International Journal of Urology (2016) doi: 10.1111/iju.

13134

Review Article

Androgen receptor splice variant 7 in castration-resistant prostate

cancer: Clinical considerations
Alan H Bryce1 and Emmanuel S Antonarakis2
1
Division of Hematology and Medical Oncology, Mayo Clinic, Scottsdale, Arizona, and 2Departments of Oncology and Urology,
Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, USA

Abbreviations & Acronyms Abstract: Constitutively-active ligand-independent splice variants of the androgen
ADT = androgen deprivation receptor are an adaptive response by prostate cancer cells to escape androgen
therapy deprivation therapy and novel androgen receptor-directed treatments. Androgen
AR = androgen receptor receptor splice variant 7 is the most common splice variant detected in clinical
ARE = androgen response biospecimens, and emerging data now suggest that the presence of tumoral androgen
element receptor splice variant 7 might be indicative of primary and acquired resistance to next-
AR-FL = full-length AR generation androgen pathway inhibitors, such as abiraterone and enzalutamide. At the
ARMOR = Androgen same time, taxane chemotherapy might retain its efficacy regardless of androgen
Receptor Modulation receptor splice variant 7 status, thus suggesting the potential for a predictive biomarker
Optimized for Response guiding treatment selection in men with metastatic castration-resistant prostate cancer.
AR-V7 = androgen receptor Herein, we review the preclinical data elucidating the structure and function of androgen
splice variant 7 receptor splice variant 7, we describe the existing clinical data using this biomarker in
ASO = antisense metastatic castration-resistant prostate cancer, and we highlight potential therapeutic
oligonucleotides strategies to target androgen receptor splice variant 7-expressing prostate cancer.
CTC = circulating tumor cell
CTD = COOH-terminal
Key words: androgen, androgen receptor splice variant 7, hormone therapy, prostate
cancer, resistance.
domain
DBD = DNA binding
domain
LBD = ligand-binding
domain
mCRPC = metastatic Introduction
castration-resistant prostate
Cumulative insights in the biology of the AR and its response to therapeutic interventions
cancer
have led to the many recent advances in AR pathway-directed therapeutics in prostate cancer.
NTD = NH2-terminal
Despite the significant initial activity of AR ligand depletion through ADT, prostate cancer
domain
cell lines remain dependent on AR-driven growth even in the castrated state.1 Studies have
OS = overall survival
shown that re-establishment of AR trascriptional activity is a critical event in the emergence
PFS = PSA progression-free
of castration-resistant disease.2 The most common adaptive response by prostate cancer cells
survival
to initial androgen deprivation is to upregulate the androgen receptor.3 Also common is intra-
PSA = prostate-specific
crine androgen production through intratumoral upregulation of CYP17.4 Thus, the current
antigen
therapeutic armamentarium for mCRPC now includes multiple AR pathway inhibitors
RT–PCR = reverse
designed to address tumoral adaptations to ADT across the treatment spectrum.
transcription polymerase
Abiraterone and enzalutamide are the current best-in-class agents for CYP17 inhibition and
chain reaction
AR inhibition, respectively, with multiple other agents currently in development. Although
these drugs have collectively extended the survival of mCRPC patients, further resistance to
Correspondence: Emmanuel S these agents inevitably develops. One mechanism of such resistance is the development of
Antonarakis M.D., Johns constitutively-active AR splice variants. Additional pathways of anti-androgen escape have
Hopkins Sidney Kimmel been recently summarized in other reviews.5 The present article will focus on the mounting
Comprehensive Cancer Center, evidence for the clinical significance of AR splice variants, and in particular, AR-V7.
1650 Orleans Street, CRB1–
1M45, Baltimore, MD 21287,
USA. Email: eantona1@jhmi. AR biology
edu
The AR protein is a 110 kDa steroid receptor transcription factor whose principal ligands are
Received 14 March 2016; testosterone and dihydrotestosterone.6 In the ligand-unbound state, the inert AR is localized to
accepted 2 May 2016. the cytoplasm where it is complexed to its chaperone protein, HSP90.7 On ligand binding, it
undergoes a conformational change that exposes the nuclear localization signal, which initi-
ates nuclear translocation of the ligand-bound AR through the nuclear pore complex.8

