Fibroblast Growth Factors and Their Receptors in Urological Cancers: Basic Research and Clinical Implications

European Urology

European Urology 43 (2003) 309±319

Fibroblast Growth Factors and Their Receptors in Urological Cancers: Basic Research and Clinical Implications M.V. Cronauer, W.A. Schulz, H.-H. Seifert, R. Ackermann, M. Burchardt* Department of Urology, Heinrich-Heine University, Moorenstrasse 5, DuÈsseldorf D-40225, Germany Accepted 26 December 2002

Abstract Because therapeutical options for advanced urological cancers are limited, the understanding of key elements responsible for invasion and metastasis is very important. It has been hypothesized that progression to malignant growth is associated with a dysregulation of growth factors and/or their receptors. In the last few years, signaling pathways of the ®broblast growth factor (FGF) family have been subject to intense investigation. Fibroblast growth factors constitute one of the largest families of growth and differentiation factors for cells of mesodermal and neuroectodermal origin. The family comprises two prototypic members, acidic FGF (aFGF) and the basic FGF (bFGF), as well as 21 additionally related polypeptide growth factors that have been identi®ed to date. FGFs are involved in many biological processes during embryonic development, wound healing, hematopoesis, and angiogenesis. In prostate, bladder, and renal cancers, FGFs regulate the induction of metalloproteinases (MMP) that degrade extracellular matrix proteins, thus facilitating tumor metastasis. Probably due to their potent angiogenic properties, aFGF and bFGF have received the most attention. However, there is increasing evidence that other FGFs also play crucial roles in tumors of the prostate, bladder, kidney, and testis. This review will discuss the different elements involved in FGF signaling and summarize the present knowledge of their biological and clinical relevance in urological cancers. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Fibroblast growth factors; Urological cancers; Clinical implications

1. Introduction Recent ®ndings have highlighted the importance of ®broblast growth factors (FGFs) in urological cancers. The expression of FGFs is increased in a substantial fraction of human prostate, bladder, renal, and testicular cancers [1±9]. Altered FGF expression can have a variety of effects, including stimulation of cell proliferation or inhibition of cell death. The FGFs represent one of the largest families of polypeptide growth and differentiation factors for cells of mesodermal and neuroectodermal origins [10]. A common characteristic of members of the FGF family is that they bind to heparan and heparan-sulfates that protect them from *

Corresponding author. Tel. ‡49-211-811-8110; Fax: ‡49-211-811-8676. E-mail address: [email protected] (M. Burchardt).

degradation [11]. To date, 23 different FGFs have been isolated [12±15]. Like other polypeptide growth factors, FGFs mediate their signals through cell surface receptor tyrosine kinases. Although more than 20 FGFs with different effects on various target cells have been identi®ed, only four FGF-receptor (FGFR) types have been isolated to date [12,16±18]. By the expression of different splice variants, it is possible for only four FGFR genes to encode a broad variety of different receptor protein isoforms to achieve the kind of diversity that is needed to respond to the many different types of FGFs and establish signaling speci®city [12,16]. An enormous number of functions are attributed to the FGF family, of which only the most important ones can be discussed here. FGFs are mitogenic for many cell types, both epithelial and mesenchymal. These properties and other activities of FGFs are essential in embryonic development. In the adult, FGFs are involved in

0302-2838/03/$ ± see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0302-2838(03)00005-8

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in¯ammatory processes, wound healing, hematopoesis, and angiogenesis [19±22]. Besides stimulating angiogenesis, FGFs have been shown to increase the invasiveness of a variety of tumor cell types from the prostate, bladder, kidney, breast, and pancreas, conferring further important properties for FGF during tumor progression [3,23±26]. Various studies analyzing the mechanisms of FGF signaling have revealed a complex signaling network between FGFs, their receptors (FGFRs), FGF-binding proteins (FGF-BPs), and glycosaminoglycans that modulate FGF signaling [27±29]. There is increasing evidence that FGFs play a crucial role in malignancies of the prostate, bladder, kidney, and testis. The following review presents the most important ®ndings in FGF signaling, discussing alterations in FGFs and FGFRs as well as the role of the FGF-BPs in urological tumors. 2. The fibroblast growth factor familyöstructure and function The FGF family consists of a group of structurally related polypeptide growth factors. To date, 23 different FGFs have been discovered (Table 1). Although all FGFs are categorized by their structure, the historical nomenclature refers to the fact that the ®rst members of the FGF family, isolated from bovine pituitary extracts, stimulated ®broblast proliferation [30]. In fact, the designation ``FGF'' is misleading since FGFs

