Noninvasive measurement of androgen receptor
signaling with a positron-emitting radiopharmaceutical
that targets prostate-specific membrane antigen
Michael J. Evansa, Peter M. Smith-Jonesb, John Wongvipata, Vincent Navarroc, Sae Kimc, Neil H. Banderc,d,
Steven M. Larsonb,e,f, and Charles L. Sawyersa,g,1
aHuman Oncology and Pathogenesis Program,bDepartment of Radiology,dDivision of Urology,eMolecular Pharmacology and Chemistry,fNuclear Medicine
Service, andgHoward Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10065; andcLaboratory of Urologic Oncology,
Weill Cornell Medical College, New York, NY 10065
Contributed by Charles L. Sawyers, April 28, 2011 (sent for review March 18, 2011)
Despite encouraging clinical results with next generation drugs
(MDV3100 and abiraterone) that inhibit androgen receptor (AR)
signaling in patients with castration-resistant prostate cancer
(CRPC), responses are variable and short-lived. There is an urgent
need to understand the basis of resistance to optimize their future
use. We reasoned that a radiopharmaceutical that measures intra-
tumoral changes in AR signaling could substantially improve our
understanding of AR pathway directed therapies. Expanding on
previous observations, we first show that prostate-specific mem-
brane antigen (PSMA) is repressed by androgen treatment in
multiple models of AR-positive prostate cancer in an AR-dependent
manner. Conversely, antiandrogens up-regulate PSMA expression.
These expression changes, including increased PSMA expression
in response to treatment with the antiandrogen MDV3100, can be
quantitatively measured in vivo in human prostate cancer xeno-
graft models through PET imaging with a fully humanized, radio-
labeled antibody to PSMA,
establish that relative changes in PSMA expression levels can be
quantitatively measured using a human-ready imaging reagent
and could serve as a biomarker of AR signaling to noninvasively
evaluate AR activity in patients with CRPC.
United States in 2011 (www.cancer.gov). One hallmark of CRPC
is reactivation of androgen receptor (AR) signaling despite cas-
trate levels of androgens in the blood (1). This insight has
rekindled interest in developing androgen deprivation therapies
with greater potency, several of which have already shown clin-
ical activity in patients with CRPC (2–4). Treatment responses,
however, are incomplete and short-lived. Given these heteroge-
neous patterns of response, new biomarkers are urgently needed
to document successful AR inhibition in tumor tissue and to
identify patients early whose tumors fail to respond.
Serum measurements of the AR-regulated, secreted protein
prostate-specific antigen (PSA) are typically used to evaluate AR
signaling in prostate cancer. PSA is highly useful for evaluating
initial response to androgen deprivation therapies and detecting
relapse (5), but reductions in serum PSA levels do not always
correlate with survival benefit in CRPC patients. Radiographic
likely a reflection of the heterogeneity of prostate cancer even
within the same patient (6). The differential sensitivity of distinct
metastatic lesions to antiandrogen therapy might be explained by
different levels of AR inhibition. Because declines in serum PSA
to determine whether AR inhibition varies at different sites.
We reasoned that these difficulties could be overcome with
a radiopharmaceutical that measures intratumoral AR signaling.
The basis for our optimism extends from previous work demon-
strating that molecular imaging tools [e.g.,18F-fluorodeoxyglucose
64Cu-J591. Collectively, these results
pproximately 27,000 patients will die of castration-resistant
11C-methionine] can capture the biological diversity of CRPC (7–
9), in addition to the emerging role of molecular imaging in the
evaluation of cancer therapies. For instance, documenting the
metabolic tumor response to imatinib with18F-FDG has greatly
simplified the clinical management of gastrointestinal stromal
tumors (10, 11).