© 2016 The Japanese Urological Association 1

AH BRYCE AND ES ANTONARAKIS

Therein, the AR forms a dimer, and binds to ARE in the pro-

moter and enhancer regions of target genes, leading to cell
growth and proliferation as well as PSA expression (Fig. 1).9
The AR is modular in its structure with discreet functional
regions across eight coding exons. Exon 1 encodes the NTD
and comprises ~60% of the AR protein; this is the transcrip-
tionally active part of the molecule and contains the AF-1
region, which is critical for the transcriptional function of
AR.10 Exons 2 and 3 each encode a zinc finger of the DBD,
respectively responsible for binding to the ARE and dimer-
ization to adjacent AR/ARE complexes.11 Exons 4–8 com-
prise the CTD, which contains the LBD and a ligand-induced
coactivator binding pocket (AF-2; Fig. 2).12,13 Of note, all
hormonal therapies for prostate cancer used in the clinic
today either directly inhibit the LBD (e.g. enzalutamide) or
indirectly target the LBD by depleting the ligands that bind
to it (e.g. abiraterone). Conversely, targeting the NTD has
been more challenging, and there are currently no approved
drugs capable of doing this.
AR splice variants
Aberrations of AR splicing were first described one-quarter
century ago in congenital androgen insensitivity syndrome.14
In prostate cancer, the occurrence of an AR isoform lacking
the LBD was first described in 22Rv1 cells that coexpressed
full-length and truncated AR proteins.15 AR splice variants in
prostate cancer were first described by Dehm et al. at the
Mayo Clinic in 2008.16 Using a castration-resistant 22Rv1
cell line derived from a mouse xenograft, the investigators
observed short-isoform AR variants in which the CTD has
been truncated. They showed that these variants are constitu-
tively active in the absence of ligands and successfully bound
AREs, thus demonstrating a new mechanism for castration-
resistant tumor growth. Simultaneous studies carried out by
Hu et al. at Johns Hopkins confirmed the presence of AR
splice variants (particularly AR-V1 and AR-V7) in tumor
samples from CRPC patients, and showed that their expres-
sion was approximately 20-fold higher in castration-resistant

T
T T
T
T
T

AR-FL
ne
bra
mem T
Cell T T

AR-V7
T
T

T T

AR-V7/
AR-V7 AR-FL AR-FL
leus Homodimer
Nuc Homodimer
T
Heterodimer
Fig. 1 Interactions between the androgen
T
receptor, its ligands and DNA. AR-FL binds to
Co-regulators Co-regulators Co-regulators testosterone in the cytoplasm, and then
translocates to the nucleus where it dimerizes
and binds to DNA to initiate transcription. AR-V7
can localize to the nucleus independent of ligand,
and dimerize with AR-FL or itself to initiate
transcription of AR-responsive target genes.

(a) 1.1 kb 5’-UTR 2.7 kb ORF 6.8 kb 3’-UTR

Translation Translation
start stop
Exons
1 2 3 4 5 67 8 Fig. 2 Androgen receptor gene structure. (a)
(b) The full-length AR gene is composed of eight
exons with a 2.7-kb open reading frame. (b) The
LBD/AF-2 AR protein components as defined by the NTD,
919 DBD and CTD. In AR splice variants, the LBD is
Zn Zn Hinge truncated, leading to ligand-independence and
1 538 627 loss of the binding site for all current AR
NTD DBD CTD inhibitors.

