Role of fibroblast growth factor receptor signaling in prostate cancer cell survival.

BACKGROUND: Expression of fibroblast growth factors (FGFs) is increased in a substantial fraction of human prostate cancers in vivo and in prostate cancer cell lines. Altered FGF signaling can potentially have a variety of effects, including stimulating cell proliferation and inhibiting cell death. To determine the biologic significance of altered FGF signaling in human prostate cancer, we disrupted signaling by expression of a dominant-negative (DN) FGF receptor in prostate cancer cell lines. METHODS: PC-3, LNCaP, and DU145 prostate cancer cells were stably transfected with DN FGFR constructs, and LNCaP and DU145 cells were infected with a recombinant adenovirus expressing DN FGFR-1. The effect of DN FGFR-1 expression was assessed by colony-formation assays, cell proliferation assays, flow cytometry, and cytogenetic analysis. Key regulators involved in the G(2)-to-M cell cycle transition were assessed by western blotting to examine cyclin B1 expression and by in vitro kinase assay to assess cdc2 kinase activity. RESULTS: Stable transfection of the DN FGFR-1 construct inhibited colony formation by more than 99% in all three cell lines. Infection of LNCaP and DU145 prostate cancer cells with adenovirus expressing DN FGFR-1 led to extensive cell death within 48 hours. Flow cytometry and cytogenetic analysis revealed that the DN FGFR-1 receptor led to arrest in the G(2) phase of the cell cycle before cell death. Cyclin B1 accumulated in DN FGFR-1-infected LNCaP cells, but cdc2 kinase activity was decreased. CONCLUSIONS: These findings reveal an unexpected dependence of prostate cancer cells on FGF receptor signal transduction to traverse the G(2)/M checkpoint. The mechanism for the G(2) arrest is not clear. Our results raise the possibility that FGF-signaling antagonists might enhance the cell death induced by other prostate cancer therapies.

Alternative splicing of fibroblast growth factor receptors in human prostate cancer.

BACKGROUND: Fibroblast growth factors (FGFs) are known to play an important role in the growth of normal prostatic epithelial cells and may promote proliferation of neoplastic prostatic epithelial cells via autocrine or paracrine mechanisms. The affinity of FGFs for FGF receptors 1-3 is critically dependent on an alternative splicing event involving the coding region for the carboxy terminal portion of the third extracellular immunoglobulin-like domain that leads to two different isoforms of each receptor (IIIb and IIIc). We therefore sought to determine whether changes in alternative splicing of FGF receptors occur in human prostate cancer. METHODS: RNAs from normal prostate and clinically localized or metastatic prostate cancers were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) followed by digestion of products with restriction enzymes specific for each FGF receptor isoform and quantitation of the relative amounts of each isoform after electrophoresis. For FGFR-2, this was correlated with immunohistochemistry to determine the localization of the protein product. RESULTS: FGFR-1 is expressed exclusively as the IIIc isoform in prostate cancer while FGFR-3 is expressed predominantly as the IIIb isoform, similar to the expression pattern in normal prostatic epithelial cells. In contrast, there was variable expression of the FGFR-2 IIIb and IIIc isoforms. In the majority of cases the FGFR-2 IIIb isoform was the predominant or exclusive isoform expressed, similar to normal epithelial cells, but in a subset of cases the IIIc isoform was increased, indicating a change in alternative splicing of FGFR-2 in some cases. CONCLUSIONS: In most cases of prostate cancer there are no changes in alternative splicing of FGF receptors, but in a subgroup there is increased expression of the FGFR-2 IIIc isoform. Given that the affinity of FGFs is highly dependent on the isoform of FGF receptor expressed, this information is critical in understanding the effects of FGFs on prostate cancer cells in vivo.

FGF-10 is expressed at low levels in the human prostate.

