From the Department of Molecular and Cellular Biology
and the ** Scott Department of Urology, Baylor College of Medicine,
Houston, Texas 77030, the ¶ Flinders Cancer
Centre, Flinders University and Flinders Medical Centre, Adelaide South
Australia 5042, and the
Institute for
Molecular Biosciences, University of Queensland, Brisbane,
Queensland 4072, Australia
Received for publication, September 7, 2000, and in revised form, November 2, 2000
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ABSTRACT |
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We have used the autochthonous
transgenic adenocarcinoma of mouse
prostate (TRAMP) model to investigate the relationship
between somatic mutation in the androgen receptor (AR) and the
emergence of androgen-independent prostate cancer. Here we report the
identification, isolation, and characterization of distinct classes of
AR variants from spontaneous prostate tumors in the TRAMP model. Using
cDNA cloning, single stranded conformation polymorphism and
sequencing strategies, 15 unique somatic mutations in the AR were
identified in prostate tumors obtained from eight TRAMP mice between 24 and 29 weeks of age. At least one mutation was isolated from each mouse. All mutations were single base substitutions, 10 were missense and 5 were silent. Nine mutations in the AR were identified in tumors
of four mice that were castrated at 12 weeks of age. Interestingly, the
majority of mutations (seven out of nine, 78%) identified in the
androgen-independent tumors colocalized in the AR transactivation domain. The remaining mutations colocalized in the AR ligand binding domain. In general, the AR variants demonstrated promoter-, cell-, and
cofactor-specific activities in response to various hormones. All AR
variants isolated in this study maintained strong sensitivity for
androgens, and four AR variants isolated from castrated mice demonstrated increased activities in the absence of ligand. The K638M
and F677S variants demonstrated increased activities in response to
androgen, and K638M also demonstrated increased response to estradiol.
In the presence of AR coactivator ARA70 the E231G variant demonstrated
increased activity in response to both androgen and estradiol. However,
in the presence of AR coactivator ARA160 the E231G variant was
selectively responsive to androgen. Collectively these analyses not
only indicate that somatic mutations in the AR gene occur spontaneously
in TRAMP tumors but also how changes in the hormonal environment may
drive the selection of spontaneous somatic mutations that provide a
growth advantage.
Androgens are essential for the development and maintenance of the
prostate and hormonal therapy. Androgen deprivation or blockade of
androgens at the level of the androgen receptor
(AR)1 remains the treatment
choice for advanced prostate cancer (1). Although this therapy usually
results in a favorable clinical response, a dramatic drop in serum
prostate specific antigen level and tumor regression, most cases
will eventually relapse with clinically defined androgen-independent
disease (2). Understanding the molecular mechanisms governing
progression of prostate cancer to androgen independence is critical to
the development of new treatment modalities.
Androgens signal via the intracellular AR, a member of the superfamily
of nuclear hormone receptors (3). Androgen binding to the AR transforms
the receptor to an active conformation and initiates translocation to
the nucleus, followed by binding to specific response elements in the
promoter regions of target genes to modulate gene expression positively
or negatively. Although it was initially thought that loss of AR
expression was a mechanism of therapy failure, recent studies have
demonstrated that despite hormone ablation therapy, the AR is expressed
at substantive levels in a majority of hormone-refractory prostate
cancers (4, 5). Subsequently, several hypotheses have been proposed to
explain how changes in the androgen signaling axis may mediate the
growth of hormone-refractory disease, including AR gene amplification (6, 7), ligand-independent activation of AR via cross-talk with growth
factor receptor pathways (8, 9), and AR gene mutations (10).
Over the past several years a number of distinct AR gene mutations have
been identified in clinical prostate cancer specimens (11-15).
Possibly the most characterized of the AR variants are those identified
in the prostate cancer cell lines LNCaP (16) and MDA PCa 2b (17, 18).
Remarkably, both the T877A variant in LNCaP cells and the L701H/T877A
double variant in MDA PCa 2b cells are promiscuous receptors in that
they are activated by steroid hormones other than androgens. Hence,
these mutations have the potential to confer a selective growth
advantage to cancer cells after hormone ablation therapy. This strongly
supports the hypothesis that mutations in the AR gene which deregulate
the androgen signaling axis can directly contribute to the development of hormone-refractory disease.
Although AR gene mutations have been identified in human prostate
cancer, it has been difficult to perform a comprehensive analysis of
their incidence and nature because of the paucity of clinical samples
representing the earliest form of the disease and the genetic and
pathologic heterogeneity of the disease. We have, therefore, used the
autochthonous transgenic adenocarcinoma of
mouse prostate (TRAMP) model (19-21) to support
further investigation of the relationship between mutations in AR and
prostate cancer. The TRAMP model (22) was generated previously using
minimal probasin (PB) Transgenic Animals--
TRAMP mice, heterozygous for the PB-T
antigen transgene, were maintained in a pure C57BL/6 background. Female
TRAMP mice were bred to nontransgenic male FVB mice (Harlan
Sprague-Dawley) to obtain transgenic and nontransgenic (C57BL/6 × FVB) F1 males for this study. Isolations of DNA from mouse tails and
polymerase chain reaction (PCR)-based screening assays to identify
transgenic mice were performed as described previously (21). All TRAMP mice were randomly assigned to two cohorts. One cohort was anesthetized and castrated through a scrotal approach at 12 weeks of age. All mice
were sacrificed between 24 and 29 weeks of age.