2 © 2016 The Japanese Urological Association


tissues compared with hormone-sensitive tissues.17 Since
then, multiple additional AR splice variants have been identi-
fied, with at least 22 now being recognized.18 Most of these
variants are characterized by loss of the CTD (and the LBD)
with maintenance of the NTD, resulting in ligand-indepen-
dent AR pathway activation. For a more extensive review of
these variants, the reader is referred elsewhere.10,19
The modular design of the AR allows variant splicing to
have a profound effect on AR function. AR genomic dele-
tions in non-coding regions drive some of the variants in
prostate cancer cell lines, but do not appear to be a mecha-
nism of AR splice variant generation in humans.20 Splice
variants have been shown to arise as a response to androgen
deprivation, and always exist in the presence of AR-FL
mRNA expression.21 This suggests that splice variants are an
adaptive response to AR-directed therapy. Cell line studies
have shown that splice variants can induce resistance to enza-
lutamide, one of the most potent anti-androgens.20 Of all the
variants, AR-V7 has been the most extensively studied, and
is the only one to be reproducibly identified in clinical sam-
ples because of its high abundance relative to other AR vari-
ants.19,22 It is generated from abnormal splicing of the third
intronic sequence of the AR pre-mRNA, resulting in abnor-
mal retention and translation of a cryptic exon that encodes a
premature stop codon 16 amino acids after exon 3.23 AR-V7
is also the first splice variant to show potential relevance as a
biomarker for response/resistance to currently available CRPC
therapies, and has been reliably measured in several body
compartments including circulating tumor cells as well as
whole-blood RNA.
AR-V7 and therapeutic resistance
Impact on androgen pathway drugs
One of the first blood-based assays to reliably detect AR-V7
was developed by Jun Luo et al. at Johns Hopkins using
CTCs. This assay used EpCAM-based CTC enrichment fol-
lowed by cell lysis to harvest mRNA, and then used a cus-
tom primer set to detect AR-V7 mRNA by RT–PCR.24 Using
this assay, Antonarakis et al. showed the ability to predict
primary resistance to abiraterone and enzalutamide in the
clinical setting.25 The investigators prospectively enrolled 62
patients with mCRPC who were about to be treated with abi-
raterone or enzalutamide as standard of care. Prior treatment
with chemotherapy as well as other androgen pathway
Table 1 Response to treatment in AR-V7-expressing prostate cancer
Prevalence of
Study Therapeutic AR-V7 (%)
Antonarakis et al.25 Abiraterone 19%
Enzalutamide 39%
Steinestel et al.27 Abiraterone or enzalutamide 64%
Todenhofer et al.28 Abiraterone 11%
Antonarakis et al.29 Docetaxel or cabazitaxel 46%
Onstenck et al.30 Cabazitaxel 55%
© 2016 The Japanese Urological Association
AR-V7 in CRPC: Clinical considerations
inhibitors was allowed, including prior abiraterone or enzalu-
tamide. AR-V7 status was measured at baseline and at sev-
eral post-treatment time-points. Patient outcomes were
assessed according to modified Prostate Cancer Working
Group 2 criteria.
A total of 71 patients were screened to identify 62 patients
with detectable CTCs (nine blood samples did not yield
CTCs). A total of 31 patients were then enrolled to each of
the abiraterone and enzalutamide arms. AR-V7 was detect-
able in 39% of the enzalutamide-treated patients and 19% of
abiraterone-treated patients. The presence of AR-V7 corre-
lated with prior AR-directed therapies, with AR-V7 being
detectable in 55% of patients who had received prior abi-
raterone, versus 9% who had not. Enzalutamide pretreatment
increased the rate of AR-V7 to 50%, compared with 15% in
enzalutamide-na€ıve patients. In all cohorts, AR-V7 presence
was also strongly associated with overexpression of AR-FL
transcripts.
Clinical responses were inversely correlated with AR-V7
presence. In the abiraterone cohort, the PSA response rate
was 0% in the presence of AR-V7 versus 68% in its absence.
Similarly with enzalutamide, the response rate was 53 versus
0% in the presence and absence of AR-V7, respectively
