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Keratinocyte growth factor expression in hormone insensitive prostate cancer Hing Y Leung, Pyush Mehta, Lisa B Gray, Anne T Collins, Craig N Robson and David E Neal Department of Surgery, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH

Cellular interactions between stroma and epithelium are important in the growth and proliferation of prostate cancer. Peptide growth factors may facilitate the progression of prostate cancer as autocrine and/or paracrine factors. Keratinocyte Growth Factor (KGF or FGF7) has a di€erentiative and proliferative e€ect on the epithelium of the developing rat prostate. We investigated if KGF may act as a paracrine agent in human prostate cancer and examined the expression of KGF and Fibroblast Growth Factor Receptors (FGFRs) (IIIb and IIIc isoforms of the FGFR1 and FGFR2 genes). Sixty-®ve percent (11 out of 17 informative cases) of prostate cancers (CaP) expressed KGF mRNA by RT ± PCR, while KGF expression was not detected in benign prostatic hyperplasia (BPH) (n=6). Upregulation of KGF expression was related to hormone insensitive tumours (P50.05). Tumour grade and stage were not associated with KGF expression. The source of KGF expression was further characterised using an in vitro primary culture model, showing its restriction to the prostatic stroma. The FGFR1IIIb isoform was expressed in all cases of prostate cancer (n=17), and FGFR1IIIc mRNA was not detected. In the BPH group, FGFR1IIIb transcripts were detected in four out of six cases. FGFR2IIIb expression was detected in ®ve of six cases of BPH and twelve out of seventeen (71%) cases of prostate cancer. In CaP, though not reaching statistical signi®cance, the persistence of FGFR2IIIb expression appeared to be associated with hormone insensitive tumours (P=0.052). FGFR2IIIc expression was present in eleven of seventeen tumours but was absent in all six cases of BPH. Functional assessment of recombinant KGF in a proliferation assay demonstrated a mitogenic e€ect of up to 100% on cultured prostatic epithelial cells. Keywords: kGF; FGF; FGFR isoform; alternative splicing; prostate cancer; paracrine loop Cancer of the prostate (CaP) is a common malignancy in males, ranking as the ®fth most common cancer in the world. Each year in England and Wales, 9000 men die of the disease. Patients with prostate cancer often present with locally advanced and/or metastatic disease, and their prognosis remains poor. Fifty percent of men with locally advanced prostate cancer have lymph node metastases on presentation and only half will survive 3 years. Only younger men with Correspondence: HY Leung Received 6 December 1996; revised 7 May 1997; accepted 7 May 1997

localised disease are suitable for radical prostatectomy and the mainstay of management of advanced disease is androgen deprivation either by medical means or orchiectomy. As many as 30% will not respond to androgen ablation in the ®rst instance and, even among the hormone sensitive tumours, an increasing proportion will subsequently progress and develop hormone insensitivity. At the end of the third year following diagnosis, only a third of the locally advanced disease and ten percent of metastatic tumours have not progressed. Peptide growth factors and their high anity tyrosine kinase receptors may contribute towards to the progression of prostate cancer (reviewed by Byrne et al., 1996). In man, the family of Fibroblast Growth Factors (FGFs) consists of nine cloned ligand genes, FGF1 to FGF9 (Klagsbrun, 1989; Tanaka et al., 1992; Miyamoto et al., 1993), and the receptor family (FGFR) comprises four members, FGFR1 to 4 (Partanen et al., 1992). More recently, FGF10 in rat (Yamasaki et al., 1996) and four FGF-homologous factors in human have been cloned (Smallwood et al., 1996). The existence of multiple ligands and receptors allows interaction between a single FGFR and multiple FGFs, and between di€erent FGFR monomers through heterodimerisation following activation by FGF (reviewed by Leung et al., 1994). The utilisation of splice variants in FGFR expression is complex and is best documented for the FGFR1 and FRGF2 genes (Eisemann et al., 1991; Crumley et al., 1991). Functional implications of the di€erent splice variants are not fully understood, but FGFR isoforms di€ering in their extracellular domains have distinct ligand binding repertoires. For instance, usage of two alternative splice exons in the second half of the third immuno-globulin loop in the extracellular domain of FGFR2 results in di€erential ligand binding, with the IIIb isoform binding KGF at high anity but not FGF2 (bFGF), and vice versa for the IIIc isoform (Miki et al., 1992). Fibroblast growth factors have been implicated in the biology of the prostate. FGF1 (aFGF) and FGF2 (bFGF), but not KGF, are mitogenic to cultured human prostatic stromal cells derived from BPH, whilst cultured benign epithelial cells respond to FGF1 and KGF, but not to FGF2 (Story et al., 1994). Cellular interactions between stroma and epithelium are important in the embryogenesis of the prostate. Cunha et al. (1983) showed that under androgen stimulation, developing epithelium derived from the urogenital sinus, though itself lacking androgen receptors, was able to di€erentiate and proliferate to mature prostatic epithelium when

