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The Journal of Clinical Endocrinology & Metabolism 88(7):2972–2982
Copyright © 2003 by The Endocrine Society
doi: 10.1210/jc.2002-022038
GENETICS OF ENDOCRINE DISEASE
Is the Achilles’ Heel for Prostate Cancer Therapy a Gain
of Function in Androgen Receptor Signaling?
IVAN V. LITVINOV, ANGELO M. DE MARZO,
AND
JOHN T. ISAACS
The Sidney Kimmel Comprehensive Cancer Center (I.V.L., A.M.D., J.T.I.), the Department of Pathology (A.M.D.), the
Cellular and Molecular Graduate Program (I.V.L., J.T.I.), and the Brady Urological Institute, The Department of Urology
(A.M.D., J.T.I.), The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231
Multiple genetic changes are required to produce prostatic
cancer cells that can metastasize and kill the patient (1, 2). The
most fundamental consequence of these molecular abnormalities is that the rate of malignant prostate cell proliferation exceeds its rate of cell death (3). It is this disruption of
cellular homeostasis that results in the continuous net
growth producing the lethality of this devastating disease.
Successful therapy for the 30,000 U.S. males dying of prostate
cancer annually will require approaches that shift the cell
kinetic balance such that the rate of prostatic cancer cell death
exceeds cell proliferation without producing unacceptable
host toxicity. To have a realistic chance of developing such
successful therapy, identification of the molecular changes
driving the imbalance in proliferation vs. death of malignant
prostate cells is critical.
Androgens are the major growth factors for the normal
prostate, and its cognate receptor is fundamental for androgen signaling within the gland (4). Prostate cancers retain
androgen receptor (AR) signaling pathways and thus are
nearly universally responsive initially to androgen ablation
therapy. Unfortunately, however, essentially all ablated patients eventually relapse. Because of this relapse, androgen
ablation therapy is not curative, no matter how complete the
ablation (5). A growing body of data has documented that
this is due to the accumulation of molecular changes inducing gain of function in the AR signaling pathways during the
progression of prostatic cancer. These gain of function
changes result in prostate cancer cells that are resistant to
androgen ablation because of their acquired ability to activate novel AR signaling pathways for their proliferation and
survival without requiring physiological androgen ligand
binding. These changes produce malignancy unique signaling pathways that, while androgen ligand-independent, are
still dependent upon AR, and this provides a therapeutic
Achilles’ heel for control of this devastating disease. The
rationale for this statement is based on the following facts.
First, the AR gene is located on the X chromosome, and thus
males have only a single copy of this gene. Second, germline
truncation mutations early in the first exon of the AR gene
result in complete androgen insensitivity syndrome because
no expression of AR protein occurs in these patients (6).
Although such complete androgen insensitivity syndrome
mutations prevent masculinization, they are not life threatening (6). This means that in prostate cancer patients with
germline wild-type AR, systemic therapy that either selectively prevents AR expression or neutralizes its signaling
ability should not be lethal to normal host tissues, except the
male accessory sex tissues. These accessory sex tissues undergo regression by standard androgen ablation without
affecting host survival. Therefore, such systemic AR-targeted
therapy would have a unique AR-dependent therapeutic
index because blocking AR signaling while eliminating the
metastatic prostate cancer cells remaining after androgen
ablation would not be life threatening. Thus, prostate cancers
should provide a paradigm for successful rational drug development based on this unique therapeutic index.
For such rational drug development to take advantage of
the Achilles’ heel of prostate cancer, identification of the
novel malignancy-acquired constitutive AR signaling pathways is critical. As a background for such research, this
review will focus on what is presently understood concerning the mechanism for androgen regulation of normal cellular homeostasis in the prostate and the genetic changes
responsible for gain of function in the AR-signaling pathways acquired during prostatic carcinogenesis and
progression.
Role of androgen in cellular homeostasis in normal prostate
Abbreviations: AR, Androgen receptor; ARE, androgen response element; BM, basement membrane; DHT, dihydrotestosterone; FGF, fibroblast growth factor; GST, glutathione-S-transferase; GSTP1, GST ␲;
HGF, hepatocyte growth factor; HGPIN, high-grade PIN; hK2, human
glandular kallikrein-2; Hsp, heat shock protein; LBD, ligand binding
domain; NE, neuroendocrine; PIA, proliferative inflammatory atrophy;
PIN, prostatic intraepithelial neoplasia; PKA, protein kinase A; PSA,
prostate-specific antigen; PSCA, prostate stem cell antigen; PSMA, prostate-specific membrane antigen; STAT, signal transducers and activators
of transcription; VEGF, vascular endothelial growth factor.