are mitogenic for many different cell types. One of the best-characterized FGFs is FGF-2, also known as basic FGF (bFGF), which can be regarded as the prototypic growth factor of the FGF family, displaying all typical features of the FGF family [10]. bFGF has a strong af®nity for heparin and glycosaminoglycans, important constituents of the extracellular matrix (ECM) [27,31]. The association of FGFs with heparan-sulfates and glycosaminoglycans of the ECM creates a local reservoir of FGFs on the cell surface and protects the growth factors from denaturation and proteolytic degradation [11,32]. Four different bFGF polypeptide isoforms can be derived from the bFGF/FGF-2 gene with molecular weights of 18 kDa, 22.5 kDa, 23.1 kDa, and 24.2 kDa, respectively. Some of the larger forms of this protein are mainly localized in the nucleus [33,34]. Different isoforms have also been isolated for FGF-1, the acidic FGF (aFGF), FGF-8, also known as androgen-induced growth factor (AIGF), and FGF-13 (Table 1). Like bFGF, FGF-1, 9, and 11±14 lack a signal peptide sequence for secretion (Table 1). There are several hypotheses to explain how FGFs are released from cells. Besides mechanical damage of the plasma membrane with subsequent release of FGFs from the cell or the formation of a complex between FGF and a carrier protein with subsequent co-secretion are discussed [35,36]. Interestingly nuclear location signals (NLS) have been identi®ed in FGF-1, FGF-3 and FGF-11±FGF-14[12]. There is experimental evidence

Table 1 Characteristics of different FGFs and their presence/activity in urological tissue Designation

Synonym

Nuclear location signal (NLS)

FGF-1 FGF-2 FGF-3 FGF-4 FGF-5 FGF-6 FGF-7 FGF-8 FGF-9 FGF-10 FGFs 11±12 FGF-13 FGF-14 FGF-15 FGFs 16±19 FGF-20 FGFs 21±22 FGF-23

aFGF bFGF Int-3 kFGF, hst-1 ± hst-2 KGF AIGF GAF KGF-2 ± ± ± ± ± ± ± ±

‡ ‡ ‡

‡ ‡ ‡ ? ? ? ? ?

Signal sequence for secretion

‡ ‡ ‡ ‡ ‡ ‡

Isoforms

Presence or activity in urological tissue

1 4

bld, pro, kid bld, pro, kid pro pro? test bld pro bld, pro bld, pro, test pro pro ? ? ? ? ? ? ? kid

7

‡

? ‡ ? ?

2 2

?

aFGF, acidic ®broblast growth factor; bFGF, basic ®broblast growth factor; kFGF, kaposi FGF; KGF, keratinocyte growth factor; AIGF, androgen-induced growth factor; GAF, glia activating factor; hst, human stomach tumors; bld, bladder; pro, prostate; kid, kidney; test, testes.

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that the NLS is necessary for FGF signaling, since removal of the NLS abrogated the mitogenic effects of FGF-1 [37]. FGFs mediate their signals through cell surface receptors to induce numerous biological effects [16± 18]. One of the best-characterized functions of FGFs is the induction of new blood vessels during early stages of tumor development [22,38±41]. In general, formation and sprouting of new capillaries involves endothelial cell proliferation and cell migration, as well as breakdown of surrounding ECM components. Together with the vascular endothelial growth factor (VEGF), FGFs are the most important regulators of these processes. During embryonic development, FGFs play a role in organogenesis, particularly in the nervous system and the limbs, but also in the development of the prostate [42±46]. FGFs play a role in the ®nal phase of wound healing and regeneration of the proximal renal tubule following chemically induced damage [47]. In different cell systems bFGF participates in the regulation of apoptosis [48±50]. Moreover, in prostate cancer cells, bFGF has been shown to confer resistance to anticancer drugs [51]. 3. FGF-receptors (FGFR)östructure and function FGFs mediate their signals through four distinct tyrosine kinase receptors, designated FGFR1±4, which share between 55% and 72% homology at the protein level [12,16±18]. FGFRs consist of an extracellular ligand-binding domain, a transmembrane domain, and an intracellular kinase domain. The ligand-binding domain contains three different immunoglobulin-like domains (designated IgI, IgII and IgIII), with the ®rst two separated by a short, highly acidic region. The ligand-binding domain is followed by a transmembrane domain and an intracellular split kinase domain (Fig. 1). The short C-terminus is one of the most divergent regions in the FGFR family. Structural changes in the IgIII domain often result in receptor isoforms with dramatically altered binding speci®cities for different FGFs [12,16]. Through the expression of different isoforms by alternative splicing, the four FGFR genes provide a mechanism that enables them to achieve unique ligand binding properties and allows a speci®c response to the many different FGF types. Upon binding to their receptors, FGFs induce receptor dimerization. The formation of the FGF/FGFR ligand±receptor complex is facilitated by heparin or glycosaminoglycans [12,27]. Following activation of the ligand±receptor complex, various signal