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combined with androgen receptor expressing stromal cells. More recently, KGF was identi®ed as an androgen-induced paracrine factor derived from prostatic stroma and found to have di€erentiating and proliferative e€ects on the epithelium of the developing rat prostate (Yan et al., 1992; Surgimura et al., 1996). The gene product of FGFR2 was found in rat benign prostatic epithelium; the presence of the exon IIIb was suggested by the strong binding to KGF and FGF1 but not to FGF2. During the development of prostate cancer in a rat model, the FGF binding characteristics of prostatic epithelium changed to a higher anity for FGF2 and FGF1, but not for KGF. This resulted from a switch of FGFR expression from the IIIb to the IIIc isoform. Such an alteration in the receptor responsiveness to FGF2 occurred along with a simultaneous over-expression of FGF2 in the malignant prostate (Yan et al., 1993), hence constituting a potential autocrine loop action. These ®ndings support the hypothesis that FGFs and their high anity receptors may contribute to the tumorigenesis of prostate cancer in the rat. We tested the hypothesis that KGF may act in a paracrine fashion in human prostate cancer and examined if the expression status of KGF and FGFRs (IIIb and IIIc isoforms of the FGFR1 and FGFR2

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genes) in a panel of resected prostate cancer may relate to the clinical progression of the disease. The cell type responsible for KGF expression were further characterized using an in vitro primary culture model. Functional assessment of KGF was performed by proliferation assay using cultured prostatic epithelium. Seventeen of the 18 prostate cancer cases and all six cases of BPH were shown to have intact RNA following ethidium bromide stained gel electrophoresis (Figure 1a) and on RT ± PCR for GAPDH expression, giving a band product of 300 bp (Figure 1b). Case 3 of the prostate cancer samples had signi®cant degradation of RNA and was excluded from the study. Of the 17 cases of prostate cancer, 11 (65%) showed KGF expression, with a PCR product of 685 bp (Figure 1c). Northern blot analysis con®rmed the expression of a 2.4 kb KGF transcript (Figure 1d). All six cases of BPH were negative for KGF mRNA expression. KGF expression was associated with hormone insensitive prostate cancer. Seven out of ten KGF positive tumours failed to respond to hormone manipulation and showed no signi®cant fall in serum PSA levels, while only one out of seven KGF negative tumours were hormone insensitive (Fisher's exact test: P50.05). The grade and stage of the tumours did not correlate with KGF expression (Tables 1 and 2).

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Figure 1 (a) ethidium bromide stained gel, (b) GAPDH expression, (c) KGF expression, (d) Northern blotting for KGF mRNA expression. The single step acid-phenol/chloroform extraction method was employed. Oligo-dT based reverse transcription was performed using the `ready to go kit'TM (Pharmacia Ltd, UK). The primer sequences were: KGF forward-CAAATGGATACTGACATGGATC, reverse-GCCATAGGAAGAAAAGTGGGCTGT; GAPDH forward-AGTCAACGGATTTGGTCGTA and reverse-AAATGAGCCCCAGCCTTCT. PCR reactions were performed in 50 ml reactions containing 50 mM KCl, 10 mM TrisCl pH 8.3, 1.5 mM MgCl2, 0.2 mM dNTPs, 1 unit of Taq polymerase (Bioline Ltd, UK). PCR protocols were optimised as follows: KGF ampli®cation (30 cycles: 948C, 1 min; 558C 1 min, 728C 1 min); GAPDH ampli®cation (25 cycles 948C 30 s, 628C 20 s, 728C 30 s). A 700 bp KGF cDNA probe was provided by Dr S Tronick (Santa Cruz Biotechnology, Inc., Santa Cruz, California, USA), and was radio-labelled using random primed labelling mixture (Finch et al., 1989). Total RNA from a KGF expressing tumour on RT ± PCR (+) and a KGF negative tumour (7) were used for Northern blotting. The arrow signi®es the expected 2.4 kb KGF transcript

