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The Prostate 28992-405 ( I 996)
REVIEW ARTICLE
Regulation of Prostatic Growth and Function by
Peptide Growth Factors
Zoran Culig, Alfred Hobisch, Marcus V. Cronauer, Christian Radmayr,
Anton Hittmair, Ju Zhang, Martin Thurnher, Georg Bartsch,
and Helmut Klocker
Departments ofUrology (Z.C.,A.Ho., M.V.C., C.R,].Z., M.T., G.B., H.K.) and Pathology
(A.Hi.), University of Innsbruck, Innsbruck, Austria
ABSTRACT:
Polypeptide growth factors are positive and negative regulators of prostatic
growth and function. Expression and biological effects of epidermal growth factor (EGF),
transforming growth factors (TGFs) a and p, fibroblast growth factors (FGFs), and insulinlike growth factors (IGFs) in the prostate have been extensively studied. EGF and TGFa,
which share the same receptor, are strong mitogens for prostatic epithelial and stromal cells.
Their paracrine mode of action in normal tissue and early-stage tumors is apparently altered
towards an autocrine stimulation in hormone-independent tumors, which gain the ability to
produce TGFa by themselves. TGFp has a dual role in the regulation of prostatic growth. It
inhibits growth of prostatic epithelial cells in culture and mediates programmed cell death
after androgen withdrawal. However, advanced prostatic carcinomas become insensitive to
the inhibitory effect of TGFP. Several members of the FGF family have been identified in the
prostate. They are mainly or exclusively expressed in the stromal cells, and stimulate the
epithelial cells. In the rat Dunning tumor model, progression is accompanied by distinct
changes in the expression of FGFs and their receptors. In the hyperplastic tissue, basic FGF
(bFGF) is accumulated. This growth factor is also a potent angiogenic inducer, expression of
which may determine the metastatic capability of a tumor. IGFs are paracrine growth stimulators in the normal and hyperplastic prostate. It is still under consideration whether
prostatic cancer cells gain the ability to produce IGF-I by themselves and thus shift to an
autocrine mode of IGF-I stimulation. Growth factors also interact with the androgen-signaling pathway. IGF-I in particular, other growth factors as well, can activate the androgen
receptor. 0 1996 Wiley-Liss, Inc.
KEY WORDS:
positive and negative growth factors, autocrine and paracrine mode of
action, epidermal growth factor, transforming growth factors, heparinbinding growth factors, insulin-like growth factors, benign prostatic
hyperplasia, prostatic carcinoma
INTRODUCTION
The prostate gland requires androgens for proliferation and maintenance of its function [l].In addition
to hormones, a whole battery of other regulators is
involved in the fine-tuning of prostatic growth and
differentiation. Among them are many polypeptide
erowth factors. which are generallv locallv produced
0 I996 Wiley-Liss, Inc.
in culture need substances other than androgens for
proliferation [2]. In this connection, epidermaigrowth
factor (EGF), transforming growth factor-a (TGFa),
transforming growth factor-p (TGFP), insulin-like
growth factors-I and -11 (IGFs), and heparin-binding
Polypeptide Growth Factors in the Prostate
Normal prostate
BPH
Early stages prostatic cancer
Fig. 1.
393
Late stages prostatic cancer
Mode of action of polypeptide growth factors in the normal, hyperplastic, and carcinomatous prostate.
growth factors have been studied most extensively.
Their compartmental localization, regulation of production and secretion, expression of their receptors,
and their biological effects have been described (Fig.
1). The aim of the present paper is to provide an
overview of current knowledge about these growth
factors and their receptors in normal, hyperplastic,
and carcinomatous prostates.
MITOGENIC EFFECTS OF EGF A N D TGFa IN
THE PROSTATE
EGF and TGFa are related polypeptides which
consist of 53 and 50 amino acids, respectively, share
about 35% sequence homology, and bind to the same
cell surface receptor [3]. It is, therefore, not surprising
that TGFa and EGF have many biological effects in
common. However, EGF is generally secreted by
both normal and malignant cells, while TGFa is predominantly produced by tumor cells. The EGF/TGFa
receptor consists of an extracellular binding domain
and a cytoplasmic part that encodes a tyrosine kinase.
Binding of one of these two growth factors to its receptor activates the intrinsic protein kinase in its
intracellular part, and leads to phosphorylation of intracellular proteins and activation of second messenger systems [4] (Fig. 1).
