Differential androgen receptor signals in different cells explain
why androgen-deprivation therapy of prostate cancer fails
Y Niu1,2,5, T-M Chang1,5, S Yeh1, W-L Ma1,3, YZ Wang4and C Chang1
1Departments of Pathology, Urology, Radiation Oncology and Wilmot Cancer Center, George Whipple Laboratory for Cancer
Research, University of Rochester Medical Center, Rochester, NY, USA;2Sex Hormone Research Center, Tianjin Institute of
Urology, Tianjin Medical University, Tianjin, China;3Sex Hormone Research Center, China Medical University/Hospital, Taichung,
Taiwan, ROC and4Department of Urological Sciences, University of British Columbia, Vancouver, Canada
Prostate cancer is one of the major causes of cancer-
related death in the western world. Androgen-deprivation
therapy (ADT) for the suppression of androgens binding
to the androgen receptor (AR) has been the norm
of prostate cancer treatment. Despite early success to
suppress prostate tumor growth, ADT eventually fails
leading to recurrent tumor growth in a hormone-
refractory manner, even though AR remains to function
in hormone-refractory prostate cancer. Interestingly,
some prostate cancer survivors who received androgen
replacement therapy had improved quality of life without
adverse effect on their cancer progression. These con-
trasting clinical data suggest that differential androgen/
AR signals in individual cells of prostate tumors can
exist in the same or different patients, and may be used
to explain why ADT of prostate cancer fails. Such a
hypothesis is supported by the results obtained from
transgenic mice with selective knockout of AR in prostatic
stromal vs epithelial cells and orthotopic transplants of
various human prostate cancer cell lines with AR over-
expression or knockout. These studies concluded that AR
functions as a stimulator for prostate cancer proliferation
and metastasis in stromal cells, as a survival factor
of prostatic cancer epithelial luminal cells, and as a
suppressor for prostate cancer basal intermediate cell
growth and metastasis. These dual yet opposite functions
of the stromal and epithelial AR may challenge the current
ADT to battle prostate cancer and should be taken into
consideration when developing new AR-targeting thera-
pies in selective prostate cancer cells.
Oncogene advance online publication, 3 May 2010;
Keywords: prostate metastasis; anti-androgen therapy;
androgen replacement therapy
Prostate cancer is the second leading cause of cancer-
related death among men in the United States (Jemal
et al., 2005). Approximately 80–90% of prostate cancers
are dependent on androgens at initial diagnosis. Since
the discovery by Huggins and Hodges (1941) that
prostate cancer progression is influenced by androgen
actions, androgen-deprivation therapy (ADT) to sup-
press androgens binding to androgen receptor (AR)
remains the major treatment regimen for the disease
(Denis and Griffiths, 2000). However, ADT ultimately
fails, and prostate cancer progresses to a hormone-
refractory (androgen-independent) state with advanced
metastasis and high morbidity and mortality.
Androgens function mainly through an axis involving
the testicular synthesis of testosterone, its transport to
target tissues (Roy et al., 1999), and the conversion by 5a-
reductase to the more active metabolite, 5a-dihydrotes-
tosterone (Shimazaki et al., 1965; Anderson and Liao,
1968; Bruchovsky and Wilson, 1968). Testosterone and
through binding and transactivating AR (Heinlein and
Chang, 2002; Heinlein and Chang, 2004; Rahman et al.,
2004; Wang et al., 2005), which involves interaction of AR
with various coregulators during prostate development
and prostate cancer progression (Heinlein and Chang,
2002; Rahman et al., 2004; Wang et al., 2005).
AR is expressed throughout prostate cancer progres-
sion and its expression persists in the majority of
patients with hormone-refractory disease (Cunha et al.,
1987; Sadi et al., 1991; van der Kwast et al., 1991;
Chodak et al., 1992; Hobisch et al., 1996; Mohler et al.,
1996; Buchanan et al., 2001), and many AR mutations
identified from hormone-refractory prostate tumors are
capable of transactivation. These observations suggest
that the eventual failure of ADT cannot be attributed
simply to the loss of AR function.
Prostate cancer patients undergoing ADT often
develop hypogonadism, which is associated with sexual
dysfunction, decreased lean body mass and muscle
life, and osteoporosis. They also have a higher risk
of developing metabolic syndrome,
cardiovascular diseases (Basaria, 2008; Taylor et al.,
2009). Several studies have indicated that androgen
reduced quality of
Received 11 December 2009; revised 10 March 2010; accepted 16 March
Correspondence: Dr C Chang, Departments of Pathology, Urology,
Radiation Oncology and Wilmot Cancer Center, George Whipple
Laboratory for Cancer Research, University of Rochester Medical,
601 Elmwood Avenue, Box 626, Rochester, NY 14642, USA.
5These authors contributed equally to this work.
Oncogene (2010), 1–12
& 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10
replacement therapy (ART) of selected patients with
castration-resistant prostate cancer led to improved
quality of life without any adverse effect on their cancer
progression for a considerable follow-up duration. Some
of the selected patients even displayed a decrease in their
serum prostate-specific antigen levels after ART (see
details in section ‘ART in prostate cancer patients’),
indicating that there might be reduced cancer progres-
sion. However, the reasons behind the failure of ADT
and why there are differential responses to androgen/
AR signals in different prostate cancer patients remain
unclear at present.
We will use this review to present our hypothesis
based on the recent findings and conclude that AR
can function as both a proliferation stimulator and a
tumor suppressor depending on its expression in indivi-
dual prostatic cells to exert its diverse and differential
functions for prostate cancer progression. These dual yet
opposite functions of AR may thus create a direct
challenge to the current ADT and promote new
therapies in the battle against prostate cancer.
Human clinical studies with ADT and ART for prostate
cancer-differential response to androgen/AR signaling
ADT in prostate cancer patients
The proliferation-stimulating function of AR is at the
center of the premise for ADT in the treatment of
prostate cancer (Huggins and Hodges, 1941). ADT with
either surgical or medical castration usually results in a
response rate of 70–80% with a 12–33 months duration
of progression-free survival (Bruchovsky, 1993). How-
ever, after an average of 24 months, the tumors almost
always recur and no longer respond to ADT (Eisenberger
et al., 1998), even though the prostate tumors still express
AR (Visakorpi et al., 2005; Mostaghel et al., 2007).
