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 [email protected])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. 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. REFERENCES 1. Isaacs JT, Coffey DS:Androgenic control of prostatic growth: Regulation of steroid levels. UICC Monogr (Prostatic Cancer) 4&112-122, 1979. 2. McKeehan WL, Adams PS, Rosser MP: Direct mitogenic effects of insulin, epidermal growth factor, glucocorticoid, cholera toxin, unknown pituitary factors and possibly prolactin, but not androgen, on normal rat prostate epithelial cells in serum-free, primary cell culture. Cancer Res 4:1998-2010, 1984. 3. Ulrich A, Coussens L, Hayflick JS, Dull TJ, Gray A, Tam AW, Lee J, Yarden Y, Liebermann TA, Schlessinger J, Downward J, Mayes ELV, Whittle N, Waterfield MD, Seeburg PH: Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A 431 epidermoid carcinoma cells. Nature 309:418-424, 1984. 4. Hill DJ: Growth factors and their cellular action. ] Reprod Fertil85:723-734, 1989. 5. Gregory H, Willshire IR, Kavanagh JP, Blacklock NJ, Chowdury S, Richards RC: Urogastrone-epidermal growth factor concentrations in prostatic fluid of normal individuals and patients with benign prostatic hypertrophy. Clin Sci 50:359-363, 1986. 6. Jacobs SC, Story MT, Sasse J, Lawson RK: Characterization of growth factors derived from the rat ventral prostate. J Urol 139:llM-1110, 1988. 7. Hiramatsu M, Kashimata M, Minami N, Sat0 A, Murayama M: Androgenic regulation of epidermal growth factor in the mouse ventral prostate. Biochem Int 17311-317, 1988. 8. McKeehan WL, Adams PS, Fast D: Different hormonal requirements for androgen-independent growth of normal and tumor epithelial cells from rat prostate. In Vitro Cell Dev Biol23:147-152, 1987. 9. Sutkowski DM, Fong C-J, Sensibar JA, Rademaker AW, Sherwood ER, Kozlowski JM, Lee C: Interaction of epidermal growth factor and transforming growth factor beta in human prostatic epithelial cells in culture. Prostate 21:133-143, 1992. 10. Levine AC, Ren M, Huber GK, Kirschenbaum A: The effect of androgen, estrogen, and growth factors on the proliferation of cultured fibroblasts derived from human fetal and adult prostates. Endocrinology 130: 2413-2419, 1992. 11. Connolly JM, Rose DP: Production of epidermal growth factor and transforming growth factor-a by the androgen-responsive LNCaP human prostate cancer cell line. Prostate 16:209-218, 1990. 12. Connolly JM, Rose DP: Secretion of epidermal growth factor and related polypeptides by the DU145 human prostate cell line. Prostate 15:177-186, 1989. 13. Fowler JE Jr, Lau JLT, Ghosh L, Mills SE, Mounzer A: Epidermal growth factor and prostatic carcinoma: An immunohistochemicalstudy. J Uroll39857-861,1988. 14. Taylor TB, Ramsdell JS: Transforming growth factor-a and its receptor are expressed in the epithelium of the rat prostate gland. Endocrinology 133:1306-1311, 1993. 15. Harper ME, Goddard L, Glynne-Jones E, Wilson DW, Price-Thomas M, Peeling WB, Griffiths K: An immunocytochemical analysis of TGFa expression in benign and malignant prostatic tumors. Prostate 23:9-23, 1993. 16. MacDonald A, Chisholm GD, Habib FK: Production and response of a human prostatic cancer line to transforming growth factor-like molecules. Br J Cancer 62: 579-584, 1990. 17. Hofer DR, Sherwood ER, Bromberg WD, Mendelsohn Polypeptide Growth Factors in the Prostate 401 ~ J, Lee C, Kozlowski JM: Autonomous growth of androgen-independent human prostatic carcinoma cells: Role of transforming growth factor a. Cancer Res 51: 2780-2785, 1991. 