KGF and FGFR in human prostate cancer HY Leung et al

Using in vitro cultured prostatic epithelial and stromal cells, KGF mRNA expression detected by RT ± PCR was shown to be restricted to prostatic stromal cells (Table 3). Stromal cells derived from both malignant and benign prostate glands expressed KGF transcripts. Primary cultured prostatic epithelial cells from both BPH and CaP showed no KGF expression. Furthermore, established prostatic epithelial cell lines, including LNCaP cancer cells and SV40 transformed epithelial PNT1a cells, did not express KGF. All 17 cases of prostate cancer were positive for FGFR1IIIb mRNA expression (Figure 2a and b). In the six cases of BPH, four expressed FGFR1IIIb at low levels (Table 2). FGFR1IIIc transcripts were not detected in either BPH or CaP. FGFR1IIIb was also expressed in LNCaP and PNT1a cells (Table 3). Twelve of 17 cases (71%) of prostate cancer expressed FGFR2IIIb (Figure 2c). Five of the six cases of BPH also expressed FGFR2IIIb isoform. FGFR2IIIc transcripts were detected in 11 of 17 tumours, but negative in all six cases of BPH (Figure 2d). The expression of FGFR2IIIc isoform did not show any association with hormone responsiveness, grade or stage of the tumours. None of the hormone sensitive tumours were FGFR2IIIb positive and FGFR2IIIb expression appeared to persist in hormone insensitive tumour. This association however did not reach statistical signi®cance (Fisher's exact test: P=0.052). No correlation was observed between KGF and FGFR2IIIb expression, with FGFR2IIIb expression in 6 of 7 KGF positive tumours and 6 of 10 KGF negative tumours. Human recombinant KGF at a concentration of 10710 mol/l enhanced proliferation of cultured prostatic

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PSA Grade (1 ± 3) T stage M stage (ng/ml) 3 3 3 2 1 2 2 3 3 3 2/3 3 3 2 2/3 3 2

T4 T3 T3/4 T2 T1 T1 T4 T4 T4 T4 T4 T4 T3 T1 T1 T1 T1

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Table 2 Case 1 2 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 BPH/1 BPH/2 BPH/3 BPH/4 BPH/5 BPH/6

Table 3

Table 1 Clinico-pathological details Case

epithelial cells by 50%. This mitogenic e€ect increased further to over 100% when KGF concentrations were 1079 mol/l and over. At concentrations below 10711 mol/l, KGF did not have any demonstrable e€ect on the growth of cultured prostatic epithelium. Human recombinant FGF2 (bFGF) was included as a comparative control in parallel experiments and did

380 141 87 52 4 72 78 17 50 17 302 16 11 18 4

Horm. Status R S R S S S R R R R R R S S S S S

Snap-frozen prostate chips were prepared following transurethral resection of the prostate (TURP). Eighteen cases of prostate cancer (CaP) and six cases of benign prostatic hyperplasia (BPH) were included in this study. The mean age of patients with prostate tumours was 72.4 years (range 57 ± 87 years). Digital rectal examination revealed locally advanced disease in ten patients with tumours invading through the prostatic capsule (TNM staging: T3 and T4 tumours); while seven patients had organ con®ned disease (T1 and T2). The mean serum prostate speci®c antigen levels in these two groups of patients with locally advanced and clinically organ con®ned tumours were 127 ng/ml (range 17 ± 380) and 17.5 ng/ml (range 4 ± 52 ng/ml) respectively. Grade 1, 2, 3 represent well, moderate and poorly di€erentiated tumours respectively; M1 signi®ed bony metastasis on isotopic bone scan; hormone status as judged by the response to hormone manipulation: R= no response, S= signi®cant drop in serum prostate speci®c antigen levels

KGF and FGFR expression in BPH and CaP samples FGF-7

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In vitro expression of KGF and IIIb isoform of FGFR1 and FGFR2