Interestingly, cell sorting of these ADT-refractory tumors
found that the prostatic epithelial basal cell marker,
cytokeratin 5 (CK5) (Bruchovsky et al., 1990; van Leender
et al., 2001) increased from 29 to 75%, an observation
consistent with the expansion of basal intermediate-like
tumor cells observed in transgenic adenocarcinoma mouse
prostate (TRAMP) mice with selective knockout of AR
(ARKO) in prostatic epithelium (pes-ARKO-TRAMP) and
inducible ARKO-TRAMP (ind-ARKO-TRAMP) mice
(see details in section ‘AR dual functions in prostate cancer
progression and metastasis’). Recent clinical findings from
254 prostate cancer patients also indicated that the
expression of nestin, which is linked to the metastatic
potential of prostate cancer cells, is present only in the
metastatic tumors of patients receiving ADT, suggesting
that treatment with ADT might result in promotion of
prostate metastatic tumors in these patients (Kleeberger
et al., 2007).
Together, these human clinical data suggest that ADT
may be effective for prostate cancer patients only in
selective prostate tumor cells and time periods, beyond
which tumors will progress into the hormone-refractory
stage with a more aggressive metastasis.
ART in prostate cancer patients
Several studies have shown that ART of hypogonadal
patients with localized prostate cancer treated with
radical prostatectomy or
effect on prostate cancer progression (Kaufman and
Graydon, 2004; Agarwal and Oefelein, 2005; Ferreira
et al., 2006; Khera et al., 2009). Fowler and Whimore,
(1981) observed that out of 52 metastatic prostate
cancer patients who received ART, 45 exhibited
increased cancer progression that could be reversed by
androgen withdrawal, whereas 7 experienced sympto-
matic benefits. Other investigators have observed that in
patients with castration-resistant metastatic prostate
cancer, ART resulted in either little adverse effect on
cancer progression or displayed some biochemical
improvement or progression (Mathew, 2008; Morris
et al., 2009; Szmulewitz et al., 2009). These clinical
studies suggested that prostate cancer patients have
differential responses to androgen/AR signals and ART
might be able to improve the quality of life in selective
prostate cancer patients with little impact on further
progression of their prostate cancers.
Differential androgen/AR signals in various prostate
tumor cells from the same patient
The above clinical data led us to hypothesize that
differential androgen/AR signals in individual cells of
the prostate tumor might exist in the same or different
patients, and may explain why ADT of prostate cancer
fails. This hypothesis is supported by the isolation
of three prostate primary tumor lines, PCa1, PCa2, and
PCa3, from the same patient that exhibited differential
responses to the androgen/AR signals after ortho-
topic implantation into androgen-supplemented vs
castrated SCID mice (Figure 1) (Wang YZ, unpublished
data). Although both PCa1 and PCa2 cells express AR
and prostate-specific antigen, PCa1 cells grew more
rapidly, whereas PCa2 cells grew more slowly in
castrated than in androgen-supplemented mice, suggest-
ing that they are androgen-suppressed and androgen-
the growth of PCa3 cells was similar in castrated vs
androgen-supplemented mice, indicating that it is
androgen insensitive. These various responses of pros-
tate cancer cells to androgen/AR signals from the same
patient are very similar to the responses of hair follicles
to androgen/AR signals from the same person (Inui
et al., 2002): positive for the hair follicles in the
mustache area, negative for the hair follicles on the
top of the skull, toward the front, and independent
for the hair follicles on the back of the head.
These contrasting effects of androgen/AR signals in
the prostate tumor cells or hair follicles from the
same person (Figure 2) strengthen our hypothesis that
differential androgen/AR signals exist in various cells
of prostate tumors to explain why ADT of prostate
The following sections will summarize various in vitro
and in vivo evidences from mice and human prostate
cancer cell lines to support such a conclusion.
respectively. In contrast,
AR dual functions in prostate cancer therapy
Y Niu et al
AR dual functions in prostate cancer progression
Recent studies using epithelial-specific, fibroblast-specific,
and smooth muscle-specific ARKO mice have indicated
that during prostate development, AR in stromal cells
functions as a proliferation stimulator, whereas AR in
epithelial basal intermediate cells functions as a suppres-
sor, and in epithelial luminal cells functions as a survival
factor. (The detailed descriptions of these AR dual
functions in normal prostate development are described
in Supplementary Information). Here, we will focus on
discussion of the similar dual AR functions in prostate
cancer progression and metastasis.
Huggins and Hodges (1941) provided the first solid
in vivo evidence for androgen/AR signals to function as a
proliferation stimulator for prostate cancer progression,
which became a common view supported by the
subsequent ADT for treating prostate cancer patients
with early success. Nevertheless, how and in which cell(s)
AR may function as a proliferation stimulator of cancer
cells remain unclear. Here, we summarize recent findings
that suggest AR in stromal cells might function as a
stimulator, whereas AR in epithelial luminal-like cells
might function as a survival factor, and in epithelial basal
intermediate-like cancer cells, AR might function as a
suppressor of prostate cancer progression and metastasis.
AR in stromal cells: a stimulator for tumor progression
Stromal–epithelial interaction remains important for
tumor progression and metastasis (Cunha et al., 2003;
Bhowmick and Moses, 2005; Condon, 2005). During
tumor progression, the stromal cells, including fibroblasts,
myofibroblasts, endothelial, and immune cells, form a
skull to the forehead, back of the head, and mustache areas of the same person have differential responses to androgens, which are
similar to the differential responses of three prostate cancer sublines isolated from the same patient shown in Figure 1.
Similar differential responses of hair follicles and prostate cancer cells to androgens/AR signals. Hair follicles in the top of
pattern of the three tumor lines transplanted orthotopically into non-obese diabetic/severe combined immunodeficient (Nod/SCID)
mice supplemented with androgen (blue line) and in castrated hosts (purple line) were compared. (a) PCa1: an androgen-suppressed
subline; (b) PCa2: an androgen-stimulated subline; and (c) PCa3: an androgen-independent subline.
