prevention_of_prostate_cancer_by_androgens_experimental_paradox_or_clinical_reality.pdf

European

Urology

European Urology 46 (2004) 285–295

Review

Preventionof Prostate Cancer byAndrogens:

Experimental Paradoxor Clinical Reality

Mich`le Algart´-G´nin a , Olivier Cussenot a,b,* , Pierre Costa c

a CeRePP/EA3104, UFR Biom´dicale, Universit´ Paris 5/Paris 7, France

b D´partement d’Urologie, Hˆpital Tenon (AP-HP), 4 rue de la Chine, 75020 Paris, France

c Urologie, Hˆ pital G. Doumergue, Nˆmes, France

Accepted 1 April 2004

Available online 10 May 2004

Abstract

Androgen replacement therapy in the aging male with partial androgen deﬁciency improved quality of life. However,

such treatment is prohibited for men with a preexisting prostate cancer. The possibility of an increased risk of prostate

cancer for healthy men has also been suggested on theoretical basis but recent experimental data showed that androgens

may act in prevention of prostate cancer. In this review, we try to evaluate beneﬁts and risks associated to a hormonal

replacement therapy in regard to recent data. Several studies analyzing the role of testosterone for prostatic epithelial

cells evidenced that testosterone acts in prostatic cell differentiation but does not have a direct role for induction of cell

proliferation. Moreover, clinical studies have shown that low free testosterone levels in serum is associated with

aggressive prostate cancer, like that has been observed in men with prostate cancer under prostate cancer chemo-

prevention by ﬁnasteride. These data suggest that an androgen pathway disruption in prostate is responsible of cell

deregulations that may be associated not only with apoptosis of differentiated prostatic cells but also with potential cell

transformation. The effects of androgens withdrawal for prostate cancer therapy induced in a short time the tumor arrest

growth. However with time, cells adapt to low levels of androgens leading to the evolution of an androgen-independent

tumor, which is more aggressive and most often fatal. The molecular mechanisms of this evolution begin to merge. A

hypothesis is that such mechanisms could be initiated in elderly men with an androgen deﬁciency. The question is raised

of whether hormonal replacement therapy could prevent prostate cancer. An encouraging recent study performed on

rats demonstrated a protective effect of DHEA for prostate cancer. However, the putative role of the normalization of

DHEA or other androgen levels in prevention of prostate cancer should be evaluated in clinical trials.

Keywords: Androgens; Hormonal replacement therapy; Prostate cancer; Prevention; Androgen receptor;

Androgen antagonists; 5a-reductase inhibitors

1. Introduction

symptoms as decreased libido, loss of muscle mass,

osteoporosis, decreased cognitive ability and depres-

sion. Multiple studies have demonstrated that the rees-

tablishment of normal hormonal levels improves quality

of life and decreases symptoms associated with the loss

of androgens. However, it has been reported that andro-

gen replacement therapy increased the growth of

already existing prostate carcinoma, meaning that

before prescription, patients should be carefully

screened to detect the presence of a preexisting tumor.

However, for healthy patients, the question is raised of

whether androgen replacement therapy could induce

Testosterone levels decrease with age and elderly men

present a partial androgen deﬁciency. The evolution of

androgen deﬁciency has been estimated to be 16.2%

(40–49 years), 20% (50–59 years), 22.6% (60–69 years)

and 26% (80 years and more). Testosterone deﬁciency is

associated with multiple deregulations that can lead to

* Corresponding author. Tel. þ 33-1-56-01-64-95;

Fax: þ 33-1-56-01-73-06.

E-mail address: olivier.cussenot@tnn.ap-hop-paris.fr (O. Cussenot).

doi:10.1016/j.eururo.2004.04.012

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M. Algart ´ -G ´ nin et al. / European Urology 46 (2004) 285–295

prostate tumor. Regarding to recent data, we made a

tentative to evaluate beneﬁts and risks of hormonal

replacement therapy for men presenting a decrease in

androgens. We reported several studies that described

the role of androgens for normal epithelial prostatic

cells. Concerning the levels of circulating testosterone in

the serum of patients with prostate cancer, we analyzed

several studies that evidenced a correlation between low

levels of testosterone for patients with a prostate tumor.