Fig. 1. Schematic representation of the FGF-receptor (FGFR). The FGFRs consist of an extracellular ligand-binding domain, a transmembrane domain and an intracellular kinase domain. The ligand-binding domain contains three different immunglobulin-like domains (designated IgI, IgII and IgIII), with the ®rst two Ig-like domains separated by a short, highly acidic region. The ligand-binding domain is followed by a single transmembrane domain and a cytoplasmatic split tyrosine kinase domain in the intracellular domain. The split kinase is followed by a short C-terminus which represents one of the most divergent regions in the FGFR-family.

transduction pathways are initiated by FGFs: elevation of intracellular calcium levels, induction of mitogenactivated protein kinase and protein kinase C pathways, stimulation of adenylate cyclase, and induction of the proto-oncogenes, myc and fos [12,52]. These intracellular signals result in various biological responses during ontogenesis, cell growth and repair processes as well as tumorigenesis. 4. The FGF-binding protein (FGF-BP) After their secretion and/or release from cells, FGFs are found tightly bound to heparansulfates and proteoglycans of the ECM. These interactions extend their

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Sbiological half-life but quench their biological activities. Two different mechanisms to explain how FGFs are released from the ECM-reservoirs have been proposed: enzymes, like heparanase, also expressed in various tumors, can degrade heparansulfate chains and the proteoglycan backbone of the ECM, thereby releasing FGFs [53]. An alternative mechanism involves the FGF-binding protein, a secreted 17 kDa protein that reversibly binds FGF preventing its degradation and conserving its biological activity [28]. Although FGFBP is almost undetectable in normal adult tissues, its expression is elevated in various tumors, including kidney cancer [28,29,54±58]. Overexpression of FGFBP in the human adrenal carcinoma cell line, SW-13, resulted in a more aggressive phenotype [56]. Downregulation of FGF-BP by ribozyme in human prostate cancer cells resulted in reduced cell growth in vitro and in vivo [55]. These observations led to the hypothesis that the regulation of FGF-BP may be as important as regulation of FGF expression. Moreover, there is evidence for a new putative FGF-BP, termed FGF-BP2, suggesting the existence of an FGF-BP family [12,29]. 5. FGF signaling in cancer SFGFs may promote tumor growth by different mechanisms: as angiogenic inducers, as mitogens for the tumor cells themselves, and as inhibitors of apoptosis. Within epithelial tumors there are several potential sources of FGFs. bFGF can be produced by tumor cells, surrounding stromal cells or tumor in®ltrating lymphocytes [3,59]. Moreover there is evidence from bladder, prostate, and renal cancer that other players in the FGF signaling cascade, such as FGFRs or FGF-BPs, can modulate the effects of FGFs during tumorigenesis. The roles of the various FGFs, FGFRs, and FGF-BP as part of a complex signaling network in urological cancers will be speci®cally discussed in the following sections. 5.1. Prostate cancer It has been hypothesized that the progression to malignant growth of the prostate is associated with a dysregulated expression of various growth factors, among them members of the FGF family. FGFs are required for development, growth, and maintenance of prostatic tissue. The prostate, the prototype of an androgen-responsive organ, requires androgen for the maintenance of its functional and structural integrity [60]. However, isolated epithelial cells do not require androgens in vitro [61]. Instead, their growth is