18. Wilding G, Valverius E, Knabbe C, Gelmann EP: Role of transforming growth factor alpha in human prostate cancer cell growth. Prostate 15:l-12, 1989. 19. Frydenberg M, Foo TMS, Jones AS, Grace J, Hensley WJ, Rogers J, Pearson BS, Raghavan D Benign prostatic hyperplasia-videoimage analysis and its relationship to androgen and epidermal growth factor receptor expression. J Urol146:872-876, 1991. 20. Ibrahim GK, Kerns B-JM, MacDonald JA, Ibrahim SN, Kinney RB, Humphrey PA, Robertson CN: Differential immunoreactivity of epidermal growth factor receptor in benign, dysplastic and malignant prostatic tissues. J Urol 149:170-173, 1993. 21. Habib FK Peptide growth factors: A new frontier in prostate cancer. Prog Clin Biol Res 357107-115, 1990. 22. Maygarden SJ, Strom S, Ware JL: Localization of epidermal growth factor receptor by immunohistochemical methods in human prostatic carcinoma, prostatic intraepithelial neoplasia, and benign hyperplasia. Arch Pathol Lab Med 116:269-273, 1992. 23. Davies P, Eaton CL: Binding of epidermal growth factor by human normal, hypertrophic, and carcinomatous prostate. Prostate 14:123-132, 1989. 24. Moms GL, Dodd JG: Epidermal growth factor receptor mRNA levels in human prostatic tumors and cell lines. J Urol 143:1272-1274, 1990. 25. Maddy SQ, Chisholm GD, Bussutil A, Habib FK Epidermal growth factor receptors in human prostate cancer: Correlation with histological differentiation of the tumour. Br J Cancer 60341-44, 1989. 26. MacDonald A, Habib FK Divergent responses to epidermal growth factor in hormone sensitive and insensitive human prostate cancer cell lines. Br J Cancer 65~177-182,1992. 27. Davidson NE, Gelmann EP, Lippman ME, Dickson RB: Epidermal growth factor gene expression in estrogen receptor-positive and negative human breast cancer cell lines. Mol Endocrinol 1:216-223, 1987. 28. Tillotson JK, Rose DP: Density-dependent regulation of epidermal growth factor receptor expression in DU 145 human prostate cancer cells. Prostate 19:53-61, 1991. 29. Schuurmans ALG, Bolt J, Mulder E: Androgens stimulate both growth rate and epidermal growth factor activity of the human prostate tumor cell LNCaP. Prostate 1255-63, 1988. 30. Jarard DF, Blitz BF, Smith RC, Patai BL, Rukstalis DB: Effect of epidermal growth factor on prostate cancer cell line PC3 growth and invasion. Prostate 24:46-53, 1994. 31. Traish AM, Wotiz HH: Prostatic epidermal growth factor receptors and their regulation by androgens. Endocrinology 121:1461-1467, 1987. 32. Schuurmans ALG, Bolt J, Veldscholte J, Mulder E: Regulation of growth of LNCaP human prostate tumor cells by growth factors and steroid hormones. J Steroid Biochem Mol Biola193-197, 1991. 33. Schuurmans ALG, Bolt J, Voorhorst MM, Blankenstein RA, Mulder E: Regulation of growth and epidermal growth factor receptor levels of LNCaP prostate tumor cells by different steroids. Int J Cancer 42:917922, 1988. 34. Veldscholte J, Ris-Stalpers C, Kuiper GGJM, Jenster G, Berrevoets C, Claassen E, van Rooij HCJ, Trapman J, Brinkmann AO: A mutation in the ligand-binding domain of the androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens. Biochem Biophys Res Commun 17: 534-540,1990. 35. Mukku VR, Stance1 GM: Regulation of epidermal growth factor receptor by estrogen. J Biol Chem 260: 9820-9824, 1985. 36. Murphy LJ, Sutherland RL, Stead B, Murphy LC, Lazarus L: Progestin regulation of epidermal growth factor receptor in human mammary carcinoma cells. Cancer Res 46528-734,1986. 