Although originally identified on the basis of its restricted
pattern of tissue expression, prostate-specific membrane antigen
(PSMA) emerged as a candidate imaging biomarker of AR ac-
PSMA expression in the LNCaP prostate cancer cell line (12,
13). Consistent with this observation, Wright et al. (14) reported
elevated immunohistochemical staining for PSMA in a small co-
hort of primary and metastatic biopsies sampled after various
androgen deprivation manipulations. Moreover, PSMA is a type
II plasma membrane protein expressed abundantly in prostate
cancer epithelia, and a substantial catalog of laboratory and
clinical imaging tools directed to this protein has been generated
(15). Collectively, these observations led us to hypothesize that
PET imaging of PSMA might be a viable strategy to measure AR
inhibition in vivo.
PSMA Is Androgen Repressed in Multiple Prostate Cancer Models.
Prior work implicating PSMA as an androgen-repressed gene is
based on a single cell line. To determine whether this biological re-
cancer celllines. PSMA is expressed in four AR-positive, hormone-
andVCaP) but not in two AR-negative prostate cancerlines (PC3
and DU145) or in two immortalized primary prostate epithelial
cell lines (BPH and RWPE1; Fig. S1). Androgen stimulation for
72 h with testosterone, 5α-dihydrotestosterone (DHT) or the syn-
LNCaP, CWR22Rv1, LAPC4, and VCaP cells comparedwith the
low androgen environment of FBS and the androgen-free envi-
Author contributions: M.J.E., N.H.B., and C.L.S. designed research; M.J.E., P.M.S.-J.,
J.W., V.N., and S.K. performed research; P.M.S.-J. contributed new reagents/analytic
tools; M.J.E., P.M.S.-J., N.H.B., and C.L.S. analyzed data; and M.J.E., S.M.L., and C.L.S.
wrote the paper.
Conflict of interest statement: The article describes a new radiotracer to monitor androgen
receptor signaling noninvasively. The various studies that demonstrate the utility of this
radiotracer include an experiment using the antiandrogen drug MDV3100. C.L.S. is a co-
inventor of MDV3100 and owns stock in the company (Medivation) that is developing the
drug for prostate cancer treatment. The article does not make any claims about the effi-
cacy of MDV3100; it merely uses it as tool to evaluate the new radiotracer. N.H.B. is the
inventor of patents related to PSMA antibodies assigned to Cornell Research Foundation.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
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ronment in charcoal-stripped serum (CSS) (Fig. 1A and Fig. S2).
Moreover, PSMA mRNA levels were reduced by androgen
treatment in LNCaP (~50% maximal reduction) and CWR22Rv1
(~80% maximal reduction), whereas PSA mRNA levels were el-
evated, as expected (Fig. 1B). These in vitro data confirm that
PSMA expression is suppressed by androgens across a panel of
AR-positive cell lines. To evaluate hormonal regulation of PSMA
xenografts in castrate male mice. Xenograft tissue harvested and
analyzed from mice 7 d after receiving an s.c. DHT pellet showed
a substantial reduction in PSMA mRNA and protein levels com-
pared with tissue derived from mice receiving no treatment. As
expected, the androgen-stimulated gene TMPRSS2 was up-regu-
lated by DHTtreatment (Fig.1C). Collectively, theseresults show
that PSMA is consistently repressed by androgen treatment in
multiple prostate cancer models.
AR Is Required for Androgen Repression of PSMA. To confirm that
we ablated AR with siRNA in LNCaP and CWR22Rv1 and sub-
sequently evaluated androgen regulation of PSMA. Whereas si-
lencing AR itself did not seem to impact basal PSMA levels at
this time point (72 h), knockdown of AR abolished PSMA repre-
ssion by DHT in both cell lines (Fig. 2A).
This observation suggested that pharmacological inhibitors of
AR might antagonize androgen-dependent PSMA suppression.