strongly dependent on different peptide growth factors. It has been hypothesized that in the prostate, growth factors are produced locally in response to androgenic stimuli, and they mediate the action of androgens [62]. These growth factors were thus termed andromedins. Several FGFs are thought to act as andromedins, i.e. FGF-7 (also known as keratinocyte growth factor, KGF), FGF-8 (androgen-induced growth factor), and FGF-10 (keratinocyte growth factor-2, KGF-2). All of them have been detected in the prostate [45,46,63±65]. The fact that FGF-7 and FGF-10 are produced exclusively in mesenchymal cells but stimulate mainly epithelial cells makes them ideal candidates for mediators of mesenchymal±epithelial interactions in the developing prostate (Fig. 2) [45,46,65]. Although FGF-7 expression is up-regulated in stromal cells after androgenic stimuli, it does not decrease following castration. Analysis of FGF-7 serum levels revealed that overall FGF-7 levels tend to be lower in patients with prostate cancer than in patients with benign prostatic hyperplasia [66]. Therefore, its role in vivo remains unclear. In vitro FGF-7 was able to induce androgen receptor activation in prostate cancer cell lines in the absence of androgens [67]. Moreover, the stimulation of prostate epithelial cells with high concentrations of FGF-7 resulted in an up-regulation of androgen receptor mRNA [68]. Recent studies reported an induction of FGF-7 and FGF-2 by interleukins in different cell systems (Fig. 2) [69±71]. In prostate induction of FGF-7 and FGF-2 by interleukin-1a and interleukin-8 is thought to play a critical role in the pathogenesis of

Fig. 2. Induction of FGFs in the prostate by androgens and interleukins. Androgenic stimuli induce the synthesis of FGF-7 and FGF-10 in prostatic stromal cells and the expression of FGF-8 in prostatic epithelial cells. All these FGFs are highly mitogenic for prostate epithelial cells. The interleukin IL-1a produced by prostatic epithelial cells induces in stromal cells the synthesis of FGF-7 which in turn stimulates the proliferation of epithelial cells. FGF-2, a mitogen for epithelial as well as stromal cells is synthesized in stromal cells upon stimulation by IL-8.

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BPH [70,71]. However the role of this regulatory mechanism in prostate cancer remains to be elucidated. The androgen-induced growth factor, FGF-8, was originally identi®ed in conditioned medium of the androgen-dependent mouse mammary Shionogi carcinoma cell line, SC-3 [72]. Neutralizing antibodies to FGF-8 abolished androgen-induced cell growth in SC3 cells. In the prostate different FGF-8 isoforms have been shown to play a key role in prostate carcinogenesis as shown by different studies [63,73±75]. In the androgen sensitive CWR22 prostate xenograft FGF-8b expression is regulated by androgenic stimuli [76]. In vitro, overexpression of FGF-8b in weakly tumorogenic prostatic cancer cells contributed to a more aggressive phenotype [74]. Analysis of clinical specimens revealed an overexpression of different FGF-8 isoforms (a, b and e) and their receptors (FGFR1IIIc, FGFR2IIIc) in human premalignant prostatic intraepithelial neoplasia (PIN) as well as in prostate cancer [73,75]. In addition overall FGF-8 expression correlated with Gleason score and stage and was associated with decreased patient survival [63]. Taken together, the data suggest a crucial role for FGF-8 in the development of prostate cancer. Probably the best-studied FGF in prostate cancer is bFGF (FGF-2). Apart from its angiogenic properties the importance of bFGF was illustrated in experimental prostatic tumors in nude mice. When injected subcutaneously, the androgen-sensitive LNCaP cells rarely form tumors in nude mice. On the other hand, tumors can be induced consistently when LNCaP cells are co-