37. Baker JB, Barsh GS, Carney DH, Cunningham DD: Dexamethasone modulates binding and action of epidermal growth factor in serum-free cell culture. Proc Natl Acad Sci USA 75:1882-1885, 1985. 38. Fiorelli G, De Bellis A, Longo A, Natali A, Constantini A, Serio M: Epidermal growth factor receptors in human hyperplastic prostate tissue and their modulation by chronic treatment with a gonadotropin-releasing hormone analog. J Clin Endocrinol Metab 68:740-743, 1989. 39. Scher HI, Sarkis A, Reuter V, Cohen D, Netto G, Petrylak D, Lianes P, Fuks Z, Mendelsohn J, CordonCardo C: Changing pattern of expression of the epidermal growth factor receptor and transforming growth factor a in the progression of prostatic neoplasms. Clin Cancer Res 1545-550, 1995. 40. Fong C-J, Sherwood ER, Mendelsohn J, Lee C, Kozlowski JM: Epidermal growth factor receptor monoclonal antibody inhibits constitutive receptor phosphorylation, reduces autonomous growth, and sensitizes androgen-independent prostatic carcinoma cells to tumor necrosis factor alpha. Cancer Res 52: 5887-5892, 1992. 41. Connolly JM, Rose DP: Autocrine regulation of DU145 human prostate cancer cell growth factor-related polypeptides. Prostate 19:173-180, 1991. 42. Sadi MV, Walsh PC, Barrack ER: Immunohistochemical study of androgen receptors in metastatic prostate cancer-comparison of receptor content and response to endocrine therapy. Cancer 673057-3064, 1991. 43. Van der Kwast TH, Schalken J, Ruizveld de Vinter JA, van Vroonhoven CCJ, Mulder E, Boersma W, Trapman J: Androgen receptors in endocrine therapy-resistant human prostate cancer. Int J Cancer 48:189193, 1991. 44. Hobisch A, Culig Z, Radmayr C, Bartsch G, Mocker H, Hittmair A: Androgen receptor status of lymph node metastases from prostatic carcinoma. Prostate (in press). 45. Hobisch A, Culig Z, Radmayr C, Bartsch G, Mocker H, Hittmair A: Distant metastases from prostatic carcinoma express androgen receptor protein. Cancer Res 55968-3072, 1995. 46. Henttu P, V i k o P: Growth factor regulation of gene expression in the human prostatic carcinoma cell line LNCaP. Cancer Res 53:1051-1058, 1993. 47. Culig Z, Hobisch A, Cronauer M V , Hittmair A, Radmayr C, Trapman J, Bartsch G, Mocker H: Androgen receptor activation in prostatic tumor cell lines by in- 402 Culig et al. sulin-like growth factor-I, keratinocyte growth factor and epidermal growth factor. Cancer Res 6454745478, 1994. 48. Culig Z, Hobisch A, Radmayr C, Hittmair A, Cronauer MV, Bartsch G, Klocker H: Regulation of androgen-receptor mediated gene transcription in the prostatic tumor cell line DU-145 by cyclic adenosine monophosphate, insulin-like growth factor-I, phorbol ester and retinoic acid. Proc Am Assoc Cancer Res 36:1618, 1995. 49. Ignar-Trowbridge DM, Teng CT, Ross KA, Parker MG, Korach KS, McLahlan JA: Peptide growth factors elicit estrogen receptor-dependent transcriptional activation of an estrogen-responsive element. Mol Endocrind 7992-998, 1993. 50. Roberts AB, Sporn MB: The transforming growth factor-ps. Peptide growth factors and their receptors. In: Sporn MB, Roberts AB (eds). ”Handbook of Experimental Pharmacology.” Heidelberg: Springer-Verlag, 1990, pp 419-472. 51. Massague J: The TGF-p family of growth and differentiation factors. Cell 49:437-438, 1987. 52. Massague J: The transforming growth [email protected] Annu Rev Cell Biol6:597-641, 1990. 53. Barnard JA, Lyons RM, Moses HL: The cell biology of transforming growth factor P. Biochim Biophys Acta 103279-87, 1990. 54. Yang EY, Moses HL: Transforming growth factor @linduced changes in cell migration, proliferation and angiogenesis in the chicken chorioallantoic membrane. J Cell Biol111:731-741, 1990. 