We report for the first time that overexpression of bFGF in human melanoma cells mediates highly increased expression of CD13 mRNA and protein, which indicates that bFGF is involved in the induction of an invasive cellular phenotype. The increased invasiveness of bFGF-overexpressing clones through Matrigel could be attributed to high CD13 expression, since the specific monoclonal antibody WM15 inhibited this process. In accordance with these effects of bFGF overexpression, we found in a panel of human melanoma cell lines a highly significant correlation between endogenous bFGF and CD13 mRNA expression as well as spontaneous metastasis formation. These findings point towards an important role for bFGF in the high metastatic potential of melanomas in patients.
For our experiments, we made use of 1F6 cells with low endogenous bFGF levels that were transfected with vectors either encoding the 18kD isoform or ALL isoforms in an attempt to distinguish between the variation in phenotypic behaviour induced by the different proteins. The 18kD bFGF isoform functions as an autocrine and paracrine growth factor and is mainly found in the cytoplasm of cells and bound to heparan sulphate proteoglycans (HSPGs) on the cell membrane. In contrast, the high molecular weight (HMW) isoforms are located in the nucleus and are thought to play a role in transcriptional regulation (Renko et al, 1990
; Bugler et al, 1991
; Florkiewicz et al, 1991
; Quarto et al, 1991
). Upon transfection, however, both 1F6-18kD and 1F6-ALL clones expressed similar amounts of CD13, which suggests that expression of the 18kD protein alone is sufficient for the induction of CD13. Moreover, changes in morphology and growth rate were similar between 1F6-18kD and 1F6-ALL clones.
Induction of CD13 expression by exogenous bFGF has previously been described in human endothelial cells and is required for vasculogenesis. In the study by Bhagwat et al (2001)
, addition of the 18kD bFGF protein to serum-starved HUVECs upregulated CD13 mRNA expression up to 2.5-fold. Although bFGF was the most potent stimulator of CD13 expression in that study, induction of CD13 protein was also observed when endothelial cells were cultured under hypoxic conditions or were treated with the angiogenic growth factors VEGF, TNFα
or IGF-1. Inhibition of CD13 expression by bestatin, amastatin or the monoclonal-anti CD13 antibody MY7 resulted in a clear reduction of capillary tube formation of HUVECs, while there was no effect of these inhibitors on proliferation (Bhagwat et al, 2001
). The authors have also reported that exogenous bFGF could induce CD13 expression in KS1767 Kaposi sarcoma cells (Bhagwat et al, 2001
). Recently, Kehlen et al (2003)
have shown induction of CD13 expression upon stimulation of serum-starved 1736 thyroid carcinoma cells with bFGF.
Repeatedly, we were not able to increase CD13 expression by stimulating serum-starved 1F6 cells with exogenous recombinant human bFGF, regardless efficient stimulation of cell proliferation (data not shown). This finding indicates that the bFGF-mediated induction of CD13 in 1F6 cells is an indirect effect of the transformation process in itself, rather than a direct effect on CD13 promoter activity. Since CD13 can be detected on melanoma cells (Elder et al, 1989
; Menrad et al, 1993
; Fujii et al, 1995
), we are the first to report that endogenous bFGF can mediate the expression and activity of this protein.
Bestatin is a well-known chemical inhibitor of CD13 aminopeptidase activity, which blocks the catalytic site of the enzyme in a competitive manner. Although bestatin is often considered to be specific for CD13, it also inhibits other aminopeptidases, such as aminopeptidase B and leucine aminopeptidases (Scornik and Botbol, 2001
). High concentrations of bestatin (150–300μM
) clearly reduced invasion of HT1080 fibrosarcoma cells and WM1158 melanoma cells through Matrigel (Menrad et al, 1993
; Fujii et al, 1996
). WM15 is a monoclonal antibody that specifically neutralises CD13 activity. WM15 has been shown to inhibit invasion through Matrigel, as described for SN12M renal-cell carcinoma, HT1080 fibrosarcoma and A375 melanoma cells (Saiki et al, 1993
). In our bFGF-overexpressing 1F6 clones, both bestatin and WM15 almost completely abrogated the enhanced invasive capacity. This observation indicates that the increased CD13 expression and activity in the bFGF-overexpressing clones is mainly responsible for the facilitated invasive potential.
The activity of the myeloid and the epithelial promoters of CD13 is believed to be mutually exclusive (Shapiro et al, 1991
). As a result, expression of CD13 protein in a particular cell type is thought to be mediated by activation of either the myeloid or the epithelial promoter. We now show that both CD13 promoters can be activated in response to bFGF overexpression. Although in 1F6 cells the basal expression of the myeloid promoter was consistently found to be higher than the activity of the epithelial promoter, both activities were increased to the same extent, up to four-fold in clones 1F6-18kD and 1F6-ALL. We do not know if the current results are specific for melanoma cells, or whether the use of the more sensitive techniques would also allow to detect upregulation of both promoters in other cell types. While initial CD13 promoter studies were performed with chloramphenicol acetyl transferase (CAT) assays and Northern blots (Shapiro et al, 1991
), we made use of RT–PCR and promoter-luciferase assays allowing us to detect the activities of both promoters in 1F6 melanoma cells and clones.