Single Stranded Conformation Polymorphism (SSCP)
Analysis--
Total RNA was isolated from tumor tissues by the cesium
chloride method as described previously (23). Reverse transcription-PCR was performed with modifications of the procedure as previously described (24). Briefly, 1 µg of RNA was reverse transcribed for
1 h at 37C using 300 ng of oligo(dT)12-18 primers
(Amersham Pharmacia Biotech) and 200 units of Moloney murine leukemia
virus reverse transcriptase (Life Technologies, Inc.) in a 20-µl
reaction containing 1 × first strand buffer (Life Technologies,
Inc.), a 500 µM concentration of each of the four
deoxyribonucleotide triphosphates (Life Technologies, Inc.), 1 mM dithiothreitol (Life Technologies, Inc.), and 40 units
of RNase inhibitor (Roche Molecular Biochemicals, Indianapolis, IN).
The reverse transcription reaction was terminated by heating for 10 min
at 95 °C, and 2 µl of the reverse transcription reaction mixture
was added to a 50-µl PCR mixture containing a 200 µM
concentration of each of the four deoxyribonucleotide triphosphates, 10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4)2SO4, 2 mM
MgSO4, 0.1% Triton-100, 200 µM appropriate sense and antisense primer sets, and 0.5 unit of Vent DNA polymerase (New England Biolabs, Beverly, MA). Initial amplification of the AR
used three primer sets (mAR-1F and mAR-3R, mAR-3F and mAR-6R, and
mAR-6F and mAR-10R) (see Fig. 1A). 30 cycles of PCR were
performed using the following conditions: 94 °C for 1 min, 65 °C
for 2 min, 72 °C for 3 min. The resulting PCR products were cloned
into EcoRV-digested pBluescript SK Sequencing Analysis--
Recombinant plasmids harboring putative
mutations as identified by SSCP analysis were sequenced. Identification
of AR mutations was performed by comparing the sequence of subcloned AR
cDNA with that of cDNA encoding C57BL/6 mouse AR (GenBank
X53779) (26) using the GCG Sequence Analysis Software Package provided
through the Baylor College of Medicine Biology Computational Resource (Houston, TX).
Expression Vectors--
The plasmid pcDNA3/HA-mAR was
constructed to express full-length wild type mouse AR with an
amino-terminal hemagglutinin antigen (HA; Santa Cruz Biotechnology,
Santa Cruz, CA) tag sequence and a consensus Kozak translation
initiation sequence (see Fig. 2B). This was accomplished by
inserting the mouse full-length AR cDNA and HA tag into CMV
expression vector pcDNA3.1 (Invitrogen Corporation, Carlsbad, CA).
To engineer the HA epitope to the amino terminus of the AR protein, the
full-length mouse AR cDNA (kindly provided by Dr. Don Tindall, Mayo
Clinic, Rochester, MN) was subcloned into the BamHI site of
pcDNA3.1. The HA tag sequence was added to the amino
terminus of the full-length AR cDNA by PCR using the primer
5'-CGGGTACCACCATGGGATACCCTTATGATGTGCCGGATTATGCCGAGGTGCAGTTAGGGCTGGA-3', which contains the sequence of the HA tag, a consensus Kozak
translation initiation sequence and a KpnI site, and the
primer mAR-6R (see Fig. 1A). The PCR product was then
digested with KpnI and HindIII and used to
replace the corresponding fragment of the wild type AR cDNA. All AR
variant expression constructs were reconstructed by the in
vitro site-directed mutagenesis system (5 Prime - 3 Prime, Inc.)
using the pcDNA3/HA-mAR plasmid and single oligonucleotide primers
containing the appropriate base substitutions:
E206K variant: 5'-GCAAGAGCCAGGAAGGCCACGGGGG3-';
A229T variant: 5'-TATCTGACAGTACCAAGGAGTTGTG-3';
E231G variant: 5'-ACAGTGCCAAGGGGTTGTGTAAAGC-3';
N384S variant: 5'-TCAAGCTGGAGAGCCCATTGGACTA-3';
V487A variant: 5'-ATCCTGGTGGAGCTGTGAACAGAGT-3';
K638M variant: 5'-ACCCATCCCAGATGATGACTGTATC-3';
F653I variant: 5'-TGTCAGCCTATCATTCTTAACGTCC-3';
F677S variant: 5'-AACCAGATTCCTCTGCTGCCTTGTT-3';
T857A variant: 5'-CTGCATCAGTTCGCTTTTGACCTGC-3'.
DNA sequence analysis was performed to verify each construct. The ARA70
and ARA160 expression plasmids PSG5-ARA70N and pCMV-ARA160FL were
kindly provided by C. Chang (University of Rochester Medical Center,
Rochester, NY) (27). The two reporter constructs PB-luciferase (PB-LUC)
and ARR3-tk-luciferase (ARR3-LUC) were kindly provided by R. J. Matusik (Vanderbilt University, Nashville, TN) (28).
Cell Culture and Reporter Gene Transactivation
Assay--
PC3 cells were cultured in Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) and 25 units/ml penicillin-streptomycin (Life Technologies, Inc.). The TRAMP cell lines C1A, C1D, and C2G were
isolated from the TRAMP C1 and C2 cell lines (29) by limited dilution
cloning and were propagated in a medium containing Dulbecco's modified
Eagle's medium supplemented with 5% Nu-serum IV (Collaborative
Biomedical Products), 5% fetal bovine serum, and 25 units/ml
penicillin-streptomycin.