(Table 1). Both PSA-PFS and radiographic PFS were nega-
tively correlated with AR-V7 status, as was OS.
When analyzing AR-V7, AR-FL levels and prior abi-
raterone/enzalutamide treatment using a multivariable Cox
model, AR-V7 status was independently predictive for PFS.
Significantly, prior use of either abiraterone or enzalutamide
before the other was not independently predictive of shorter
PFS after accounting for AR-V7. Given the many reports
showing poor outcomes with sequential use of abiraterone
and enzalutamide or vice versa, this suggests that identifica-
tion of specific mechanisms of resistance including AR-V7
can predict for lack of efficacy of the second AR-directed
agent.26 Conversely, absence of AR-V7 after treatment with
abiraterone or enzalutamide might help inform which patients
might have the best chance of responding to sequential AR-
targeting therapies.
The negative prognostic impact of AR-V7 detection on
response to AR-directed therapies has subsequently been con-
firmed in at least two additional studies. In the first study,
Steinestel et al. also used a CTC-derived mRNA-based
AR-V7 assay in mCRPC patients starting treatment with
abiraterone or enzalutamide.27 The authors detected AR-V7
PSA response in AR-V7
+ vs AR-V7 patients (%) AR-V7 assay
0% vs 68% (P < 0.01) CTC-derived mRNA (AdnaTest; Qiagen, Hilden, Germany)
0% vs 53% (P < 0.01)
7% vs 63% (P = 0.01) CTC-derived mRNA (AdnaTest; Qiagen, Hilden, Germany)
0% vs 42% (P = 0.04) Whole-blood mRNA (PAXgene; PreAnalytiX,
Hombrechtikon, Switzerland)
41% vs 65% (P = 0.19) CTC-derived mRNA (AdnaTest; Qiagen, Hilden, Germany)
8% vs 22% (P = 0.70) CTC-derived mRNA (CellSearch; Janssen, Horsham, PA, USA)
3
AH BRYCE AND ES ANTONARAKIS
mRNA in 18 of 37 patients (48%) with evaluable CTCs. In
this diverse patient population (where prior therapies ranged
from treatment-na€ıve patients, to patients previously treated
with ADT alone, to ADT and chemotherapy, ADT and abi-
raterone or enzalutamide, and ADT plus chemotherapy plus
abiraterone or enzalutamide), AR-V7 was not detected in any
patients who were treatment-na€ıve, and was more prevalent
with more lines of prior therapy. This supports the idea that
AR-V7 might be an adaptive response to hormonal therapy.
With regard to prognostic significance, 10 out of 14 patients
(71%) who were AR-V7-negative responded to subsequent
hormonal therapy, whereas only one out of 15 AR-V7-posi-
tive patients (7%) responded to the next hormonal therapy.
In a more recent study, Todenh€ ofer et al. developed a
whole-blood RNA assay to detect AR-V7 mRNA from
mCRPC patients starting abiraterone treatment.28 In that
study, the authors used 2.5 mL of whole blood in PaxGene
tubes to carry out RT–PCR for a panel of transcripts includ-
ing AR-V7. Samples were collected from patients who had
received prior docetaxel chemotherapy, but had not received
abiraterone or enzalutamide. All patients were then treated
with standard-of-care abiraterone. Four out of 37 patients
(11%) expressed AR-V7. The proportion of patients with a
50% decline in their PSA was 0% versus 42% for AR-V7-
positive versus negative patients. The median PFS and OS
were also significantly inferior in AR-V7-positive patients
compared with negative patients (0.7 months vs 4.0 months
for PFS, and 5.5 months vs 22.1 months for OS). One poten-
tial advantage of the whole-blood RNA approach is that the
AR-V7 status can be determined even in patients who do not
have detectable CTCs, in whom the two previously described
assays would not have yielded an evaluable result.
Impact on taxane chemotherapy
At present, there are conflicting data about the prognostic
impact of AR-V7 detection on response to taxane chemother-
apy. Antonarakis et al. studied a series of 37 patients in a
similar fashion to the abiraterone/enzalutamide study dis-
cussed above.29 That study evaluated men with mCRPC who
had progressive disease, detectable CTCs and were about to
receive docetaxel or cabazitaxel chemotherapy as standard of
care. Prior abiraterone or enzalutamide were allowed, and all
patients receiving cabazitaxel had previously received prior
docetaxel.