We also described new studies analyzing the molecular

mechanisms involved in response to a decrease of

androgens. Such mechanisms induced cell deregulations

and are responsible for the evolution of tumors to

androgen-independent tumors. However, a question is

whether such mechanism could be initiated in elderly

men with a deﬁciency in androgens, suggesting that

hormone normalization may prevent prostate cancer.

The potential role of hormonal replacement therapy

in the prevention of prostate cancer is also evaluated

in regard to new data with animals treated with DHEA.

one plays a key role in the development and

maintaining of prostate. The androgen receptor is

activated by two ligands; testosterone and dihydrotes-

tosterone, this latter one binding the androgen receptor

with a higher afﬁnity [1] . The interaction of steroids

with their speciﬁc receptors induces the binding of

such activating receptors to promoter region of genes.

Steroid receptors participate directly to the induction of

transcription of genes ( Fig. 1 ). However, the activation

of cells by steroids can also induce a so-called ‘‘non-

genomic’’ (second messenger mediated) action of ster-

oid receptors via the MAP kinase [2] or a ‘‘non-

genomic’’ action of non-classic steroid receptors; G

protein coupled receptors (GPCR) increasing intracel-

lular concentration of Ca 2 þ ( Fig. 1 ). Such mechanisms

have been described for hormones as glucocorticoids,

progesterone, estrogens, androgens, neurosteroids,

mineralcorticoids, vitamin D3 and the thyroid hor-

mones T3 and T4 [3] . Testosterone has been described

to act through or independently of intracellular andro-

gen receptor in cells of the immune system as described

in studies performed on macrophages and T lympho-

cytes. Benten et al. described testosterone signaling in

macrophages IC-21 lacking intracellular androgen

receptor but exhibiting a membrane form of testoster-

one receptor. Treatment of such cells by testosterone

induces a rapid and transient increase of intracellular

2. Therole ofandrogens onepithelialcells

proliferation anddifferentiation

Testosterone is the most abundant hormone circulat-

ing in males. Through the androgen receptor, testoster-

Fig. 1. Summary of testosterone mechanisms for cell induction. ‘‘Genomic’’ action of androgens: interaction of DHT (after testosterone conversion by 5a

reductase) with androgen receptors (AR) induces the binding of such activating receptors to promoter region of genes (A). ‘‘Non-genomic’’ action of

androgen receptor: interaction of DHT with androgen receptors induces activation of MAPK. MAPK activates second messengers and transcription factors to

regulate speciﬁc gene expression (B). ‘‘Non-genomic’’ action of non-steroid receptors: AR receptor induces G protein coupled receptors (GPCR), leading to

increase of intracellular Ca 2 þ release from intracellular Ca 2 þ stores. The link between GPCR activation by testosterone and induction of transcription factors

has to be determined (C).

M. Algart ´ -G ´ nin et al. / European Urology 46 (2004) 285–295

287

concentration of Ca 2 þ . It is well documented that the

intracellular elevation of calcium in cells of the

immune system induces the activation of transcription

factors such as NF-AT, Jun kinase and NF-kB. Acti-

vated transcription factors participate to the regulation

(activation or inhibition) of the transcription of speciﬁc

genes. However, activation of transcription factors in

response to testosterone binding GPCR has not yet

been demonstrated. The physiological signiﬁcance of

the signal triggered by testosterone via a membrane

receptor in remains to be elucidated [4] .

Many studies have demonstrated the role of andro-

gens in prostate growth and differentiation. However

these functions involved complex interactions between

multiple growth factors and receptors with the different

cell type constituting the prostate. The paracrine role of

growth factors expressed by stromal, epithelial and

neuroendocrine cells regulates differentiation and

growth of the prostate [5] .

The role of growth factors in prostate development

and maintaining has been largely studied; however, the

exact contribution of androgens still remains unclear.

Several studies analyzed the role of androgens on

proliferation and differentiation for isolated epithelial

cells from prostate. To analyze the effect of androgens

on isolated cells, human prostatic epithelial cells

(HEPC) derived from normal, benign (BPH), primary

cancer and metastatic cells were treated with increas-

ing concentrations of dihydrotestosterone (10 11 to

10 7 M). The treatment including different doses of

DHT had no effect on the proliferation of normal cells

or cells from benign or metastatic prostate cancer.