Three tumor sublines from the same prostate cancer patient had differential growth responses to androgen. The growth
AR dual functions in prostate cancer therapy
Y Niu et al
microenvironment supporting the progression, survival,
and metastasis of the tumor. In the normal human
prostate, the stroma is constituted mainly of smooth
muscle cells expressing AR. In prostatic carcinoma, the
tumor stroma is constituted mainly of fibroblastic and
myofibroblastic cells (Cunha et al., 2003), suggesting that
cell-transition changes have occurred in both the stroma
and the epithelium during tumorigenesis. Earlier tissue
recombination studies by Thompson et al. (1993) have
indicated that prostatic tumorigenesis indeed requires
changes in both the stroma and the epithelium, a notion
supported by the results of Cunha and co-workers (Olumi
et al., 1999; Wang et al., 2001; Cunha et al., 2003; Ricke
et al., 2006). Thus, prostate cancer-associated fibroblasts
could promote tumor transformation of the epithelial
BPH-1 cells that are immortalized with SV40 Tag (Olumi
et al., 1999; Wang et al., 2001). Similarly, urogenital
mesenchymes from rat and mouse could also elicite tumor
transformation in BPH-1 cells in the presence of
testosterone and 17b-estradiol (E2) (Wang et al., 2001;
Ricke et al., 2006). However, cancer-associated fibroblasts
in a castrated host and urogenital mesenchyme from
testicular feminized mice (a mouse without functional
AR) were unable to promote tumor transformation from
BPH-1 cells (Ricke WA, personal communication).
In addition, knockdown of AR in cancer-associated
fibroblasts isolated from tumors of TRAMP mice reduces
their ability to promote BPH-1 colony formation in soft
agar (Chang C et al., unpublished data). These observa-
tions indicate that the stromal AR is required for prostate
AR-negative epithelial PC-3 cells were co-cultured
with human prostatic stromal WPMY1-v or WPMY-
ARsi cells (Niu et al., 2008a) to investigate the function
of the stromal AR in tumor progression and metastasis.
The results from the co-culture system indicated that
knockdown of AR in WPMY1-ARsi cells resulted in the
co-cultured PC-3 cells being less invasive in a Boyden
chamber invasion assay than PC-3 cells with vector-
transfected WPMY1-v cells (Niu et al., 2008a). After
orthotopic inoculation in nude mice, PC-3 cells also
produced smaller primary and pelvic lymph node (PLN)
metastatic tumors when combined with WPMY1-ARsi
than with WPMY1-v cells (Niu et al., 2008b). Similar
in vivo androgen/AR-dependent tumor growth were also
observed using other AR-negative prostate cancer cell
lines (Marques et al., 2005; Halin et al., 2007), and
LNCaP cells might grow more aggressively on ortho-
topic transplantation after being co-inoculated with rat
urogenital mesenchyme or bone marrow fibroblasts
(Gleave et al., 1991). Together, these in vitro and
in vivo findings suggested that the stromal AR might
function as a proliferation stimulator to promote
prostate tumor progression and metastasis.
AR in epithelial cells: an anti-apoptosis survival
factor and a suppressor of proliferation for cancer
The TRAMP mouse model is a prostate cancer
model that develops prostate tumors spontaneously at
10–12 weeks of age (Gingrich et al., 1991). TRAMP mice
were mated with the floxed AR transgenic mice to obtain
TRAMP mice carrying the floxed AR transgene, which in
turn were crossed with probasin-Cre mice to generate
prostate epithelial-specific (pes)-ARKO-TRAMP mice
(Niu et al., 2008a,b). Prostatic epithelial cells develop
from stem cells through proliferation and differentiation
into basal and intermediate cells that finally differentiate
into epithelial luminal cells (Litvinov et al., 2003; Tokar
et al., 2005). AR is expressed in about 50% of the basal
cells and all of the luminal cells in mouse prostate
(Mirosevich et al., 1999; Niu et al., 2008a). Similar to pes-
ARKO mice (Wu et al., 2007), knocking out AR from
these epithelial cells in pes-ARKO-TRAMP mice and
others resulted in increased apoptosis of CK8-positive
epithelial luminal cells (18%) as compared with those
(2%) from wild-type TRAMP mice at 16 weeks of age
(Niu et al., 2008a), suggesting that AR in the epithelial
luminal cells may function as a survival factor to protect
prostate cancer cells from apoptosis.
However, knocking out the epithelial AR in pes-
ARKO-TRAMP mice also resulted in increasing num-
bers of proliferating cells in CK5-positive-basal cells,
including the CK5/CK8-double-positive-basal inter-
mediate cells. The consequence of increased apoptosis
in luminal cells and increased proliferation in basal
intermediate cells then resulted in less differentiated yet
larger primary tumors in the ventral prostate of pes-
ARKO-TRAMP mice than tumors from 16-week-old
TRAMP mice (Niu et al., 2008b). The primary prostate
tumors of pes-ARKO-TRAMP mice exhibited a higher
population of CD44-positive (Liu et al., 2004; Bhatia
et al., 2005) and CK5/CK8-positive (van Leenders and
Schalken, 2003; Bhatia et al., 2005) intermediate-like
cells than those found in wild-type TRAMP mice (Niu
et al., 2008a). Incidentally, CD44-positive, but AR-
negative prostate cancer cells purified from human
prostate cancer xenografts were also enriched in
tumorigenic and metastatic progenitor cells (Patrawala
et al., 2006). These results indicated knocking out the
epithelial AR might lead to cell population changes
and decreased secretory luminal cells in the prostates
of pes-ARKO-TRAMP mice (Niu et al., 2008a). These
results suggested that epithelial AR might function
as a proliferation suppressor in epithelial basal inter-
mediate cells and a survival factor in epithelial luminal
cells. As prostate carcinoma arises from epithelial
cells, the opposite functions of the epithelial AR in
different epithelial cells could then affect prostate
cancer progression in TRAMP mice by favoring survival
of differentiated tumor epithelium while suppressing
proliferation of epithelial basal intermediate cells,
thereby retarding tumor progression to a more malig-
AR in epithelial cells: a suppressor of prostate cancer
The AR signals in prostate cancer epithelial cells also
influence tumor metastasis. Thus, the size of metastatic
AR dual functions in prostate cancer therapy
Y Niu et al
tumors in PLNs of pes-ARKO-TRAMP mice was larger
than those from wild-type TRAMP littermates at the
age of 24 weeks. In addition, more prostate cancer
metastatic foci were observed within the livers of pes-
ARKO-TRAMP than in TRAMP mice (Niu et al.,
2008a). AR-negative PLN metastatic tumor isolated
from pes-ARKO-TRAMP mice was more invasive than
those from TRAMP mice in vitro. Importantly, restor-
ing the expression of AR by transfection could reduce
the invasiveness of PLN tumors from pes-ARKO-
TRAMP mice (Niu et al., 2008a). An early study also
found that poorly differentiated PLN metastatic tumors
in castrated TRAMP mice were more aggressive than
tumors from intact TRAMP mice (Gingrich et al.,
1991). Therefore, it may be concluded that the epithelial
AR also functions as a suppressor of prostate tumor
invasion and metastasis.