CTCs were sampled at baseline, at the time of clinical or
biochemical response, and at progression. The primary end-
point was a PSA response of ≥50%, with the sample size cal-
culated to detect a 30% difference in response rate (45% in
AR-V7 negative, 15% in AR-V7 positive). PSA-PFS and
radiographic PFS as well as OS were the secondary
end-points.
The study enrolled 30 men treated with docetaxel and
seven men treated with cabazitaxel. A total of 17 of 37
patients (46%) had detectable AR-V7 at baseline. AR-V7
was positive in seven of 14 men (50%) who had received
prior abiraterone or enzalutamide, and eight of 15 men (53%)
who had received both. Just two of eight men (25%) who
had received neither drug had detectable AR-V7.
4 © 2016 The Japanese Urological Association
PSA response was seen in seven of 17 (41%) AR-V7 posi-
tive patients, and 13 of 20 (65%) of AR-V7 negative patients.
Using multivariable logistic regression modeling, AR-V7 did
not predict for PSA response with an odds ratio of 0.39
(P = 0.31). PSA-PFS was also not significantly different
between AR-V7-positive and negative men, at 4.5 months
and 6.1 months, respectively. Radiographic PFS was also not
significantly different, at 5.1 months and 6.9 months. When
these differences were corrected for prior use of abiraterone
and/or enzalutamide, AR-V7 remained non-predictive for any
of the clinical outcomes. Based on these data, the authors
concluded that AR-V7 expression did not appear to influence
responses to taxane chemotherapies to the same degree that it
influenced responses to abiraterone and enzalutamide. As a
result, they suggested that AR-V7 might serve as a treatment
selection marker to help guide treatment decisions: in AR-
V7-positive men, taxanes would appear to be more effective
than AR-directed therapies; whereas in AR-V7-negative men,
AR-targeting drugs would be equally effective as taxanes.
These results were corroborated by a separate group in a
subsequent study evaluating AR-V7 in the setting of cabazi-
taxel chemotherapy. To this end, Onstenck et al. developed a
CTC-derived mRNA-based AR-V7 assay and prospectively
evaluated 29 patients embarking on therapy with cabazi-
taxel.30 AR-V7 was detected in 16 of 29 patients (55%) who
had at least 10 CTCs per 7.5 mL of blood. Consistent with
the results using docetaxel described above, AR-V7 positivity
did not confer any difference in PSA response rate, CTC
response rate, PFS or OS. The cumulative clinical data to
date therefore suggest that taxane efficacy might be indepen-
dent of AR-V7 detection.
In contrast, recently reported cell line data suggest that AR
splice variants could help circumvent AR blockade by tax-
anes.31 Zhang et al. first showed induction of AR-V7 in tax-
ane-resistant LNCaP95 and 22Rv1 cell lines. They then
transfected AR-V7 and ARv567es variants into AR-V-null
LNCaP cells and tested for sensitivity to taxanes. Their data
showed increased cell viability of AR splice variant trans-
fected cells over AR-FL cells lines across increasing doses of
taxane exposure, as well as correspondingly higher AR tran-
scriptional activity in splice variant-expressing cells. They
then used enhanced green fluorescent protein-tagged AR-V7
and AR-FL to study the cellular localization of the proteins.
Their results suggest that AR-V7 is able to spontaneously
translocate to the nucleus in a microtubule-independent man-
ner, whereas AR-FL cannot. Consequently, taxane exposure
resulted in the accumulation of AR-FL in the cytoplasm with
no such impact on AR-V7. Finally, these authors showed that
the microtubule-binding domain of AR-FL is contained
within the LBD. Thus, AR-V7 does not bind to the micro-
tubules and, more intriguingly, AR-V7 can complex with
cytoplasmic AR-FL to allow AR-FL to traffic to the nucleus
in a microtubule-independent manner.
Thus, the full impact of AR-V7 on taxane efficacy is still
being elucidated. That the effect of AR-V7 positivity is far
greater in AR-directed therapies than taxanes appears clear,
but it might be that there is partial resistance to taxane
chemotherapy. The collective clinical data discussed above
are not sufficiently powered to detect small differences in