1 day prior to transfection, cells were seeded in six-well culture
plates at a density of 1 × 105 cells/well. Cells were
transfected with 2 µg of appropriate plasmid DNA using Superfect
reagent (Qiagen, Inc, Valencia, CA) according to the manufacturer's
instructions. After 24 h, the cells were washed and fed with
phenol-free Dulbecco's modified Eagle's medium containing 10%
dextran-coated charcoal-stripped fetal bovine serum (Hyclone)
containing hormones as required. 24 h later cells were washed
twice with phosphate-buffered saline and lysed in 150 µl of lysis
buffer containing 0.2 M Tris-HCl (pH 8.0), 0.1% Triton X-100. The luciferase and Western Immunoblots--
Cultured cells were lysed in 40 mM Tris-HCl (pH 7.0), 1 nM EDTA, 4% glycerol,
10 mM dithiothreitol, 2% SDS, and protease inhibitors. Protein concentrations were determined by the Bio-Rad protein assay.
Proteins were resolved by electrophoresis through a 7.5% SDS-polyacrylamide gel and transferred to nitrocellulose membrane. The
membranes were probed with a mouse monoclonal antibody specific for HA
and a goat anti-mouse IgG horseradish peroxidase conjugate. AR protein
was displayed by a reaction with Supersignal Chemiluminescent Substrate
using the ECL kit (Amersham Pharmacia Biotech).
Homology Modeling--
A model of the human holo-AR LBD was
constructed based on the crystal structure coordinates of the human
progesterone receptor LBD in a complex with progesterone (30), as
described elsewhere (31). The structure of the retinoid X receptor LBD
(32) was used as the starting point for modeling the apo-AR LBD.
Initially a threading based model was constructed in SPDV 3.5 (33) and a structural alignment generated. Portions of the receptor which aligned well with the retinoid X receptor were subjected to further minimization as for the holo-AR. Testosterone and the glucocorticord receptor-interacting protein-1 (GRIP1) peptide were docked into the AR
model by superimposing the AR LBD model onto crystal structures of the
progesterone receptor LBD-progesterone (30) and estrogen receptor
LBD-estrogen-GRIP1 peptide (34) complexes. An energy-minimized structure for testosterone was constructed using ISIS/Draw and Sculpt 3 (35) and aligned with progesterone by a Maximal Common Substructure
Search (36). Electrostatic and van der Waals interactions of
testosterone and the GRIP1 peptide with the AR LBD were minimized locally using Monte Carlo minimization (37); Van der Waals interactions were modeled using a modified Lennard-Jones function between atoms within 6 Å of each other, and electrostatic interactions were modeled
with a distance-dependent dielectric between atoms within 10 Å.
Hormones--
The synthetic androgen R1881 was purchased from
NEN Life Science Products. 17 Spontaneous Mutation in AR in Primary Prostate Tumors--
To
investigate the possibility that somatic mutation in the AR occurred
during the natural history of prostate cancer in the TRAMP model,
full-length AR cDNA was first amplified from tumor tissue by
reverse transcription-PCR using three overlapping primer sets and the
error-free Vent DNA polymerase. The resulting AR cDNA fragments
were then subcloned and analyzed by SSCP to detect mutations (38).
Putative mutations in the AR cDNA fragments identified by the
appearance of bands of altered mobility on the SSCP gels (Fig.
1B) were confirmed by DNA
sequence analysis. In total, 15 somatic mutations in the AR were
identified in prostate tumors isolated from eight individual TRAMP mice
that ranged in age from 24 to 29 weeks. Of these, 9 mutations were
identified in the tumors from castrated mice and 6 in tumors from
intact mice. All mutations were single base substitutions. The majority of the mutations (10 out of 15; 67%) were missense mutations, and the
remaining 5 were silent (Table I). No
frameshifts or deletions were observed, and no mutations in the AR DNA
binding domain were identified. No mutations were detected in parallel reactions using RNA isolated from testis.
In this study, we chose to analyze 10 unique and independent clones for
each AR cDNA fragment by SSCP. At least one variant AR was
identified in each tumor examined. Hence it is reasonable to state that
at least 10% of cells in each tumor likely harbored transcripts
encoding a variant AR. Interestingly, nine of the AR variants were
isolated from tumors of TRAMP mice that were castrated at 12 weeks of
age, and the majority (seven out of nine; 78%) colocalized in the AR
transactivation domain. In contrast, all six AR variants isolated from
the tumors of four intact TRAMP mice colocalized to the AR LBD (Fig.
2A). These observations
demonstrate that mutation of the AR is a spontaneous event in the
natural history of prostate cancer in the TRAMP model. Furthermore,
although the incidence of mutations in any given tumor is ~10%, the
nature of the mutations appears to be a function of the hormonal status of the animal.
Transcriptional Activity of the AR Variants in PC3 Cells--
To
determine the consequence of mutation in the AR gene, the
transcriptional activities of the AR variants identified in the TRAMP
tumors were characterized. To this end, an expression vector pcDNA3/HA-mAR was first constructed to carry a full-length wild type mouse AR cDNA modified with a consensus Kozak translation sequence and the HA epitope tag at the amino terminus (Fig.
2B). The missense mutations identified from TRAMP tumors and
the T857A variant corresponding to the human AR in LNCaP cells were
subsequently introduced into the pcDNA3/HA-mAR using in
vitro site-directed mutagenesis. The identity and integrity of
each construct were confirmed by DNA sequencing (data not shown).
Western blot analysis was used to confirm that each construct encoded a
full-length HA-tagged AR. No significant variability in the level of
expression was observed for these constructs (Fig. 2C).