55. Roberts AB, Sporn MB, Assoian RK, Smith JM, Roche NS: Transforming growth factor type @: Rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci USA 83~4167-4171,1986. 56. Welch DR, Fabra A, Nakajima M: Transforming growth factor p stimulates mammary adenocarcinoma cell invasion and metastatic potential. Proc Natl Acad Sci USA 877678-7682, 1990. 57. Kehrl J H Transforming growth factor-p: An important mediator of immunoregulation. Int J Cell Cloning 9:439-450, 1991. 58. Wilding G: Response of prostate cancer cells to peptide growth factors: Transforming growth factor-p. Cancer Surv 11:147-163, 1991. 59. Moses HL, Yang EY, Pietenpol JA: TGFP stimulation and inhibition of cell proliferation: New mechanistic insights. Cell 63:245-247, 1990. 60. Fine A, Goldstein RH: The effect of transforming growth factor beta on cell proliferation and collagen formation by lung fibroblasts. J Biol Chem 2623973902, 1987. 61. Centrella M, McCarthy TL, Canalis E: Transforming growth factor beta is a bifunctional regulator of replication and collagen synthesis in osteoblast-enriched cell cultures from fetal rat bone. J Biol Chem 262:28692874, 1987. 62. Hubbs AF, Hahn FF, Thomassen DG: Increased resistance to transforming growth factor beta accompanies neoplastic progression of rat tracheal epithelial cells. Carcinogenesis (Lond) 101599-1605, 1989. 63. Knabbe C, Lippman ME, Wakefield LM, Flanders KC, Kasid A, Deryndc R, Dickson RB: Evidence that transforming growth [email protected] a hormonally regulated negative growth factor in human breast cancer. Cell 48:417-428, 1987. 64. Liu C, Tsao MS, Grisham JW: Transforming growth factors produced by normal and neoplastically transformed rat liver epithelial cells in culture. Cancer Res 48~850-855, 1988. 65. Jennings MT, Maaunas RJ, Carver R, Bascom CC, Juneau B, Misulis K, Moses HL: TGF-p1 and TGF-p2 are potent growth regulators for low grade and malignant gliomas in vitro: Evidence in support of an autocrine hypothesis. Int J Cancer 49:129-139, 1991. 66. Pircher R, Julien P, Lawrence DA: p-transforming growth factor is stored in human blood platelets as a latent high molecular weight complex. Biochem Biophys Res Commun 13630-37, 1986. 67. Lyons RM, Gentry LE, Purchio AF, Moses HL: Mechanism of activation of latent recombinant transforming growth factor @l by plasmin. J Cell Biol 110:13611367, 1990. 68. Grainger DJ, Kemp PR, Metcalfe JC, Liu AC, Lawn RM, Williams NR, Grace AA, Schofield PM, Chauhan A: The serum concentration of active transforming growth factor$ is severely depressed in advanced atherosclerosis. Nature Med 1:74-79, 1995. 69. Sporn MB, Roberts AB, Wakefield LM, de Crombrugghe B: Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Biol 105:1039-1045, 1987. 70. Franzen P, ten Dijke P, Ichijo H, Jamashita H, Schulz P, Heldin CH, Miyazono K Cloning of a TGFP type I receptor that forms a heteromeric complex with the TGFP type II receptor. Cell 75681-692, 1993. 71. Lin HY, Wang XF, Ng-Eaton E, Weinberg RA, Lodish HF: Expression cloning of the TGF-p type I1 receptor, a functional transmembrane serine/threonine kinase. Cell 68775-785, 1992. 72. Laiho M, Weis FMB, Boyd FT, Ignotz RA, Massague J: Responsiveness to transforming growth factor p (TGF-P) restored by genetic complementation between cells defective in [email protected] I and 11. J Biol Chem 266:9108-9112, 1991. 73. Ewen ME, Sluss HK, Whitehouse LL, Livingston DM: TGF-P inhibition of Cdk4 synthesis is linked to cell cycle arrest. Cell 74:1009-1020, 1993. 