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The Prostate Supplement 6:9-12 (I 996)
Provocative Aspects of Androgen Genetics
Terry R. Brown
Department of Population Dynamics, Division of Reproductive Biology, johns Hopkins
University School of Hygiene and Public Health, Baltimore, Maryland
ABSTRACT: Androgens play a key role in prostate structure and function, leading to the
hypothesis that effects of the hormone are an important component in the development of
prostatic disease. Differences in serum testosterone levels and 5a-reductase activities between ethnic and racial groups have been implicated in the variable incidence of prostate
cancer among certain populations. Androgen receptors transduce the steroid signal within
cells, but attempts to correlate differencesin receptor levels with prostatic disease have been
unsuccessful. However, molecular studies of androgen receptor gene structure have recently provided new insights toward defining a genetic basis for the pathology associated
with three diseases-spinal bulbar muscular atrophy, breast carcinoma, and prostate cancer-affecting middle-aged and older men. In summary, epidemiologic data on androgen
biosynthesis, metabolism, and action of androgens and molecular genetic analysis of gene
structure have led to a new understanding of the interrelationshipsbetween environmental
and genetic factors that may impact on the incidence of certain pathologic conditions in
men. 0 19%Wiley-Liss, Inc.
KEY WORDS:
testosterone, dihydrotestosterone, Sa-reductase, androgen receptor,
prostate
INTRODUCTION
Androgens promote normal male sex differentiation and development, pubertal masculinization, and
initiation of spermatogenesis, and maintenance of
male sexual function. Testosterone is the primary androgen secreted by the testes, but its conversion by
the enzyme, 5a-reductase, to the more biologically
active metabolite, 5a-dihydrotestosterone (DHT), is
critical for differentiation and development of the
male external genitalia and prostate. Both testosterone and DHT bind to the intracellular androgen receptor to regulate androgen-dependent cellular differentiation and function. The role of testosterone
and DHT in maintenance of normal prostatic function
and in the pathobiology of prostatic carcinoma and
benign prostatic hyperplasia has been the focus of
many studies [l].At the center of these studies have
been measurements of 5a-reductase enzyme activity
and intracellular DHT concentrations, and of the intracellular concentration and subcellular localization
of the androgen receptor. Pharmacologic approaches
to prevention or treatment of prostatic diseases has
relied on inhibitors of 5a-reductase and antiandro0 I996 Wiley-Liss, Inc.
gens to prevent the conversion of testosterone to
DHT and to block the transcriptional activation of
gene transcription by the androgen receptor, respectively. A few recent studies of 5a-reductase activity
and the androgen receptor have suggested that the
production of DHT and the molecular structure of the
androgen receptor may provide an unexpected genetic basis for several pathologic conditions in men.
~CX-REDUCTASE
ACTIVITY
The incidence of prostate cancer varies widely between countries and ethnic groups. Black Americans
have the highest incidence rates worldwide, whereas
native Japanese have among the lowest. The reasons
for this risk differential are unknown; however, cir-
Received for publication January 16, 1995; accepted October 5,
1995.
Address reprint requests to Dr. Terry R. Brown, Department of
Population Dynamics, Division of Reproductive Biology, Johns
Hopkins University School of Hygiene and Public Health, 615
North Wolfe Street, Baltimore, MD 21205.
10
Brown
Androgen Receptor
Prostate Cell
R608K
blood
etfect
blood
Fig. I. Androgen metabolism and effects on prostate cells. Testosterone (T), the primary androgen secreted by the testes into
blood, enters prostate cells, where it can bind directly to the
androgen receptor (AR) or be converted by the Sa-reductase
enzyme to the more potent androgen, 5a-dihydrotestosterone
(DHT). DHT binds t o AR with higher affinity than T. AR regulates
androgen effects in cells. Measurement of DHT or of the Sareduced products, 3a-androstanediol glucuronide and androsterone glucuronide. provides an index of androgenic potency.
culating testosterone concentrations are higher in
young black men than in young white men and may
help explain the underlying differences in subsequent prostate cancer incidence between these two
populations [2]. By comparison (Fig. l), serum testosterone concentrations of young Japanese men are
not significantly different from those in young white
or black men. In one study, both black and white men
had significantly higher serum levels of 5a-reduced
androgen products, androstanediol glucuronide, and
androsterone glucuonide, than were found in Japanese men [2]. In a second study, both precursor androgens (dehydroepiandrosterone sulfate and androstenedione) and 5a-reduced androgen products
(androstanediol glucuronide and androsterone glucuronide) were higher in the serum of Caucasian men
than of Chinese men [3]. These studies provide circumstantial evidence for genetic differences in androgenic activity among men of different racial backgrounds that may have a role in prostatic carcinoma.