To test this hypothesis, LNCaP-AR cells (parental LNCaP over-
expressing wild-type AR) were treated with androgen deprivation,
and the effect on PSMA surface expression was assayed by FACS
analysis with fluorescently labeled J591, a fully humanized mAb
that targets an extracellular epitope of PSMA (16, 17). MDV3100,
an experimental antiandrogen developed in castration-resistant
models of prostate cancer (18), increased PSMA expression com-
pared with vehicle control after 7 d, with further up-regulation
deprivation) resulted in larger overall up-regulation of PSMA
but only after 14 d. Similar results were observed with parental
LNCaP cells (Fig. S3). In CWR22Rv1 cells, MDV3100 treatment
as did harmol hydrochloride (10 μM), a natural product inhibitor
of AR that is not ligand competitive (19) (Fig. S4). In summary,
these results document the role of AR in androgen-dependent
regulation of PSMA and demonstrate that pharmacologic modu-
lation of AR signaling is faithfully reflected by changes in relative
7–14 d of androgen deprivationsuggests that stable AR knockdown
may be required to see similar effects using RNAi targeting AR.
Androgen Repression of PSMA Can Be Quantitatively Imaged with
PET. We next explored whether androgen-dependent changes in
PSMA expression are sufficiently large to be quantitatively im-
aged in vivo with PET. To this end, bilateral s.c. CWR22Rv1
were imaged by PET with64Cu-J591 (Fig. 3 A and B). All xeno-
grafts had approximately equivalent basal incorporation of64Cu-
J591 [average standardized uptake value (SUV)mean= 39.7 ±
LNCaP and CWR22Rv1 (22Rv1) for 72 h with the endogenous androgens testosterone (Test., 10 nM) and DHT (10 nM) or the synthetic androgen R1881 (0.1 nM)
decreases PSMA protein levels compared with low androgen conditions (FBS and CSS). In CWR22Rv1 cells, AR levels increase after androgen treatment com-
pared with vehicle control, suggesting agonist-mediated receptor stabilization and minimal hormone degradation during incubation. (B) PSMA mRNA levels
are suppressed after 72 h of treatment with androgens in LNCaP and CWR22Rv1, consistent with transcriptional regulation of PSMA by hormone treatment. As
anticipated, mRNA levels of PSA, an AR target gene, increase in response to androgen challenge, confirming the bioactivity of hormone treatment at this time
point. (C) Subcutaneous xenografts of LNCaP and CWR22Rv1 derived in castrate male mice were harvested 7 d after implantation of a DHT pellet or no surgical
treatment (No Tx). Immunoblot (Upper) and quantitative PCR analysis (Lower) shows that PSMA is expressed in PCa xenografts, and expression is reduced by
DHT treatment. The androgen-stimulated gene product TMPRSS2 is up-regulated by DHT, confirming the bioactivity of the pellet dose.
Androgens suppress PSMA in multiple prostate cancer cell lines and xenografts. (A) Incubation of the hormone-responsive prostate cancer cell lines
Evans et al.PNAS
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6.5%ID/mL]. Twenty-four hours after imaging, mice were ran-
domized into equal cohorts receiving (i) no treatment, (ii) a s.c.
testosterone pellet, or (iii) an s.c. DHT pellet. After 6 d, the
groups were again injected with64Cu-J591, imaged 16 h after in-
jection, and subsequently euthanized for ex vivo tissue analysis. A
modest reduction in tumor uptake of64Cu-J591 was observed
7 d after the initial injection in the group receiving no treatment
(0.81 ± 0.08), likely owing to the presence of residual antibody
from scan 1. Nevertheless, testosterone and DHT treatments
greatly reduced the incorporation of64Cu-J591 in tumor tissue
compared with no-treatment control (0.52 ± 0.1 and 0.45 ± 0.1
respective ratios, P < 0.01; Fig. S5 shows a plot of correlated PET
and biodistribution data, and Table S1 lists SUVmeanand bio-
distribution data from the tumor tissue of the full mouse cohort).
Moreover, biodistribution data revealed that64Cu-J591 uptake
was unaffected in androgen-insensitive host tissues, indicative of
Table S2 lists biodistribution values from the full mouse cohort).