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inoculated with a matrix absorbed with bFGF [77]. Clinically bFGF is implicated in benign and malignant growth of the prostate. Basic FGF levels are increased in serum and urine of patients suffering from proliferative disorders of the prostate [3,78,79]. Immunohistochemical analysis of tissue specimens from patients with prostate cancer showed that bFGF is mainly expressed in carcinomatous areas (Fig. 3), indicating an enhanced production of bFGF by tumor cells [3,64]. Interestingly, patients in whom the disease progressed showed a dramatic increase in serum bFGF levels [3]. Furthermore, the importance of FGFs for prostate cancer progression was demonstrated by an in vitro experiment in which the prostatic cell line, PNT1a, was transfected with bFGF cDNA. This transfection led to the acquisition of an anchorage-independent phenotype of PNT1a cells. Recently, bFGF was shown to downregulate androgen receptor protein in LNCaP cells [80]. In the rat Dunning tumor system, tumor progression is associated with changes in the FGF receptor-2 (FGFR2). In early stages of prostate cancer, the predominant receptor form is the FGFR2IIIb isoform, which preferentially binds KGF/FGF-7. The progression from a benign to a malignant cell is accompanied by a switch to the FGFR2IIIc isoform, which no longer recognizes FGF-7 but has a strong af®nity for bFGF. This switch is followed by activation of the bFGF, FGF-3 and FGF-5 genes [81]. The loss of the FGFR2IIIb isoform is generally accompanied by concurrent reduction of FGFR2 gene expression in general and an abnormal activation of the FGFR1 gene [82].

Fig. 3. Immunohistochemical detection of bFGF in tissue sections of carcinomatous prostatic tissue. Note the strong cytoplasmatic expression of bFGF in carcinoma cells, whereas benign epithelia are negative. (Cronauer et al. [3], reproduced with permission of Wiley-Liss Inc., a subsidiary of John Wiley & Sons, Inc.)

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Restoration of the FGFR2 expression in highly malignant FGFR1 expressing rat prostatic tumor cell lines resulted in an inhibition of cell growth [83]. In contrast, disruption of both FGFR2 as well as FGFR1 mediated signaling in human prostate cancer cell lines lead to extensive cell death in vitro [84]. Although the results of both studies concerning the effects of FGFR2 in human and rodent prostate are controversial, both ®ndings revealed a dramatical dependence of prostate cancer cells on FGF-signaling. Recently Ropiquet et al. demonstrated that FGF-6, although undetectable in the normal prostate, was elevated in a subset of PIN lesions and in prostate cancer. Moreover, the authors found FGF-6 to be mitogenic for primary epithelial and stromal cells of the prostate as well as for several prostate cancer cell lines [4]. Analysis of FGF-9 revealed that this growth factor is quantitatively the predominant FGF in the human prostate [85]. Although FGF-9 is a potent mitogen for prostatic epithelial and stromal cells, its role in prostate cancer remains to be elucidated. In addition to their angiogenic properties, a further mechanism by which FGFs in¯uence prostate tumor metastases is the induction of promatrilysin, a metalloproteinase (MMP). MMPs belong to a group of enzymes that are responsible for the degradation of ECM components, a main step in tumor invasion. Several MMPs are overexpressed in prostate cancer [86]. Acidic FGF is a very potent inducer of promatrilysin in prostate cancer cell lines. At higher concentrations, bFGF, FGF-8 and FGF-9, also induced MMP expression [87,88]. A further important ®nding is that aFGF and bFGF confer broad resistance to anti-cancer drugs in prostate cancer cells [51]. Accordingly, FGF-inhibitors, like suramin, enhanced the anti-tumor activity of chemotherapeutics like doxorubicin in PC-3 cells [89]. Taken together, there is substantial evidence that enhanced expression of FGFs, contributes to a more aggressive phenotype in prostate cancer. 5.2. Bladder cancer Bladder cancer is the second most common malignancy of the genitourinary tract. The majority of bladder carcinomas are low-grade papillary carcinomas that frequently recur, but rarely progress. The remainder is highly invasive and prone to metastasis. In cases with metastatic spread, prognosis is generally poor. There is an urgent need to understand the mechanisms of metastasis and invasion in bladder cancer. Early reports showed that aFGF is detectable in transitional cell carcinoma (TCC) by immunohistochemistry [2]. In further studies aFGF was frequently elevated in tissue extracts and in the urine of bladder