The first studies on EGF in prostatic tissue and
fluids suggested that this growth factor has a mitogenic role in the prostate gland. In fact, human prostatic secretions were found to contain the highest
EGF levels of all biological fluids [5]. Large amounts
of EGF are also present in prostatic tissue; it was iden-
tified as one of two major growth factors in extracts of
the rat ventral prostate [6]. EGF is androgen-regulated; androgen withdrawal by castration in mice is
followed by a reduction in prostatic EGF levels
which, however, can be restored by administration of
testosterone [7J In cultured epithelial and stromal
cells EGF proved to be one of the most potent stimulators of proliferation. Primary epithelial cells do not
respond to androgen stimulation in vitro [2]. However, they express great amounts of functional EGF
receptor and proliferate, if EGF is present in the medium [8,9]. One of the mechanisms which may account for this stimulation is an increase in the expression of the c-fos protooncogene [lo].
EGF expression in prostatic carcinoma was studied
in prostatic tumor cell lines and in human specimens.
EGF was detected in medium from both androgenresponsive LNCaP and androgen-independent DU145 cells [11,12]. The latter, however, secreted 14-fold
greater amounts of EGF than did LNCaP cells. In prostatic tumors, immunoreactive EGF was present in
about 70% of the specimens investigated [13]. Therefore, locally-produced EGF is thought to stimulate
prostatic tumor growth.
EGF-related TGFa protein is secreted by the epithelial cells of the ventral and lateral lobes of the rat
prostate gland [14]. In normal human prostatic tissue
TGFa level has not been measured. Harper et al. [15]
analyzed TGFa immunostaining intensity in formaIin-fixed sections obtained from human benign prostatic hyperplasia (BPH) and prostatic carcinoma specimens. In most BPH samples they observed very low
394
Culig et al.
TGFa immunoreactivity. With regard to prostatic carcinomas, there was a tendency towards increased
staining intensity in specimens from advanced tumors. These results supported the hypothesis that
increased expression of this growth factor reflects a
more malignant phenotype of the tumor. Consistent
with this hypothesis, autocrine production of TGFa is
characteristic of all three prostatic tumor cell lines,
LNCaP, PC-3, and DU-145, which are derived from
metastatic lesions [ll, 16-18].
Knowledge about the effects of EGF/TGFa on the
prostate is incomplete without an understanding of
both expression and regulation of the EGF receptor
(EGFR) in normal, hyperplastic, and carcinomatous
prostates. This is important for predicting cellular response to autocrine or paracrine growth factors. EGFR
expression in hyperplastic and carcinomatous tissue
has been studied with several techniques such as binding assays, immunohistochemistry, and RNase protection assay. However, these investigations do not
provide unequivocal data. Frydenberg et al. [19] analyzed EGFR expression in BPH tissue by means of
immunohistochemistry, and found that it was present
in 81% of specimens. Though this observation was
supported by other investigators [20-221, it is in contrast to a previous publication according to which
EGFR was present in a very low number of homogenized BPH specimens [23]. In BPH and prostatic intraepithelial neoplasia, EGFR immunoreactivity is
localized in the basal cells, which do not contain androgen receptors (-)
[20]. Since ARs are present in
the luminal cells of prostatic epithelium, it was assumed that androgen and EGF exert their effects on
different cell subpopulations in the prostate.
Several authors have reported a lower number of
EGFR-positive cells in malignant than in BPH tissue
[20-221. This was attributed to rapid receptor turnover. Conversely, Moms and Dodd [24] found that
EGFR mRNA levels in carcinoma specimens and in
tumor cell lines were slightly higher than those in
BPH tissue. In two studies, EGFR mRNA levels correlated with tumor stage and grade [23,24]. Contrarily, Maddy et al. [25] observed an inverse correlation
between EGFR levels and tumor grade. On account of
the histological heterogeneity of human prostate cancers, which may be encountered even within a single
biopsy specimen, correlations between EGFR content
and tumor grade must always be interpreted with
some reservation. The findings in tumor cell lines
also support the assumption that EGFR expression
increases with malignant potential. Androgen-independent DU-145 cells express about 10 times more
EGFR than androgen-sensitiveLNCaP cells [26]. Similar data were reported for estrogen-responsive and
estrogen-unresponsive mammary tumor cell lines
[27l. In DU-145 cells, the amount of EGFR protein
correlates inversely with cell density [28]. Subconfluent cells exhibit higher levels of EGFR than confluent
cells. Since androgen- independent cell lines are more
aggressive than androgen-responsive ones, one may
presume that this expression of high EGFR levels associated with the production of great amounts of EGF
reflects an autocrine mechanism through which these
cells overcome androgen-dependency. The observation that LNCaP cells, unlike DU-145 cells, are stimulated by exogenous EGF is in line with this hypothesis [26]. Obviously, DU-145 cells produce enough
EGF for maximal autocrine stimulation, whereas LNCaP cells do not. The situation in LNCaP cells is complicated by some as-yet unidentified factors present
in serum, which were shown to modulate LNCaP
responsiveness to exogenous EGF [29]. In PC-3 cells
another remarkable response to EGF was observed.
Although EGF was found to have only a minor proliferative effect on these cells, it did stimulate their
invasive potential. This was obviously due to stimulation of the extracellular protease uPA [30].