Recently, induction of CD13 expression by bFGF in endothelial cells was shown to be regulated by two Ras-mediated signalling pathways that have been implicated in the transition of quiescent to active endothelium: the mitogen-activated protein kinase (MAPK) and phosphoinositide-3 kinase/AKT (PI-3K/AKT) pathways (Bhagwat et al, 2003
). Petrovic et al (2003)
have demonstrated in activated endothelial cells that CD13 transcription was induced by RAS/MAPK-mediated phosphorylation of the transcription factor Ets-2 resulting in increased activity of the epithelial CD13 promoter. In our bFGF-overexpressing 1F6 clones, phospho-AKT and phospho-p38 MAPK levels were increased as compared to basal levels in parent 1F6 cells, while basal phospho-ERK1/2 MAPK was not further increased (Fontijn et al
, unpublished data). We are further investigating the possible contribution of the PI-3K/AKT and p38 MAPK signalling pathways in the increased CD13 expression in bFGF-overexpressing 1F6 clones.
In general, high CD13 expression in solid tumours in patients is considered as an unfavourable factor. Few clinical studies are available, among which are observations in pancreatic carcinoma, colon carcinoma and thyroid carcinoma, which will be described shortly. CD13 protein expression was detected in 48% of pancreatic carcinoma patients and was significantly associated with a shorter median survival (Ikeda et al, 2003
). The disease-free and overall survival rate for patients with CD13-positive colon carcinoma was significantly lower than that for patients without CD13 expression (Hashida et al, 2002
). Kehlen et al (2003)
have shown that CD13 expression in undifferentiated thyroid carcinomas was higher than that in papillary or follicular thyroid carcinomas, suggesting that it is a marker for differentiation. Clinical studies on CD13 expression in cutaneous melanoma are yet to be carried out.
It can be hypothesized that the presence of CD13 in melanoma patients will also be associated with poor prognosis. As a rationale, it has been shown in vitro
that inhibition of CD13 aminopeptidase activity can reduce the invasive capacity of WM1158 and A375M melanoma cells through Matrigel (Menrad et al, 1993
; Saiki et al, 1993
). Upon transfection of CD13 into A375M and A2058 melanoma cells, increased degradation of type IV collagen and invasion in ECM have been observed (Fujii et al, 1995
). These CD13-transfected cells showed a significantly augmented lung-colonising potential in nude mice (Fujii et al, 1995
). The observations are in agreement with our data that 1F6 cells could hardly invade through Matrigel, but their invasive capacity is greatly facilitated in the case of CD13 overexpression. As proof of concept, in our panel of human melanoma cell lines, we found a clear correlation between bFGF and CD13 mRNA and protein expression (). High bFGF and CD13 protein expression could be detected in melanoma cell lines BRO and BLM, being aggressive cell lines on the basis of a high in vitro
and in vivo
growth rate. Furthermore, BLM and BRO cells grown as subcutaneous xenografts in nude mice have a very high rate of spontaneous metastasis formation (Lockshin et al, 1985
; Van Muijen et al, 1991
Bestatin has been studied for its therapeutic usefulness in the clinic in the past, since the compound was considered to have immunopotentiating activity (Ota, 1991
). In a prospective randomised trial in adult nonlymphocytic leukaemia, prolongation of remission duration and survival was achieved with bestatin added to maintenance chemotherapy in elderly patients (Ota et al, 1986
). Prospective randomised clinical trials in various solid tumours contained either too few patients or were negative to demonstrate a beneficial effect on survival (Ota, 1991
). In a recent, randomised double-blind placebo-controlled trial by Ichinose et al (2003)
, bestatin was given for its aminopeptidase inhibiting, immunostimulant and antitumour activity to patients with completely resected stage I squamous-cell lung carcinoma. Of the 402 patients that entered the study, it appeared that the overall survival and cancer-free survival were both significantly different in favour of the bestatin-treated group. Since we found a clear correlation between bFGF and CD13 expression and invasiveness of melanoma cells, the incorporation of bestatin in a clinical trial in stage II melanoma patients should be considered with the aim of preventing the development of metastases and to improve survival.
In conclusion, our data indicate that high bFGF expression in human melanoma cells is a major mechanism of the upregulation of CD13 resulting in enhanced invasive capacity and metastatic behaviour. Selective inhibition of CD13, or even of bFGF itself, should therefore be an attractive strategy for the treatment of advanced melanoma, but likely also for adjuvant treatment in stage II disease.