74. Atfi A, Samperez S, Jouan P: Secretion of transforming growth factor-p by the immature rat prostate. Prostate 24:149-155, 1994. 75. McKeehan WL, Adams PS: Heparin-binding growth factodprostatropin attenuates inhibition of rat prostate tumor epithelial cell growth by transforming growth factor type beta. In Vitro Cell Dev Biol24:243246, 1988. 76. Kyprianou N, Isaacs J T Expression of transforming growth factor-p in the rat ventral prostate during castration-induced programmed cell death. Mol Endocrinol3:1515-1522, 1989. 77. Kyprianou N, Isaacs JT: Identification of a cellular receptor for transforming growth factor-p in rat ventral prostate and its negative regulation by androgens. Endocrinology 123:2124-2131, 1988. 78. Anzano MA, Smith JM, McCune BK, Danielpour D, Sporn MB: Induction of transforming growth [email protected] p3 in rat prostate following castration. J Cell Biochem [Suppl] 16:105, 1992. 79. Story MT, Hopp KA, Meier DA, Begun FP, Lawson Polypeptide Growth Factors in the Prostate RK: Influence of transforming growth factor pl and other growth factors on basic fibroblast growth factor level and proliferation of cultured human prostate-derived fibroblasts. Prostate 23:183-197, 1993. 80. Shain SA, Lin AL, Koger JD, Karaganis AG: Rat prostate cancer cells contain functional receptors for transforming growth [email protected] 126:818-825, 1990. 81. Steiner MS, Barrack E R Expression of transforming growth factor-pl in prostate cancer. Endocrinology 1355940-2247, 1994. 82. Steiner MS, Barrack ER Transforming growth factor-pl overproduction in prostate cancer: Effects on growth in vivo and in vitro. Mol Endocrinol6:15-25, 1992. 83. Matuo Y, Nishi N, Takasuka H, Masuda Y, Nishikawa K, Isaacs JT, Adams PS,McKeehan WL, Sato G H Production and signihcance of TGF-p in AT-3 metastatic cell line established from the Dunning rat prostatic adenocarcinoma. Biochem Biophys Res Commun 166: 840-847,1990. 84. Merz VW, Miller GJ, Krebs T, Timme TL, Kadmon D, Park SH, Egawa S, Scardino IT, Thompson TC: Elevated TGF-p1 and p3 mRNA levels are associated with ras myc-induced carcinomas in reconstituted mouse prostate: Evidence for a paracrine role during progression. Mol Endocrinol5:503-513, 1991. 85. Schuurmans ALG, Bolt J, Mulder E: Androgens and transforming growth factor p modulate the growth response to epidermal growth factor in human prostate tumor cells (LNCaP). Mol Cell Endocrinol6O:lOl104, 1988. 86. Wilding G, Zugmeier G, Knabbe C, Flanders KC, Gelmann E: Differential effects of transforming growth factor beta on human prostate cancer cells in vitro. Mol Cell Endocrinol62:79-87, 1989. 87. Watts RG, Ware JL: Isolation and characterization of transforming growth factor beta response variants from human prostatic tumor cell lines. Prostate 21: 223-237, 1992. 88. Thompson TC, Truong LD, Timme TL, Kadmon D, McCune BK, Flanders KC, Scardino PT, Park SH: Transforming growth factor p l as a biomarker for prostate cancer. J Cell Biochem 1654-61, 1992. 89. Truong LD, Kadmon D, McCune BK, Flanders KC, Scardino IT,Thompson TC: Association of transforming growth factor-pl with prostate cancer: A immunohistochemical study. Hum Pathol 24:4-9, 1993. 90. Bang Y, Kim SJ, Danielpour D, OReilly MA, Kim Ky, Myers CE, Trepel JB: Cyclic AMP induces transforming growth factor 82 gene expression and growth arrest in the human androgen-independent prostate carcinoma cell line. Proc Natl Acad Sci USA 89:35563560, 1992. 