Many studies have addressed the effect of androgens on EGFR regulation, which seems to be different
in normal and carcinomatousprostates. In the rat ventral prostate, androgens downregulate EGFR level by
&fold, as was demonstrated by DHT treatment of
castrated rats [31]. Conversely, an inverse pattern of
EGFR regulation was observed in the LNCaP tumor
cell line [18,32,33]. Schuurmans et al. [32,33] reported
that in these cells EGFR levels were increased by the
synthetic androgen methyltrienolone as well as by
estrogens and progesterone. Due to the altered specificity of their mutant AR, LNCaP cells exhibit the
same response to estrogens, progesterone, and androgens [MI. Indeed, the effect on EGFR expression
was found to correlate directly with affinity for the AR,
which indicates that it is mediated via the AR. In
another LNCaP subline derived from a fast-growing
colony, this pattern of AR-mediated EGFR expression
was not observed [26]. Stimulation of EGFR in response to steroids is not restricted to prostate tumor
cell lines but is also seen in other hormone-dependent
cell lines [35-371. Regulation of EGFR expression in
prostatic tissue appears to be more complex. In patients with advanced prostatic carcinoma, long-term
administration of luteinizing hormone-releasing hormone, which lowers androgen levels, is accompanied
by an increase in EGFR levels [38].
Recently, a comprehensive report on alterations of
the EGF/TGFa loop following progression of prostatic
carcinoma has been published [39]. Primary tumors
from patients undergoing radical prostatectomy and
specimens from metastases of patients subjected to
endocrine therapy were investigated by means of im-
-
Polypeptide Growth Factors in the Prostate
munohistochemistry. In primary prostatic tumors,
EGFR immunostaining was localized in the epithelial
cells, and TGFa staining in the stromal cells, while
coexpression of the ligand and the receptor by epithelial tumor cells was observed in nearly 80%of the
specimens obtained from hormone-refractory metastases. These findings suggest that in primary tumors, a paracrine pattern of growth factor stimulation
predominates, whereas in androgen-independent disease there is a shift towards an autocrine stimulatory
loop. On this basis, therapeutic strategies capable of
counteracting these EGF/TGFa effects on prostatic tumor cells should be developed. In vitro tumor growth
was shown to be slowed down by application of either
anti-TGFa or anti-EGFR antibodies [17,40,41]. The
monoclonal anti-EGFR antibody not only reduced receptor phosphorylation and inhibited proliferation of
both PC-3 and DU-145 cells, but also led to sensitization of androgen-independent prostatic carcinoma
cells to tumor necrosis factor a [MI.
One of the tasks of prostate cancer research is
to characterize communication between different signaling pathways, above all between the AR and
growth factor transduction cascades. This is particularly important because of the presence of ARs in
androgen-independent tumors, which suggests an active androgen-signaling cascade in advanced androgen-deprived tumors [42-451. There are some data
supporting an interaction between the EGF/TGFa
pathway and the androgen-signaling transduction
cascade. EGF and TGFa downregulate the steadystate mRNA and protein levels of prostatic acid phosphatase and prostate-specific antigen (PSA), two protein markers of prostatic function [46].Thus, both EGF
and TGFa have an inhibitory effect on the expression
of these two androgen-stimulated proteins. Conversely, EGF was reported to substitute for steroid
hormones by activating the transactivation function of
androgen or estrogen receptors, and can, therefore,
replace estrogen in estrogen-responsive tissues [47491 (Fig. 2). Further studies on AR activation by
growth factors and its sigruficancefor prostatic biology
will probably provide interesting details.
TRANSFORMING GROWTH FACTOR+:
A BIFUNCTIONAL REGULATOR
IN THE PROSTATE
The well-conserved TGFP family consists of five
TGFP isoforms, TGFPl-5, and several related proteins including activins and inhibins [50]. Isoforms
1-3 are expressed in mammalian cells. TGFP polypeptides contain 112 amino acids and share about
80% sequence homology [51]. In vivo, TGFP stimulates angiogenesis [52-541, wound healing [55], and
1
GF
I
I G F-I
KGF
EGF
f
.
