgen sensitivity, (ii) the LNCaP AR tomodel promiscuous receptor activation by nonclassical ligands, and(iii) AR-E231G to represent alterations in AR function.

MethodsConstruction of the Transgene. The cDNA fragments encoding WTmouse AR, E231G (human E251G), and T857A (human T877A)bearing N-terminal hemagglutinin (HA) epitope tags were excisedfrom pCDNA3�HA-AR-WT, pCDNA3�HA-mAR�E231A, andpCDNA3�HA-mAR�T857A (5) with KpnI and PstI, converted toblunt ends with T4 polymerase, and ligated into a Pb-kbpa vectorwith a �426 to �28 fragment of rat probasin (rPB) digested withEcoRI and blunt ended with T4 polymerase to yield rPB-AR-WT,rPB-AR-E231G, and rPB-AR-T857A, respectively. For microin-jection, plasmids were digested with NotI and XhoI and subjectedto agarose gel electrophoresis, and transgenes were recovered byQIAEX II (Qiagen, Valencia, CA). Linear fragments were intro-duced by pronuclear injection into FVB embryos (22).

Screening of Transgenic Mice. Mouse-tail DNA isolation and PCRscreening were as described (23). The P1 primers used to screen for

positive transgenic mice were P1F (5�-CTTGTCAGTGAGGTC-CAGATACCTACAG-3�) and P1R (5�-ATCCGGCACATCATA-AGGGTATCCCATG-3�). The point mutation E231G was con-firmed by sequencing fragments amplified by P2 primers P2F(5�-ACCCTTATGATGTGCCGGATTATGCC-3�) and P2R (5�-GGCGTAACCTCCCTTGAAAGAGGA-3�). The point muta-tion T857A was confirmed by sequencing fragments amplified byP3 primers P3F (5�-TGCTGCTCTTCAGCATTATTCCAGT-3�)and P3R (5�-GGTTTTGGGTATTAGGGTTTCCAAA-3�).

RT-PCR. Tissue RNA was extracted with the RNeasy kit (Qiagen)and digested with DNase I (Invitrogen). RT-PCR used 1 �g of totalRNA as described (5). The P2 primers were used for transgene-specific transcripts. Primers specific for ribosome protein L19 wereused for controls (24).

Ribonuclease Protection Assay. The antisense probe was a 215-bpfragment containing the HA sequence and 170-bp AR N-terminalsequence amplified with 5�-CGCGGATCCCGGCTACCACCAT-GGGATACCC-3� (forward) and 5�-CGGGATCCTGCCTCT-

Fig. 1. Generation and identification oftransgenic mice. (A) The AR-N-terminal sig-nature (ANTS) sequence was identifiedfrom multiple AR sequence alignments byusing a CLUSTALW algorithm (42) with hu-man AR as the profile sequence. Align-ment revealed a conserved region ofamino acids corresponding to mouse resi-dues 229–242. Blue, identical; yellow, con-served; white, nonhomologous. (B) TheAR-E231G transgene. The E231G mutation(GAG � GGG) was introduced into a con-struct carrying an HA epitope-tagged WTmouse AR cDNA (AR-WT) between the rPBwith a rabbit �-globin fragment with asmall intron and a bovine growth hormonepoly(A) signal sequence. Arrows indicatethe primer pairs. The DNA sequences wereconfirmed in germ-line DNA. (C) The AR-T857A transgene. The T857A mutation(ACT � GCT) was created as described in B.(D) Expression analysis of AR-E231G andAR-WT transgenic mice. Tissue RNA at 12weeks of age was analyzed by RT-PCR withP2 primers. Primers for L-19 were includedas internal controls. Nontransgenic litter-mates were the controls (NT). Transgeneplasmid DNA was the positive control (�).Transgenes were expressed predomi-nantly in ventral prostate in independentlines. Expression of AR-E231G was primar-ily detected in the ventral prostate, butsome expression was detected in the dor-solateral prostate. TE, testis; SV, seminalvesicle; SP, spleen; LU, lung; LV, liver; KD,kidney; BL, bladder; H, heart; MU, muscle;TH, thymus; BR, brain; M, DNA molecularweight markers. (E) RNase protection anal-ysis. Probe was hybridized with RNA fromVP lobes of AR-T857A, AR-E231G, AR-WT,and NT mice. Protected probe was sepa-rated by acrylamide gel. (F) Expression ofAR-WT, AR-E231G, and AR-T857A protein.Extracts prepared from the DLP, VP, and APlobes of transgenic and NT littermateswere fractionated by SDS�PAGE andprobed with anti-HA (Upper) or anti-GAPDH (Lower) antibodies. Transgeneprotein was detected only in the VP lobesof transgenic mice.