The normal human prostate is a tubular-alveolar gland
composed of a well developed stromal compartment containing nerves, fibroblasts, infiltrating lymphocytes and macrophages, endothelial cell capillaries, and smooth muscle
cells surrounding glandular acini composed of a two-layered
(i.e. basal and secretory luminal) epithelium (Fig. 1). Scattered throughout this epithelial compartment are occasional
neuroendocrine (NE) cells that are characterized by expres-
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FIG. 1. Cellular heterogeneity within the normal prostate.
Histological architecture of the prostate is comprised of
blood vessels that provide nutrients including androgens to
the fibrous stromal layer, which primarily consists of fibroblasts and smooth muscle cells, and to the epithelial
layer. Epithelium can be subdivided into a basal epithelium, which contains AR-negative proliferating cells, and
secretory luminal epithelium, which consists of fully differentiated AR and p27 Kip1 positive, nonproliferating
cells.
sion of chromogranin A, serotonin, and neuron-specific enolase, but not AR (7). Functionally, the epithelium is composed
of multiple stem cell units (8 –14) (Fig. 2). In an individual
stem cell unit, the stem cell that has the capacity for unlimited
self-renewal characteristically expresses ␣2␤1-integrins (15)
but only rarely proliferates to provide progeny that differentiate to become either transit-amplifying or NE cells (10,
11). The stem and the majority of the transit-amplifying cells
are believed to be located in the basal epithelial layer (Figs.
1 and 2). A subset of basal cells represent stem cells, and the
remainder represent transit-amplifying cells.
Basal cells express prostate stem cell antigen (PSCA), the
p53-related p63 protein, c-Met, the plasma membrane receptor for hepatocyte growth factor (HGF), and the prosurvival
protein bcl-2 (16 –19). A subset of these cells shows proliferative activity as evidenced by positive staining for Ki-67,
and these presumed transit-amplifying cells, which are also
referred to as intermediate cells in the prostate, either do not
express AR or express it at very low levels (20, 21). During
the hierarchical expansion of prostatic epithelium cells, a
minority of stem cell progeny differentiate into NE cells that
secrete neuroendocrine peptides like bombesin, calcitonin,
and PTH-related peptide (22). The majority of the stem cell
progeny become transit-amplifying cells that eventually terminally differentiate into mature secretory luminal cells that
are nonproliferative and positive for AR and p27Kip1cyclindependent kinase inhibitor (20, 21, 23) (Fig. 2). Because of this
hierarchical expansion, these nonproliferating AR/p27Kip1positive secretory luminal cells are quantitatively the major
subtype of epithelial cells present in the normal prostate.
These AR/p27Kip1-positive secretory luminal cells also express the prostate-specific differentiation markers, prostatic
specific acid phosphatase, prostate-specific antigen (PSA),
NKX 3.1, human glandular kallikrein-2 (hK2), prostatespecific membrane antigen (PSMA), and PSCA, as well as
vascular endothelial growth factor (VEGF) (25–29). The transcriptional expression of these prostate-specific differentiation marker genes is enhanced by occupancy of the AR with
physiological androgen [dihydrotestosterone (DHT)] and
the subsequent binding of the occupied AR to androgen
response elements in the promoter and enhancer sequences
of these genes within the nuclei of these secretory luminal
cells (27, 29 –32).
In contrast to the regulation of transcription of these prostate differentiation marker proteins, AR in the nuclei of these
secretory luminal cells does not directly regulate the survival
of these luminal cells, nor does it positively regulate the
proliferation and survival of the prostatic epithelial stem and
transit-amplifying cells. Instead, the survival of the secretory
luminal cells and the proliferation of the transit-amplifying
cells requires the androgen-dependent production of peptide
growth factors by the prostatic stromal cells (33–35). These
processes are initiated by testosterone diffusing from the
capillary bed in the stromal compartment of the normal
prostate across the basement membrane (BM) to enter the
basal epithelial cells. These basal cells express type I and II/5
␣-reductase proteins that enzymatically convert testosterone
to 5 ␣-DHT (36). Once formed, DHT diffuses both into the
secretory luminal cells in the epithelial compartment and
back across the BM to the smooth muscle cells and fibroblasts
in the stromal compartment. Secretory luminal cells also
express type I/5 ␣ reductase activity (36), thus further increasing their cellular level of DHT above that provided by
the basal cells. Within these secretory luminal epithelial cell
nuclei, this enhanced level of DHT binds to the AR and
directly transcriptionally up-regulates the expression of the
prostate-specific differentiation markers (prostatic specific
acid phosphatase, PSA, hKh2, PSCA, NKX3.1, and PSMA)
(16, 25, 27, 29 –32) and also VEGF indirectly (28). These secretory luminal cells also express TGF␤1 (37). These growth
factors diffuse across the BM to affect stromal cells. Specifically, VEGF affects the survival of the stromal endothelial
cells (28), and TGF␤1 inhibits stromal cell proliferation and
induces smooth muscle differentiation and neuronal trophism (38, 39).