The ability of the AR variants to transactivate AR-responsive reporter
gene constructs was first evaluated in PC3 cells, an AR-deficient human
prostate cancer cell line. In these experiments, two androgen-regulated
reporter constructs, PB-LUC and ARR3-LUC, were employed. PB-LUC carries
the naturally occurring androgen-regulated rat complex probasin
promoter sequence spanning
The ligand specificity of each AR variant was next examined by
transactivation analysis. Remarkably, the wild type AR and all AR
variants except for T857A exhibited a similar induction by 1 nM R1881 when assayed with PB-LUC (Fig. 3B). In
contrast, the K638M and F677S variants showed 1.5-2-fold better
response than wild type AR (p < 0.001) in the presence
of 1 nM R1881 (Fig. 3G) when assayed on
ARR3-LUC, whereas T857A demonstrated a significantly lower response
than wild type AR (p < 0.001) on either PB-LUC or
ARR3-LUC (Fig. 3, B and G). No induction of any
murine AR variant was detected in response to stimulation with 10 nM dexamethasone using either reporter constructs (Fig. 3,
C and H). As predicted from a previous study
(11), the T857A variant demonstrated a 2-3-fold greater induction than
wild type AR (p < 0.001) in response to 10 nM progesterone with PB-LUC and ARR3-LUC (Fig. 3,
E and J). In response to 10 nM
17 Transcription Activity of the AR Variants in TRAMP
Cells--
Three TRAMP cell lines, C1A, C1D, and C2G, were used to
address the possibility that cellular context could influence AR
function. These cell lines were all derived from a single primary TRAMP tumor. Whereas the C1A cells are not tumorigenic, the C1D cells are
tumorigenic but not metastatic, and the C2G cells are both tumorigenic
and metastatic.2 Although
endogenous AR can be detected in these cells by Western blotting, the
level of AR is insufficient to drive expression of transfected PB-LUC
or ARR3-LUC reporter genes (data not shown).
In these experiments, the activity of three AR variants, E231G, K638M,
and T857A, were compared with exogenous wild type receptor. As shown in
Fig. 4, consistent with the study in PC3
cells and a previous report (11), the T857A variant was found to
activate ARR3-LUC by different hormones regardless of cell context
(Fig. 4, A-C). In contrast, the E231G variant exhibited
3-fold higher induction than wild type AR (p < 0.001)
in C1D cells in the presence of 1 nM R1881 (Fig.
4C) but lower induction than wild type AR in both C1A and
C2G cells (Fig. 4, A and B). In response to
R1881, the K638M variant exhibited a 2-fold increase in activity
compared with wild type AR (p < 0.001) in C1A cells
but lower activity than wild type AR in C1D cells (Fig. 4, A
and C). No difference in activity was observed between wild
type AR, E231G and K638M variants in response to stimulation by
progesterone and 17
Based on these data it was clear that cell context could influence the
function of the AR variants. In all three cell lines, both E231G and
K638M variants exhibited induction similar to that of wild type AR in
the presence of 1 nM R1881 when ARR3-LUC was replaced by
PB-LUC. However, T857A exhibited a 2-5-fold increased induction
compared with wild type in response to 1 nM R1881, 10 nM progesterone, and 10 nM 17 Functional Interaction of the AR Variants with Known AR
Cofactors--
To investigate the consequence of mutation on the
interaction between AR with known cofactors, we performed an additional series of transactivation experiments in PC3 cells. In these studies, the abilities of the E231G, K638M, and T857A variants and wild type AR
to transactivate PB-LUC in presence of ARA70 and ARA160 were examined.
Both cofactors have previously been shown to interact physically with
wild type AR and functionally enhance transactivation activity of the
AR in the presence of certain ligands (40). As shown in Fig.
5, ARA70 enhanced transcriptional
activity of wild type AR and AR variants to 6-12-fold over base line
in the presence of 1 nM R1881. Remarkably, the E231G
variant exhibited 2-fold greater activity than wild type AR
(p < 0.001) in the presence of ARA70 (Fig.
5A). ARA70 has also been reported to be the mediator of AR
response to estrogen stimulation (41), and our transactivation data
confirmed this observation. Upon transfection of wild type AR or AR
variants in the absence of ARA70, only marginal induction was detected
in the presence of 10 nM 17 Homology Modeling of the AR Variants--
To investigate the
mechanism of altered ligand responsiveness of the F677S (TRAMP) and
T857A (LNCaP) AR variants observed in transactivation analysis, we
applied a homology modeling protocol to construct models of the apo-
and holo-AR LBD. A similar model has been shown to predict the recently
solved AR crystal structure accurately (31). Analysis of the AR
structure in that study determined that the threonine residue mutated
in the LNCaP AR (i.e. T857A) forms one of the two hydrogen
bond partners with the 17 Naturally occurring somatic mutations have been identified in
human AR isolated from prostate cancer specimens (42). However, there
is considerable debate in the literature concerning the incidence and
frequency of mutation in the AR gene (43) For the most part, this can
be attributed to variability in analytic methodology and the
heterogeneity of the clinical population and their clinical history.
Generally, these studies have focused on the LBD of the AR. However, we
(44) have examined all eight exons of the AR gene in 25 primary
prostate cancer patients and discovered mutations in 13 (52%) of the
tumors. Surprisingly, one-half (50%) of the mutations were located in
exon 1. This not only demonstrates that the transactivation domain of
the AR is a target for somatic mutation, but also provides a mechanism
to explain the low frequencies of mutation observed in other reports. In this study we report identification of 15 somatic mutations in the
AR gene from primary prostate tumors of TRAMP mice between 24 and 29 weeks of age. Of these, 7 mutations (47%) were also colocalized in
exon 1. Clearly, these data confirm that exon 1 encoding the
transactivation domain of the AR is indeed a target for somatic
mutations in both human and murine primary prostate cancer.