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GCTGTAAACAGGCG-3� (reverse) primers. The fragments weredigested with KpnI and BamHI and cloned into KpnI- and BamHI-digested pDP18-T7�T3 (Ambion). The [�-32P]UTP-labeled probewas synthesized by in vitro transcription (BD Biosciences, FranklinLakes, NJ) with T7 polymerase. RPA was performed with 5 �g oftotal ventral prostate RNA. Protected probes were separated by10% acrylamide gel and identified by exposure to XAR5 film(Kodak).

Histology. The genitourinary tract consisting of the bladder, ure-thra, seminal vesicles, and prostate was harvested as a complex. Theprostate dorsal lobe (DP lobe), prostate lateral lobe (LP lobe),prostate ventral lobe (VP lobe), and prostate anterior lobe (APlobe) were dissected under a microscope. Tissues collected atnecropsy were fixed in 4% paraformaldehyde in PBS for 4 h andtransferred to 70% (vol�vol) ethanol overnight. Specimens wereprocessed through graded alcohols, embedded in paraffin, sec-tioned at 5 �m, and mounted on ProbeON-Plus slides (Fisher).Sections for histological analysis were stained with hematoxylin�eosin (H&E).

Immunohistochemistry. Paraffin sections were baked overnight at55°C and dewaxed. Antigens were retrieved by boiling in 10 mMsodium citrate (pH 6.0) for 15 min and cooled for 1 h at 25°C.Endogenous peroxidase activity was quenched by a 10-min immer-sion in a solution of 3% H2O2 in methanol. Blocking was performedwith the Dako LSAB system for 5 min at 25°C. Primary antibodiesand working conditions were as follows: HA antibody (Covance,Richmond, CA), 1:500, overnight at 4°C; Ki-67 antibody (VectorLaboratories), 1:1,000, 1 h at 25°C; and AR antibody (UpstateBiotechnology, Lake Placid, NY), 1:60, overnight at 4°C. Productswere visualized with Dako LSAB kit and the chromogen 3�3�-diaminobenzidine tetrahydrochloride (BioGenex Laboratories,San Ramon, CA). Primary antibodies were replaced with normalrabbit serum for negative controls. Sections were counterstainedwith methyl green.

Immunoblotting. Mouse tissues were homogenized in ice-coldRIPA buffer (40 mM Tris-HCl, pH 7.0�1 nM EDTA�4% glycer-ol�10 mM DTT�0.2% SDS�2 mM PMSF�20 �g/ml aprotinin�5�g/ml leupeptin�5 �g/ml pepstatin�1 mM NaF), by using a 9-mmPolytron homogenizer (Brinkmann). Protein concentrations weredetermined by the Bradford assay. Briefly, 50 �g of protein wasresolved through a 7.5% SDS-polyacrylamide gel and transferred tonitrocellulose. Membranes were probed with antibodies specific forHA at 1:500 (H-11, Covance) and subsequently with a goatanti-mouse IgG horseradish peroxidase conjugate. AR protein wasdetected by Supersignal chemiluminescent substrate (AmershamPharmacia). Membranes were stripped and probed with rabbitpolyclonal antibody for AR at 1:500 (N-20, Santa Cruz Biotech-nology) and anti-GAPDH antibody at 1:5,000 (Ambion). Immu-noblots were developed as before and quantified with the AlphaIm-ager system (Alpha Innotech, San Leandro, CA).

Structural Analysis of AR Mutations. Molecular model 3-dimensionalstructures representing WT or mutant AR-NTD peptide sequenceswere constructed by using CHEMSITE PRO (ChemSW, Fairfield,CA). Nonpolar hydrogen atoms were added, and the structure wassolvated in a box of single-point charge water molecules andsubjected to energy minimization (20 steps of steepest descent witha cutoff for nonbonded interactions of 10 Å). For each peptide, 10independent molecular dynamic simulations were performed atconstant temperature (300 K) over 100 ps. The rms deviation(rmsd) values for all atoms were calculated by using the relevantminimized starting structures as templates, and solutions werecolored according to displacement from the starting minimizedstructure. Existing structures for the human AR (Protein Data

Bank ID code 1I37) and its LNCaP variant (Protein Data Bank IDcode 1I38) were analyzed by using SPDBV (Version 3.7).