395
0
ANDROGEN
MITOGENIC
EFFECT
Fig. 2. Coupling of growth factor- and androgen-signaling pathways in the prostate. Three polypeptide growth factors, IGF-I,
KGF, and EGF, stimulate AR activity and thus influence activation of
the androgen signal transduction pathway [47]. GF, growth factor;
GFR growth factor receptor; AIG, androgen-inducible gene.
invasion and metastatic spread [56], and suppresses
the immune response by inhibiting lymphocytes
[57,58]. Hence, TGFP displays stimulatory as well as
inhibitory effects on cell proliferation. Which effect
predominates, depends on cell type and concentration of TGFP. The majority of stromal cells are mitogenically stimulated by TGFPs, whereas most cells of
epithelial origin are inhibited by these growth factors
[59-611. Loss of responsiveness to TGFP is believed
to be a major factor in tumor formation [62]. TGFP
regulates the synthesis and turnover of components
of the extracellular matrix, stimulates protease inhibitors, and inhibits the tissue plasminogen activator
[52]. TGFP is synthesized as a latent precursor molecule [63-651. In vitro its activation can be achieved by
transient acidification or alkalization, application of
heat, or treatment with chaotropic agents [66]. The
mechanism of TGFP activation in vivo is still poorly
understood. TGFPs are probably activated by proteases such as plasmin [67-691.
Virtually all cells contain TGF receptors and binding proteins [52]. Three TGFP receptors have been
detected in mammalian cells, and type 1and I1 receptors are involved in TGFP signal transmission [59].
They form a heterodimer complex [70,71]. Receptor
I11 is probably a binding protein [72]. After binding of
TGFP to its receptor, a seridthreonin protein kinase
localized in the C-terminus of receptor I1 is activated,
which, in turn, triggers a signal cascade that finally
results in inhibition of cyclin-dependent protein kinases [73].
The most intriguing observation regarding the ac-
396
Culig et al.
~
~~
In prostatic carcinoma, TGFP also acts as a bifunction of TGFP in the prostate is that this growth factor
has a dual role in the regulation of cell growth and
tional regulator of cell growth; both negative and positive proliferative effects of TGFP have been obviability. There is evidence that TGFP can act both as
served. In AXC/SSh cancer cells the effects of TGFP
a negative and a positive growth factor. Negative efon thymidine incorporation were concentration-defects have been observed predominantly in normal
pendent [80]. Thymidine incorporation was inhibited
prostatic tissue. Rat ventral prostate cells in culture
at low concentrations only. In the sublines MATproduce and secrete a factor that inhibits the growth
LyLu, AT2, G, HI, and H of the Dunning tumor sysof PC-3 cells, which immunoblot analysis has shown
tem, TGFPl mRNA levels were found to be higher
to be a protein similar to TGFP [74]. The major anthan in normal prostates [81]. In normal tissue and in
tagonist of positive growth factors in prostatic epithetwo well-differentiated Dunning tumor cell lines
lial cell cultures is TGFP [9,75], which is of special
TGFP was localized in the stroma, while it was disinterest for prostatic physiology because of its central
tributed homogeneously in poorly-differentiated turole in programmed cell death. Castration-induced
mors. This indicates that aggressive tumor cells acandrogen deprivation causes a dramatic decrease in
quire the ability to produce their own TGFP. It was
AR expression in the rat ventral prostate, which is
demonstrated that these cells are also able to activate
followed by DNA fragmentation, formation of apopthe latent TGFPl precursor. These findings suggest
totic bodies, and, finally, involution of the prostate
gland. During this process, expression of several
an autocrine stimulatory TGFP loop in advanced
stages of prostatic carcinoma. Experimental induction
genes, including those encoding TGFP, is upreguof an autocrine loop by stable transfection of MATlated. An increase in TGFP level was observed within
LyLu cells with an expression vector that encodes
a day after castration, reaching a maximum 4 days
latent TGFPl yielded the same results. MATLyLu tuafter androgen withdrawal [76]. At the same time, the
mors overexpressing TGFPl were shown to be larger
expression of testosterone-repressed prostatic mesand to produce more metastases as compared to consage (TIU'M-2), the classic apoptotic marker of the
trol tumors [82]. If grown for a long period of time in
prostate, increased. Following administration of anthe presence of TGFP, AT-3 cells, another Dunning
drogen to castrated rats after 4 days of androgen
tumor subline, undergo a change in phenotype [83].
withdrawal, TGFP levels promptly returned to norSubsequently, they stimulate the growth of cultured
mal. Administration of TGFP into the rat ventral
osteoblasts, which probably contributes to the formaprostate also led to a decrease in prostatic DNA contion of osteoblastic bony metastases.