There is evidence that the incidence of mutation in AR may be higher in
the advanced stages of prostate cancer (45), supporting the clonal
selection hypothesis that cells harboring distinct mutations in the AR
would have a growth advantage under certain selective pressures. Taplin
and co-workers (46) were able to detect mutations in the AR in 5 of 16 (31%) patients who were treated by androgen ablation and the
anti-androgen flutamide. All 5 mutations in the AR were in codon 877, and these AR variants were strongly activated by flutamide but
demonstrated sensitivity to the anti-androgen bicalutamide. In
contrast, only one mutation in codon 890 of the AR was isolated from 17 patients (6%) treated with androgen ablation alone, and this AR
variant was not stimulated by flutamide. These data not only
demonstrate that mutations in the AR are detected following flutamide
treatment, but also suggest that the selective pressure of flutamide
treatment may determine the nature of mutation in the AR. In our
studies, the majority (7 out of 9, 78%) of the AR variants isolated
from castrated TRAMP mice colocalized in the AR transactivation domain
(i.e. exon 1), whereas all of the mutations from the intact
TRAMP mice were colocalized in the AR LBD. This indicates that the
hormonal status influences the nature of the somatic mutations in AR in
primary autochthonous prostate tumors. Furthermore, these data suggest
that some mutations in the AR detected in tumors from castrated TRAMP
mice might have originated in cells at a time when they were still
androgen dependent. In this study, all mutations in the AR exon 1 were
isolated from androgen-independent tumors, suggesting that although the
patients did not receive androgen ablation in the clinical study (44), it is possible that the cells harboring mutations in exon 1 were already androgen-independent. It is also possible that castration by
orchiectomy may apply different selective pressures from castration by
anti-androgens such as flutamide, bicalutamide, and luteinizing hormone-releasing hormone agonist treatment. These data underscore the
need to undertake a comprehensive molecular study of the consequences of alternative hormonal therapies to understand better how mutations in
the AR mechanistically contribute to the emergence of
androgen-independent disease.
Alteration of the ligand specificity or affinity is one possible
mechanism through which the AR variants may functionally contribute to
the emergence of androgen-independent prostate cancer. In fact, most AR
variants isolated from clinical prostate cancer demonstrated broadened
ligand specificity that can significantly increase transcriptional
capacity in response to estrogen, progesterone, or even paradoxically
anti-androgens (11, 16). In this study we have confirmed that the LNCaP
T857A variant could be activated by estrogen and progesterone, and we
demonstrated that this activity was independent of the promoter
context. We also found that the K638M variant isolated from an
androgen-independent tumor demonstrated greater response to both
androgen and estradiol stimulation compared with the wild type AR. In
contrast to T857A and K638M variants, the F677S variant lost the
response to estrogen but increased the response to androgen. The model
of the holo-AR LBD clearly demonstrates that these mutations result in
AR molecules with significant conformational changes in the LBD which
likely function in ligand dependent manner and provides a mechanism to
explain the altered response of the AR variants to androgenic and
nonandrogenic ligands.
Ligand binding specificity or affinity does not alone reflect the
functional capability of the AR. Recently, the
"ligand-receptor-cofactors" tripartite system has been proposed to
explain the molecular interactions of steroid receptors (47, 48). It is
now generally accepted that interactions between ligand-receptor
complexes and cofactors are essential for AR function, selectivity, and
sensitivity. In this study we demonstrated that the LNCaP T857A variant
exhibited cell-specific activities (Fig. 5). We also demonstrated that
ARA70 could synergistically enhance the AR transcription activity more than 10-fold in the presence of either R1881 or 10 nM
17 As evident from our observations, the specificity and biological
diversity of the steroid hormone receptor family can be generated at
the level of cofactors. In addition, these observations suggest that
structural or functional changes of AR coregulators could also modulate
the androgen signaling axis and may contribute to the
progression of prostate cancer. In fact, amplification and/or overexpression of steroid hormone receptor cofactors, such as A1B1 (49)
and ASC-2 (50), have been reported recently in breast and other human
cancers. Hence, concomitant changes in coactivators or corepressors in
addition to AR are also likely to be important molecular events in
prostate cancer progression. It will therefore be necessary to develop
suitable autochthonous models that carry specific complements of
cofactors and AR variants. Such models can then be used to assess
comprehensively the contribution of a specific AR variant to the
initiation, progression, and metastasis of prostate cancer under
various hormonal pressures to a better understanding the causal
relationships between AR variants and prostate cancer.
Some AR mutations were identified to be silent in this study. These
mutations, like the missense mutations, were located in either the AR
transactivation domain or the LBD. Silent mutations have been reported
in clinical prostate cancer at high frequency (44). It will be
interesting in later studies to examine the functional consequence of
these silent mutations and address the possibility that these mutations
could influence AR mRNA stability or translational regulation.