ResultsGeneration of Transgenic Mice Harboring AR Variants. To investigatethe role of the AR NTD and test the hypothesis that changes in ARsignaling could cause spontaneous prostate cancer in vivo, wegenerated independent lines of transgenic mice with prostate-specific constructs encoding WT-AR, AR-E231G, and AR-T857A(Fig. 1 B and C). We identified multiple independent founderscarrying AR-WT (three lines), AR-E231G (seven lines), andAR-T857A (five lines) and confirmed the presence of the E231G(Fig. 1B) and T857A (Fig. 1C) mutations in the germ line ofrespective founder mice.

Tissue-specific expression of all transgenes was confirmed inindependent lines by RT-PCR. Consistent with previous data, theminimal probasin promoter reproducibly directed transgene ex-pression to the epithelial compartment of the ventral prostate (Fig.1D). We were unable to detect transgene expression in other organs(Fig. 1D). As shown in Fig. 1E, steady-state transcript analysisdemonstrated all transgenes were expressed at similar levels. Anal-ysis of tissue extracts procured from independent lines of transgenicmice confirmed VP lobe-restricted AR protein expression (Fig. 1Fand data not shown). We were unable to detect expression inprostate tissues of nontransgenic littermates under identicalconditions.

Consequence of AR Variant Expression in Transgenic Mice. Theconsequence of AR-WT, AR-E231G, and AR-T857A expressionwas first examined at 12 weeks of age. As shown in Fig. 2, the DP,LP, VP, and AP lobes appeared normal in the nontransgeniclittermates (Fig. 2 A–D) and the AR-WT mice (Fig. 2 E–H).Whereas the DP, LP, and AP lobes of AR-E231G mice wereunremarkable (Fig. 2 I, J, and L), we readily observed epithelialhyperplasia and dysplasia in the VP lobes of all AR-E231G mice(Fig. 2K). The lesions in AR-E231G mice typically presented asdisorganized layers of epithelial cells in contrast to the single regularlayers observed in the nontransgenic and AR-WT mice. In contrast,we were unable to detect a significant phenotype in the AR-T857Amice (Fig. 2 M–P).

The histopathologic features observed in AR-E231G mice wereconsistent with the mouse prostatic intraepithelial neoplasia phe-notype and resembled the prostatic intraepithelial neoplasia lesionsin other transgenic models (25). The frequent papillary lesions withtufting and intraepithelial lumen formation, cellular atypia withprominent enlarged and hyperchromatic nuclei of variable sizes andshapes were reproducibly observed to be perfectly concomitantwith transgene expression (Fig. 2 Q–T). Whereas expression ofAR-WT (Fig. 2V) and AR-T857A (Fig. 2W) were readily detectedin the VP lobe, these glands appeared normal and unremarkable upto 50 weeks of age. As shown in Fig. 2X, the level of proliferationwas very low in the epithelial compartment of the nontransgenicand AR-WT mice at 12 weeks of age, consistent with the prolif-eration normally observed in mouse prostate epithelium (26). Incontrast, the Ki-67 index in the VP lobe of AR-E231G wasstatistically greater (P � 0.05) than in nontransgenic littermates.We were unable to detect a significant difference in proliferationindex in the DLP between any of the genotypes. These observationsconfirm that AR-E231G specifically promoted epithelial prolifer-ation leading to the rapid development of prostatic intraepithelialneoplasia in the ventral prostate.