prognosis, and thus the clinical results might not necessarily
be in conflict with the cell line data. Further studies to answer
this question are ongoing and are described in more detail
below.
Serial measurement of AR-V7
In accordance with the evidence that AR-V7 is an inducible
biomarker, the Johns Hopkins group has recently shown that
AR-V7 status can change through an individual patient’s
treatment trajectory. In the initial abiraterone/enzalutamide
study, 42 men with undetectable AR-V7 at baseline had sub-
sequent blood available for analysis. Of these, six men (14%)
ultimately converted to AR-V7-positive during treatment with
enzalutamide (four cases) or abiraterone (two cases). These
patients had worse outcomes than those who remained AR-
V7-negative throughout therapy. Interestingly, none of the
patients with positive AR-V7 status at baseline converted to
negative during treatment with abiraterone/enzalutamide.
In another study from the same group, the investigators
evaluated 25 men with sequential assessments of AR-V7 sta-
tus. The study focused on men that had received at least four
separate AR-V7 assessments across at least two systemic ther-
apies for metastatic prostate cancer. Treatments might have
included any of ADT, abiraterone, enzalutamide or taxane
chemotherapy. The purpose was to evaluate the change in
AR-V7 over time.21 Of the 25 men, 14 had AR-V7 in at least
one blood sample. Of those, two were hormone-sensitive at
study entry. Three patients were AR-V7-positive in all of their
samples. The other 11 patients experienced at least one
change in their AR-V7 status. Five patients converted from
AR-V7-negative to positive during treatment. One reverted
from AR-V7-positive back to negative. The remaining five
experienced both a conversion and a reversion at different
time-points. All reversions from positive to negative AR-V7
status occurred during taxane chemotherapy, whereas conver-
sions from negative to positive status occurred primarily with
hormonal agents (ADT as well as abiraterone/enzalutamide).
AR-V7 reversions were also seen in the taxane study men-
tioned previously.29 In that study of 37 men, 21 patients had
paired CTCs at baseline and at progression. Nine were AR-
V7-negative at baseline, of which only one (11%) converted
to AR-V7-positive on taxane progression. Of the 12 men
who were AR-V7-positive at baseline, seven (58%) converted
to negative with taxane therapy. These reversions are particu-
larly interesting, as they theoretically suggest the possibility
that chemotherapy might re-establish sensitivity to AR-direc-
ted agents if initial resistance was driven by the emergence of
AR-V7. Formal evaluation of this hypothesis is ongoing, and
the clinical significance of transitions in AR-V7 status (posi-
tive to negative, or vice versa) remains poorly understood at
this time.
Impact of AR-V7 in different therapeutic
contexts
Although the evidence suggests that at least part of the effect
of taxane chemotherapy on prostate cancer is exerted through
the androgen axis through impaired AR trafficking, and this
© 2016 The Japanese Urological Association
AR-V7 in CRPC: Clinical considerations
could indicate that AR variants might affect taxane efficacy,
no such evidence is present in the current clinical data.32,33
The prognostic utility of AR-V7 appears limited to abi-
raterone and enzalutamide, with the impact on chemotherapy
perhaps existing as a byproduct of pretreatment. AR-V7-posi-
tive patients might benefit from chemotherapy, but receive lit-
tle to no benefit from AR-directed agents. In contrast, AR-
V7-negative patients appear to benefit equally from either
chemotherapy or AR-directed agents.29
Similarly, there is a differential impact of treatment type
on the expression of ARV7. Whereas the androgen pathway
inhibitors will induce the expression of AR-V7, chemother-
apy can often suppress AR-V7 expression and even cause
reversion of AR-V7-expressing disease back to AR-V7-nega-
tive disease. These two trends combine to suggest the possi-
bility of rational sequencing of drugs based on AR-V7
expression. The evidence to date favors the use of taxane
chemotherapy if AR-V7 positivity develops as a result of abi-
raterone or enzalutamide for mCRPC. Similarly, if AR-V7 is
expressed after taxane chemotherapy, then abiraterone and
enzalutamide are less optimal, and next-line treatment should
consist of further chemotherapy or another non-AR therapy.
In contrast, if AR-V7 negativity can be restored by
chemotherapy, then abiraterone of enzalutamide sensitivity
might also be theoretically restored.34 However, despite these
intriguing hypotheses, additional prospective trials will first