Binding of DHT to the AR within the nuclei of these stromal smooth muscle cells inhibits their expression of certain
cytokines like TGF␤1 (40 – 41) while enhancing their secretion
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Litvinov et al. • Genetics of Endocrine Disease
FIG. 2. Stem cell model of prostatic epithelial cell compartmentalization. The prostate gland consists of a number of stem cell units that arise
from one stem cell. Such a stem cell is located in the basal epithelial layer of the prostate and, upon division, gives rise to a population of
transit-amplifying cells. The latter divide in the basal layer, and a fraction of them differentiate and move into the secretory luminal epithelial
layer. As transit-amplifying cells differentiate and move into a secretory luminal layer from the basal layer, they acquire expression of a number
of genetic markers, as indicated. **, Low-level retention of expression by a subset of transit-amplifying (i.e. intermediate) cells; ⫹, expression
of marker; ⫺, lack of detectable expression of marker.
of andromedins (i.e. androgen-induced stromal peptide
growth factors) (42, 43). These andromedins diffuse back
across the BM into the epithelial compartment where they
interact with their specific cognate plasma membrane receptors of the secretory luminal cells generating intracellular
signaling (e.g. down-regulation of TG␤ receptors) needed to
repress the apoptotic death pathway in the secretory luminal
cells (44). Binding of the andromedins to the plasma membrane receptors of the transit-amplifying epithelial cells can
recruit them into the cell cycle. If a sufficient systemic androgen level is not chronically maintained (e.g. after androgen ablation), then the level of DHT-occupied AR within
prostatic stromal cells decreases to a level unable to maintain
adequate expression of the stromally derived andromedins
and unable to repress expression of TGF␤1 (40, 41). Without
adequate andromedins, prostatic transit-amplifying epithe-
lial cells remain proliferatively quiescent in Go and do not
enter the cell cycle, whereas in the prostatic secretory luminal
epithelial cells, lack of sufficient andromedins results in the
up-regulation of expression of type I and II TGF␤1 receptors
(41). The enhanced levels of TGF␤1 receptors in these secretory luminal cells are activated by the enhanced levels of
TGF␤1 ligand produced by stromal cells after androgen ablation (40, 41). This enhanced TGF␤1 receptor signaling activates the energy-dependent apoptotic cascade within the
secretory luminal cells inducing their death (40, 44, 45). This
apoptotic cascade involves changes in the intracellular free
calcium level, caspase and nuclease activation, and degradation of the secretory luminal cells into apoptotic fragments
(45– 47). Because secretory luminal cells are the source of
VEGF production in the prostate, their death results in a
lowering of the prostatic VEGF levels (28). This lowering of
Litvinov et al. • Genetics of Endocrine Disease
the tissue VEGF levels results in the activation of the apoptotic death of a subset of stromal endothelial cells, reducing
tissue blood flow (48).
Although secretory luminal cells undergo apoptosis after
androgen ablation, the basal stem and transit-amplifying
cells do not (49). A possible explanation for this observation
is that prostatic stromal cells express HGF (18). HGF expression by these stromal cells is not regulated by androgen
occupancy of the AR in these stromal cells (50). Basal stem
and transit-amplifying cells constitutively express c-MET,
the plasma membrane cognate receptor for HGF, whereas
secretory luminal cells do not (18). Such c-MET signaling is
inhibitory for basal cell apoptosis and proliferation (18).
Thus, after androgen ablation, the prostatic stromal cells
continue to supply adequate levels of HGF to bind to and
induce signaling by the c-MET receptors of the basal cells,
thus both blocking activation of apoptosis and inhibiting
proliferation of these basal cells (18).
AR signaling in prostate epithelium functions as a growth
suppressor and differentiation inducer
When normal prostatic tissue is used to establish in vitro
cultures, only the transit-amplifying cells (i.e. intermediate
cell type) continue to proliferate during the subsequent several passages (51). Low passage cultures of these transitamplifying cells (i.e. intermediate cell type) have a higher rate
of proliferation (i.e. ⱖ50% proliferation per day) when grown
in vitro in serum free defined media (52). Cells in such low
passage cultures do not express AR and thus are not affected
by adding androgen to the culture media. These cells are
dependent, however, on a critical mixture of peptide growth
factor andromedins in the media for their survival and a high
rate of proliferation (52). In contrast to the high proliferation
rate in in vitro cultures, only 0.2% of the epithelial cells are
proliferating per day in normal prostatic tissue in vivo although these cells are exposed continuously to maximal
physiological levels of andromedins present in non-androgen-ablated hosts (3). These observations raise the issue of
how the in vivo proliferation of the transit-amplifying cells
becomes restricted to allow only homeostatic renewal and
not net continuous prostatic epithelial growth although the
level of the stromally produced andromedins remains constantly high in the presence of physiological androgen levels.