The TRAMP model is the first transgenic prostate cancer model to
display somatic mutations in AR. Interestingly, many of these mutations
map to the regions known to be a "hot spot," such as exon 4, in
strong correlation with data for the human AR gene. This indicates that
the TRAMP model is uniquely suited to elucidate how deregulation of the
androgen signaling axis mechanistically contributes to the progression
of prostate cancer. We anticipate that ongoing studies with the TRAMP
model will elucidate the molecular nature whereby the AR can facilitate
prostate cancer progression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
426/+28 regulatory sequence to direct SV40
early gene (T and t antigens; Tag) expression to prostatic epithelium in a developmentally and hormonally regulated fashion. By 10-12 weeks
of age, male TRAMP mice generally develop prostatic intraepithelial neoplasia and/or well-differentiated prostate cancer. All TRAMP males
ultimately will develop prostate adenocarcinoma that metastasizes to
distant sites, primarily the lymph nodes and lungs. This generally occurs by 24-30 weeks of age (21). After androgen ablation at 12 weeks
of age, 70-80% TRAMP males will ultimately develop
androgen-independent disease. These mice typically develop poorly
differentiated adenocarcinoma and exhibit twice the incidence of
metastasis as intact littermate (20). The parallel development of
clinical prostate cancer and tumors in TRAMP mice, the comparable
emergence of hormone-refractory disease after androgen ablation, and
the highly conserved nature of the AR suggested that the TRAMP model
could be used to investigate the relationship between mutations in the
AR gene and prostate cancer. Here we report the identification and
characterization of distinct classes of AR variants isolated as
spontaneous somatic mutations during prostate cancer progression in
TRAMP. We demonstrate that hormonal status influences the nature of the
mutations in AR and that these AR variants display unique functional
properties. This study establishes in the TRAMP model a link between
deregulation of the androgen signaling axis and prostate cancer progression.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
vector
(Stratagene, Inc). 10 clones from each AR cDNA fragment were
isolated and used for SSCP analysis essentially as described previously
(25). Thus, a total of 30 clones was analyzed for each tumor sample.
Briefly, 10 sets of overlapping primers (see Fig. 1A) were
used spanning the full-length of the AR cDNA. 30-cycle PCR was
performed on the plasmid DNA in reactions containing a 200 µM concentration of each of the four deoxyribonucleotide
triphosphates, 1.5 µCi of [
-32P]dCTP (3,000 Ci/mmol,
ICN Biochemicals, Irvine, CA), 10 mM KCl, 20 mM
Tris-HCl (pH 8.8), 10 mM
(NH4)2SO4, 2 mM
MgSO4, 0.1% Triton-100, 200 µM appropriate
sense and antisense primer sets, and 0.5 unit of Vent DNA polymerase.
After an initial denaturing step of 2 min at 94 °C, the cycle
parameters were as follows: 1 min at 94 °C, 2 min at 65 °C, and 3 min at 72 °C. After amplification, the radiolabeled PCR products
were diluted 1:10 in loading buffer that contained 95% formamide
(v/v), 50 mM EDTA, 20 mM NaOH, and 0.05% each
of xylene cyanol and bromphenol blue. Samples were denatured at
100 °C for 10 min, quick frozen on dry ice, thawed slowly on wet
ice, and fractionated on a nondenaturing 6% polyacrymide gel (40:1
acrymide:bisacrylamide) containing 10% glycerol in 0.5 × Tris
borate-EDTA buffer at 4 °C. The gels were transferred to filter
paper and exposed to x-ray film (Kodak X-Omat AR5) at
80 °C. Point mutations were identified by a shift in the relative mobility of the PCR fragments compared with wild type AR controls. Testis RNA from each animal was used to control for germ line mutations.
-galactosidase activities were assayed using 20-µl aliquots of the extracts. The luciferase activity was
corrected for transfection efficiency by normalization against
-galactosidase activity. The values shown represent the means of at
least three independent experiments. Data processing was performed
using the Microsoft Excel program, and statistical analyses were
performed using two-way analysis of variance followed by post
hoc comparisons with Fisher's protected least significant difference test.
-Estradiol, progesterone, and
dexamethasone were purchased from Sigma.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (39K):
[in a new window]
Fig. 1.
SSCP analysis. Panel A,
primer sequences and PCR product sizes in base pair (bp).
Panel B, representative SSCP gel. RNA samples
isolated from TRAMP tumors were analyzed by reverse transcription-PCR
and SSCP, fractionated on a 7% acrylamide gel, and exposed to film.
The arrow demonstrates aberrantly migrating species in
fragment amplified by primer set 7. T, testis RNA;
C, tumor RNA.
Mutations identified in the AR gene in TRAMP tumors
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Fig. 2.
Hormonal status influences the nature of
somatic AR mutations in primary prostate cancer. Panel
A, shown is a schematic structure of the mouse AR indicating the
location of the mutations identified from TRAMP mice. The majority of
mutations (seven out of nine; 78%) detected in tumors obtained from
the mice castrated at 12 weeks of age were colocalized in the AR
transactivation domain, and the majority of mutations (six out of
eight; 75%) in the LBD clustered in the exon 4 encoded region.
Light boxes, intact mice; boldface boxes,
castrated mice. Panel B, a full-length cDNA encoding the
wild type C57BL/6 mouse AR (mAR) was fused in-frame to the
HA sequence carrying a consensus Kozak translation initiation signal
and inserted into the KpnI-BamHI sites of
pcDNA3.1 vector. The integrity of the construct was confirmed by
automated DNA sequence analysis. MCS, multiple cloning site.
Panel C, recombinant wild type (wt) and mutated
androgen receptor were transfected into PC3 cells. Cell extracts were
prepared and fractionated by SDS-polyacrylamide gel electrophoresis,
transferred to nylon membranes, and probed with anti-HA antibody. PC3
cells transfected with pcDNA3.1 vector alone were used as a
control.
286 to +28 fused to the luciferase
reporter. This sequence represents the native chromosomal sequence
immediately upstream of the rat probasin structural gene and has been
shown to impart both tissue and hormone specificity (39).
ARR3-tk-luciferase (ARR3-LUC) carries three repeats of the rat probasin
AR response element-1 and -2 region (
244/
96) ligated in tandem
upstream of the minimal thymidine kinase (tk) enhancer element fused to
the luciferase reporter (28). The semisynthetic ARR3-LUC has been
demonstrated previously to be more sensitive to AR signals than PB-LUC.