91. Esch F, Baird A, Ling N, Ueno N, Hill F, Denoroy L, Klepper R, Gospodarowicz D, Bohlen P, Guillemin R Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the aminoterminal sequence of bovine brain acidic FGF. Proc Natl Acad Sci USA 82:6507-6511, 1985. 92. Saksela 0, Moscatelli D, Sommer A, Rifkin DB Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytk degradation. J Cell BiollO7743-751, 1988. 93. Ingber DE, Folkman J: Mechanochemical switching 94. 95. 96. 97. 98. + 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 403 between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: Role of extracellular matrix. J Cell Biol 109:317-330, 1990. Partanen J, Makela TP, Eerola E, Korhonen J, Hirvonen H, Claesson-Welsh L, Alitalo K: FGFR-4, a novel acidic fibroblast growth factor receptor with a distinct expression pattern. EMBO J 10:1347-1354, 1991. Givol D, Yayon A: Complexity of FGF receptors: Genetic basis for structural diversity and functional specificity. FASEB J 6:3362-3369, 1992. Mansson P-E, Adams I?, Kan M, McKeehan WL: Heparin-binding growth factor gene expression and receptor characteristics in normal rat prostate and two transplantable rat prostate tumors. Cancer Res 49: 2485-2494, 1989. Mori H, Maki M, Oishi K, Jaye M, Igarashi K, Yoshida 0, Hatanaka M: Increased expression of genes for basic fibroblast growth factor and transforming growth factor type 82 in human benign prostatic hyperplasia. Prostate 16:71-80, 1990. Story MT, Sasse J, Jacobs SC, Lawson RK: Prostatic growth factor: Purification and structural relationship to basic fibroblast growth factor. Biochemistry 36: 3843-3849, 1987. Mydlo JH, Bulbul MA, Richon VM, Heston WDW, Fair WR: Heparin-binding growth factor isolated from human prostatic extracts. Prostate 12543-355, 1988. Story MT, Livingston B, Baeten L, Swartz SJ, Jacobs SC, Begun FP, Lawson IUC Cultured human prostatederived fibroblasts produce a factor that stimulates their growth with properties indistinguishable from basic fibroblast growth factor. Prostate 15:355-365, 1989. Nishi N, Matuo Y,Kunitomi K, Takenaka I, Usami M, Kotake T, Wada F Comparative analysis of growth factors in normal and pathologic human prostates. Prostate 13:39-48, 1988. Begun FP, Story MT, Hopp KA, Shapiro E, Lawson RK: Regional concentration of basic fibroblast growth factor in normal and benign hyperplastic human prostates. J Urol 153839-843, 1995. Muller WJ,Lee FS, Dickson C, Petters G, Pattengale P, Leder P: The int-2 gene product acts as an epithelial growth factor in transgenic mice. EMBO J 9:907-913, 1990. Deering RE, Bigler SA, Brown M, Brawer M K Microvascularity in benign prostatic hyperplasia. Prostate 26:lll-115, 1995. Yan G, Fukabori Y, McBride G, Nikolaropolous S, McKeehan WL: Exon switching and activation of stromal and embryonic fibroblast growth factor (FGF)-FGF receptor genes in prostate epithelial cells accompany stromal independence and malignancy. Mol Cell Biol 13~4513-4522, 1993. Matuo Y, McKeehan WL, Yan GC, Nikolaropoulos S, Adams €5,Fukabori Y, Yamanaka H, Gaudreau J: Potential role of HBGF (FGF) and TGF-p on prostate growth. Adv Exp Med Biol324107-114, 1992. Nakamoto T, Chang C, Li A, Chodak GW: Basic fibroblast growth factor in human prostate cancer cells. Cancer Res 52:571-577, 1992. Gleave ME, Hsieh JT, von Eschenbach AC, Chung L W Prostate and bone fibroblasts induce human prostate cancer growth in vivo: Implications for bidi- 404 Culig et al. rectional tumor-stromal cell interaction in prostate carcinoma growth and metastasis. J Urol 1471151-1159, 1992. 