One explanation is that AR signaling in the nuclei of the
prostatic secretory luminal cells and the subset of ARexpressing transit-amplifying cells actively inhibits proliferation of these cells even in the presence of continuous andromedin stimulation (53–55). This mechanism has been
documented experimentally using both human (54) and rodent (55) prostate epithelial cells. These latter studies have
demonstrated that when AR-negative intermediate prostatic
epithelial cells are transgenically induced to express AR and
then exposed to physiological levels of androgen, their in
vitro proliferation is profoundly inhibited even in the presence of andromedins with no effect upon cell survival (54,
55). These results demonstrate that for nonmalignant prostatic epithelial cells, the ligand-occupied AR functions as a
growth suppressor via its ability to inhibit andromedin
induced proliferation. While functioning as a growth sup-
J Clin Endocrinol Metab, July 2003, 88(7):2972–2982 2975
pressor, such AR signaling also induces differentiation of
these transit-amplifying cells from an intermediate to a
secretory luminal cell phenotype (54, 55). This AR-mediated
inhibition of andromedin-induced proliferation appears to
be related to AR-induced up-regulation of the p27Kip1 cyclindependent kinase inhibitor (56 –58). The mechanism for this
up-regulation in normal prostatic epithelial cells involves
enhanced stability of the p27Kip1 protein secondary to ARinduced transcriptional repression of expression of the E3
ubiquitin ligase Skp2 involved in p27Kip1 degradation (58, 59).
Cell of origin for prostatic carcinogenesis
Although it is clear that prostate cancer arises from the
epithelial compartment, the identification of the specific epithelial cell subtype in which the carcinogenic process initiates has only recently been the focus of study. Currently, the
precursor for most peripheral zone prostatic carcinomas is
thought to be high-grade prostatic intraepithelial neoplasia
(HGPIN) (60). It is believed that HGPIN arises from lowgrade PIN, which in turn is thought to arise within normal
prostate epithelium (Fig. 3). The cell type of origin for
HGPIN, however, is still incompletely understood. A widely
held view of carcinogenesis is that the common carcinomas
generally arise in self-renewing tissues in which dividing
cells acquire somatic genetic alterations in growth regulatory
genes. In normal human prostate epithelium, most cell division takes place in the basal cell compartment in which the
tissue stem and presumably the transit-amplifying cells reside (20, 21). The majority of secretory luminal cells do not
normally proliferate and are the terminally differentiated
cells that perform the androgen-regulated differentiated
functions of the prostate, such as PSA production and secretion. Both prostate cancer and HGPIN cells possess many
phenotypic and morphological features of secretory luminal
cells (i.e. cytokeratin 8 and 18, PSA, hK2, PSMA, and AR
expression), yet they also contain features of the basal transitamplifying cell compartment such as c-Met expression, DNA
replication, and extensive self-renewal (61– 64) (Fig. 2). Thus,
in carcinoma these stem-cell and transit-amplifying cell-like
features have been shifted up from the basal into the secretory luminal compartment (23, 64). It has been postulated
that the cell of origin for prostate cancer is an intermediate,
prostatic epithelial cell, presumably derived from the basal
transit-amplifying population that undergoes the initial malignant molecular changes that allow gene expression and
morphological features of both basal and secretory luminal
cells (23, 61– 64).
The site of these phenotypically intermediate initiated cells
appears not to be random within the prostate. Instead, they
are enriched in sites of focal glandular atrophy in which the
atrophic appearing luminal epithelial cells are quite proliferative and often surrounded by inflammation within the
gland. Therefore, these sites have been termed proliferative
inflammatory atrophy (PIA) (65). On the basis of the following lines of evidence, these PIA lesions are proposed to be an
intermediate transition stage to HGPIN and/or early prostatic carcinoma: 1) as compared with normal-appearing epithelium, PIA is highly proliferative; 2) PIA contains many
proliferating cells in the luminal layer, which is similar to
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Litvinov et al. • Genetics of Endocrine Disease
FIG. 3. New model for prostatic carcinogenesis. A number of molecular and morphological changes take place as normal prostatic epithelium
proceeds to form invasive incurable prostatic carcinoma. Such changes are associated and could be caused by inflammation, diet, and other
environmental stresses. Inherent genetic factors are also playing an important role in cancer initiation and progression.