Relative influences of the stromal and epithelial AR
on prostate cancer progression and metastasis
The ind-ARKO-TRAMP (Niu et al., 2008b) mice were
then produced to assess the consequence of simulta-
neously knockdown of both the stromal and epithelial
AR that have opposite functions in prostate cancer
progression and metastasis. The knockout AR in ind-
ARKO-TRAMP mice is mediated by Mx1-Cre, which is
interferon inducible and can be activated by injection of
polyinosinic–polycytidic acid to induce endogenous
interferon and thus activate the Cre recombinase in
various tissues (Ku ¨ hn et al., 1995) including the prostate
(Wang et al., 2006; Niu et al., 2008b). After injection
of polyinosinic–polycytidic acid in 12-week-old mice,
AR mRNA expression in the prostate was found
knocked down by 50% in the stroma and 60% in the
epithelium in 16-week-old ind-ARKO-TRAMP mice
compared with polyinosinic–polycytidic acid-injected
control TRAMP mice. Knocking down prostatic AR
expression at this early stage resulted in smaller and less-
differentiated primary prostate tumors in ind-ARKO-
TRAMP than in the control TRAMP mice through
16–24 weeks of age. The tumors of ind-ARKO-TRAMP
mice had lower proliferation rates and higher apoptosis
rates than tumors of the control TRAMP mice. More-
over, the tumors of ind-ARKO-TRAMP mice had
decreased CK8-positive luminal-like cells and increased
CK5- and CD44-positive-basal cells and CK5/CK8-
double-positive-basal intermediate-like cells than tu-
mors of the control TRAMP mice (Niu et al., 2008b).
Despite the fact that both pes-ARKO-TRAMP and
ind-ARKO-TRAMP mice had higher epithelial luminal
cell apoptosis and expansion of basal intermediate-like
cells, pes-ARKO-TRAMP mice produced larger PLN
metastatic tumors, whereas knocking down AR at an
early stage in ind-ARKO-TRAMP mice produced
smaller metastatic tumors than the control mice. These
contrasting observations suggest that the stromal AR
may have more dominant functions during prostate
cancer progression at an early stage.
Metastatic tumors were also compared at the time
when primary tumors had reached 1cm2in TRAMP
(20 weeks), pes-ARKO-TRAMP (18 weeks), and
differentiated tumors of TRAMP mice developed small
metastatic tumors in the PLN. The poorly differentiated
tumors of pes-ARKO-TRAMP mice developed much
larger PLN metastatic tumors and metastasized into
multiple organs, whereas those of ind-ARKO-TRAMP
mice were smallest and they metastasized into the
seminal vesicle and liver. Thus, loss of the epithelial
AR promotes prostate cancer progression and meta-
stasis as shown in pes-ARKO-TRAMP, whereas con-
current knockdown of the stromal and epithelial AR at
an early stage can override these effects of the epithelial
ARKO, thereby retarding growth of the tumor and
suppressing their metastasis as shown in ind-ARKO-
More importantly, ind-ARKO-TRAMP mice with
early knockdown of prostatic AR had longer survival
time than wild-type TRAMP and pes-ARKO-TRAMP
mice (Niu et al., 2008b). However, the dominance of the
stromal AR over the epithelial AR function diminished
when ARKO in ind-ARKO-TRAMP mice was induced
after the primary tumor has progressed for some time.
Thus, when knockdown of AR in ind-ARKO-mice was
induced at 20 weeks of age (and not at 12 weeks of age as
mentioned above), the sizes of primary and PLN
metastatic tumors developed after 24 weeks of age were
similar between ind-ARKO-TRAMP and TRAMP mice,
suggesting that the relative influences of the stromal and
epithelial AR signals on prostate cancer progression and
metastasis can vary with the progression of the tumor
(Niu et al., 2008b).
Together, these observations not only support the notion
that the epithelial AR functions as a tumor suppressor for
prostate cancer progression and metastasis, but alsoindicate
that the stromal AR may function as a stimulator of the
prostate cancer progression and metastasis.
weeks) mice. The well-
Dual AR functions in human prostate cancer cell lines
The dual yet opposite AR functions to influence prostate
cancer cell proliferation and metastasis are also confirmed
in various human prostate cancer cell lines as follows.
PC-3 cells: AR functions as suppressor of proliferation
The PC-3 cell line was originally isolated from a human
bone marrow prostate metastastic tumor (Kaighn et al.,
1979). PC-3 cells express CK5 and CK8/18, but not AR.
Thus, PC-3 cells are basal intermediate-like tumor cells
(van Bokhoven et al., 2003) that are highly tumorigenic.
Early studies have reported that ectopic expression of AR
driven by a strong viral promoter in PC-3 cells resulted in
androgen-dependent suppression of cell proliferation
(Yuan et al., 1993; Garcia-Arenas et al., 1995; Heisler
et al., 1997). This androgen-suppressed cell growth in PC-
3 cells transfected with AR was confirmed later by
Litvinov et al. (2004, 2006) with a modified expression
vector for the ectopic AR expression. Interestingly, using
AR dual functions in prostate cancer therapy
Y Niu et al
a natural human AR promoter to drive the expression of
human AR in PC-3 cells, Altuwaijri et al. (2007) observed
a slight androgen-induced cell growth in the resultant PC-
3-AR9 cells. These observations indicate that ectopic
expression of the AR in PC-3 cells may modulate their
growth in vitro in a manner dependent on the AR-
expressing vector. Therefore, the growth properties of
prostate cancer cells in vitro might not adequately
represent the behavior of the cells in vivo.