be required before AR-V7 testing is ready to be used for clin-
ical decision-making.
Ongoing clinical studies
Biomarker validation of AR-V7
The foremost priority for establishing the clinical utility of
AR-V7 as a predictive marker is a randomized, prospective
clinical trial with AR-V7 as an integral biomarker. The exist-
ing preliminary data, while compelling, are not sufficiently
robust to establish AR-V7 as a definitive standard-of-care test
at this time. Hopefully, the recently opened PRIMCAB study
(NCT02379390) will be able to satisfy this need.
PRIMCAB will enroll 274 chemotherapy-na€ıve men with
mCRPC who develop progression within 6 months of begin-
ning abiraterone or enzalutamide treatment. Patients will then
be randomized to receive cabazitaxel or the other AR-directed
drug (abiraterone-resistant men will receive enzalutamide, and
vice versa). AR-V7 in CTCs will be assessed in all patients
using the Johns Hopkins CTC-enriched mRNA-based assay.
The primary end-point of this trial is radiographic PFS. If the
robust differences seen in the single institution data are correct,
then this study should be sufficiently powered to establish the
ability of the AR-V7 assay to predict for treatment selection of
chemotherapy versus AR-directed therapy in this context.
For a detailed account of additional trials aiming to clinically
qualify the AR-V7 biomarker in a variety of therapeutic con-
texts, we refer the readers to a recent review on this topic.35
Therapeutic targeting of AR-V7
Multiple novel agents are currently being developed as poten-
tial non-chemotherapeutic approaches to overcome AR splice
5
AH BRYCE AND ES ANTONARAKIS
variant-driven disease. Galeterone (formerly TOK-001) is one
potential AR-V7-targeting drug that is the most advanced in
its clinical development. In addition to inhibiting the CYP17
enzyme as well as the AR ligand-binding domain, galeterone
is able to accelerate the degradation of AR-FL and AR-V7 in
a proteasome-dependent fashion.36 These encouraging pre-
clinical data have led to the design of the phase III
ARMOR3-SV clinical trial (NCT02438007), which is a regis-
tration study testing galeterone versus enzalutamide in AR-
V7-expressing mCRPC patients. In the phase II ARMOR2
study, splice variant expression was indirectly assessed using
an immunohistochemical protein-based assay to detect AR C-
terminal loss. Seven of the enrolled patients tested positive
by this assay, of which six (86%) experienced a >50% PSA
reduction, indirectly suggesting that galeterone might have
activity in AR-V7-positive patients.37 The ARMOR3-SV
study will utilize a RT–PCR AR-V7 assay, with 148 AR-V7-
positive men then being randomized equally to galeterone
versus enzalutamide; the primary end-point is centrally-
assessed radiographic PFS.
ASO targeting the AR have also shown the ability to target
AR splice variants, and AZD 5312 is now being tested in a
phase I study (NCT02144051). Gleave et al. have reported
cell line data and in vivo mouse data using three ASOs tar-
geting exon 1, intron 1 and exon 8 of AR pre-mRNA.38
Enzalutamide-resistant cell lines, which express both AR-FL
and AR-V7, were cultured and transfected with AR-ASO,
then assessed for proliferation and AR-FL and AR-V7
expression levels. In vivo studies in patient-derived tumor
xenografts were carried out with MR49F LNCaP. As
expected, exon 1 and intron 1 targeted ASOs successfully
reduced expression of AR-V7 and AR-FL, whereas the
exon 8 ASO was specific for AR-FL. All of the AR-ASOs
successfully induced apoptosis in AR-dependent cell lines,
with no impact on the control group of AR-negative PC3 cell
lines. Although there was a differential effect on AR-FL and
AR-V7 according to the ASO used, there was no differential
effect on cell death, thus suggesting that most AR-driven cell
growth is dependent on AR-FL. Interestingly, selective
knockdown of AR-V7 (without knockdown of AR-FL) inhib-
ited cell growth in only some of the AR-V7-expressing cell
lines, suggesting that the biological significance of AR-V7
varies across different cell lines. The in vivo studies similarly
showed equal tumor reduction using exon 1 and exon 8
ASOs, with the expected selectivity for knockdown of AR-
V7 and AR-FL. Here again, the suggestion is that biological
activity of the AR-V7-positive tumors is largely driven
through AR-FL.
Selective targeting of the AR-NTD is another strategy that
is theoretically attractive for inhibiting AR splice variants as
well as AR-FL. All known AR splice variants maintain the
full-length NTD. Furthermore, as the NTD contains the ARE
binding portion of the AR, all transcriptionally active AR
variants must maintain an intact NTD. One family of mole-