109. Thalmann GN, Anezinis PE, Chang SM, Zhau HE, Kim E, Hopwood VL, Pathak S, von Eschenbach AC, Chung LWK: Androgen-independent cancer progression and bone metastasis in the LNCaP model of human prostate cancer. Cancer Res 54:2577-2581, 1994. 110. Pienta KJ, Isaacs WB, Vindivich D, Coffey DS: The effects of basic fibroblast growth factor and suramin on cell motility and growth of rat prostate cancer cells. J Urol 145:199-202, 1991. 111. Gospodarowicz D, Ferrara N, Schweigerer L, Neufeld G: Structural characterization and biological functions of fibroblast growth factor. Endocr Rev 8:95-114,1987. 112. Bigler SA, Deering RE, Brawer MK: A quantitative morphometric analysis of the microcirculation in prostate carcinoma. Hum Pathol 24220-226, 1993. 113. Weidner N, Carroll PR, Flax J, Blumenfeld W, Folkman J: Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Pathol 143:401420, 1993. 114. Katz AE, Benson MC, Wise GJ, Olsson CA, Bandyk MG, Sawczuk IS, Tomashefsky P, Buttyan R Gene activity during the early phase of androgen-stimulated rat prostate regrowth. Cancer Res 49:5889-5894, 1989. 115. Harris SE, Smith RG, Zhou H, Mansson PE, Malark M: Androgens and glucocorticoids modulate heparinbinding growth factor-I mRNA accumulation in DDTl cells as analyzed by in situ hybridization. Mol Endocrin013:1839-1844, 1989. 116. Hall JA, Harris MA, Malark M, Mansson PE, Zhou H, Harris SE: Characterization of the hamster DDT-1 cell aFGF/HBGF-I gene and cDNA and its modulation by steroids. J Cell Biochem 43:17-26, 1990. 117. Geller J, Sionit LR, Baird A, Kohls M, Connors KM, Hoffman RM: In vivo and in vitro effects of androgen on fibroblast growth factor-2 concentrations in the human prostate. Prostate 25:206-209, 1994. 118. Zuck B, Goepfert C, Nedlin-Chittka A, Sohrt K, Voight KD, Knabbe C: Regulation of fibroblast growth factor-like proteins in the androgen-responsive human prostate carcinoma cell line LNCaP. J Steroid Biochem Mol Biol 41659-663, 1992. 119. Yan G, Fukabori Y, Nikolaropoulos S, Wang F, McKeehan WL: Heparin-binding keratinocyte growth factor is a candidate stromal to epithelial cell andromedin. Mol Endocrinol6:2123-2128, 1992. 120. Miki T, Bottaro DP, Fleming TP, Smith CL, Burgess WH, Chan AML, Aaronson SA: Determination of ligand-binding specificity by alternative splicing: Two distinct growth factor receptors encoded by a single gene. Proc Natl Acad Sci USA 89246-250, 1992. 121. Chang SM, Chung LWK Interaction between prostatic fibroblast and epithelial cells in culture: Role of androgen. Endocrinology 125:2719-2727, 1989. 122. Alarid ET, Rubin JS, Young P, Chedid M, Ron D, Aaronson SA, Cunha GR Keratinocyte growth factor functions in epithelial induction during seminal vesicle development. Proc Natl Acad sd USA 91:10741078, 1994. 123. Daughaday WH, Rotwein P: Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr Rev 1068-91, 1989. -~ ~ ~ ~ 124. Murphy LJ, Friesen HG: Differential effects of estrogen and growth hormone on uterine and hepatic insulin-like growth factor I gene expression in the ovariectomised hypophysectomised rat. Endocrinology 122~325-332,1988. 125. Rechler MM, Nisley S P Insulin-like growth factor/somatomedin receptor subtypes: Structure, function and relationships to insulin receptors and IGF carrier proteins. Horm Res 24152-159, 1986. 126. Czech MP: Signal transmission by the insulin-like growth factors. Cell 59:235-238, 1989. 127. Cohen P, Peehl DM, Lamson G, Rosenfeld R Insulinlike growth factors (IGFs), IGF receptors, and IGFbinding proteins in primary cultures of prostate epithelial cells. J Clin Endocrinol Metab 73:401-47,1991. 