To test whether AR in PC-3 cells may also function as
both stimulator and suppressor observed in mouse
models, the growth, invasive, and metastatic properties
of PC-3 cells carrying the empty vector (PC-3-v cells)
and PC-3-AR9 cells were compared. PC-3-AR9 cells
were found less invasive in an in vitro invasion assay and
produced less osteolytic lesions in a bone-wafer resorp-
tion assay than PC-3-v cells (Niu et al., 2008a). When
these cells were inoculated into the tibia of athymic nude
mice, it was found that PC-3-v tumors grew more
invasively and aggressively than PC-3-AR9 tumors,
suggesting that knockin of a functional human AR in
PC-3 cells resulted in suppression of their invasion
in vitro and in vivo.
Furthermore, PC-3-v or PC-3-AR9 cells were ortho-
topically injected into the anterior prostate of nude
mice. Consistent with the above findings, mice injected
with PC-3-v cells developed bigger prostate primary
tumors that were less differentiated (Niu et al., 2008b)
and larger PLN metastatic tumors (Niu et al., 2008a)
than mice inoculated with PC-3-AR9 cells. Moreover,
when PC-3-v or PC-3-AR9 cells were co-cultured with
WMPY1 stromal cells (Webber et al., 1999) and
orthotopically transplanted in nude mice, PC-3-AR9
co-cultured cells still produced smaller primary and
metastatic tumors than PC-3-v co-cultured cells, sug-
gesting that even in the presence of stromal AR
stimulation, the suppressor function of the epithelial
AR remains effective. Using transfectants of PC-3 cells
with an inducible AR-expressing transgene, Nelius et al.
(2007) also observed that induction of AR expression in
an inducible PC-3-ARþline resulted in an androgen-
dependent decrease in invasion in vitro and decreased
tumorigenecity because of decreased microvascular
density that led to increased tumor cell apoptosis after
subcutaneous inoculation in nude mice.
Together, these results showed that loss of prostatic
epithelial AR results in the development of more
invasive and metastatic prostate tumors and gain-
of-AR function reverses these characteristics. Thus,
the above human prostate cancer cell observations
are consistent with pes-ARKO-TRAMP mice data
and strongly indicate that prostatic epithelial AR
functions as a suppressor of prostate tumor growth
LNCaP cells: AR could function as proliferation
stimulator and suppressor
The LNCaP cells, which were isolated from a lymph node
prostate metastatic tumor (Horoszewicz et al., 1983) and
express a mutated AR(T877A) (Veldscholte et al., 1990),
are luminal-like tumor cells (van Bokhoven et al., 2003).
These cells may respond to androgen/AR signals differ-
entially depending on different environments (Olea et al.,
1990) and exhibited variants. For example, the AR-
expressing LNCaP-FGC and LNCaP-LNO cell lines
isolated from the same lymph node metastatic tumor
(Horoszewicz et al., 1983) exhibited different and opposite
proliferation responses toward androgen (Olea et al.,
1990, Soto et al., 1995). Proliferation of LNCaP-FGC
cells was stimulated by a low concentration and
suppressed by a high concentration of androgen, whereas
proliferation of LNCaP-LNO cells was suppressed by
physiological concentrations of androgen. These different
cell phenotypes might represent adaptive changes in
prostate cancer cells during tumor progression.
The differential responses to androgen seen in various
LNCaP cells was confirmed later using androgen-
dependent LNCaP cells that were cultured in long-term
absence of androgen (Kokontis et al., 1994) or after
prolonged numbers of passages (480) (Igawa et al.,
2002) to develop sublines with androgen-independent
proliferation. Addition of androgen to these androgen-
independent variants of LNCaP cells suppressed pro-
liferation (Kokontis et al., 1998) and promoted apop-
tosis (Joly-Pharaboz et al., 2000). Such adaptive changes
from growth stimulator to suppressor have also been
observed in LNCaP xenografts in vivo after castration of
the host (Thalmann et al., 2000; Zhou et al., 2004; Chuu
et al., 2006).
Down-regulation of AR with anti-sense oligonucleo-
tides (Eder et al., 2000) or siRNA (Ha ˚ a ˚ g et al., 2005;
Liao et al., 2005) may result in the suppression
of cell proliferation or promotion of apoptosis in
both androgen-dependent and androgen-independent
sublines of LNCaP cells. Eder et al. (2002) found that
an AR-specific anti-sense oligonucleotide could suppress
the growth of LNCaP xenografts. Down-regulation
of the AR (through siRNA) in LNCaP cells also
suppressed their invasion in vitro (Chang C et al.,
Interestingly, recurrent androgen-independent tumors
developed from orthotopic primary tumors of LNCaP
cells after castration of SCID mice hosts exhibited
increased proliferation and decreased apoptosis com-
pared with the androgen-dependent primary tumors of
LNCaP cells, a change that is associated with decreased
AR protein expression (Zhou et al., 2004). Thus, it
seems that once progressed to androgen-independent
state in vivo, LNCaP cells might be relieved from
epithelial AR suppression because of down-regulation
of the AR.
CK5-negativeand CK8/18-positiveand thus
CWR22Rv1 cells: AR functions as proliferation
stimulator and suppressor
The CWR22Rv1 prostate cancer cell line, derived from a
recurrent tumor (Nagabhushan et al., 1996) after ADT
of a CWR22 xenograft originally established from a
human prostate primary tumor (Wainstein et al., 1994),
is an AR and CK8/18-expressing tumor cell line (van
AR dual functions in prostate cancer therapy
Y Niu et al
Bokhoven et al., 2003). CWR22rv1-ARþ/?cells were
generated after knocking down AR in CWR22rv1 cells
by genomic recombination. CWR22rv1-ARþ/?cells
expressed much less AR with negligible AR transactiva-
tion and displayed suppressed growth rate compared
with the parental CWR22rv1-ARþ/þcells (Chang et al.,
paper in preparation). In contrast, CWR22rv1-ARþ/?
cells produced bigger primary and PLN metastatic
tumors than CWR22rv1-ARþ/þcells on orthotopic
transplantation (Niu et al., paper in preparation),
suggesting that the AR functions observed in cell lines
in vitro might not accurately represent the AR functions
CWR22rv1-ARþ/?cells were more invasive than
the parental cells in vitro. Using an AR-specific
siRNA to knockdown the AR in CWR22rv1-ARþ/þ
cells also rendered the resultant CWR22rv1-ARþ/þ-
ARsi cells more invasive than the parental cells
transfected with scrambled RNA (Niu et al., 2008a),
whereas expression of a functional human AR in
CWR22rv1-ARþ/?cells through a retroviral vector
with the empty vector (Niu et al., 2008a). Together,
these observations support the notion that the AR in
prostatic cancer epithelial cells functions as a suppressor
of prostate cancer metastasis.