The consequence of expression of AR-WT, AR-E231G, andAR-T857A was then examined at �50 weeks of age. Although weobserved no significant difference in pathologic grade betweenAR-WT, AR-T857A mice, and control littermates at 1 year, all(5�5; 100%) of the AR-E231G mice demonstrated primary pros-tate tumors with associated lymphocytic invasion (Fig. 3). Theepithelial origin of these tumors was confirmed by analysis for

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E-cadherin (Fig. 3B), and expression of the transgene was concom-itant with the adenocarcinoma (Fig. 3 C and D). Moreover,metastatic deposits were detected in the lungs of all of the mice (Fig.3 E and F). These deposits were found to express the transgene (Fig.3G) but not NKX2.1, a specific marker of lung epithelial differen-tiation (Fig. 3H). Inflammation associated with the adenocarci-noma in AR-E231G mice was not related to the NH2-HA epitopebecause AR-WT and AR-T857A also carried this sequence. In all,these observations support a direct causal relationship betweenAR-E321G expression and malignant prostate cancer. To ourknowledge, this is the first report of a somatic mutation able toconvert the AR, or any other steroid receptor, into a potentoncogene sufficient to cause invasive and metastatic prostatecancer in vivo. These data also functionally demonstrate that theARNSM is a critical region of AR and provide an alternative to thehypothesis that increased AR sensitivity alone initiates cancer.

Modeling the WT and Variant AR NTD. Because the AR NTD has notbeen crystallized or induced to form an ordered structure suitablefor structural determination via NMR, dynamic simulation wasused to further study the E231G mutation. Modeling the A229Tand E231G substitutions was performed on the local helical struc-ture of a peptide encompassing the predicted NTD signaturesequence (228NAKELCKAVSV238) and assumed that the peptide

was exposed to solvent and adopted an alpha-helical structure aspreviously predicted (19). Comparison of 10 independent molec-ular dynamic simulations for each AR peptide solvated in explicitsingle-point charge water demonstrated a significantly (P � 0.05)higher positional fluctuation (rmsd) for the WT than either Thr-229or Gly-231 mutant peptides (Fig. 4 A–C). The position of these twosubstitutions at the N terminus of the helix may reinforce thehelix-generated dipole with subsequent stabilization as has previ-ously been demonstrated for substitution of glutamate from glu-tamine at the N terminus in a model helix (27). Therefore, bothThr-229 and Gly-231 mutations may stabilize the secondary struc-ture in the vicinity of the ARNSM that could enhance accessoryprotein recruitment (19). This is supported by in vitro analysisdemonstrating enhanced ligand-specific responses of AR-E321G toARA70 and ARA160 (5). It will be important to fully elucidate howAR-E231G induces deregulated growth given the evolutionarypressure to maintain the encompassing ARNSM sequence.

The LBD of mouse AR-WT and AR-T857A have identicalamino acid sequences to the human WT and LNCaP variants,respectively, and can be represented by the solved crystal structuresof these receptors (28, 29). Detailed analysis of these structuresdemonstrated that the T857A substitution had very little effect onoverall LBD structure with an rmsd of only 0.41 Å for all atomswithin 6 Å of bound ligand (excluding Thr�Ala-877) and a peptide

Fig. 2. Pathobiology of transgenic mice. Paraf-fin sections (5 �m) were prepared from NT (A–D),AR-WT (E–H), AR-E231G (I–L), and AR-T857A(M–P) mice at 12 weeks of age. Representativesections stained with H&E are shown for DP lobe(A, E, I, and M), LP lobe (B, F, J, and N), VP lobe (C,G, K, and O), and AP lobe (D, H, L, and P). Histo-logical features consistent with prostatic intra-epithelial neoplasia were observed in the VPlobes of AR-E231G mice (K, Q–T). The anti-HAantibody was used to probe serial sections of VP(T) and NT (U), AR-WT (V) and AR-T857A mice(W). Brown nuclei indicate immunoreactivity.Sections were counterstained with methylgreen. All �20 except S and T, which were �40original magnification. (X) Increased prolifera-tion in the VP lobes of AR-E231G transgenic mice.An anti-Ki67 antibody was used on sections of VPlobes from NT, AR-WT, and AR-E231G mice at 12weeks of age. Quantitation of Ki-67 positive cellswas from at least three mice for each group. *,Significant difference between AR-E231G andNT and AR-WT, P � 0.05 by Student’s t test.

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backbone deviation of only 0.81 Å over the whole molecule (datanot shown). However, the LNCaP mutation causes a significantrearrangement of the hydrogen bonding network between receptorand bound ligand (Fig. 4D), resulting in a lowered constraint on theligand D ring and lowering selectivity of the AR-LBD for ligandswith differing D ring substituents at the 17� position. In addition,the LNCaP mutation results in an �5% increase in volume of theligand-binding pocket volume, which may explain the ability to bindprogesterone.