Androgen Deprivation Therapies Increase
Xenografts. Having shown that androgen repression of PSMA
can be quantitatively imaged, we asked whether up-regulation of
PSMA expression can be detected with
treatment with antiandrogens. The preclinical studies of MDV3100
were conducted in LNCaP-AR xenografts and were predictive of
clinical activity; therefore, we conducted our PSMA imaging
studies using the same model. Intact male mice were inoculated
with bilateral s.c. LNCaP-AR xenografts, and basal uptake of
64Cu-J591 was determined (average SUVmean= 30.5 ± 9.3%ID/g).
Mice were then randomized into cohorts receiving (i) vehicle
(daily oral gavage), (ii) castration, or (iii) MDV3100 (daily oral
gavage, 10 mg/kg). Tumor volumes decreased by 10–20% 7 d
64Cu-J591 Uptake in
64Cu-J591 PET after
male mice and imaged by PET with64Cu-J591 24 h before manipulation (scan 1). Animals were again injected with64Cu-J591 6 d after hormonal manipulation
(Test., DHT) or no treatment (No Tx), and scan 2 was acquired 16 h after injection on day 7. The ratio of SUVmeanvalues (scan 2/scan 1) shows substantial
reduction in64Cu-J591 incorporation in the xenografts exposed to testosterone (Test.) and DHT treatment. (B) Representative transverse and coronal slices
(scan 2) of animals bearing CWR22Rv1 xenografts showing visibly reduced uptake in tumors exposed to androgens compared with no treatment. The
positions of the tumors are indicated with arrows. (C) Ex vivo biodistribution data (%ID/g) of the tumors and selected host tissues show that the tumors alone,
and not androgen-insensitive tissues, respond to hormonal manipulation. Fig. S5 shows a graphical representation of the correlation between calculated
SUVmeanvalues and activity measurements acquired ex vivo, Table S1 lists the SUVmeanand biodistribution data from the full cohort of mice, and Table S2 lists
the activity measurements (%ID/g) from the host tissues. *P < 0.01 compared with No Tx controls.
Detection of androgen repression of PSMA in prostate cancer xenografts by PET. (A) Bilateral s.c. CWR22Rv1 xenografts were established in castrate
nontargeted (NT) or AR-directed (AR) siRNA pools and treated with vehicle (EtOH) or DHT (10 nM). Whereas basal expression of PSMA is unaffected by AR
knockdown in either cell line at this time point, AR silencing inhibits PSMA suppression by DHT, thus suggesting a role for AR in this process. Cells were
transfected with 100 nM of siRNA, DHT challenge was initiated 24 h after transfection, and cells were harvested 48 h after androgen treatment. (B) Hormone
withdrawal and antiandrogen treatment increase PSMA expression in LNCaP-AR in vitro. Cells were plated in media containing 10% (vol/vol) FBS and treated
with vehicle, MDV3100 (10 μM), or the media was replaced with 10% (vol/vol) CSS (indicated as “-”). After 7 or 14 d, cells were harvested and incubated with
Alexa Fluor 488-labeled J591, and PSMA expression was analyzed by FACS. Mean fluorescent intensities (MFI) were calculated and show that MDV3100 up-
regulates PSMA expression by 7 d, whereas the effects of hormone withdrawal were observed between 7 and 14 d.
Genetic and pharmacological inhibition of AR blocks androgen suppression of PSMA. (A) LNCaP and CWR22Rv1 (22Rv1) were transfected with
| www.pnas.org/cgi/doi/10.1073/pnas.1106383108Evans et al.
after castration or MDV3100 treatment, as expected (Fig. 4A).
After 6 d, the groups were again injected with64Cu-J591, imaged
16 h after injection, and subsequently euthanized for ex vivo
tumor analysis. Whereas the relative change in intratumoral
uptake of64Cu-J591 in cohorts receiving no treatment or cas-
tration was minimal (ratios of 1.10 ± 0.2 and 1.13 ± 0.2, re-
spectively), MDV3100 treatment significantly increased the
vehicle (1.43 ± 0.2, P < 0.05; Fig. 4 B and C; Fig. S5 shows a plot
of correlated PET and biodistribution data, and Table S3 lists
SUVmeanvalues from the full mouse cohort).