PC346C cells: AR functions as proliferation stimulator
PC346C, established from a human primary tumor
through xenograft (van Weerden et al., 1996; van Weerden
and Romijn, 2000), is an androgen-dependent prostate
cancer cell line expressing wild-type AR and CK8/18 (van
Bokhoven et al., 2003). After long-term androgen ablation,
another androgen-independent subline, PC346DCC, with
a 95% decrease in AR expression, was generated from
PC346C cells. On orthotopic transplantation, PC346DCC
tumors grew more rapidly in intact than in castrated hosts,
suggesting stromal influence in addition to androgen-
independent growth (Marques et al., 2005). Treatment of
PC346C cells with the anti-androgen, hydroxyflutamide,
results in two new sublines: one (PC346Flu1) with over-
expression of the AR and its proliferation suppressed by
androgen in vitro was more tumorigenic in castrated than
in intact nude mice, whereas the other (PC346Flu2) with
the AR(T877A) mutation behaved similarly to LNCaP
cells (Marques et al., 2005). These contrasting results again
showed that androgen/AR signals could be either a
proliferation stimulator or suppressor in similar human
prostate cancer cells.
Disadvantage of using a single human prostate cancer cell
line to study prostate cancer progression and metastasis
From the results described above, it should be noted
that data obtained solely from in vitro studies of human
prostate cancer cell lines might not reliably predict the
in vivo AR functions in prostate cancer progression
and metastasis. For example, PC-3 and PC345DCC cells
that either lack AR or with minimal AR have been
regarded as androgen-independent cells. It is generally
believed that a prostate cancer with this type of cells
would not respond to ADT. The results presented in
sections ‘PC-3 cells: AR functions as suppressor of
proliferation and metastasis’ and ‘PC346C cells: AR
functions as proliferation stimulator and suppressor’,
however, indicate these cells are still capable of
responding to stromal AR signals for growth and/or
metastasis. Furthermore, PC-3 cells with stable transfec-
tion of AR cDNA under different promoters may yield
different results on androgen treatment (section ‘PC-3
cells: AR functions as suppressor of proliferation and
metastasis’). CWR22rv1-ARþ/?cells also had lower
in vitro growth rate, yet produced bigger primary and
PLN metastatic tumors in vivo than its parental AR-
positive CWR22rv1-ARþ/þcells (section ‘CWR22Rv1
cells: AR functions as proliferation stimulator and
Finally, several current available human prostate
cancer cell lines were generated from long-term culture
in the absence of androgen, which may not represent the
in vivo human prostate condition based on the reports
by Titus et al. (2005) showing that even after ADT
treatment, the human prostate tissues still have about
1–3nM 5a-dihydrotestosterone, which is approximately
10% of the normal level and is still sufficient to elicit AR
Together, these contrasting examples clearly point out
the importance of conducting in vivo animal studies of
prostate tumors to delineate the pathophysiologic func-
tions of AR in prostate cancer progression and metastasis.
AR dual functions in relation to the altered hormone
sensitivity in prostate cancer progression during ADT
It has been proposed that cancer arises from neoplastic
transformation of stem cells (Reya et al., 2001). Thus,
prostate cancer can be considered to derive from
neoplastic transformation of prostate stem cells to form
prostate cancer stem cells that generate progenitor cells,
which then progress sequentially into CK5-positive-
basal cells, CK5/CK8-positive-basal intermediate cells,
and then CK8-positive luminal cells (Figure 3) (Litvinov
et al., 2003). Recent studies have indicated that prostate
cancer progression from prostate cancer stem cells might
involve different stem/progenitor cells with different
levels of AR expression at different stages with varying
proliferation and differentiation potentials (Figure 3).
The distribution of these cells in prostate cancer might
vary with patients and tumor stages and thus have
different sensitivities toward AR-differential signals and
may respond to ADT differentially (see Supplementary
Information for more details).
Other possible explanations involving differential AR
functions in various prostatic cells to influence hor-
mone sensitivity during ADT include (a) AR somatic
mutations, (b) altered interactions between AR and AR
coregulators, (c) neuroendocrine
prostate cancer cells, (d) epithelial-mesenchymal transi-
AR dual functions in prostate cancer therapy
Y Niu et al
tion (EMT) of prostate cancer cells, (e) development of
ligand-independent activation of AR by growth factors
or protein kinases, and (f) changes in AR expression
between primary and metastatic prostate tumors. All
these phenotypic changes undoubtedly would alter
androgen/AR signals and affect the outcome of ADT
(see Supplementary Information for more detailed
descriptions of these changes).
The impact of AR dual functions on current clinical ADT
Challenge to current ADT
The fact that the stromal and epithelial ARs have
opposite functions to modulate prostate cancer cell
proliferation and metastasis undoubtedly is a main
challenge for current ADT to treat prostate cancer
patients. As ADT suppresses androgens binding to
whole body AR, including both the stromal and
epithelial AR, its treatment effect would depend on
which cell’s AR has a more significant function in a
given stage of prostate cancer progression. At early
stages of prostate cancer when tumors are dependent
predominantly on stromal AR-modulated signals for
growth and metastasis, ADT would result in regression
of the tumor because of increased apoptosis of the
tumor epithelial luminal and stromal cells, with much
less effect on cancer stem/progenitor cells and some
(Figure 3). Subsequently, prostate cancer stem/progeni-
tor cells and their transit amplifying progenitors/basal
cells will adapt to the new AR signals during ADT and
develop, either with AR somatic mutations, or altered
AR to AR coregulators ratio, or increasing neuroendo-
crine differentiation, and/or EMT into more aggressive
tumors that have different responses to AR signals.