Furthermore, adding the anti-androgen Casodex 1 to

cell media did not induce growth inhibition of cell

proliferation. The absence of effects of androgens for

metastatic cell lines is likely to be related to androgen

independent growth of tumoral cells. For normal and

BPH primary cells, androgens have no direct effects

on proliferation, however primary cells may have

decreased expression of androgen receptor in culture

[6] . Also, several studies have demonstrated the

absence of direct mitogenic effects of DHT on rodent

prostatic epithelial cells. A systematic analysis of

normal rat prostate epithelial cells demonstrated a

direct mitogenic effect of several hormones, including

insulin, epidermal growth factor, glucocorticoids and

prolactin. However this analysis didn’t reveal a mito-

genic effect of DHT [7] . A second study submitted

primary cultured epithelial cells derived from the rat

dorso-lateral prostate to a treatment of DHT (10 10 to

10 6 M). Interestingly, cell proliferation was signiﬁ-

cantly inhibited at the physiological dose of 10 9 M

[8] . These data suggest that DHT is not directly

involved in the proliferation of isolated epithelial cells.

The discrepancy between experiments performed on

isolated epithelial cells demonstrating that DHT had no

effect on isolated epithelial cells and in vivo studies

suggesting a role for androgens in proliferation and

differentiation [9,10] may reﬂect an indirect action of

androgens on prostate epithelium for proliferation. In

the prostate, growth of epithelial cells may be the

consequence of a multi-hormonal contribution.

However, a role for androgens in prostate differen-

tiation has been largely described [10] . The differen-

tiated functions of primary cultures of human prostate

epithelial cells have been analyzed. Such isolated cells

in culture lose secretory differentiated functions and

androgen responsiveness, as well as the capacity of re-

aggregation in the ﬁrst 2 or 3 weeks. The authors

showed that a combined treatment of androgens and

retinoid is able to preserve low level of PSA (Prostatic

Speciﬁc Antigen) for at least 40 days. Cells also

exhibited the capacity of re-aggregation and still

express androgen receptors [11] . The participation of

androgens to prostate differentiation is supported by

multiple studies and is mediated by the high range of

genes activated in response to the binding of testoster-

one or dihydrotestosterone to the androgen receptor.

Recently, a cDNA microarray analysis identiﬁed two

dozen of androgen responsive genes, including pro-

teins involved in metabolism, chaperoning, trafﬁcking,

cell cycle apoptosis, protein synthesis, structural and

extracellular matrix proteins. Also novel proteins were

induced, which functions remain to be elucidated [12] .

These data suggest a role of androgens in epithelial cell

differentiation, without a direct mitogenic effect.

Interactions between stromal and epithelial cells are

essential for prostate differentiation and growth. In

normal prostate, growth of epithelial cells is dependent

of growth factors secreted by stromal cells and exerting

paracrine effects. However, epithelial cells derived to

malignant tumor growth independently from stroma.

Several growth factors have been identiﬁed to be

secreted by stromal cells and to stimulate proliferation

of epithelial cells. Studies performed on prostate devel-

opment or on isolated cells suggest that Fibroblast

growth factor, FGF-7 (KGF), acts as an andromedin

that mediates the indirect control of epithelial cells by

androgens. Androgens induced expression of FGF-7 by

stromal cells and, through paracrine control, FGF-7

induces proliferation of epithelial cells [13] . Regula-

tion of FGF-7 by androgens was not identiﬁed in vivo,

as demonstrated by RNAse protection assays experi-

ments. However, experiments suggest that FGF-7 and

androgen receptor pathway may interact since antian-

drogens can block FGF-7 stimulated development [14] .

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FGF-10 is the second candidate identiﬁed to act as an

andromedin. FGF-10 is secreted by stromal cells and

induced proliferation of epithelial cells. As for FGF-7,

androgens induced expression of FGF-10 from stromal

isolated cells, however in vivo experiments should be

performed to determine if androgens directly induced

FGF-10 gene expression or if androgen receptor path-

way may interact with FGF-10 [15] . Also, androgens

action on epithelial cells may be linked to several

pathways, including FGF-7 and FGF-10.

tion of testosterone from patients presenting a prostate

tumor. However, the fact that the authors of this study

observed an increase of testosterone level after pros-

tatectomy suggests that factors secreted by the cells

from the tumoral prostate may inﬂuence testosterone

levels [19] .