64Cu-J591 in the xenografts compared with
Recent clinical success with two next-generation therapies that
target AR signaling in CRPC, abiraterone and MDV3100, high-
lights the importance of developing noninvasive tools to quanti-
tatively monitor the state of AR pathway activity in patients.
Despite their promise, responses to these compounds are het-
erogeneous andoften transient. Reasons fortreatment failure are
unclear. Although changes in serum PSA levels can serve as
asurrogateforARactivity, thisapproachcannotdetect variability
in response of independent lesions within the same patient. Here
we provide proof of concept in clinically validated xenograft
models that cell-surface PSMA expression is AR dependent and
can be quantitatively assessed by PET using a humanized mono-
clonal antibody cleared for clinical use.
of AR, assesses receptor occupancy but not downstream activity.
Recent studies of18F-FDHT PET in CRPC patients treated with
MDV3100 found that tumors in nearly all patients showed a de-
crease in18F-FDHT binding, indicating that MDV3100 can oc-
cupy the AR ligand-binding domain and preclude
binding. However, these
correlate with declines in serum PSA or tumor response (3).
dose of antiandrogen required for complete blockade of androgen
binding to AR, but it cannot assess AR pathway activity. By
quantitatively assessing expression of a downstream AR target
gene,64Cu-J591 PET may identify those patients whose tumors
retain AR activity despite blockade of the AR ligand-binding do-
main and therefore would be ideal candidates for additional, or-
thogonal therapies to fully inhibit AR signaling.
18F-FDHT PET “responses” did not
18F-FDHT PET may have utility in optimizing the
The molecular basis for down-regulation of PSMA expression
by AR remains unclear. Noss et al. (12) localized the DHT-
mediated suppression of PSMA to an enhancer region, but no
androgen response elements have been identified. Recent AR
ChIP-Seq reveals four peaks of AR binding among multiple
introns of PSMA in LNCaP (20). Functional studies are needed to
determine whether these sites mediate AR repression.
The molecular imaging strategy described here could have
near-term clinical impact because the unlabeled J591 antibody
has already been optimized for use in patients (21, 22). Recent
success imaging preclinical prostate cancer models with
labeled rather than64Cu-labeled J591 (23) adds further confi-
dence that this mAb could be readily adapted for a feasibility
study in patients. Another clinical implication of our finding that
PSMA is up-regulated in response to antiandrogen therapy is that
a toxin-conjugated PSMA-targeted mAb could be an effective
combination therapy with antiandrogens. Indeed, J591 has been
adapted for radioimmunotherapy, and Ab–drug conjugates and
therapeutic doses are well tolerated in patients (24, 25).
Materials and Methods
Detailed information is provided in SI Materials and Methods.
Antibody Radiolabeling. The monoclonal antibody J591 was modified with
1,4,7,10-tetraazacyclododecane-N,N′,N″,N′′′-tetraacetic acid (DOTA), by direct
coupling of one of the four carboxylic acid groups of DOTA to the primary
by adding 10 μL of64CuCl2to 150 μL of DOTA-J591 (3.3 mg/mL, 1.0 M NH4OAc),
diethylenetriaminepentaacetic acid (pH 7.0) was added and the solution in-
mL column of P6 Bio-Gel (Bio-Rad) with an eluant of 1% BSA/saline. The re-
sultant64Cu-DOTA-J591 had a specific activity of 1.5 GBq/mg (220 MBq/μmol),
an immunoreactivity of >90%, and a radiochemical purity of >99.8%.