DiscussionAlthough mechanisms have been proposed to explain how AR mayfacilitate prostate cancer (for review, see refs. 30 and 31), our newdata support the hypothesis that AR is a protooncogene and thatabrogation of the classical AR signal pathway by mutation or

hormonal perturbation can facilitate the transformed state. Indeed,these data are consistent with a report that immunization againstluteinizing hormone-releasing hormone proteins was unable todecrease prostate cancer in a transgenic model (32) as well as theProstate Cancer Prevention Trial report showing that androgensignal titration by finasteride-mediated inhibition of 5�-reductasetype II could select for more advanced disease (33). Although theAR status in the patients receiving finasteride therapy was notdetermined, our data suggest the possibility that mutations will befound in the AR gene or other components of the androgensignaling axis. Observations that tumors in patients receiving in-termittent androgen blockade and cyclic androgen ablation andtestosterone replacement therapy progress slower than for contin-uous androgen ablation (34–36) further support the hypothesis thatmaintaining an intact and functional AR signaling axis can bebeneficial.

The paradoxical ability of the AR to both drive prostate growthand limit prostate cancer progression may represent compartment-specific roles within the prostate microenvironment (37). We haveproposed that the consequence of androgen action in the prostatestroma is to elaborate polypeptide growth factors critical for thegrowth and survival of the epithelial compartment and that abro-gation or supplementation of such signals in vivo can lead toprofound phenotypes (38). Conversely, the consequence of andro-gen action in the epithelial compartment is to suppress proliferationand maintain terminal differentiation and cellular function. By ourscheme, systemic androgen ablation not only abrogates the elabo-ration of stromal factors that support epithelial growth, but alsoremoves a differentiation signal that constitutes selective pressurefor the proliferation of epithelial cells or progenitors that had,through somatic mutation or other mechanisms, acquired theability to grow in a stromal independent fashion. This model notonly explains the initially dramatic response observed in prostatecancer patients to hormone withdrawal, it also provides a paradigm

Fig. 3. Pathobiology of prostate cancer in AR-E231G mice. (A–D) Adjacentsections (5 �m) were prepared from AR-E231G mice at 50 weeks of age. (A)Representative section of VP lobes stained with H&E shows adenocarcinomawith lymphocytic infiltration. (B) Analysis with anti-E-cadherin antibodiesdemonstrates E231G tumors to be of epithelial origin. (C) Analysis withanti-AR-specific antibodies demonstrates collocation of AR expression in ad-enocarcinoma. (D) Analysis with anti-HA antibody demonstrates expression oftransgene collocates with tumor and AR expression (compare with C). (E–H)Sections of lungs of AR-E231G mice at 50 weeks stained with H&E demon-strating minimal residual glandular structure of a metastatic deposit (E) and apoorly differentiated lung deposit from an independent AR-E231G mouse (F).(G) Stained section adjacent to that in E with anti-HA specific antibodiesdemonstrates expression of the AR-E231G transgene in the metastatic lesion.(H) Stained section adjacent to that in G with anti-NKX2.1-specific antibodydemonstrates the metastatic lesion is not of lung origin.

Fig. 4. Structural analysis of AR mutations. (A–C) Three-dimensional molec-ular model structures representing WT or mutant AR-NTD peptide sequencesconstructed by using CHEMSITE PRO. Nonpolar hydrogen atoms were added, andthe structure was solvated in a box of single-point charge water molecules andsubjected to energy minimization (20 steps of steepest descent with a cutofffor nonbonded interactions of 10 Å). Ten independent molecular dynamicsimulations, performed at constant temperature (300 K) over a period of 100ps, are shown for each peptide. rmsd values for all atoms were calculated byusing the relevant minimized starting structures as templates, and solutionsare colored according to displacement from the starting minimized structure(blue, minimal displacement; red, maximum displacement). For each mutant,the WT peptide is shown in pink. Average rmsd (�SD) of the 10 solutions foreach peptide is indicated. Solvent is not shown. (D) Existing structures for thehuman AR (Protein Data Bank ID code 1I37) and its LNCaP variant (Protein DataBank ID code 1I38) were analyzed by using SPDBV (Version 3.7). The environ-ment within 6 Å of bound ligand is shown in magenta or aqua, with boundligand shown in blue and hydrogen bonds represented by dashed green lines.