cancer patients [1]. In an experimental study, NBT-II bladder carcinoma cells transfected with aFGF cDNA gave rise to well-vascularized and rapidly growing tumors in contrast to untransfected or mock-transfected cells [90]. Elevated bFGF levels are found in the urine of bladder cancer patients displaying a strong association with clinical parameters like tumor size and vascularization [79,91±93]. bFGF is almost undetectable in normal bladder tissue as well as in most super®cial bladder cancer tissue specimens [94]. By comparing cells derived from an invasive bladder carcinoma (EJ) with cells from a non-invasive bladder carcinoma (RT4) in vitro, Allen and Maher reported high bFGF expression in the aggressive EJ cell line and no bFGF expression in the non-invasive RT4 cells [95]. In a further in vitro experiment the introduction of bFGF cDNA into bladder carcinoma cells markedly enhanced their invasive potential. The increased metastatic potential was accompanied by increased expression of metalloproteinases (MMP-2, MMP-9) [23]. This ®nding is in agreement with a clinical study showing that MMP-2 and MMP-9 are more strongly expressed in invasive than in super®cial TCCs [96]. The results suggest that overexpression of bFGF contributes to a more aggressive phenotype in bladder cancer. KGF/FGF-7 has been shown to stimulate the proliferation of normal urothelium and bladder carcinoma cells in vitro [97,98]. However, decreased expression of the KGF receptor, FGFR2IIIb, was observed during progression of human TCCs [99]. The decrease in FGFR2IIIb was not accompanied by an increase in FGFRIIIc, as it is in prostate cancer [81,82]. Analysis of the FGFR3 gene demonstrated a high prevalence of FGFR3-activating mutations in human bladder and cervical carcinomas [100]. FGFR3 mutations occurred in 41% of bladder tumors, making it one of the most frequently mutated genes in bladder cancer [101]. Most interestingly, activating FGFR3 mutations were restricted to papillary, non-invasive tumors [102,103]. The authors conclude that FGFR3 mutations identify a cohort of bladder cancer patients with favorable disease characteristics. 5.3. Renal cancer A common characteristic of renal cancer is its hypervascularity. Therapeutical options for this malignancy are limited once metastasis has occurred. Informations regarding the role of FGFs in renal cancer are sparse. Initial studies showed increased bFGF levels in the urine and serum of renal cancer patients [79,91,104]. After resection of the tumor-affected kidney, bFGF serum levels dropped to nearly zero within 2 weeks [104].

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In patients suffering from advanced renal cancer, increased bFGF serum levels were associated with poor survival and a higher frequency of pulmonary metastases [6,24]. High expression of bFGF and the ratio of MMP to E-cadherin, were stronger predictors for metastasis than either tumor size, grade, or ploidy [105]. Transfection of bFGF cDNA into RenCa mouse renal cancer cell lines increased their invasive potential in an in vitro tumor cell invasion assay [23]. Subsequent zymography showed a marked increase in MMP2 in these cells. Accordingly, when injected into nude mice, transfected RenCa cells formed more than 10 times as many metastatic nodules in the lung as the non-transfected cells. Analysis of FGF and FGFR mRNAs in bladder and in renal carcinoma cell lines revealed expression of bFGF, aFGF, FGF-5, and FGF-8 [106]. Quantitative real-time RT-PCR showed that FGF-5 was overexpressed in most renal carcinomas [107]. The newest member of the FGF family, FGF-23, plays an important role in the kidney by modulating renal tubular phosphate transport [108]. Expression of FGF-23 is probably one of the major causes for tumor-induced osteomalacia [109]. Another mechanism by which renal cells can modulate their need for FGFs is via secretion of an FGFbinding protein. There is evidence that FGF-BP modulates the activity of bFGF in pediatric renal diseases [58]. It will be important to elucidate the exact function of FGF-BP in FGF-mediated angiogenesis in adult renal cancer. 5.4. Testicular cancer The incidence of malignant tumors of the testis is increasing rapidly in industrialized countries. While the etiology of testicular cancer remains unknown, both congenital and acquired factors have been associated with tumor development. Non-seminomatous components within testicular germ cell tumors affect their prognosis to varying degrees. These components are well known to mimic embryonic tissue. Because FGFs are known to be involved in embryonic development and differentiation, several studies were performed to elucidate the role of FGFs in testicular cancer. Early studies suggested that FGF-4 (hst-1) is overexpressed in different germ cell tumors [110,111]. Strohmeyer et al. found signi®cant association between FGF-4 and tumor stage in non-seminomas; 53% of local tumors, 58% of tumors with nodal metastases, and 82% of tumors with distant metastatic spread showed an overexpression of FGF-4 [111]. A recent immunohistochemical study on primary testicular germ cell tumors showed predominant expression of FGF-4,