ANDROGEN RECEPTOR
The androgen receptor is a member of the superfamily of ligand-activated nuclear transcription factors
that mediates androgen action at the transcriptional
level. Cloning of the cDNA and deduction of the structure of the androgen receptor have contributed significantly to our understanding of the molecular basis
of androgen action. The androgen receptor protein is
composed of 919 amino acids and, like all members of
this family, it is structurally organized as three domains; an N-terminal transactivation domain, two
zinc-finger motifs within a central DNA-binding domain, and a C-terminal ligand binding domain (Fig.
2). The gene encoding the androgen receptor consists
of eight exons, the first encoding the large N-terminal
Fig. 2. Mutations in the androgen receptor alter androgen action. In spinal bulbar muscular atrophy, the polymeric glutamine
region in the transcriptional activation domain is expanded from
the normal number of glutarnine residues ( I 1-30) to more than 40;
in male breast cancer, two different amino acid substitutions have
been identified in the second zinc finger of the receptor; and in
prostate cancer, several different amino acid substitutions in the
steroid-binding domain have been associated with altered cell responsiveness t o antiandrogens, progesterone and adrenal androgens.
domain, the second and third encoding each of the
two zinc fingers required for binding to DNA, and the
fourth through eighth exons encoding the hormone
binding domain. The gene is localized to the X chromosome at q11-12.
Whereas in vitro deletion mutagenesis of the AR
cDNA has confirmed the importance and role of the
functional domains of the androgen receptor, more
detailed understanding of its structure and function
has derived from the naturally occurring mutations
present in subjects across the phenotypic and clinical
spectrum of the androgen insensitivity syndromes [4].
The mutations of the androgen receptor gene associated with the various forms of androgen insensitivity
inevitably result in loss of function, either partial or
complete, and are manifested by abnormalities in sex
differentiation. The present discussion of genetic alterations in the androgen receptor focuses on potential
“gain of function” mutations associated with extension of the polymorphic glutamine repeat region of the
N-terminal transactivation domain in spinal bulbar
muscular atrophy (Kennedy syndrome), mutation of
the DNA-binding domain in male breast carcinoma,
and somatic cell mutations in the ligand-binding domain from prostate tumors (Fig. 2).
X-Linked Spinal Bulbar Muscular Atrophy
X-linked spinal bulbar muscular atrophy is a form
of adult-onset progressive motor neuron degenerative
disease uniquely associated with male hypogonadism
(gynecomastia, oligospermia, testicular atrophy, impotence) [5]. Affected men have a characteristic expansion of the trinucleotide repeat, (CAG),, in the 5’
translated portion of the androgen receptor gene from
Genetics of Androgen Action
a normal, polymorphic length of n = 11-31 to n 240.
The resulting androgen receptor protein has an expanded polyglutamine tract in its N-terminal transactivation domain and is postulated to lose an intrinsic
function that leads to a mild form of androgen insensitivity; however, it also appears to gain a novel extrinsic function that destroys motor neurons. The unexplained mechanism that culminates in this form of
neuron-specific death is the prototype for three different adult-onset autosomal dominant forms of neuronopathies caused by (CAG), expansions in the
genes associated with Huntington disease, spinocerebellar ataxia, type 1, and dentatorubropallidoluysian
atrophy [6]. A number of mechanisms, including reduction in transcriptional activity due to a conformational aberration in the androgen receptor protein,
altered translation initiation efficiencies, or differential
proteolytic processing of the receptor protein, have
been proposed to explain the involvement of the receptor in this neurodegenerative disease.