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for the subsequent emergence of hormone therapy-resistant diseasefrom either a non-AR stem population or one already expressingthe AR. Given that hormone ablation selects for AR mutations andthese mutations can cause the development of aggressive andmetastatic disease, it could be argued that maintaining an intactandrogen signaling axis would be of benefit to most patients.Collectively, these studies suggest that prevention trials based solelyon androgen titration paradigms should proceed cautiously.

Because AR protein is overexpressed in advanced prostatecancer, increased AR signaling may contribute to tumor progres-sion and emergence of the hormone-independent phenotype (39).However, studies on the LNCaP cell line indicate that althoughlow-level activation of (albeit mutated) AR can facilitate cellgrowth, hyperactivation can lead to cell death (40), consistent withour hypothesis that AR has properties consistent with a tumorsuppressor and that abrogation of AR action is associated withproliferative disease. This is reminiscent of the case of p53. Indeed,we show that enforced expression of AR-WT did not causesignificant prostate disease in vivo. Our current data show thatincreased expression of WT AR or increased AR sensitivitythrough mutation (AR-T857A) in the presence of endogenous ARcannot initiate the prostate cancer pathway.

The findings of this study also address the functional role ofmutations such as AR-T857A. It is known that the LBD of mouseAR-WT and AR-T857A have identical amino acid sequences to thehuman WT and LNCaP variants, respectively, and can therefore berepresented by the solved crystal structures of these receptors (28,29). As shown in Fig. 4, the T857A substitution causes a significantrearrangement of the hydrogen bonding network between receptorand bound ligand, resulting in a lowered constraint on the ligand Dring. This consequently lowers selectivity of the AR-LBD forligands with different D ring substitutions at the 17� position, whichtogether with a �5% increase in volume of the ligand-bindingpocket volume, explains the capacity of the mutant receptor to bindligands such as progesterone. However, analysis of these structuresindicates that T857A has very little effect on overall LBD structurewith an rmsd of only 0.41 Å for all atoms within 6 Å of bound ligand(excluding Thr�Ala-857) and a peptide backbone deviation of only0.81 Å over the whole molecule (data not shown). We thereforepropose that AR-T857A did not promote prostate cancer in mice

because this substitution does not alter receptor function per se, butallows it to be activated indiscriminately. In contrast, we havepreviously demonstrated that E231G provides AR with a true gainof function, namely an increased baseline activity in the absence ofhormonal signals and an enhanced functional response to thecoregulators (5). That the AR-NTD adopts a loose flexible ar-rangement with limited structural content has so far precludedcrystal analysis of this region of the receptor. However, chemicalcleavage has defined a few small regions of structure within theAR-NTD, including an alpha helix that spans the highly conservedARNSM and the E231 residue, as critical determinants of ARtranscriptional activity (19). Changes in structural order across theARNSM, as suggested by modeling of the E231G substitution, arepredicted to result in an altered capacity of the AR-NTD toassemble components of the transcription complex (19), includingthe CHIP E3 ligase that was recently shown to interact directly withthis conserved region (21). That enforced expression of AR-E231Gbut not AR-T857A in the mouse prostate results in tumor forma-tion underscores the more dramatic consequence of the NTDsubstitution with respect to receptor function. Our results supportother evidence that programs regulated by transcription fac-tors, including steroid receptors, are critically dependent on thenature of multiprotein complexes recruited during all phases ofactivation (41).

In summary, we have presented an example that a variantnuclear steroid receptor is sufficient for the development ofcancer, confirmed a critical domain in the AR-NTD that may actthrough coregulators to modulate AR function, and distin-guished true gain of function AR variants from those that act tofacilitate promiscuous ligand binding. Importantly, the AR-E231G transgenic mouse represents a new model of prostatecancer that is independent of potent oncogenes or supraphysi-ological levels of steroids. This model will facilitate preclinicaltesting of emerging strategies that aim to inhibit the growth ofprostate tumors by directly targeting the AR.

We thank Deborah Ng for administrative assistance and RebeccaMontgomery and Caroline Castile for technical support. We also thankDr. Jeffrey M. Rosen for helpful discussions. This work was supportedby CaP CURE (to G.H. and N.M.G.) and National Cancer InstituteGrants CA73747 and CA84296 (to N.M.G.).

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