tent, which, however, was much less pronounced
The role of TGFp was also studied in experimenthan that observed after castration. Nevertheless, this
tally-induced prostatic carcinomas in mice [MI. In
experiment confirmed the crucial role of TGFP in the
this model system, epithelial and mesenchymal tisprogrammed death of prostatic cells. The finding that
sues from the urogenital sinus were separately inthe TGFp receptor is under negative androgen confected with a retrovirus containing ras and myc ontrol is also consistent with the proposed role of TGFP
as a growth-inhibiting factor in normal prostates [n].cogenes. After recombination of both components
and grafting to the renal capsule, poorly-differentiUnresponsiveness of tumor cells to androgen ablaated adenocarcinomas developed. In these tumors
tion, as seen in the rat Dunning tumor, seems to be
the two TGF isoforms, TGFPl and TGFP3, were
accompanied by alterations in TGFp regulation. In
found to be markedly elevated, whereas TGFP2
contrast to the normal rat prostate, no increase in
mRNA levels remained unchanged. These results
TGFP levels was observed in rat Dunning tumors folalso suggest a stimulatory role of TGFPs in advanced
lowing castration. Another possible explanation for
tumors. Human prostatic tumor cell lines do not exthe differences in TGFP expression patterns between
hibit a uniform pattern of inhibition by exogenous
normal and malignant prostates of castrated rats is
TGFP. This growth factor does not slow down prothat Dunning tumors are developed from the dorsal
liferation of androgen-sensitive LNCaP cells, but anlobe of the rat prostate, which does not show a castration-induced increase in TGFp expression [78].
tagonizes the growth-stimulatory effect of EGF on
these cells [85]. The androgen-independent cell lines
In fibroblasts, another compartment of the prosPC-3 and DU-145 produce TGFp in an autocrine mantate, TGFP1, at a concentration of 5 ng/ml, causes
ner. Nevertheless, exogenous TGFPl was shown to
inhibition of growth in vitro [79]. Furthermore, TGFPl
inhibit their proliferation [86]. By contrast 1-LN-E
counteracts the mitogenic stimuli of bFGF in culture.
The mechanism through which it inhibits the effects of
cells, a PC-3 subline, are resistant to TGFpl-mediated
inhibition of growth, which suggests that in the very
bFGF is still unclear. Possibly, a decrease in TGFPl
relative to bFGF promotes the proliferation of human
late stages of prostatic carcinoma TGFp does not act
as an inhibitor any longer [87]. Current knowledge
prostatic stromal cells.
Polypeptide Growth Factors in the Prostate
about TGFP expression in tumor specimens is rather
limited. Immunohistochemical studies revealed that
extracellular staining for TGFPl is more intense in
prostate cancer than in normal or hyperplastic tissues
188,891. However, detailed studies correlating TGFP
expression with stage, grade, and markers of cell proliferation have not been performed as yet.
In summary, studies on TGFP expression and
function in prostate cancer indicate that advanced
prostatic tumors escape the inhibitory effects of
TGFp. Therefore, administration of TGFP is limited to
stages which precede overexpression of this growth
factor. A therapeutic strategy currently being tested
in vitro consists of the treatment of prostatic cells
with cyclic adenosine monophosphate analogs,
which increase TGFP levels and thus cause growth
arrest [90].
FIBROBLAST GROWTH FACTORS IN
THE PROSTATE: KEYS TO
STROMAL-TO-EPITHELIAL
CELL INTERACTION
The fibroblast growth factor family consists of at
least seven members and their related peptides [91].
Since all of them bind to heparin, they are also known
as the heparin-binding growth factor family. As demonstrated with basic fibroblast growth factor (bFGF,
FGF2), binding to heparane sulfate protects them
from being degraded by proteases [92]. Acidic FGF
(aFGF, FGF1) and bFGF, for which this growth factor
family was originally named, consist of 155 amino
acids and share approximately 55% amino acid sequence homology [91]. Four members of the FGF
family (FGFs 3-6) are oncogene products. FGF7 was
found to be a growth factor for keratinocytes and was
therefore termed keratinocyte growth factor (KGF).
FGFs stimulate proliferation of various cells of mesodermal, neuroectodermal, ectodermal, and endoderma1 origin. The influence of aFGF and bFGF on the
proliferation of endoepithelial cells, such as those
found in capillaries, demonstrates the importance of
this growth factor family for angiogenesis [93]. Since
tumor spread hinges on the formation of new blood
vessels, many studies have focused on the role of
FGFs in the induction of tumor vascularization.
The FGF receptor family consists of four genes
which exhibit structural heterogeneity [94]. These are
FGFRl (the flg gene product), FGFR2 (the bek gene
product), FGFR3, and FGFR4. Due to alternative
splicing there are several variants of these receptors,
which complicates their analysis [95]. Basically, FGF
receptors consist of an extracellular binding region, a
transmembrane region, and a cytosolic tyrosine kinase domain.
397
High levels of aFGF were detected in the developing rat prostate [96]. Fourteen weeks after birth its
expression begins to decrease and is undetectable at
35 weeks. In the human prostate, expression of aFGF
is either low or undetectable [97-991, whereas bFGF is
produced in large amounts. Two independent studies have identified bFGF as the main growth factor
produced by human prostatic fibroblasts [100,101].In
prostatic cell cultures, bFGF stimulates the growth of
both epithelial and mesenchymal cells [8,100]. Although the latter produce their own bFGF they do not
abandon their response to exogenous growth factor.