PET Imaging. Animal studies were carried out under Protocol 06-07-012 ap-
proved by the MSKCC Institutional Animal Care and Use Committee. Institu-
tional guidelines for the proper, humane use of animals in research were
followed.Bilateral s.c.xenografts of CWR22Rv1 or LNCaP-AR were establishedin
the flanks of castrate or intact male mice, respectively. At tumor volumes of 200
mm3, the animals were injected with 30 MBq of64Cu-J591 (20 μg IgG, 200 μL) in
the tail vein. After 16 h, the animals were sedated using 1.5% isofluorane
(Baxter Healthcare) and imaged with a microPET camera (Concorde Micro-
systems). Ten-minute acquisitions were collected with an energy window of
change in tumor volumes show that LNCaP-AR xenografts, established in intact male mice, regress after castration (Orch.) or daily oral gavage of MDV3100
(10 mg/kg). Tumor volume measurements were recorded at day 0 and after the final treatment on day 7. (B) To evaluate the effect of antiandrogen therapy
on64Cu-J591 incorporation, mice were imaged 24 h before the initiation of therapy (scan 1), and after 6 d of therapy mice were again injected with64Cu-J591
and imaged by PET 16 h after radiotracer injection (scan 2). SUVmeanvalues were calculated, and the ratio for each tumor (scan 2/scan 1) is reported. The
change in64Cu-J591 uptake associated with vehicle treatment and castration was minimal, but MDV3100 induced a measurable increase in64Cu-J591 binding,
consistent with the in vitro data. (C) Representative transverse and coronal slices (scan 2) of animals bearing LNCaP-AR xenografts showing increased
intratumoral uptake of64Cu-J591 in tumors exposed to MDV3100 compared with castration or vehicle. The positions of the tumors are indicated with arrows.
Fig. S5 shows a graphical representation of the correlation between calculated SUVmeanvalues and activity measurements of tumor tissue ex vivo, and Table
S3 lists the SUVmeanand biodistribution data from the full cohort of mice. ADTs, androgen deprivation therapies. *P < 0.05 compared with vehicle.
64Cu-J591 PET detects up-regulation of PSMA expression in prostate cancer xenografts in response to androgen deprivation therapies. (A) Percentage
Evans et al. PNAS
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| vol. 108
| no. 23
350–750 keV, and a coincidence-timing window of 6 ns was used. Twenty-four Download full-text
hours after the initial image, the mice were manipulated (castrates >were
treated with an s.c. testosterone or DHT pellet; intacts were castrated or gav-
aged daily with vehicle or 10 mg/kg MDV3100 for 7 d). After 6 d the mice were
injected with64Cu-J591 and were imaged 16 h after injection. Tumor size was
7. At the end of the last PET scan the animals were euthanized with CO2. The
major organs were removed and counted in a gamma counter with a known
sample of the %ID. Region-of-interest analysis of the acquired images was
maximum pixel value was corrected for partial volume effects according to the
size of the tumor and normalized to the injecteddose to give the percentage of
the injected dose per mL of tumor (%ID/mL).
ACKNOWLEDGMENTS. We thank Valerie Longo and Pat Zanzonico of the
Small-Animal Imaging Core Facility at Memorial Sloan-Kettering Cancer
Center (MSKCC) for providing technical services and Thomas Voller of
Washington University School of Medicine (St. Louis, MO) for the
production of64Cu. The Small Animal Imaging Core Facility was supported
by the National Institutes of Health (R24 CA83084 and P30 CA08748); pro-
CA86307). M.J.E. was supported by an R25T training grant in molecular im-
aging (R25-CA096945) from the National Institutes of Health. C.L.S. was sup-
ported by the Howard Hughes Medical Institute. S.M.L. and P.M.S.-J. were
supported by the Ludwig Center for Cancer Immunotherapy at MSKCC and
by National Cancer Institute Grant P50-CA86483. N.H.B., V.N., and S.K. are
supported by the David H. Koch Foundation and the Peter M. Sacerdote
64Cu was supported by the National Institutes of Health (R24
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