Breast Cancer
Breast cancer is very rare in men, and the etiology
is unknown. Factors such as hypoandrogenism in
Klinefelter syndrome, testicular atrophy, orchitis, undescended testes, testicular trauma, and infertility
have been determined as risk factors for male breast
cancer. Recently, two germline mutations in the androgen receptor gene of subjects with breast cancer
and partial androgen insensitivity have been reported
[7,8]. Both mutations occurred within exon 3 encoding the second zinc finger of the DNA-binding domain of the androgen receptor. Each was caused by a
point mutation in adjacent codons causing amino
acid substitutions of argm7-*glu and arg608+1ys. The
mechanism by which these mutant receptors can lead
to breast cancer is unknown, as the function of these
mutant receptors has not been characterized in a
transfection system. One could postulate that these
amino acid substitutions within the DNA-binding domain might lead to alternative recognition of hormone response elements such that their respective
androgen specificity of transactivation might be altered.
Prostate Cancer
Prostate cancer is one of the most common cancers
in men. The growth of prostatic carcinomas is sensitive to androgen and hormonal manipulation has
been used for its treatment. About 75% of prostate
cancers initially respond to endocrine therapy; however, more than one-half of responders gradually become resistant to this therapy. Changes in tumors
from an androgen-responsive to an androgen-resis-
II
tant state have been explained by adaptation or clonal
selection of cancer cells. Thus, relapsed tumors consist primarily of androgen-independent cells. These
cells contain variable levels of androgen receptor by
ligand binding assays and often retain immunocytochemically detectable androgen receptor protein.
These findings suggest that structural abnormalities
may arise in the androgen receptors of tumor cells to
alter their function.
A mutation in the androgen receptor of LNCaP
cells, derived from a metastatic lesion in the lymph
node of a subject with prostate cancer, provided the
first evidence that such mutations might exist in prostate tumor cells [9]. Subsequently, our laboratory was
the first to idenbfy a somatic cell mutation in an organ-confined tumor of a subject with prostatic carcinoma [lo]. The mutation &A of codon 730 was
present in approximately 50% of cells from the tumor
specimen, whereas only the normal coding sequence
was identified in DNA from peripheral lymphocytes
of the same individual. The mutation in LNCaP cells
occurs in codon 877 (thwala) of exon 8 and the mutation in exon 5 of our patient caused substitution of
valnO+met. The mutation of thr877+ala leads to paradoxical stimulatory activity of the androgen receptor
in the presence of the antiandrogen, hydroxyflutamide. Recent studies have shown that this same mutation is present in the prostate tumors from other
subjects, suggesting that this region is hypermutable
during the evolution of tumors to the metastatic form
and from androgen-responsive to androgen-independent [ll].One subject with the AR gene mutation,
thrsV+ala, in a metastatic tumor also had a second
mutation, leu701+his, in the primary tumor [12].
In an androgen-unresponsive tumor from another
subject, a G-A transition occurred in codon 715 of
the AR gene, causing the substitution of met for Val
1131. In transfection assays, this mutation did not significantly alter transactivation by androgens such as
dihydrotestosterone or mibolerone but increased the
relative ability of progesterone and the adrenal androgens, androstenedione and dehydroepiandrosterone, to induce androgen-responsive gene transcription. These findings suggest that the mutant receptor
leads to a gain of function, rather than a loss. Another
AR gene alteration that contracted the number of glutamine repeats from 24 in some cells to 18 in others,
occurred in the tumor from a subject with a paradoxical response to flutamide therapy [14]. The functional significance of this example of genetic instability in tumor cells is unknown.
SUMMARY
Taken together, these results suggest an involvement of AR mutations in tumor progression, rather
I2
Brown
than in tumor promotion. Functional characterization
indicates an increase in receptor activity related to
alterations in its ligand-binding properties. Whereas
most specimens investigated represent primary lesions of early and intermediate stages, further examination of highly malignant prostatic lesions and tumor metastases is required to determine the full
extent of androgen receptor involvement in tumor
progression and the design of adequate therapeutic
modalities.
ACKNOWLEDGMENT
The work performed in our laboratory was supported by grant DK43147 from the National Institutes
of Health.
REFERENCES
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