As shown in Fig. 3, the basal
transcriptional activities of the E206K, A229T, E231G, N384S, and T857A
variants were about 1.5-2-fold over that observed for the wild type on
the complex reporter PB-LUC (p < 0.001) (Fig. 3A). Note that, except for T857A, all of these variants were
isolated from tumors of castrated mice. Surprisingly, when the
semisynthetic ARR3-LUC reporter was employed, only the T857A variant
demonstrated an elevated basal transcriptional activity (Fig.
3F).
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Fig. 3.
Ligand specificity and transcriptional
activities of the AR variants in PC3 cells. PC3 cells were
transfected with a DNA mixture containing 0.5 µg of the mutant or the
wild type AR (wtAR), 1.0 µg of PB-LUC or ARR3-LUC, and 0.5 µg of CMV- -galactosidase plasmid as an internal reference. 24 h after transfection, the medium was changed to contain 10%
charcoal-stripped serum with or without the hormones tested as
indicated. The LUC activity was normalized against the
-galactosidase activity and is presented as the fold increase over
the basal level. The values are the means ± S.D. of at least
three independent experiments.
-estradiol, all of the AR variants except F677S induced expression
of both PB-LUC and ARR3-LUC, and the K638M and T857A variants induced
ARR3-LUC constructs to a significantly greater extent than the wild
type (p < 0.001). It was interesting to note that the
F677S variant did not respond to 10 nM 17
-estradiol on
either PB-LUC or ARR3-LUC (Fig. 3, D and I),
suggesting that this mutation likely defines a region of the AR
required for estrogen stimulation. These data clearly demonstrate that
the AR variants display specific and unique deviation from the activity
of the wild type receptor.
-estradiol on ARR3-LUC in the three TRAMP cell
lines (Fig. 4, A-C).
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Fig. 4.
Ligand specificity and transcriptional
activities of the AR variants in TRAMP cells. TRAMP cells (C1A,
C2G, and C1D) were transfected with a DNA mixture containing 0.5 µg
of the mutant or the wild type AR (wtAR), 1.0 µg of PB-LUC
or ARR3-LUC, and the 0.5 µg of CMV- -galactosidase plasmid as an
internal reference. After transfection, cells were incubated in the
medium containing 10% charcoal-stripped serum with or without the
hormones tested as indicated. The LUC activity was normalized against
the
-galactosidase activity and is presented as the fold increase
over the basal level. The values are the means ± S.D. of at least
three independent experiments. DEX, dexamethasone.
-estradiol in
C1A cells but not in C2G and C1D cells (Fig. 4, D-F).
Therefore, cellular context was found to influence the function of the
well characterized LNCaP T857A variant. Collectively, the E231G, K638M,
and T857A variants exhibited multiple activity profiles in the three
different TRAMP cell lines using the two different but related promoter systems. These observations suggest that these cell lines may express
different complements of coactivators or corepressors that subsequently
and profoundly influence the transcriptional activities of the AR variants.
-estradiol. The addition of
ARA70, however, further enhanced the transcriptional activity of wild
type and AR variants to 5-20-fold (Fig. 5B). It was
interesting to note that the addition of ARA70 resulted in a higher
induction on the E231G variant compared with wild type
(p < 0.001). In contrast, K638M and T857A variants
exhibited 4- and 2- fold less induction than wild type AR
(p < 0.001) in the presence of ARA70 and 10 nM 17
-estradiol, respectively (Fig. 5B). When
ARA70 was replaced by ARA160, the enhancement of AR activity was
minimal except as observed for the E231G variant in the presence 10 nM R1881 (Fig. 6A). ARA160 did not enhance
the activity of either wild type or the AR variants in the presence of
10 nM 17
-estradiol (Fig. 6B). Surprisingly,
the E231G variant, isolated from a tumor of a castrated mouse,
exhibited 3-fold more activity in response to androgen in the presence
of ARA160 and 2-fold more activity in response to either androgen or
estrogen in the presence of ARA70. Hence, the E231G variant was able to
distinguish R1881 from 17
-estradiol in the presence of ARA160. These
data clearly demonstrate that mutations in the AR can significantly
influence functional interactions between AR and coregulators to modify
promoter- and cell-specific patterns of gene expression.
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Fig. 5.
Functional interaction of the AR variants
with ARA70. PC3 cells were transfected with a DNA mixture
containing 400 ng of PB-LUC, 400 ng of CMV- -galactosidase plasmid,
150 ng of AR, or 450 ng of ARA70. The total DNA was adjusted to 2 µg
with pcDNA3.1 empty vector. After transfection, cells were
incubated in medium containing 10% charcoal-stripped serum with or
without the hormones tested as indicated. The LUC activity was
normalized against the
-galactosidase activity and is presented as
the fold increase over the basal level. The values are the means ± S.D. of at least three independent experiments.
View larger version (19K):
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Fig. 6.
Functional interaction of the AR variants
with ARA160. PC3 cells were transfected with a DNA mixture
containing 400 ng of PB-LUC, 400 ng of CMV- -galactosidase plasmid,
150 ng of the mutant or wild type AR (wtAR), or 450 ng of
ARA160. The total DNA was adjusted to 2 µg with pcDNA3.1 empty
vector. After transfection, cells were incubated in medium containing
10% charcoal-stripped serum with or without the hormones tested as
indicated. The LUC activity was normalized against the
-galactosidase activity and is presented as the fold increase over
the basal level. The values are the means ± S.D. of at least
three independent experiments.