128. Perkel VS, Mohan S, Baylink SJ, Linkhart TA: An inhibitory insulin-like growth factor binding protein (IN-IGFBP)from human prostatic cell conditioned medium reveals N-terminal sequence identity with bone derived IN-IGFBP. J Clin Endocrinol Metab 71:533535, 1990. 129. Barni T, Vannelli BG, Sadri R, Pupilli C, Ghiandi P, Rizzo M, Selli C, Serio M, Fiorelli G: Insulin-like growth factor-I (IGF-I) and its binding protein IGFBP-4 in human prostatic hyperplastic tissue: Gene expression and its cellular localization. J Clin Endocrinol Metab 78:778-783, 1994. 130. Cohen P, Peehl DM, Baker B, Liu F, Hintz RL, Rosenfeld RG: Insulin-like growth factor axis abnormalities in prostatic stromal cell from patients with benign prostatic hyperplasia. J Clin Endocrinol Metab 79:14101415, 1994. 131. Iwamura M, Sluss PM, Casamento JB, Cockett A X Insulin-like growth factor I: Action and receptor characterization in human prostate cancer cell lines. Prostate 22:243-252, 1993. 132. Connolly JM, Rose DP: Regulation of DU145 human prostate cancer cell proliferation by insulin-like growth factors and its interaction with the epidermal growth factor autocrine loop. Prostate 24167-175, 1994. 133. Bhaumick B, George D, Bala RM: Potentiation of epidermal growth factor-induced differentiation of cultured human placental cells by insulin-like growth factor-I. J Clin Endocrinol Metab 74:1005-1011, 1992. 134. Clemmons DR Multiple hormones stimulate the production of somatomedin by cultured human fibroblasts. J Clin Endocrinol Metab 58:850-856, 1984. 135. Centrelia M, McCarthy TL, Canalis E: Receptors for insulin-like growth factors-I and -11 in osteoblast enriched cultures from fetal rat bone. Endocrinology 126: 39-44,1990. 136. Bichell DP, Rotwein P, McCarthy TL: Prostaglandin E2 rapidly stimulates insulin-like growth factor-I gene expression in primary rat osteoblastic cultures: Evidence for transcriptional control. Endocrinology 133: 1020-1028, 1993. 137. Pietrzkowski Z, Mulholland G, Gomella L, Jameson BA, Wernicke D, Baserga R Inhibition of growth of prostatic cancer cell lines by peptide analogues of insulin-like growth factor 1. Cancer Res 53:1102-1106, 1993. 138. Polychronakos C, Janthly U, Lehoux J-G, Koutsilieris M: Mitogenic effects of insulin and insulin-like growth factors on PA-III rat prostate adenocarcinoma cells: PolYDeDtide Growth Factors in the Prostate Characterization of the receptors involved. Prostate 19~313-321, 1991. 139. Cohen P, Peehl DM, Stamey TA, Wilson KF, Clemmons DR, Rosenfeld RG: Elevated levels of insulinlike growth factor-binding protein-2 in the serum of prostate cancer patients. J Clin Endocrinol Metab 76: 1031-1035, 1993. 140. Kanety H, Madjar Y, Dagan Y, Levi J, Papa MZ, Pariente C, Goldwasser B, Karasik A: Serum insulin-like growth factor binding protein-2 is increased in patients with prostatic carcinoma: Correlation with serum prostate-specific antigen. J Clin Endocrinol Metab 77229-233, 1993. 405 141. Cohen P, Grawes HCB, Peehl DM, Kamarli M, Giudice LC, Rosenfeld RG: Prostate-specific antigen is an insulin-like growth factor-binding protein-3 protease found in seminal plasma. J Clin Endocrinol Metab 75: 1046-1053, 1992. 142. Marcelli M, Haidacher SJ, Plymate SR, Birnbaum RS: Altered growth and insulin-like growth factor-binding protein-3 production in PC3 prostate carcinoma cells stably transfected with a constitutively active androgen receptor complementary deoxyribonucleic acid. Endocrinology 136:lMO-1048, 1995.