DU145 LNCaP PNT1a 18 cultured epithelial cells from BPH 18 cultured epithelial cells from CaP 18 cultured ®broblast from BPH 18 cultured ®broblast from CaP

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PNT1a, a SV40 transformed prostatic epithelial cell line derived from normal prostate, was a gift from Professor N Maitland (University of York, York, UK). The established prostatic cancer cell line LNCaP and DU145 were obtained from the American Type Culture Collection. Prostate tissues obtained following TURP was used for primary culture to study the epithelial and stromal cells separately (Collins et al, 1994). In brief, prostate chips were minced into 1 mm cubes and incubated overnight in RPMI-1640 (Life Technologies, Ltd, Scotland), containing 5% foetal calf serum (Life Technologies) and collagenase type 1 at 200 IU (Lorne Laboratories, UK). Following washings and repeated centrifugation (400 g for 20 min each time), stromal and epithelial cells were separated to over 95% purity (as assessed by immuno-¯orescence for cyto-keratin, vimentin and a-actin (Collins et al, 1994), and were plated as individual cultures for subsequent analysis. Stromal cells were maintained in RPMI-1640 medium with 10% FCS, penicillin (100 units/ml) and streptomycin (100 mg/ml). Epithelial cells were cultured in complete WAJC-404 serum free medium supplemented with insulin (2.5 mg/ ml), dexamethasone (1 mM), epidermal growth factor (10 ng/ml), bovine pituitary extract (25 mg/ml), cholera toxin (10 ng/ml), heparin (25 mg/ml), penicillin (100 units/ml) and streptomycin (100 mg/ml). Cultured stromal and epithelial cells were harvested for experimentation within the ®rst two passages

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KGF and FGFR in human prostate cancer HY Leung et al

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Figure 2 FGFR mRNA expression (a) RT ± PCR for FGFR1IIIb and IIIc, (b) Southern analysis using FGFR1IIIb-speci®c oligonucleotide probe on RT ± PCR products (subsequent hybridization with FGFR1IIIc-speci®c probe gave no signals, data not shown), (c) FGFR2IIIb mRNA expression, (d) FGFR2IIIc mRNA expression. PCR primers: FGFR1IIIb forwardCATTCGGGGATTAATAGCTC, FGFR1IIIc forward-ACTGCTGGAGTTAATACCAC, FGFR1 reverse-GGAGTCAGCAGACACTGT; FGFR21IIIb forward-CACTCGGGGATAAATAGTT, FGFR2IIIc forward-ATTCTATTGGGATATCCTTT, FGFR2 reverse-ACTCGGAGACCCCTGCCA. PCR conditions were carried out as follows: FGFR1 IIIb and IIIc isoforms (30 cycles: 948C 1 min, 558C 20 s, 728C, 1 min); FGFR2 IIIb and IIIc isoforms (30 cycles: 948C 1 min, 558C 20 s, 728C, 30 specify.) Southern blots were prepared using capillary transfer onto Hybond N+ membrane (Amersham International, Plc) and probed with a corresponding internal oligonucleotide to the receptor isoforms for which the PCR was performed (FGFR1: IIIb-GGATGCGGAGGTGCTGACCCTGTTCAATGT and IIIc-CAAAGAGATGGAGGTGCTTCACTTAAGAAA; FGFR2: IIIb-ATATAGGGC AGGCCAACCAGTCTGCCT and IIIc-ACTCTGCATGGTTGACAGTTCTGC. Following pre-hybridization blocking, the ®lters were hybridized at 508C for 2 h with the corresponding 32P-radiolabelled oligonucleotide probe in a bu€er containing 56SSC, 56Denhardt's solution, 0.5% sodium dodecyl sulphate, and 1 mg/ml salmon sperm DNA. The ®lters were then washed in successive washes of 26SSC/0.1% SDS twice followed by a single wash in SSC/0.1% SDS once at 508C. Filters were exposed by autoradiography for 3 to 4 h