PIN; 3) many of the luminal cells in PIA have decreased
expression of the p27Kip1 cyclin-dependent kinase inhibitor
although they express AR; 4) PIA contains many cells with
phenotypic features of intermediate cells, which have been
proposed as the target cells for carcinogenesis in the prostate;
5) PIA contains very few cells undergoing apoptosis, with
many cells in the luminal layer expressing bcl-2; 6) PIA shows
increased expression in the carcinogen-detoxifying enzyme,
glutathione-S-transferase ␲ (GSTP1), and GST ␣ in many of
the cells, consistent with a stress response to an increased
oxidative burden; and 7) finally, PIA shows frequent morphological transitions to PIN and frequently occurs adjacent
to small cancers (66).
On the basis of these findings, a new model of prostate
carcinogenesis has been proposed whereby chronic and
acute inflammation, in conjunction with dietary and other
environmental factors, targets prostate epithelial cells for
injury and destruction (Fig. 3). Increased proliferation occurs
as a regenerative response to lost epithelial cells. The increased proliferation occurs in cells with a transit-amplifying
or intermediate phenotype (Refs. 62 and 64; and van Leenders, G., and A. M. DeMarzo, personal communication). In
this process, GSTP1 expression is elevated in many of the
cells in PIA as a genome-protective measure. Although elevated in many of the cells in PIA, GSTP1 expression is
eventually lost in some cells as the result of aberrant methylation of the CpG island of the GSTP1 gene promoter (67).
Indeed, such aberrant methylation of the GSTP1 promoter is
one of the earliest molecular abnormalities characteristic of
prostate cancer cells (67). This heritable epigenetic alteration
places these cells at increased risk for the accumulation of
additional genetic damage, with acceleration of the neoplastic process toward PIN (67). One of these additional genetic
changes involves telomerase shortening by PIN cells. This
appears to increase their genetic instability, driving further
genetic damage producing invasive cancers (64).
Gain of function changes convert AR from a growth
suppressor to an oncogene during prostatic carcinogenesis
During the initiation of prostate carcinogenesis, there are
distinct “hard-wiring” changes in the AR signaling pathways. Normally, the proliferating transit-amplifying cells in
the basal epithelial layer do not express the AR or express
only low levels of AR. As discussed, during their maturation,
these cells eventually express higher levels of AR. Once a
critical AR level is reached, the occupancy of AR by its ligand
inhibits proliferation of these cells and induces their differentiation into secretory luminal cells. In contrast, the intermediate type of proliferating cells in PIA variably expresses
higher levels of AR, and such AR expression is further enhanced in proliferating cells in HGPIN (66). This indicated
that hard-wiring changes occur in the AR-signaling pathways even at this early stage of cancer development because
now AR-expressing cells are proliferating and are not growth
arrested. This gain of function ability now allows the AR to
engage the molecular signaling pathways stimulating the
proliferation and survival of these initiated prostatic cells
directly. Unlike the paracrine situation in the normal prostate
in which such growth regulation is initiated by AR binding
to genomic sequences in the nuclei of stromal cells, during
prostatic carcinogenesis, genomic AR binding within the
transformed cells themselves activates this growth regulation. Because of these hard-wiring changes, there is a conversion from paracrine to autocrine AR-signaling pathways
in invasive prostate cancer (68, 69). These gain of function
hard-wiring changes pathologically allow androgen/AR
complexes to bind to and enhance expression of survival and
proliferation genes that physiologically are not affected by
these complexes in either normal transit-amplifying or secretory luminal cells (68, 69). In addition, such gain of function AR oncogenic signaling no longer represses but instead
stimulates Skp2 expression. Such Skp2 enhanced expression
results in down-regulation of p27Kip1 protein, enhancing proliferation of these cancer cells (70).
Litvinov et al. • Genetics of Endocrine Disease
Even with these hard-wiring changes, activation of these
pathological growth-promoting (i.e. oncogenic) pathways
can still be dependent on the binding of androgen to its
receptor in the nuclei of these neoplastic cells themselves, (i.e.
androgen and AR dependent), or they can be constitutive (i.e.
independent of the binding of physiological androgens to the
receptor), but still requiring AR functioning in the nuclei of
these malignant cells to enhance the transcription of both
secretory markers and also growth promoting genes, (i.e.
androgen independent but still AR dependent).
