Carcinogenesis vol.19 no.6 pp.1153–1156, 1998
SHORT COMMUNICATION
Fibroblast growth factor receptor expression reflects cellular differentiation in human oral squamous carcinoma cell lines
C.S.Drugan, I.C.Paterson and S.S.Prime1 Division of Oral Medicine, Pathology and Microbiology, Department of Oral and Dental Science, University of Bristol, Lower Maudlin Street, Bristol BS1 2LY, UK 1To
whom correspondence should be addressed Email:
[email protected]
This study examined the expression of fibroblast growth factor receptor 2 (FGFR 2) splice variants, IIIb and IIIc, in normal and malignant human oral keratinocytes and in normal oral fibroblasts by RT-PCR using both exon-specific primers and primers common to both FGFR 2 isoforms. Fibroblasts expressed exclusively FGFR 2/IIIc whilst the normal and malignant keratinocytes co-expressed FGFR 2/IIIb and FGFR 2/IIIc. Well-differentiated keratinocytes expressed proportionally more FGFR 2/IIIb than IIIc whereas the poorly-differentiated cells expressed more FGFR 2/IIIc than IIIb. The normal and malignant keratinocytes, but not fibroblasts, expressed an additional amplification product, which consisted of both IIIb and IIIc of FGFR 2 joined by an extra base pair and with the intronic sequence removed. The results indicate that the expression of FGFR 2 isoforms reflects the degree of cellular differentiation in normal and malignant human oral keratinocytes and that receptor complexes of FGFR 2/IIIb and IIIc may regulate ligand–receptor interactions.
The fibroblast growth factor (FGF*) family is composed of a series of heparin binding growth factors of which 12 members have been identified (1–5). The proteins are structurally-related single chain polypeptides, which range in size from 20–30 kDa and which share an overall amino acid homology of 30– 55% with conservation of two cysteine residues (6–9). The peptides bind specifically to fibroblast growth factor receptors (FGFR), which are tyrosine kinase receptors that share a common structural arrangement and which are the products of four distinct receptor genes (FGFR 1–4; 10). Alternative splicing of exons that encode the second half of immunoglobulin domain III of FGFR 1, FGFR 2 and FGFR 3 give rise to membrane-spanning proteins with different ligand binding affinities (10). The specificity of ligand binding of FGFR 2 resides in the second half of the third immunoglobulin domain where two alternatively spliced exons, termed IIIb and IIIc, encode this region of the receptor protein (10; Figure 1). The keratinocyte growth factor (KGF) receptor is encoded by the IIIb splice variant of FGFR 2 and binds KGF (FGF-7) and FGF-1 (acidic-FGF) with equal high affinity. By contrast, the IIIc splice variant binds FGF-1 and FGF-2, but not KGF (11). In general, FGFR 2 exon IIIb (KGFR) is expressed in epithelial *Abbreviations: FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptors; KGF, keratinocyte growth factor. © Oxford University Press
cells (11–13) and exon IIIc is present in mesenchymal and endothelial cells (11,13,14). Evidence is accumulating that FGF receptors are closely involved in tumour development and progression (1), but their exact function is unclear. The expression of FGFR 2, for example, appears to be variable with both loss of expression and over-expression having been demonstrated in a variety of different tumours (14–22). The situation has not been clarified with regard to the isoforms of FGFR 2 because exon IIIb can be expressed alone, exon IIIc can be expressed alone, or both exons can be independently co-expressed (15,18). We have shown that the mitogenic response of a series of oral malignant keratinocyte cell lines to KGF broadly correlates with the degree of cellular differentiation: well-differentiated keratinocytes are stimulated more by KGF than their less differentiated counterparts (23). These studies have been extended to show that loss of tumour cell differentiation leads to a loss of KGF-induced mitogenesis and increased stimulation by FGF-2 (unpublished observations). What is not known, however, is whether this variation in response of the malignant keratinocyte cell lines to members of the FGF family is related to the expression of specific FGFR 2 isoforms. The purpose of the present study, therefore, was to examine the expression of FGFR 2 mRNA using RT-PCR in human malignant oral keratinocyte cell lines and to compare the presence of FGFR 2 variants with normal oral keratinocytes and fibroblasts. Normal human oral keratinocytes and fibroblasts were obtained from fresh tissues collected from patients undergoing routine surgical procedures. Malignant cell lines and normal oral keratinocytes and fibroblasts were cultured as described previously and have been characterized with regard to differentiation and response to KGF (23,24; Table I). Mitomycin Ctreated 3T3 fibroblasts (European Collection of Animal Cell Cultures, UK) were used in cultures of the normal oral keratinocytes in order to assist the growth of the primary keratinocytes and to limit host fibroblast contamination (25). Primers specific for