The transition of prostate cancer into a more
aggressive phenotype under low androgen conditions
has been reported during tumorigenesis in Nkx3.1-Pten
mutant mice (Banach-Petrosky et al., 2007). Continuing
suppression of androgen at castration level through
ADT would accelerate tumor metastasis because of
ablation of the metastasis suppressor function of AR in
the basal intermediate cells, thereby resulting in failure
of ADT. Consistent with this view is the observation of
Kleeberger et al. (2007) that the expression of nestin, a
tumor metastasis marker, in prostate cancer cells is
associated with ADT, and is mainly detected in
refractory tumors while almost undetectable in tumors
from patients without ADT.
their transit amplifying progenitors-basalcells
Target the stromal AR at an early stage
It is apparent that ADT will have some beneficial effects
on early prostate cancer, but will eventually fail in view
of the dual functions of AR signals in prostate cancer
sequentially from prostate (cancer) stem/progenitor cells (AR-negative) to transit amplifying cells with basal cell character (CK5-
positive/CK8–negative and AR-negative), to intermediate cells with both basal and luminal cell character (CK5-positive/CK8-positive
with either AR-positive or AR-negative), and then to luminal cells (CK5-negative/CK8-positive and AR-positive). Targeting epithelial
AR through either knockout of epithelial AR or suppression of androgens binding to whole body AR signals as current ADT does,
may result in decreased luminal cells and increased basal intermediate cells, which may be due to increased basal transit amplifying cells
derived from prostate cancer stem cells and/or transition from luminal cells. ADT may also lead to increasing tumor-associated
macrophage (TAM) (Mercader et al., 2001) and decreased stromal cells, including the cancer-associated fibroblasts (CAF).
Differential AR signals in different prostate cancer cells on ADT treatment. The prostatic epithelial tumor may be developed
AR dual functions in prostate cancer therapy
Y Niu et al
progression (Figure 3). Therefore, it may be better to
target the AR at an early stage of prostate cancer
when the progression and metastasis of the tumor is
predominantly dependent on the stromal AR function.
This approach is best exemplified by the timing of
inducing ARKO in ind-ARKO-TRAMP mice described
in section ‘Relative influences of the stromal and
epithelial AR on prostate cancer progression and
(Niu et al., 2008b). Thus, because of the dual functions
of AR, treatment of prostate cancer patients with ADT
may have good response during the early stage of
cancer, but worsen the prognosis during the later stages
of cancer progression when they are less dependent on
the stromal AR-modulated signals for growth, a
scenario apparently observed in castrated TRAMP mice
(Johnson et al., 2005) as well as in ind-ARKO-TRAMP
mice after induction of ARKO at an early or late stage
(Niu et al., 2008b). This also explains why some patients
can benefit from intermittent treatment with cycles of
androgen deprivation and supplementation (Akakura
et al., 1993; Crook et al., 1999; Hurtado-Coll et al.,
2002). For example, combined androgen blockade
treatment of advanced prostate cancers provided at best
a 5% increase in 5-year survival rate over ADT alone
(Schmitt et al., 2001), but does not cure the disease. On
the other hand, therapies aiming at maximal eradication
of cancer cells including the cancer stem cells and their
progenitors, such as radical prostatectomy and radiation
therapy in conjunction with ADT, should provide a
better clinical outcome than ADT alone, particularly in
localized prostate cancer.
Strategies targeting the tumor stroma (Bouzin and
Feron, 2007; Hofmeister et al., 2008) also should be
beneficial to prostate cancer patients, as exemplified by
the better treatment efficacy of combining therapy with
ADT and anti-angiogenic agents targeting the stromal-
derived vascular factors (Johansson et al., 2007) in the
rat Dunning prostate cancer model. Similarly, combin-
ing ADT with Trk tyrosine kinase inhibitors targeting
the receptor for stroma-derived AR-independent nerve
growth factor was reported to prolong tumor regression
in the rat prostate cancer model (George et al., 1999).
However, if the disease recurs after these treatments, the
adjuvant ADT may still fail because the recurrent tumor
may become less dependent on the stromal AR for
growth and more invasive because of abrogation of the
metastasis suppressor function of the epithelial AR.
Concomitant treatment of AR antagonism
with anti-metastasis agents
As ADT may promote the development of metastatic
prostate tumors, it would be better to combine ADT
with anti-metastatic agents or agents capable of
suppressing EMT to improve the treatment effect. In
metastatic tumors of pes-ARKO-TRAMP and PC-3-v
cells, the expression of various tumor metastasis-related
genes were significantly changed in favor of invasion
and metastasis as compared with the tumors of TRAMP
mice and PC-3-AR9 cells, respectively. Thus, several
pro-metastasis genes such as cyclooxgenase-2, matrix
metaloproteinase-9, interleukin-6, and tumor necrosis
factor-a were elevated, whereas anti-metastasis genes
such as neutral endopeptidase and the cell cycle
inhibitor P27 (Kip1) were decreased in the tumors of
pes-ARKO-TRAMP mice and PC-3-v orthotopic grafts
compared with the tumors of their AR-expressing
counterparts (Niu et al., 2008a). Part of these effects
of AR seemed to be mediated through the suppression
of TGFb1, as expression of AR in prostate cancer cells
resulted in suppression of TGFb1 expression and over-
expression of TGFb1 resulted in increased invasiveness
and similarly altered expressions of the metastasis-
related genes mentioned above (Niu et al., 2008a).