To appreciate the therapeutic relevance of these mechanistic distinctions, an understanding of the cellular heterogeneity and responsiveness of prostate cancer cellular subtypes is required. Androgen ablation therapy, whether by
surgical or medical means, induces the elimination of only
androgen-dependent prostate cancer cells because these cells
require a critical level of physiological androgen for their
continuous proliferation and survival (68, 69, 71). Unfortunately, androgen ablation is not curative because, once clinically detected, prostate cancers are heterogeneously composed of clones of androgen-dependent cancer cells and also
malignant clones that are androgen independent (72). These
latter cells are androgen independent because androgen occupancy of their nuclear AR is not required for their survival
(72). There are two basic subtypes of such androgen-independent prostate cancer cells. One subtype retains a sensitivity to androgen occupancy of its nuclear AR to enhance its
rate of cell proliferation, although such occupancy is not
required for its survival. Thus, these cells are androgenindependent/sensitive because their rate of growth is inhibited but not prevented by androgen ablation. The other subtype is termed androgen independent/insensitive because
androgen ablation decreases neither their rate of proliferation nor survival (72).
These last two subtypes of malignant clones are not eliminated by standard androgen ablation, and thus these are the
malignant cells that eventually kill the patient (72). It had
been assumed that, after androgen ablation, such androgen
independent/insensitive prostate cancer cells no longer express AR and that in such androgen independent/sensitive
cells, the expressed AR had no function in regulating survival. This assumption was based on earlier observations that
the majority of serially passaged rodent and human (i.e. PC-3,
DU-145) in vitro cell lines established from androgen ablation
failing hosts consistently did not express AR. In contrast to
this experimental situation, more than 90% of prostatic cancers obtained directly from patients failing androgen ablation actually overexpress AR (73–75). In approximately 30%
of such progressing prostatic cancer, this overexpression is
associated with genetic amplification (76, 77) and in 10 – 40%
with AR mutations (78, 79).
These clinical results strongly implicate continual involvement of AR in the stimulation of proliferation and/or inhibition of death, even in ligand- (i.e. androgen) independent
prostate cancer cells resistant to androgen ablation. This is
supported by a growing body of experimental studies using
prostate cancer model systems that have documented that
manipulations which interfere with AR expression, nuclear
translocation, and/or appropriate genomic binding, inhibit
proliferation and induce apoptosis of ligand-independent
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(i.e. androgen ablation resistant) AR-expressing prostatic
cancer cells (80 – 82). Thus, targeted inhibition of these ligand-independent AR-signaling pathways should provide
rational drug development for androgen ablation-resistant
prostatic cancers. To develop such approaches, however, a
critical level of understanding of the molecular mechanism(s)
for how such ligand-independent oncogenic AR signaling is
required.
Molecular signaling pathways induced by AR
ARs are ligand-dependent zinc finger DNA binding proteins whose genomic binding coordinates formation of transcriptional complexes at the regulatory elements of targeted
genes. The AR gene is located on the long arm of the X
chromosome (i.e. Xq11.2) and encodes a protein with three
critical domains: 1) an N-terminal domain involved in homotypic dimerization and binding with other transcriptional
coactivator or corepressor proteins; 2) a DNA binding domain with two zinc finger binding motifs and hinge region;
and 3) a C-terminal steroid ligand binding domain (LBD),
which is also involved in homotypic dimerization and coactivation binding (Fig. 4). This latter C-terminal LBD domain is also where 90-kDa heat shock protein (i.e. Hsp-90)
dimers bind to stabilize the AR protein during its folding,
subsequent to its synthesis (83). Specific interaction with
androgenic ligands results in the conformational activation
of AR. This allows the dissociation of the Hsp-90 dimer
proteins and thus the binding and dimerization of the occupied AR ; Ref. (84) to androgen-response elements present
in the promoter and enhancer regions in AR-regulated genes
(27, 29 –32).
This initial genomic AR binding allows further binding to
specific regions of the bound AR by additional nuclear proteins (i.e. transcriptional coactivator proteins like SRC-1,
ARA 70, etc., and general transcription factors like TFIIF and
H) to produce transcriptional complexes that can activate or
repress specific gene expression (85). For activation, formation of an active transcriptional complex is required, resulting in site-directed chromatin remodeling via histone acetylation and methylation that enhances target gene expression
(85– 89) (Fig. 5). SRC-1 is a member of the p160 transcriptional
coactivator gene family that includes SRC-1, TIF2 (also
termed GRIP-1 and SRC-2), and p/CIP (also termed RAC3,
ACTR, AIBI, and SRC-3) (89). Cell-free in vitro transcription
and in vivo experiments have indicated that the SRC-1 family
members enhance AR-dependent transactivation of nuclear
genes (85– 89), the mechanism for such enhancement involves binding of p160 proteins to the DNA bound AR (Fig.
5). This allows the p160 to acetylate histones via its histone
acetyltransferase activity. Additional coactivators with histone acetyltransferase activity such as CBP, p300, or p/CAF
also bind to the p160/AR complex. This results in chromatin
remodeling and additional binding of general transcription
factors such as TBp and TIFIIB with the AR coactivation
complexes (85– 89) (Fig. 5).