KGF and FGFR in human prostate cancer HY Leung et al

not show any mitogenic e€ect on the growth of the cultured prostatic epithelial cells (Figure 3). Using an in vitro primary culture model to separate epithelial and stromal cells, prostatic stromal cells were con®rmed to be the exclusive source of KGF expression, in keeping ®ndings from other workers (Stroy et al., 1994; Ittman and Mansukhani, 1997). However, using in situ hybridization with a digoxigenin-labelled oligonucleotide probe, McGarvery and Stearns (1995) detected KGF expression at low level in the epithelium of BPH and at higher levels in the high grade malignant epithelium. Despite repeated experiments with RT ± PCR for KGF, we did not observe any evidence of such `ectopic' KGF expression in the prostatic epithelial cells, including primary cultured epithelial cells from BPH and CaP, as well as the established cell lines (LNCaP, DU145 and PC3), and the transformed PNT1a cells (Table 3). Although KGF expression was not detected in cDNA extracted from BPH specimens, it was detected in cultured stromal cells from both BPH and prostate cancer. KGF expression in cultured stromal cells derived from BPH may represent cellular responses to in vitro culture conditions, similar to the upregulation of KGF expression observed during wound healing and active in¯ammation (Werner et al., 1992a). KGF functions via its interaction with the tyrosine kinase FGF receptors. We have demonstrated mRNA expression of the IIIb isoform of both FGFR1 and FGFR2 in the prostate. The FGFR2IIIb isoform has been shown to bind to KGF at high anity and has been previously referred to as KGFR (Werner et al., 1992b). Comparing the sequence homology between the IIIb exons of the human FGFR1 and FGFR2 genes, we and others (Werner et al., 1992b) reasoned that FGFR1IIIb is likely to interact with KGF to give rise to signal transduction activities. We have therefore examined the expression of FGFR1 and FGFR2 in our study. During the course of this study, KGF was shown to interact with the FGFR1IIIb isoform only extremely weakly (Ornitz et al., 1996). Formation of heterodimers between FGFR2IIIb and other FGFR isoforms that do not interact with KGF directly at high anities may nevertheless result in signal transduction activities. FGFR1 expression in BPH, when compared to normal prostate, has been shown to be upregulated (Story et al., 1994 and Hamaguchi et al., 1995). In our study, FGFR1 expression in CaP was at higher levels than in BPH. Of the 17 informative tumour cases, all expressed FGFR1IIIb. FGFR2IIIb expression was detected in 12 of 17 cases of CaP and ®ve of six cases of BPH. The co-expression of KGF and the IIIb isoforms of FGFR1 and FGFR2 suggests a potential paracrine loop. This was supported by our ®nding of a mitogenic e€ect of KGF on cultured prostatic epithelial cells. We have observed that FGFR was expressed in cultured prostatic epithelium and KGF

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Figure 3 Proliferation assay using human recombinant KGF and FGF2 on cultured prostatic epithelial cells. Using primary cultured prostate epithelial cells from BPH, the e€ects of exogenous recombinant human KGF (R&D) were assessed in a thymidine incorporation experiment. Primary cultured prostatic epithelial cells were trypsinized and plated at a density of 16104 cells per well in a 96 cell plate (Corning) with WAJC-404 serum free medium supplemented with insulin (2.5 mg/ml), dexamethasone (1 mM), penicillin (100 units/ml) and streptomycin (100 mg/ ml) along with a range of concentrations of recombinant KGF. All treatment conditions were performed in triplicate. Following a period of culture for 3 to 4 days, [3H]Thymidine incorporation was performed and DNA synthesis measured as described previously (Collins et al., 1994). Human recombinant FGF2 (bFGF) was used in parallel as an internal control

had a signi®cant mitogenic e€ect on prostatic epithelium. In addition, FGF2 did not have any e€ect. This is in keeping with previous reports (Story et al., 1994; Peehl et al., 1996) and further supports the mutually exclusive nature of the IIIb and IIIc isoforms at the functional level. In summary, we have demonstrated signi®cant upregulation of KGF expression in hormone resistant prostate cancer and con®rmed its role as a potential paracrine growth factor. Future work on the heterodimerisation of FGFR monomers and the consequences of their signal transduction activities will allow a better understanding of the role of FGFs in prostate cancer. Abbreviations FGF, ®broblast growth factor; FGFR, ®broblast growth factor receptor; BPH, benign prostatic hyperplasia; CaP, prostate cancer. Acknowledgements This project was funded by a grant from the Harker Foundation, University of Newcastle upon Tyne. The authors thank Wendy Robson for tissue collection and Kate Gilmore for technical assistance.

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