Growth stimulation by bFGF plays a role in the development of BPH, which is essentially a proliferative
disorder of the stroma. In BPH tissue the level of
bFGF is sigruficantly higher than in normal prostatic
and carcinomatous tissue [97,101,102]. Transgenic
mice which overproduced the bFGF-related int-2 oncogene in the prostatic tissue were shown to develop
epithelial prostatic hyperplasia. These findings were
interpreted as evidence that FGFs play a role in the
pathogenesis of BPH [103]. However, it is still unknown at which stage@)of BPH development bFGF
exerts its effects. Consequently, an appropriate therapy that counteracts the effects of bFGF has not been
developed yet. Furthermore, the increased microvessel density observed in BPH tissue suggests that
bFGF has a role in angiogenesis [104].
Early research on prostatic carcinoma focused on
the expression of FGFs. More recent studies have also
evaluated the function of these mitogens in prostatic
neoplasms and in the changes occurring during tumor progression. The slow-growing, androgen-responsive, nonmetastatic Dunning R3327 PAP tumor
predominantly produces aFGF. In contrast, the fastgrowing, androgen-independent, metastatic variant
AT-3 expresses both aFGF and bFGF [96]. The same
pattern of expression can be observed in embryonic
tissue, which reflects the embryonal properties of
some advanced prostatic carcinomas. In the Dunning
tumor model, progression is characterized by increasing cellular independence from paracrine FGFs. Advanced tumor cells switch to autocrine stimulation
and start producing their own growth factors. In this
way they become independent of the supply by the
stromal cells [105]. Activation of bFGF (FGF2), FGF3
(int-2), and FGF5 genes has been observed in Dunning tumor progression. Growth factor independence was accompanied by a shift in the expression
of the FGF receptor 2 gene from exclusive expression
of exon IIIb to expression of exon IIIc. The exon IIIbcontaining receptor isoform is an epithelial-specific
isoform which has a high affinity for stromal cellderived FGF7 (KGF), whereas the isoform containing
exon IIIc recognizes and responds to bFGF. Inhibition
398
Culig et al.
of bFGF translation by application of antisense oligonucleotides was shown to slow down growth of
the AT-3 Dunning tumor, which is also in line with
autocrine bFGF growth stimulation [106].
Like the Dunning tumor system, the highly metastatic androgen-independent human prostatic carcinoma cell lines PC-3 and DU-145 produce large
amounts of bFGF [107]. These cells are also able to
form tumors and metastasize in nude mice. By contrast, the androgen-sensitive human prostatic cancer
cell line LNCaP does not synthesize bFGF or form
tumors in nude mice. Only when coinoculated with
bone or prostatic fibroblasts, which express large
amounts of bFGF, do they form carcinomas. The tumors are initially androgen-sensitive, but progress towards androgen-insensitivity during propagation
[108,109]. In this model, the stromal cells could be
replaced with matrigel, which also contains high levels of bFGF. This finding, and the fact that in vitro
bFGF stimulates LNCaP growth in a dose-dependent
manner, indicate that bFGF is an important growth
factor in tumor formation. In this coinoculation
model there are probably other growth factors besides bFGF which have the same effect, since it was
not possible to inhibit the effect of the stromal cells
with anti-bFGF antibodies [108].
Apart from its mitotic effect on prostatic cells,
bFGF also seems to contribute to metastatic spread
since it enhances cell motility, stimulates angiogenesis, and exerts an influence on the extracellular matrix.Increased cell motility was observed when MATLyLu and LNCaP cells were treated with bFGF [110].
This effect could be blocked by suramin, which is a
growth factor receptor antagonist. Like TGFP, bFGF
regulates the turnover of the extracellular matrix by
modulating its proteases and promoting the synthesis of collagen, fibronectin, and proteoglycans [1111.
This seems to enhance the ability of bFGF-producing
cells to escape from the primary tumor and to invade
other tissues. Due to its angiogenic property, bFGF
promotes vascularization of both primary tumors and
metastases. In prostatic carcinoma, microvessel density is in fact elevated; nevertheless, a sigruficant correlation with tumor grade or stage could not be established [112,113].
FGFs and androgens were found to influence each
other in the prostate. In the rat prostate, bFGF expression was upregulated by androgens. This finding
was confirmed in vitro in the steroid-responsive hamster smooth muscle tumor cells DDTl and in LNCaP
cells, but not in primary prostatic epithelial cells [1141181. Keratinocyte growth factor (KGF, FGF7), another member of the FGF family, is also regulated by
androgens. KGF was detected exclusively in prostatic
stromal cells, while its receptor was present on pro-
static epithelial cells. As was to be expected from the
presence of the KGF receptor, this growth factor is
mitogenic for epithelial but not for stromal cells
[119,120]. Stromal cells of the prostate are known to
be primary targets of androgen action during organogenesis. Therefore, the existence of factors which mediate the effect of androgens from the stroma to the
epithelium was postulated [1211. KGF is considered
to be such a stromal-to-epithelial cell andromedin. In
the morphogenesis of the seminal vesicle, KGF can
substitute for testosterone [122]. In prostatic carcinoma cells in culture, KGF can also substitute for testosterone by activating the AR through an as-yet unknown mechanism. This was demonstrated to cause
induction of an androgen-regulated gene in the absence of androgen hormones [47] (Fig. 2).