On the basis of this overview of normal AR signaling, the
AR-signaling cascade in prostate cancer cells can become
constitutively active (i.e. independent of circulating androgen) via several genetic and epigenetic mechanisms. With
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Litvinov et al. • Genetics of Endocrine Disease
FIG. 4. Organization of the AR gene,
mRNA, and protein. AR is coded by a
180-kb gene located on the long arm of the
X chromosome (i.e. 11q11.2) and, hence, is
present as a single gene copy per cell. The
gene is encoded by 8 exons as denoted by
8 boxes in the figure. After transcriptional
processing, the polyA mRNA is 4.3 kb,
which is translated into a 919-amino acidlong protein. A number of functional domains are recognized in AR protein: 1)
N-terminal transactivating domain; 2)
DNA binding domain (DBD) and hinge
region (HR); and 3) a C-terminus LBD.
Besides the aforementioned modular domains, a number of sequence motifs are
believed to be important for proper AR
function, including two nuclear import
signals (NLS) located in the hinge region
and in the C-terminal ligand binding domain, and Hsp-90 binding site located in
the C-terminal LBD.
FIG. 5. Overview of transcriptional complex
organized by AR at the ARE of the promoter/
enhancer region of the prostate-specific antigen (PSA) gene. AR assembles a transcription initiation complex on the promoter/
enhancer regions of androgen-regulated
genes like PSA. Ligand-bound AR undergoes
a DNA-dependent dimerization in the nucleus and recruits a number of transcription
factors that acetylate histones, recruit additional transcription factors, and DNA polymerase and initiate transcription of the target gene.
regard to genetic mechanisms, gain of function AR mutations
occur in low frequency (i.e. ⬍10%) in primary prostate cancer
(78). In contrast, the cells in distant metastases and recurrent
prostate cancer after androgen ablation have a higher level
(i.e. ⱖ10%) of AR mutations (78, 79). Further molecular analysis using both human and rodent prostate cancer tissue has
demonstrated that mutations in the N-terminal region are
more common in AR prostate cancer tissue from androgen
ablation failing hosts, whereas mutations in the C-terminal
ligand binding domain are more common before androgen
ablation (78, 79). N-terminal mutations potentially allow AR
binding interaction with specific transcriptional coactivators
without the requirement for occupancy of the ligand binding
domain of the AR. C-terminal AR mutations have been documented to allow new physiological endogenous nonandrogenic steroids (e.g. estrogens, glucocorticoids, and progestins) to bind to the LBD of AR-inducing formation of
functional transcriptional complexes (90, 91).
Additional epigenetic mechanisms also can allow such
productive formation of constitutive AR transcriptional complexes without androgen ligand binding. For example, it has
been documented that expression of AR at relatively low
levels results in strong ligand-dependent activation, whereas
high levels of AR lead to androgen-independent transcriptional activation (92). Because approximately 90% of androgen ablation failing prostate cancers overexpress AR (75–77),
Litvinov et al. • Genetics of Endocrine Disease
this could provide a mechanism for their resistance. In addition, experimental studies have demonstrated that there is
cross talk between the AR and the signaling pathway induced by costimulation of pathways involving protein kinase A (PKA) and/or certain peptide growth factors (93–97).
These studies have demonstrated that when AR is not expressed, signaling induced by PKA or certain peptide growth
factors (e.g. IL-6) is unable to stimulate the transcription of
prostate-specific marker genes like PSA, by androgen-independent prostate cancer cells. In contrast, when AR is expressed ectopically, PKA and IL-6 can now induce the expression of these marker genes constitutively even in an
androgen-depleted environment (93, 95–98).
For this latter effect of ligand-independent AR signaling by
IL-6, physical interaction between N-terminal domain of AR
with SRC-1 coactivator is critical (98). Although such physical AR/SRC-1 interaction does not require phosphorylation
of SRC-1 or ligand binding, transcriptional signaling by the
AR/SRC-1 complex does require phosphorylation of SRC-1
by MAPK activated in the signaling cascade initiated by IL-6
binding to its plasma membrane receptor expressed on prostate cancer cells (98). Besides activating the MAPK pathways,
such IL-6 binding to its receptor activates the janus-kinasesignal transducers and activators of transcription (STAT)-3
pathway (99, 100). This activation results in activated januskinase phosphorylating STAT-3 proteins in the cytoplasm.
Once phosphorylated, STAT-3 dimerizes and then is transported into the nucleus. Dimerized STAT-3 can bind ligandfree AR, allowing the complex to be translocated into the
nucleus where it can induce transcription (99). Such STAT3/AR activation allows resistance to androgen ablation by
AR-expressing prostate cancer cells both in vitro and in vivo
(100).
