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 . 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 . 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  (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 . 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 . 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 . 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 . 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 . In normal human prostatic tissue TGFa level has not been measured. Harper et al.  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.  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 . In BPH and prostatic intraepithelial neoplasia, EGFR immunoreactivity is localized in the basal cells, which do not contain androgen receptors (-) . 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  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.  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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . Recently, a comprehensive report on alterations of the EGF/TGFa loop following progression of prostatic carcinoma has been published . 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 .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 . Isoforms 1-3 are expressed in mammalian cells. TGFP polypeptides contain 112 amino acids and share about 80% sequence homology . In vivo, TGFP stimulates angiogenesis [52-541, wound healing , 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 . GF, growth factor; GFR growth factor receptor; AIG, androgen-inducible gene. invasion and metastatic spread , 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 . TGFP regulates the synthesis and turnover of components of the extracellular matrix, stimulates protease inhibitors, and inhibits the tissue plasminogen activator . 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 . 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 . Three TGFP receptors have been detected in mammalian cells, and type 1and I1 receptors are involved in TGFP signal transmission . They form a heterodimer complex [70,71]. Receptor I11 is probably a binding protein . 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 . 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 . 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 . The major anthan in normal prostates . 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 . 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 . 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 . 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 . tagonizes the growth-stimulatory effect of EGF on these cells . 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 . Furthermore, TGFPl inhibit their proliferation . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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  (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 . 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 . 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.  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 . 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.  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  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 . No IGF was detected in conditioned medium from DU-145 cells . 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 . In contrast to the results mentioned above, Pietrzkowski et al. , 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 . 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 . 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  (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. 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