INSULIN-LIKE GROWTH FACTORS IN NORMAL
A N D HYPERPLASTIC H UMA N PROSTATES
SHIFT TO AUTOCRINE STIMULATION
The IGF system is characterized by complex interaction between the two growth factors IGF-I and -11,
their receptors, high-affinity binding proteins, and
proteases. IGFs are polypeptides with an amino acid
sequence and functional homology with insulin.
IGF-I consists of 70 and IGF-I1 of 67 amino acids [1231.
In contrast to insulin, IGFs are produced locally in
many tissues and are considered to be autocrine and
paracrine growth factors. The liver is the main site
where these growth factors and their binding proteins are synthesized in humans. Their biosynthesis
is controlled by growth hormone. In steroid hormone-sensitive organs there may also be other control mechanisms of IGF synthesis. For example, estrogen enhances IGF-I expression in the rat uterus
[124]. Two types of IGF receptor have been described
[125,126]. Each of them binds both growth factors,
but with different affinity. Type I receptors have an
approximately 3-fold higher affinity for IGF-I than for
IGF-I1 [126]. Conversely, type I1 receptors preferably
bind IGF-11. IGFs were first studied in other organs
before their action on the prostate was analyzed. Currently there are several studies which provide evidence that these growth factors have mitogenic effects on the prostate, and that their expression
undergoes changes in proliferative prostatic disease.
In primary culture, prostatic epithelial cells exhibit
a proliferative response to both IGFs and insulin, and
they secrete IGF-binding proteins (IGFBPs) into medium [127,128]. They also express IGF-receptor I, the
affinity of which determines the effect of the individual growth factors. IGF-I was found to be a more
potent growth factor than IGF-I1 or insulin. IGF-I1
achieves the same level of stimulation as IGF-I at a
Polypeptide Growth Factors in the Prostate
10-fold higher concentration, and insulin at a 500-fold
higher concentration. The fact that IGF-I and IGF-I1
could not be detected in conditioned medium from
prostatic epithelial cells suggests that IGFs, which are
produced in the stroma, act as paracrine growth factors in normal prostatic epithelium. This pattern of
IGF expression appears to be unchanged in BPH tissue. Barni et al. [129] found that in hyperplastic prostates IGF-I mRNA was localized exclusively in the
stromal cells, whereas IGF binding protein-4 mRNA
was produced mainly in the epithelial compartment.
However, quantitative alterations in the expression of
IGFs and their binding proteins may occur [130]. Stroma1 cells derived from patients with BPH were reported to overexpress mRNA for IGF-II and IGF-enhancing binding protein-5, whereas the mRNA for
IGF-inhibiting binding protein-2 was reduced [1301.
On the protein level, concentration of IGF-11 peptide
was not increased in conditioned medium from stromal cells.
In prostatic carcinoma the mode of IGF action is
not yet fully understood. The data available are contradictory; whether IGFs continue to act as paracrine
growth factors or switch to autocrine stimulation is
still an unsettled issue. Iwamura et al. [131] studied
the effects of exogenous IGF-I in androgen-responsive and -unresponsive tumor cell lines. IGF-I stimulated DNA synthesis in PC-3 and DU-145 cells, while
no such effect was observed in LNCaP cells. Interestingly, IGF-I showed a synergistic effect with dihydrotestosterone (DHT) in LNCaP cells. In this study
the three human cell lines did not secrete IGF-I into
their culture media, which does not suggest an autocrine mode of action. C O M O ~and
~ Y Rose [132] analyzed the IGF system in DU-145 cells in more detail.
They identified type I IGF receptors on these cells,
and assessed the secretion of IGFBPl. Both IGF-I and
IGF-11 were found to stimulate thymidine incorporation into DU-145 cells. It is of interest that addition of
anti-EGF receptor antibodies reversed the growth
promoting effects of both IGFs and halted the secretion of IGFJ3P1, which indicates the existence of a link
between the signaling pathways of EGF and IGF in
the prostate, as already demonstrated in other organs
[133]. No IGF was detected in conditioned medium
from DU-145 cells [132]. These results lead to the hypothesis that IGFs, unlike TGFa and bFGF, continue
to act as paracrine growth factors in advanced prostatic carcinoma. IGF secretion by prostatic and bone
fibroblasts may, therefore, influence the growth of
both normal and malignant prostatic tissue [134,135].