cules potentially exploiting this strategy are the EPI com-
pounds. These are stereoisomers of EPI-001, which
covalently bind to the AR-NTD, and inhibit coactivator
recruitment and transactivation of AR target genes.39 Cell line
studies with AR-dependent LNCaP cells and AR-independent
6 © 2016 The Japanese Urological Association
PC3 cell lines show selective inhibition only of LNCaP cells
with exposure to EPI molecules. In addition, VCaP xenograft
mouse models, which express AR-V7 in addition to AR-FL,
are also inhibited by the EPI compounds. To this end, EPI-
506 is an oral prodrug of EPI-002 that has now entered
phase I testing in patients with mCRPC who have been pre-
viously treated with abiraterone, enzalutamide or both
(NCT02606123).
Another epigenetic strategy to inhibit the AR-NTD is to
target BRD4. BRD4 is a bromodomain/BET family protein
that interacts with the AR-NTD to facilitate AR-mediated
gene transcription as part of a super-enhancer complex.
Promising preclinical data have shown the efficacy of BRD4
targeting in CRPC cell lines and xenografts with reduction in
cell growth and AR-driven transcription.40,41 Multiple BET
inhibitors are currently being tested in phase I clinical trials,
including several trials that involve mCRPC patients
(NCT01587703, NCT02259114).
Another promising agent discovered through large scale
drug screening is the antihelminthic agent, niclosamide.
In vitro and in vivo studies using CRPC cell lines showed
downregulation of AR-V7 with exposure to niclosamide. This
appears to occur through proteasome-dependent protein
degradation of AR-V7. Furthermore, the addition of niclosa-
mide to enzalutamide led to inhibition of enzalutamide-
resistant cell lines.42 On this basis, an ongoing trial is testing
the combination of niclosamide and enzalutamide in abi-
raterone-refractory patients who test positive for AR-V7
(NCT02532114). A favorable result in this pilot study would
trigger a randomized phase II study of enzalutamide versus
enzalutamide plus niclosamide in AR-V7-positive prostate
cancer.
Yet another potential approach to target AR-V7-positive
CRPC might involve the use of combined immune check-
point blockade with ipilimumab plus nivolumab. It has
recently been proposed that colorectal and other cancers that
harbor DNA mismatch repair defects (i.e. microsatellite insta-
bility) might respond more favorably to immune checkpoint
blockade, because these cancers have a higher mutational
load that might create a larger number of neo-epitopes that
could be recognized by an activated immune system.43 Along
these lines, emerging molecular data are beginning to suggest
that AR-V7-positive prostate cancers might be associated
with higher numbers of mutations in homologous recombina-
tion and DNA mismatch repair pathways.44 To this end, the
Johns Hopkins group has recently initiated a small single-
center phase II study of ipilimumab plus nivolumab for
AR-V7-positive prostate cancer (NCT02601014); this trial
will also test for tumoral PD-L1 expression using immunohis-
tochemistry, and will also undergo somatic genetic sequenc-
ing to evaluate potential DNA repair mutational signatures.
Conclusion
Accumulating evidence suggests that AR-V7 is a common
adaptive response to androgen-directed therapies in mCRPC,
and might be a more relevant marker of therapeutic resistance
to abiraterone and enzalutamide than to taxane chemothera-
pies. Although the exact clinical utility of AR-V7 as a

biomarker and the impact on various available therapies
needs to be further established in prospective well-powered
trials, efforts are already underway to develop novel thera-
peutic strategies targeting AR-V7-positive disease. In addi-
tion, the optimal method to detect and quantify AR-V7
remains unclear, and multiple efforts are underway to mea-
sure this biomarker in tissue, CTCs, whole-blood RNA and
cell-free tumor mRNA. Finally, the clinical relevance of AR-
V7 must always be interpreted in the context of multiple
other competing and overlapping resistance mechanisms at
play in patients with metastatic castration-resistant prostate
cancer.
Acknowledgments
ESA has received funding from the Prostate Cancer Founda-
tion (PCF), the Patrick C. Walsh Fund, and NIH grants R01
CA185297 and P30 CA006973.
Conflict of interest
ESA has served as a paid consultant/advisor for Janssen,
Astellas, Sanofi, Dendreon, Essa and Medivation; has
received research funding to his institution from Janssen,
Johnson & Johnson, Sanofi, Dendreon, Exelixis, Genentech,
Novartis and Tokai; and is a co-inventor of a biomarker tech-
nology that has been licensed to Tokai. AHB declares no
conflict of interest.
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