The serum androgen bioactivity (ABA) was quanti-

ﬁed by using a recombinant cell-based bioassay. This

functional assay included the association of testoster-

one to the androgen receptor followed by the binding of

this complex to the promoter of a reporter gene. The

results showed a decrease in androgen bioactivity for

patients with low Gleason score ( 5) whereas the level

of free testosterone was not changed, as described

above. This bioactivity is more decreased in the serum

of patients with a high Gleason score ( 8). One of the

explanations for the discrepancy between the level of

free testosterone and the bioactivity detected may

reﬂect the presence of androgen antibodies in the

serum of patients presenting a prostate cancer. How-

ever, the androgen bioactivity is inversely proportional

to the tumor grade and volume [20] .

Finally, a study performed on a large cohort of 879

patients was realized, following different parameters

during several years. Pretreatment total testosterone

was measured before prostatectomy and this analysis

could be used as prognostic in patients with prostate

cancer. This analysis conﬁrmed that low levels of

testosterone can predict aggressive disease. The level

of this hormone appeared inversely proportional to the

tumor grade [21] .

A recent study (Prostate Cancer Prevention Trial)

[22] performed on a large cohort of 18882 men within 7

years analyzed the potential effect of ﬁnasteride, a 5a-

reductase type 2 inhibitor, on prostate cancer preven-

tion. In the trial, men chosen were 55 years and

presented a normal digital rectal examination and a

PSA level not exceeding 3.0 ng per millimetter. Men

received ﬁnasteride (5 mg per day) treatment or pla-

cebo. Men underwent annual digital rectal examination

and PSA measurements, and when abnormal examina-

tions were observed a biopsy was performed. The rate

of prostate cancer diagnosed was higher in the placebo

group (24%) compared to the group receiving ﬁnaster-

ide (18%). However, patients receiving ﬁnasteride

exhibited a higher proportion (37%) of tumors with

worse Gleason score (7–10), compared to the placebo

group (22%). The difference of cancer incidence which

appears early in the trial suggests that ﬁnasteride could

inhibit the development of emerging cancer. As the

difference between the ﬁnasteride and placebo groups

increases with time, this suggests that ﬁnasteride

postpones the clinical emerging of prostate cancer.

3. Low levels of testosterone are

detectedinthe serum of patients

with prostate cancer

Testosterone levels decrease with age and elderly

men present a partial androgen deﬁciency, while pros-

tate cancer incidence increases with the age [16] .

Several studies analyzing the level of total testosterone

in patients with a prostate cancer showed controversial

results. Recently, reports that discriminated free and

total testosterone evidenced that a low level of free

testosterone in the serum of patients can be associated

with a prostate cancer compared with healthy men in

the same range of age. Interestingly, a decrease in free

testosterone level has been observed on 14% of elderly

men belonging to a group with a cancer of the prostate

(Gleason score 6 or 7) undetectable with digito-rectal

examination and presenting normal PSA level [17] .

The same group investigated whether the level of free

testosterone could have an incidence on prostate cancer

by analyzing biopsy from men with low versus normal

free testosterone level. The study showed that patients

with low free testosterone have an increased mean

percent of biopsies that revealed cancer (43% versus

22%) with higher Gleason score of 8 or greater (7 of 64

versus 0 of 48). Moreover the authors did not evidence

changes in the level of total testosterone. In agreement

with these results, the authors raised the possibility that

level of free testosterone in the sera of patients could be

a marker of aggressive prostate cancer [18] .

The levels of both total and free testosterone have

been compared in the serum of patients exhibiting a

moderate or high grade prostate cancer; it appear that a

low level of testosterone was detected for patients with

a high grade tumor, and normal levels are observed in

patients presenting only a moderate grade of tumor.

Testosterone levels have also been followed in a group

of patients presenting a high grade prostate cancer.

After radical prostatectomy patients exhibit a higher

level of both total and free testosterone. Different

hypotheses have been proposed to explain the ﬂuctua-

M. Algart ´ -G ´ nin et al. / European Urology 46 (2004) 285–295

289

However, the emergence of prostate cancer with a

higher grade for patients treated with ﬁnasteride may

be in favor of the hypothesis that decrease of DHT is

associated with more aggressive tumors [22] . Even if a

wrongly pejorative interpretation of the Gleason score,

induced by Finasteride, has been suggested.

tor gene has also been reported in prostate tumorigenic

cells deprived for steroids [25] . Hormone receptors,

following activation, bind DNA to activate gene tran-

scription together with co-activator complex having

histone acetyltransferase activity [26] . Several co-acti-

vators or repressor molecules have been identiﬁed to

interact with androgen receptors for optimal activation

of transcription. Brady et al. showed the role of co-

activator Tip60 which is a histone acetyltransferase that

interacts directly with the androgen receptor to

enhance the transcriptional activity of this molecule.