It is interesting to note that IGF-I levels in bone, i.e.,
the primary landing site of metastases from prostatic
carcinoma, are high [136]. In contrast to the results
mentioned above, Pietrzkowski et al. [137], reported
399
that all three tumor cell lines grow in serum-free medium without the addition of exogenous growth factors such as IGFs, since thgy produce large amounts
of these polypeptides themselves. Treatment of prostatic cancer cells with peptide analogs of IGF-I that
act as receptor antagonists slowed down their
growth. These results suggest the existence of an
IGF-I autocrine mechanism in which the overexpressed peptide activates its receptor on the same
cell. Differences in cell culturing, the use of various
radioimmunoassay kits for IGF-I determination, and
interference of growth factors with binding proteins
may account for the controversial findings obtained
in human prostatic tumor cell lines. Unfortunately,
no data are available on IGF-I expression and action
in human prostate cancer tissue. IGF receptors were
also identified in rat PA-111 prostatic tumors, which
trigger lytic and blastic reactions in the skeleton. In
these tumors, both IGFs and insulin were found to
stimulate DNA synthesis and cell proliferation in a
dose-dependent manner [1381.
Changes in serum IGFBP levels were observed in
patients with prostate cancer [139]. IGFBP2, the predominant form of IGFBP secreted by prostatic epithelial cells, was elevated, whereas IGFBPS levels were
This finding was confirmed by both
decreased [la].
radioimmunoassay and Western ligand blot analysis.
The decreased IGFBP3 level may have been due to
proteolitic cleavage by the serine protease PSA,
which was shown to cut IGFBP3 [141]. Possibly, this
cleavage results in increased bioavailability of IGF-I
and in a potentiation of its effects.
A recent study has demonstrated that even in the
absence of androgen, IGF-I, at a concentration of 50
ng/ml, is capable of activating the AR in cotransfected
DU-145 cells [47] (see Fig. 2). Also, at lower concentrations, IGF-I potentiated the effects of very low concentrations of androgen on AR-mediated reporter
These IGF-I effects were inhibited
gene activity [MI.
by the nonsteroidal antiandrogen casodex, which indicates that they are mediated through the AR. This
synergism between androgens and IGF-I in AR activation may be of importance, particularly in advanced prostatic carcinoma, when testicular androgens are suppressed but small amounts of androgen
are still supplied by the adrenals. Another recently
published study also provides evidence that IGFs interact with the androgen-signaling system. Marcelli
et al. [ l a ] stably transfected PC-3 cells with an expression vector encoding a constitutively active AR,
and studied the growth characteristics of these cells.
Original PC-3 cells did not respond to IGF stimulation, whereas AR-expressing cells displayed a proliferative response. Unlike original PC-3 cells, the stably
transfected subline did not express IGFBP3. Identifi-
400
Culig et al.
cation of the mechanism underlying the interaction
between the androgen and IGF-signaling cascades is
an issue which should be addressed in future studies.
Since nearly all primary prostatic tumors and their
metastases express the AR protein, one might expect
the main impact of communication between IGF and
androgen transduction to occur in the advanced
stages of prostatic carcinoma, when the androgen
supply is dramatically reduced during androgenwithdrawal therapy [U-45].
CONCLUSION
A variety of growth factors has been studied in rat
and human prostates. All of them, with the exception
of TGFP, are believed to be positive growth factors.
TGFP has a dual function in the regulation of prostatic growth. In normal prostatic tissue, in BPH, and
probably in the early phases of prostatic carcinogenesis, it acts as an inhibitor of prostatic growth and as
an antagonist of growth-promoting factors. Yet, in
advanced prostatic cancer, it stimulates tumor cell
proliferation. Growth factors such as EGF, TGFce, and
IGFs are secreted in a paracrine manner in normal
prostates and in benign prostatic proliferative disorders. EGF and TGFce switch to an autocrine pattern of
secretion in the late stages of prostatic carcinoma.
Whether this also holds true for IGFs remains to be
determined. This shift to autocrine secretion reflects
the crucial role of growth factors in advanced prostatic carcinoma. Furthermore, there is evidence that
bFGF, one of the main growth factors in BPH, also
acts in an autocrine manner in hormone-independent
prostate cancer.
One of the main tasks of future research will be to
develop therapeutic agents which inhibit paracrine
and aurocrine growth factor pathways without producing undesirable side effects. Currently, our efforts
must focus on a detailed characterization of the communication between androgen- and growth factorsignaling pathways. Studying each transduction cascade alone has provided a wealth of valuable data on
prostatic growth and function, but many of the open
questions concerning prostate cancer cannot be settled unless we gain a better understanding of the interaction between these pathways.
ACKNOWLEDGMENTS
Work done in our laboratory was supported by
grant SFB F203 from Austrian Research Funds. The
authors thank Monica Trebo for editorial assistance.
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