Moreover, TGFb1 is known as an inducer of cancer
cells to undergo EMT that is linked to the process of
As patients with recurrent prostate cancer usually
die of metastaticdisease,
extended if tumor metastasis can be delayed or
(Baritaki et al., 2009) or EMT-related genes downstream
to the epithelial AR signaling or targeting neuroendo-
crine factors should improve the clinical results of
TGFb1 and/or its receptor have been under clinical
trial (Pinkas and Teicher, 2006). In addition, current
available agents targeting Akt, COX-2, MMP-9, Vita-
min E derivatives, or other relevant anti-metastasis
agents in conjunction with ADT as earlier proposed
(Miyamoto et al., 2005) may have clinical benefits for
patients to battle metastatic prostate cancer, and may
represent new treatment strategies to combat this deadly
Future direction: Targeting AR in selective prostate cells
It is apparent that successful treatment of prostate
cancer will rely on complete eradication of prostate
cancer cells. This treatment objective can be achieved by
prostatectomy when the disease is localized. Unfortu-
nately, detection of prostate cancer at such an early
stage is less frequent than more advanced diseases. As
prostate cancer progresses with continuous interactions
among various AR-positive stromal, epithelial, and
infiltrating cells within the tumor microenvironment
(Figure 3) targeting only those cells (such as stromal
and/or luminal cells) with AR functions as positive
roles to promote prostate cancer progression may
be worthy of development in the future to battle
prostate cancer. Recent studies (Yang et al., 2007;
Chang et al., paper in preparation) showing ASC-J9
could degrade AR more aggressively in stromal and
luminal cells that resulted in suppression of prostate
refractory tumors with little side effects or toxicity may
shed new light on our hope to treat prostate cancer.
Alternatively, developing new nanoparticles carrying
anti-AR compounds that only recognize and kill those
AR dual functions in prostate cancer therapy
Y Niu et al
prostate cells with AR-positive roles will be another new
hope in the future.
Conflict of interest
ASC-J9 was patented by the University of Rochester,
the University of North Carolina, and AndroScience
Corp., and then licensed to AndroScience Corp. Both
the University of Rochester and C Chang own royalties
and equity in AndroScience Corp.
This work was supported by the George Whipple Professor-
ship and NIH grant CA122840 and NSFC grant project
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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)
AR dual functions in prostate cancer therapy
Y Niu et al
S1. Dual roles of the AR in normal prostate development
S1a. AR in stromal cells: functions as a proliferation stimulator and
Previous tissue recombination studies by Cunha et al (2004) have indicated
that prenatal development of the prostate is dependent on androgen/AR signals
from the urogenital mesenchyme (UGM)/stroma to induce prostatic
organogenesis and subsequent ductal morphogenesis and to maintain epithelial
cell survival, whereas the epithelial AR is required for epithelial terminal
differentiation. Prostatic epithelium can also induce UGM to differentiate into
smooth muscle. These reciprocal stromal-epithelial interactions remain operative
in adult prostate to maintain prostate cellular homeostasis (Cunha et al., 2004).
The prostatic stroma is constituted of fibroblasts, smooth muscle cells,
endothelial cells, and immune cells (Cunha et al., 2004). A recent study of
fibroblast-specific ARKO (fsp-ARKO, Yu et al, manuscript submitted) mice
indicated that reducing AR signals in stromal fibroblasts resulted in under-
development of the prostate gland with a decrease in proliferation and
substantial increase in apoptosis in the epithelium, which was associated with
decreased expression of several stromal paracrine growth factors in fps-ARKO
compared to wild type mice. The levels of epithelial differentiation markers were
markedly reduced in the prostate of fps-ARKO mice, suggesting loss of luminal
epithelial cells. Smooth muscle cell-specific ARKO (sm-ARKO) male mice have
also been generated and found to display decreased epithelial infolding with
decreased epithelial cell proliferation and IGF-1 expression in the prostate
(Chang et al., unpublished observation), and IGF-1 has been suggested as an
important proliferation stimulator and survival factor of prostatic epithelial cells
(Ohlson et al., 2006). Furthermore, after combining knockout of the stromal AR in
a fps- and sm-double ARKO mice, Lai et al. (unpublished observation) found
increased epithelial apoptosis and decreased proliferation of CK-5+ basal
intermediate cells as well as CK8+ epithelial cells. These observations thus
strongly support the consensus view derived from previous tissue recombination
studies (Cunha et al., 2004) that prostatic stromal AR is an important positive
regulator of epithelial cell proliferation and survival in prostatic development and
S1b. AR in epithelial cells: a survival factor for luminal cells and a
suppressor for basal intermediate cell proliferation
There are three major types of prostate epithelial cells, luminal cells (CK5-
/CK8+), basal intermediate cells (CK5+/CK8+), and basal cells (CK5+/CK8-). The
role of epithelial AR in adult prostate could not be delineated through the tissue
recombination study using embryonic tissues with a short period of observation
(4-6 weeks). By crossing floxed AR mice (Yeh et al., 2002) with probasin-Cre
mice (Wu et al., 2001), Wu et al (2007) have generated prostate epithelial
specific (pes)-ARKO mice. Consistent with increasing probasin-Cre expression,
the epithelial AR levels gradually decreased in the prostate of pes-ARKO mice Download full-text
beginning at 6-weeks old, and were below detection at 24 weeks of age. The
ventral prostates of pes-ARKO mice in comparison with those of wild type mice
were enlarged at 24-weeks or older and displayed a progressive decrease in
epithelial height, loss of glandular infolding, and an increase in epithelial luminal
cell apoptosis through 6-32 weeks of age. As the pes-ARKO mice matured,
CK5/CK8-double positive (Wu et al., 2007) epithelial basal intermediate cell
populations increased during puberty and then remained elevated, while the
CK8/ CK18-positive epithelial luminal cell population declined. In contrast, in wild
type animals, the basal cell number declined with age, whereas the luminal
CK8/18-positive cells population remained stable. These observations strongly
suggest that the epithelial AR is an important survival factor for epithelial luminal
cells. Thus, the survival of epithelial luminal cells appears to require AR signals
being maintained in both stromal and epithelial cells, since lacking either one
resulted in apoptosis.
Further examination of pes-ARKO mice at 24-weeks-old indicated that the
prostate glands had one layer of undifferentiated epithelial cells with increased
proliferation of CK5-positive basal cells. In addition, there was an expansion of
CK5/CK8 double positive cells characteristic for basal intermediate cells
increasing from <10% to >50% of total epithelial cells in wild type and pes-ARKO
Knocking-in a functional AR in pes-ARKO mice via transgenic strategy to
generate (T857A)-pes-ARKO double transgenic mice showed the normal