-hydroxy group of testosterone and the
synthetic androgen, R1881 (31). Mutation of the threonine residue to
alanine results in the loss of one of the hydrogen bond partners for
the 17
-hydroxy group but does not affect the ability of the receptor
to orient ligand into the pocket (31), which probably accounts for the reduced activity of the LNCaP variant in response to R1881 observed in
our transactivation analysis. Our homology model of the T857A AR
variant also demonstrated that the threonine residue composes a large
portion of the ligand binding pocket surface and that substitution of
this residue for alanine results in changes in the shape and volume of
the pocket (Fig. 7, A and
B). Bulkier ligands could be accommodated within the larger
pocket, providing a mechanism for the altered response of the T857A AR
variant observed with progesterone and 17
-estradiol (Fig. 3,
D-J). In contrast to the LNCaP AR, the F677S AR variant
exhibited a marked reduced activation by progesterone and
17
-estradiol but enhanced activation by R1881. Our model of the
holo-AR LBD revealed that the phenylalanine residue at position 677 neither forms part of the ligand binding pocket nor plays a major
structural role in the receptor (Fig. 7, A and B;
data not shown). However, the aromatic ring is conserved in the
divergent receptor for retinoic acid which has a tryptophan residue in
this position (data not shown). Comparison of the holo- and apo-AR
models clearly demonstrates that ligand binding results in significant
conformational changes in this area of the LBD, which brings the
aromatic side chain of the phenylalanine residue into a position where
it may act as a trap door preventing premature escape of the bound
ligand (Fig. 7C). The loss of the aromatic side chain in the
F677S variant could facilitate more rapid dissociation of the weakly
bound 17
-estradiol and progesterone, providing an explanation for
the loss of response to these steroids observed in transactivation
analysis (Fig. 3, D-J). When the homology model was used to
compare the binding of the synthetic androgen R1881 to the AR LBD with
testosterone, significant differences in the disposition of these
ligands in the pocket were observed (Fig. 7, D and
E). These differences suggest that the effect of the phenylalanine residue in controlling ligand escape will depend on the
nature of the bound ligand. The effect of the F677S mutation on ligand
binding kinetics of these and other ligands will be determined in
subsequent studies. Docking of the nuclear receptor box
LXXLL peptide of the p160 coactivator GRIP1 to the AR in the homology model showed that neither the T857A nor F677S mutation is
likely to influence the ability of the AR to interact with this class
of coactivators. However, as shown by the coactivator analysis for
several mutations (Figs. 5 and 6), the precise effect of these mutants
may only be apparent in the presence of the appropriate cofactor.
Unfortunately, as the crystal structure of the amino-terminal transactivation domain has not been solved for the AR, or indeed any of
the nuclear receptors, predictive modeling was only possible for
mutations occurring in the LBD.
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Fig. 7.
Homology modeling of the AR LBD. Shown
are MOLSCRIPT/Raster3D ribbon diagrams of the AR LBD with the ligand
binding pocket cavity represented in grid format, one grid point
representing 1.4 Å and a similar probe size. Panel A,
structure of the wild type (wt) AR LBD with testosterone
docked in the ligand binding pocket. The side chains of amino acids
residues 857 and 677 are shown in stick form. Panel B,
structure of the LBD and ligand binding pocket of the testosterone
bound T857A (LNCaP) AR variant. The side chains of amino acids residues
857 and 677 are shown in stick form. Panel C, comparison of
the ligand binding pocket and associated structures of the apo-AR LBD
(red) superimposed on the holo-AR LBD (blue)
bound to testosterone (colored according to
Corey-Pauling-Koltun). The phenylalanine residue at position 677 is
depicted in stick form. Panels D and E,
disposition of the synthetic androgen R1881 and testosterone,
respectively, in the ligand binding pocket of the wild type AR LBD.
mut., mutant.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-estradiol; and the K231G, K638M, and T857A variants demonstrated
differential activities in a ligand-dependent fashion in
the presence of ARA70. Whereas ARA160 could only slightly increase the
AR transcriptional activity in the presence of R1881, this was not
observed in the presence of 17
-estradiol. Remarkably, the E231G
variant exhibited increased activity in response to androgen but not to
estrogen in the presence of ARA160. It was important to note that the
E231G variant could discriminate between androgen and estrogen in the presence of cofactor. This suggests that selective cofactors, such as
ARA160, can determine ligand specificity of AR variants.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Chawnshang Chang for the PSG5-ARA70N and pCMV-ARA160FL plasmids, Dr. R. J. Matusik for the PB-LUC and ARR3-LUC reporter plasmids, Dr. Don Tindall for the mouse cDNA clone, and Dr. Jeffrey Rosen for helpful discussions. We also thank Caroline Castille and Rhonda Chaplin for animal husbandry and Alvenia Daniels for secretarial support.
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FOOTNOTES |
---|
* This work was supported by Grant CA73747 from the NCI, National Institutes of Health (to N. M. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dept. of Urology, University of Pittsburgh, 5200 Center Ave., G34, Pittsburgh, PA 15232.
To whom correspondence should be addressed: Dept. of Molecular
and Cellular Biology, Baylor College of Medicine, One Baylor Plaza,
Suite M626, Houston, TX 77030. Tel.: 713-798-3819; Fax: 713-798-8012; E-mail: normang@bcm.tmc.edu.
Published, JBC Papers in Press, November 3, DOI 10.1074/jbc.M008207200
2 B. A. Foster, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: AR, androgen receptor; TRAMP, transgenic adenocarcinoma mouse prostate; PB, probasin; PCR, polymerase chain reaction; SSCP, single-stranded conformational polymorphism; mAR, mouse androgen receptor; HA, hemagglutinin antigen; CMV, cytomegalovirus; LUC, luciferase; tk, thymidine kinase; LBD, ligand-binding domain; GRIP1, glucocorticord receptor-interacting protein-1.
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REFERENCES |
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