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FGF-8, and FGFR1 in non-seminomatous and highly proliferative components of the tumors [8]. Analyzing human teratocarcinoma cells (Tera-2) in vitro, Pertovaara et al. showed that undifferentiated cells expressed mRNAs for all four known FGFRs [112]. The induction of differentiation in Tera-2 cells by retinoic acid led to the loss of FGFR4 expression and to down-regulation of FGFR3 and FGFR2 transcripts, whereas the FGFR1 mRNA level remained unaltered. The down-regulation of FGFRs resulted in a substantial decrease of FGF-4 binding sites [112]. Additional in vitro experiments with teratoma cells showed that low concentrations of bFGF stimulated their proliferation, whereas higher concentrations induced cell migration [9]. Taken together, the recent clinical and in vitro observations suggest that FGF-4 and FGF-8 and, to a minor extent bFGF, are involved in the course of testicular cancer. 6. Clinical and preclinical studies involving FGFs Because FGFs, FGFRs, and FGF-BPs are components of a complex signaling-network in both normal and malignant urological tissues they represent potential markers and/or targets for therapy of urological tumors. As determined by ELISA, aFGF as well as bFGF levels are elevated in the urine and serum of tumor patients suffering from malignant tumors of the bladder, prostate, and kidney [3,79,91±93,104]. However, due to their interference with in¯ammatory processes, the speci®city of aFGF and bFGF serum levels as potential tumor markers is limited. However, determination of intratumoral aFGF and bFGF by immunohistochemistry or in situ hybridization may be helpful in identifying which tumors are likely to progress [3,26,113]. To date, clinical approaches involving FGF signaling have focused on the angiogenic properties of aFGF and bFGF. It has been hypothesized that the anti-angiogenic properties of thalidomide metabolites are due to an interference with the FGF signaling cascade. Using a cornea micropocket assay, D'Amato et al. were able to demonstrate that thalidomide is indeed an inhibitor of bFGF-induced angiogenesis [114]. A ®rst phase II trial with thalidomide showed some bene®cial effects on patients suffering from androgen-independent metastatic prostate cancer who had failed multiple therapies [115]. Moreover, in a further phase II trial, 3 of 18 renal cancer patients showed a partial response, another three patients demonstrated a stabilization of disease after thalidomide treatment [116]. Suramin, a polysulfonated napthylurea, is a pharmacological growth factor antagonist that interferes with

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FGF signaling pathways. In clinical trials, ef®cacy of the compound has been reported in cancers of the prostate, kidney, and bladder [117±121]. Apart from its direct effect on tumor cells, suramin inhibits tumor growth through its ability to block bFGF-induced endothelial cell proliferation [121,122]. In prostate cancer cells aFGF and bFGF induced a broad resistance to anticancer drugs [51]. Based on this observation, the treatment of prostate cancer cells with low doses of suramin dramatically enhanced the antitumor activity of chemotherapeutics, like doxorubicin [89]. When treated with interferon-a or -b, expression of bFGF was down-regulated in human renal, bladder, and prostate carcinoma cell lines [123]. Accordingly, bFGF expression, angiogenesis, and growth of human bladder carcinoma cells, implanted into mice was inhibited by systemic administration of interferon-a [124]. Experimental gene therapy trials in athymic nude mice showed that the growth of ectopically implanted cell lines derived from TCC could be inhibited by

adenoviral-mediated antisense bFGF-DNA gene transfer [125]. The recent observation that FGFR signaling is absolutely necessary for the survival of prostate cancer cells in vitro supports the potential of therapies targeting a disruption of the FGF signaling cascade [84]. There is a growing body of evidence that FGFs play a key role in the growth and progression of urological tumors. Currently, the majority of clinical and experimental studies target bFGF. Our expanding knowledge of the FGF family strongly suggests that FGFs are valuable targets for novel cancer diagnostics and therapies in urological tumors. Acknowledgements The authors wish to thank Dr. Lynn Janulis for critically reading the manuscript. This work was supported by the Forschungskomission der Medizinischen FakultaÈt der Heinrich-Heine UniversitaÈt, DuÈsseldorf and Action LionsÐVaincre le Cancer, Luxembourg.

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