Rational drug development based on gain of function
changes in AR signaling pathways
These previously discussed genetic and epigenetic mechanisms provide a framework for rational drug development
for androgen ablation failing prostate cancer cells. The most
obvious way to prevent the gain of function AR-dependent
signaling is to prevent the expression of the AR protein itself.
Presently, this can be achieved experimentally by a variety
of molecular (e.g. antisense, small interfering RNA, Hammerhead ribozyme) (80 – 82) and small molecule approaches
(e.g. HSP-90 antagonists) (101). Although the nucleic acidbased approaches are potentially more specific for AR expression down-regulation, they are presently harder to develop clinically. The small molecule approaches have
identified that agents like geldanamycin can bind to the
Hsp-90/AR complex enhancing the degradation of the AR
protein by proteasomal targeting (101). Because Hsp-90
chaperones a series of additional proteins other than AR (e.g.
raf, Akt, etc.), such Hsp-90 antagonists may suffer from the
problem of their therapeutic index because they will downregulate more than just AR in normal as well as cancer cells.
An alternative approach is not to down-regulate AR directly but instead to inhibit the ability of AR to interact and
bind with the critical coactivators required for the formation
of productive transcriptional complexes. Because these are
J Clin Endocrinol Metab, July 2003, 88(7):2972–2982 2979
malignancy-acquired gain of function interactions, these interactions should be unique to androgen ablation-resistant
prostatic cancer cells, and thus inhibiting these interactions
should not produce host toxicity. To develop such inhibitors,
in vitro cell line model systems are critical because they allow
for both mechanistic studies for therapeutic target discovery
and rapid throughput screening to identify compounds for
drug development. It has been assumed that understanding
the interaction of the specific coactivators with AR at these
prostate-specific differentiation genes (i.e. PSA, hK2, PSMA)
in these model systems would also provide an understanding of the mechanisms whereby AR interactions regulate the
genes for proliferation and survival of malignant prostate
cells.
In a significant number of such model cell lines, however,
there is a dissociation between androgen-responsive regulation of malignant growth vs. regulation of the expression
of prostate-specific markers PSA and hK2 (102). These results
emphasize that tumor growth and the expression of the
differentiation-specific marker genes are independently regulated molecular events even if they share a requirement for
androgen and/or AR function. Additional independent
mechanisms can occur in prostate cancer cells for regulation
of expression for even the highly related PSA and hK2 genes
(102). Thus, the use of prostate-specific differentiation
marker gene regulation as surrogate model systems for clarifying AR signals of proliferation and survival of prostate
cancer is highly problematic. Future studies need to clarify
the mechanisms for androgen ligand-independent/ARdependent regulation of the genes that directly affect the
proliferation and survival of androgen ablation resistant
prostate cancer cells.
Conclusions
Clarifying how AR regulates growth-promoting genes
within prostate cancer cells without the requirement for
binding its physiological ligand DHT is critically needed.
This should be possible because androgen response elements
(AREs) are also present in growth regulatory genes. For
example, AREs have been documented in the promoters of
the cyclin-dependent kinase inhibitor p21 (103) and the fibroblast growth factor (FGF)-8 gene (104). Both p21 and
FGF-8 are growth promoting for prostate cancer cells resistant to androgen ablation (105–107), whereas in contrast p21
is not expressed by normal prostate epithelial cells (108) and
FGF-8 is not an andromedin produced by normal prostate
stromal cells (106). These latter results again demonstrate
that the AR-signaling pathways become hard-wired differently in androgen ablation resistant prostate cancer cells.
Although such a malignant acquisition allows these cells to
become potentially lethal, it also creates an Achilles’ heel for
their elimination. This raises the possibility that these latter
growth-related genes can be used as more appropriate genetic model systems for screening small molecule inhibitors
of gain of function AR signaling, characteristic of androgen
ablation-resistant prostate cancer cells. Using such a screen,
rational drug development should be able to convert theory
to therapy for this devastating disease.
2980
J Clin Endocrinol Metab, July 2003, 88(7):2972–2982
Acknowledgments
We appreciate the secretarial support of Dorothea K. Stieff and thank
Don Vindivich for help in preparation of the figures.
Received December 26, 2002. Accepted April 4, 2003.
Address all correspondence and requests for reprints to: John T.
Isaacs, Ph.D., Johns Hopkins Oncology, 1650 Orleans Street, Cancer
Research Building 1M44, Baltimore, Maryland 21231-1000. E-mail:
[email protected].
This work was supported by National Institutes of Health Grant
DK52645.
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