BACKGROUND: Fibroblast growth factors (FGFs) are known to play an important role in the growth of normal prostatic epithelial cells. FGF-10 is a secreted growth factor that binds to FGF receptor-2 IIIb, which is expressed in prostatic epithelial cells and thus can potentially act as a growth factor for these cells. Prior work has indicated that FGF10 may play an important role in the development of the rat prostate, but its role in the adult human prostate is unclear. METHODS: Expression of FGF10 in human prostate tissue and primary cultures of prostatic epithelial and stromal cells was assessed by reverse-transcriptase PCR (RT-PCR) and Northern blotting. Growth response to FGF10 was assessed by the addition of recombinant FGF-10 to primary cultures of prostatic epithelial and stromal cells. RESULTS: FGF10 is expressed at levels detectable by RT-PCR and can act as a growth factor for prostatic epithelial cells, but is not active as a growth factor for stromal cells. However, FGF10 is expressed at extremely low levels relative to FGF7, which has a similar biological activity. CONCLUSIONS: While FGF10 may play a role in prostatic development, it is unlikely to play a major role in prostate growth in normal or hyperplastic adult human prostate, due to its extremely low expression compared to FGF7.

Increased expression of fibroblast growth factor 6 in human prostatic intraepithelial neoplasia and prostate cancer.

Fibroblast growth factors (FGFs) are known to play an important role in the growth of normal prostatic epithelial cells. In addition to their effects on proliferation, FGFs can promote cell motility, increase tumor angiogenesis, and inhibit apoptosis, all of which play an important role in tumor progression. To determine whether FGFs are overexpressed in human prostate cancers, we analyzed 26 prostate cancer RNAs by reverse transcription-PCR for expression of FGF3, FGF4, and FGF6, which cannot be detected in normal prostate tissue by this technique. Fourteen of 26 prostate cancers expressed FGF6 mRNA. No expression of FGF3 or FGF4 was detected. An ELISA of tissue extracts of normal prostate, high-grade prostatic intraepithelial neoplasia (PIN), and prostate cancer for FGF6 showed that this growth factor was undetectable in normal prostate but was present at elevated levels in 4 of 9 PIN lesions and in 15 of 24 prostate cancers. Immunohistochemical analysis with anti-FGF6 antibody revealed weak staining of prostatic basal cells in normal prostate that was markedly elevated in PIN. In the prostate cancers, the majority of cases revealed expression of FGF6 by the prostate cancer cells themselves. In two cases, expression was present in prostatic stromal cells. Exogenous FGF6 was able to stimulate proliferation of primary prostatic epithelial and stromal cells, immortalized prostatic epithelial cells, and prostate cancer cell lines in tissue culture. FGF receptor 4, which is the most potent FGF receptor for FGF6, is expressed in the human prostate in vivo and in all of the cultured cell lines. Thus, FGF6 is increased in PIN and prostate cancer and can promote the proliferation of the transformed prostatic epithelial cells via paracrine and autocrine mechanisms.

FGF7 and FGF2 are increased in benign prostatic hyperplasia and are associated with increased proliferation.

PURPOSE: To determine if overexpression of FGF7 and FGF2 occurs in benign prostatic hyperplasia (BPH) and if so, whether such overexpression is correlated with increased proliferation of epithelial and/or stromal cells. MATERIALS AND METHODS: The FGF7 and FGF2 content of protein extracts of normal peripheral zone, normal transition zone and hyperplastic prostatic tissues were determined by enzyme-linked immunoabsorption assay. Proliferation of epithelial and stromal cells was assessed by immunohistochemistry with anti-Ki67 antibodies on frozen sections of the same tissues used for protein extraction. The in vitro effects of FGF7 and FGF2 on proliferation were assessed by addition of recombinant growth factor to primary cultures of prostatic epithelial and stromal cells. RESULTS: We have found that both FGF7 and FGF2 are overexpressed in hyperplastic prostate in comparison to normal peripheral and transition zone tissue. FGF7 is a potent mitogen for epithelial cells in culture. Consistent with these in vitro effects, quantitative analysis of cellular proliferation by Ki67 immunohistochemistry revealed a strong correlation of epithelial proliferation with FGF7 content in BPH tissue, consistent with a key role for this growth factor in driving the abnormal epithelial proliferation in BPH. FGF2 is mitogenic for stromal cells in culture and there was a weaker correlation of FGF2 content with increased stromal proliferation. CONCLUSION: Overexpression of FGF7 and FGF2 may play an important role in the abnormal cellular proliferation seen in benign prostatic hyperplasia.