Tip60 is a co-activator speciﬁc for class I nuclear

hormone receptor, also including estrogen and proges-

terone [27] . These authors also evidenced, following

androgen withdrawal, the increased expression of both

mRNA and protein of Tip60 with a major localization

in the nucleus in androgen-independent tumors.

Also, experiments using the prostate tumor cell

line LNCaP demonstrated the same result; up-regula-

tion and nuclear localization of Tip60. In such cells

Tip60 functionally binds the promoter region of PSA.

These studies demonstrated that in conditions of depri-

vation of androgens, cells adapt the regulation of

molecules to bypass the signals regulated by testoster-

one. However the upregulation of functional co-acti-

vators, less speciﬁc than hormones, may have other

unexpected effects affecting cell proliferation or dif-

ferentiation [27,28] .

Another characteristic of androgen-independent

tumors is the expression of neuroendocrine patterns

of differentiation. By secretion of neuropeptide,

neuroendocrine cells exert auto/paracrine activities

involved in proliferation, transformation and metasta-

sis [29,30] . Several studies demonstrated that

withdrawal of androgens is responsible for the differ-

entiation of prostatic epithelial cells in neuroendocrine

cells [31,32] . However the mechanisms of this change

in cell population still remained unclear. Inhibition of

androgen receptor expression, by the method of silen-

cing RNA (siRNA), induced biochemical and morpho-

logical changes associated with the neuroendocrine

differentiation process on LNCaP cells. Thus, the

androgen receptor is directly responsible for the repres-

sion of an intrinsic neuroendocrine differentiation pro-

cess in androgen responsive tumoral cells. The

alteration of androgen receptor or the withdrawal of

testosterone cells may be linked to the neuroendocrine

differentiation in prostate cancer [33] .

These studies analyzed the molecular mechanisms

that may be involved in the androgen-independent

evolution of a prostate tumor. It appears that a with-

drawal in androgens induced the adaptation and/or

selection of cells responsive to very low levels

4. Deregulations of tumorigenic

prostatecellsinducedby thedecrease

ofsteroidlevelsleading to

hormone independence

At early stages of prostate cancer, cells are depen-

dent of androgens for growth and survival. One of the

ﬁrst treatments for patients with advanced prostate

cancer is the androgen ablation, hence inducing at

early stages of the treatment a tumor regression by

the induction of apoptosis for epithelial cells. However,

tumors still progress in an androgen-independent way,

more aggressive, leading to metastasis and in death.

Multiple studies try to elucidate whether the androgen

ablation is responsible for the evolution of the prostate

cancer to androgen-independent disease.

One of the effects of steroid ablation is the selection

of prostatic cells sensitive to low levels of androgens.

The prostatic cancer cell line LNCaP (obtained from a

lymph node metastatic prostate cancer), which

expresses an androgen receptor mutated in the ligand

domain, has been cultured in conditions with a steroid

free medium in long term. Such treated cells respond

strongly to low levels of androgens compared to

LNCaP cells cultured in the presence of steroid. The

level of response is evaluated by measuring the fold

induction of a reporter gene controlled by a promoter

region containing androgen responsive elements [23] .

The molecular mechanisms of androgen indepen-

dence are largely studied, several modiﬁcations have

been evidenced, such as ampliﬁcation of the androgen

receptor gene in approximately 30% of the tumors or

missens mutations in the androgen binding domain of

the androgen receptor [24] which led to an hypersen-

sitive receptor activated by a wide range of steroid

hormones or antiandrogen molecules. Also, some

growth factors as insulin-like growth factor (IGF),

keratinocyte growth factor (KGF), and epidermal

growth factors (EGF) can activate the androgen recep-

tor in the absence of androgens and induced genes

normally regulated by steroid hormones. Intracellular

molecules involved in signal transduction such as

AKT and MAPK, generally upregulated in cancer,

also bypass the androgen pathway and induce multiple

cellular deregulations. The ampliﬁcation of the recep-

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