FGF7/KGF triggers cell transformation and invasion on immortalised human prostatic epithelial PNT1A cells.

Fibroblast growth factor 7 (FGF7/KGF) is synthesized exclusively by fibroblasts in normal tissues; it acts as a potent mitogen on epithelial cells, through interaction with the FGF7-specific receptor FGFR2/IIIb. To examine the importance of this growth factor both to prostate physiology and to prostate-cancer progression, we have tested the exogenous effect of FGF7. Thus, by mimicking the paracrine pathway (on proliferation, growth in soft agar and invasion) on the human prostatic epithelial cell line PNT1A positively checked for FGFR2/IIIb expression, FGF7 significantly enhanced cell proliferation at an optimal concentration of 7.5 x 10(-11) M, but no significant invasion or growth in soft agar were observed. To confirm FGF7 properties on human prostatic epithelial cells, we constitutively expressed FGF7 by transfecting PNT1A cells with FGF7-cDNA. The FGF7-transfected clones, PNT1A/ FGF7-T5 and PNT1A/FGF7-T6, were stable and expressed FGF7. Analysis of the FGF7-autocrine loop on the non-tumorigenic epithelial cells PNT1A showed acquired invasive potential in in vitro extracellular-matrix migration assays, specifically inhibited by an FGF7-neutralizing antibody, and over-expressed factors implicated in the migration process: the metalloproteinase MMP-1 and the plasminogen activator uPA. Taken together, these results demonstrate a role for FGF7 in triggering invasion of human prostatic epithelial cells. Furthermore, these FGF7-transfected clones exhibited functional and physiological differences from the original PNT1A cell line: anchorage-independent growth, growth in serum-free media and increased proliferation. These data confirm the oncogenic function of FGF7 in prostate progression potentially acting through paracrine and/or autocrine regulatory pathways.

Alterations in expression of basic fibroblast growth factor (FGF) 2 and its receptor FGFR-1 in human prostate cancer.

Fibroblast growth factors (FGFs) play an important role in the growth and maintenance of the normal prostate. There is increasing evidence from both animal models and analysis of human prostate cancer cell lines that alterations of FGFs and/or FGF receptors (FGFRs) may play an important role in prostate cancer progression. To better define the role of FGF2 and FGF7 in human prostate cancer in vivo, we have quantified these two growth factors in clinically localized human prostate cancers and uninvolved prostate by ELISA and Western blotting and determined their localization by immunohistochemistry. The expression of two of the primary receptors for these growth factors, FGFR-1 and FGFR-2, were also analyzed by immunohistochemistry and Western blotting in these same samples. We have found that FGF2 is significantly increased in prostate cancers when compared with uninvolved prostate and that the FGF2 is present in the stromal fibroblasts and endothelial cells but not the cancer cells. In addition, we have observed overexpression of both FGFR-1 and FGFR-2 in the prostate cancer epithelial cells in a subset of prostate cancers and that such overexpression is correlated with poor differentiation. Thus, there is both an increase in FGF2 concentration in prostate cancers and an increased expression of a receptor capable of responding to this growth factor, establishing a potential paracrine stimulation of prostate cancer cells by the surrounding stromal cells, which may play an important role in prostate cancer progression.

FGF9 is an autocrine and paracrine prostatic growth factor expressed by prostatic stromal cells.

Polypeptide growth factors, including members of the fibroblast growth factor (FGF) family, play an important role in the growth and maintenance of the normal prostate. We have found that FGF9 is expressed at high levels in the normal peripheral and transition zone of the human prostate. Analysis of FGF9 production by primary cultures of prostatic epithelial and stromal cells has shown that FGF9 is produced and secreted by the prostatic stromal cells. Neither of these processes appears to be modulated by androgens. Production of FGF9 by stromal cells in vivo was confirmed by immunohistochemistry. FGF9 is a potent mitogen for both prostatic epithelial and stromal cells in culture and is a more potent mitogen for these cells than either FGF2 or FGF7, two other FGFs expressed in the human prostate. FGF9 is an abundant secreted growth factor that can act as both a paracrine mitogen for epithelial cells and an autocrine mitogen for stromal cells. Western blot analysis of tissue extracts from the normal and hyperplastic transition zone shows that FGF9 is present at two to threefold higher levels in the hyperplastic transition zone. Overexpression of this paracrine and autocrine growth factor may play an important role in the epithelial and stromal proliferation in benign prostatic hyperplasia.

Upregulation of endothelin 1 and its precursor by IL-1beta, TNF-alpha, and TGF-beta in the PC3 human prostate cancer cell line.

Increasing evidence indicates that endothelin 1 (ET-1) is implicated in prostate tumour progression. However, data on ET-1 regulation in human prostate and prostate cancer cell lines are lacking. In this study, regulation of ET-1 and its precursor big ET-1, using PC3 cells, a human bone metastatic prostatic carcinoma cell line, was addressed. ET-1 and big ET-1 assays demonstrated greater secretion of both peptides in the presence of 10% fetal calf serum (FCS) as compared with 0.5% FCS. Incubation of PC3 cells in the absence and presence of various cytokines and growth factors known to be implicated in prostate stroma-epithelium interactions, revealed that IL-6, FGF7/KGF and FGF2/bFGF had no effect on ET-1 and big ET-1 secretion, whereas interleukin 1beta (IL-1beta), tumour necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) stimulated their secretion in a concentration-dependent manner. Binding experiments indicated the presence of specific ET-1 receptors in PC3 cells: Kdapp = 1.1 x 0.2 x 10(-10)M, Bmax = 2660 +/- 390 sites/cell. Data analysis demonstrated the presence of only the ETA receptor subtype in PC3 cells. In conclusion, our results indicate that the implication of ET-1 in prostate cancer is likely to be mediated via paracrine/autocrine control of cell factors.

Constitutive expression of FGF2/bFGF in non-tumorigenic human prostatic epithelial cells results in the acquisition of a partial neoplastic phenotype.

Fibroblast growth factor 2 (FGF2), also known as basic fibroblast growth factor (bFGF), belongs to the FGF family, which consists of at least 9 closely related members. FGF2 is a potent mitogen for fibroblasts derived from normal prostate and, to a lesser extent, for prostatic epithelial cells. Its role in the physiology of the normal prostate seems to be limited to stromal cells, whereas in prostate cancer FGF2 may also have an autocrine/paracrine effect on epithelial cells. In order to better understand the effects of FGF2 on the prostatic epithelium, especially its role in the progression of prostate cancer by establishing an autocrine-stimulation loop, we transfected FGF2 cDNA into a human prostatic epithelial cell line, PNT1A, immortalized with SV40 large-T antigen. This cell line is non-tumorigenic and expresses a high-affinity FGF2 receptor, FGFR1/flg. We characterized 3 independent FGF2-transfected clones and found that the establishment of an FGF2 autocrine loop on these cells led to (i) serum-independent growth, (ii) increased proliferation and (iii) anchorage-independent growth. Such results argue in favor of the possible action of FGF2 on progression of prostate cancer via an FGF2 autocrine loop on epithelial cells.

Identification of endothelin receptors in normal and hyperplasic human prostate tissues.

Specific endothelin-1 (ET1) binding sites have been demonstrated in membranes derived form normal (NP) and benign hyperplasic (BPH) human prostate using an 125I-ET1 binding assay. 125I saturation experiments and Scatchard analysis demonstrated the existence of a homogeneous population of binding sites with high affinity (Kd(app)) and density (B(max)), respectively, 106 +/- 15 pM and 1086 +/- 399 fmol/mg protein for NP (n = 5) and 168 +/- 26 pM and 964 +/- 445 fmol/mg protein for BPH (n = 5). We demonstrated the presence of two subtypes of ET1 receptors, ETA and ETB, by means of the following ET1 competitors: ET2, ET3, and BQ123 (which is selective for the ETA receptor), and IRL1620 and sarafotoxine c (S6c) (which are selective for the ETB receptor). The displacement curves allowed us to conclude that the large majority (85%) of the ET1 receptors in normal and hyperplasic human prostate are of the A subtype.