the exon splice variants of the 3rd immunoglobulin domain of FGFR 2 were designed according to the published sequence (26,27; Figure 1). Total RNA was prepared using the RNeasy total RNA kit (Qiagen, UK) and first strand cDNA was synthesized from 5 µg total RNA using the SuperScript pre-amplification system (Gibco). The cDNA was amplified using 30–40 PCR cycles using a standard PCR protocol. PCR amplification was repeated with all primer combinations on at least three occasions using different RNA samples. All of the malignant cell lines and normal keratinocytes expressed the IIIb exon of FGFR 2, as demonstrated by amplification with the primer pairs 1 & 4, 2 & 4 and 2 & 6. Both the malignant and normal keratinocytes also expressed the IIIc exon of FGFR 2 as demonstrated by amplification with the primer pairs 3 & 5 and 3 & 6 (Figure 2). The product of the 3 & 6 primer pair was inconsistently amplified in H357 and was only faintly visible in both H103 and normal 1153
C.S.Drugan, I.C.Paterson and S.S.Prime
Fig. 1. Schematic representation of fibroblast growth factor receptor 2. (A) General structure of the fibroblast growth factor receptor. Ig-I, II, III immunoglobulin like domains; TM, transmembrane domain; JM, juxtamembrane region; Tk, tyrosine kinase domains (10). (B) Configuration of exons in the 3rd immunoglobulin domain of FGFR 2 and the location of the PCR primers used for analysis (26,27). Primer 1, 59-AACGGCAGTAAATACGGGC-39; primer 2, 59-CACTCGGGGATAAATAGTTCC-39; primer 3, 59-GCCGCCGGTGTTAACAC-39; primer 4, 59-TTGCTGTTTTGGCAGGAC-39; primer 5, 59-TGAAAGGATATCCCAATAGAATTACC-39; primer 6, 59-CCAGGTAGTCTGGGGAA-39. The predicted product size for the primer pair combinations were 1 & 4, 198 bp; 2 & 4, 147 bp; 1 & 5 , 171 bp; 3 & 5, 120 bp; 1 & 6, 244 bp; 2 & 6, 193 bp; 3 & 6, 190 bp; 2 & 5, 0 bp in cells expressing the relevant exon. Representative PCR amplification products from the above primer pair combinations were confirmed by DNA sequencing.
Fig. 3. PCR amplification using primers 1 & 5. Ethidium bromide-stained agarose gel of PCR products amplified with primers 1 & 5 from malignant and normal (N) keratinocyte cultures. Two PCR products were amplified. The smaller product, which corresponded to amplification of exon IIIc of FGFR 2 with the predicted size of 171 base pairs, was detected in five of the keratinocyte cell lines and in normal keratinocyte cultures. In repeat experiments, FGFR 2 exon IIIc was faintly visible in H103. Amplification of exon IIIc of FGFR 2 in H357 using primers 1 & 5 was only obtained with 40 PCR cycles. The size of the larger amplification product (319 base pairs) was consistent with co-expression of exons IIIb and IIIc spliced together without the intervening intron. This product was detected in all of the keratinocyte cell lines and in normal keratinocytes. The blank lane (Bl) is a simultaneous control amplification with no DNA template. The lane marked M indicates base pair size markers.
Table I. Characteristics of the tumour-derived keratinocyte cell lines Cell line
Cellular differentiationa
Stimulation by KGFb (%)
Stimulation by FGF-2c
H103 H157 H314 H357 H376 H400 H413
Moderately differentiated Moderately differentiated Poorly differentiated Well differentiated Moderately differentiated Moderately differentiated Well differentiated
216% 166% 129% 183% 184% 205% 251%
Not done Not done 506% 127% 316% Not done 211%
aPrime
et al. (24); bDrugan et al. (23); cunpublished observations.
Fig. 2. PCR amplification using primers 3 & 6. Ethidium bromide-stained agarose gel of FGFR 2 exon IIIc PCR products from malignant and normal (N) keratinocyte cultures and from normal oral fibroblasts (F). The product was of the predicted length of 190 base pairs for exon IIIc and was detected in five of the keratinocyte cell lines and in normal fibroblast cultures. The product was only faintly visible in H103 and normal keratinocytes and was not present in H357. In repeat PCR experiments, inconsistent amplification of exon IIIc was obtained with H357 and, when present, only faint amplification was detectable. The blank lane (Bl) is a simultaneous control amplification with no DNA template. The lane marked M indicates base pair size markers.
keratinocytes. By contrast, H314 expressed more IIIc than the other keratinocyte cell lines. When the cDNA was amplified using primers 1 & 5, flanking exons IIIb and IIIc of FGFR 2, 1154
Fig. 4. PCR amplification of normal oral fibroblast cDNA. Ethidium bromide-stained agarose gel of PCR products from normal oral fibroblasts. For each primer pair, fibroblast cDNA (F) and a control sample with no DNA template (Bl) were used. PCR products were amplified with primers 1 & 5, 1 & 6, 3 & 5 and 3 & 6. The bands were of the expected size for exon IIIc of FGFR 2. No products were amplified with primers 1 & 4, 2 & 5 and 2 & 6, which demonstrates that fibroblasts did not express exon IIIb of FGFR 2 or exons IIIb and IIIc in tandem. The lane marked M indicates base pair size markers.
two products were amplified, namely, a 171-base-pair product consistent with the IIIc splice variant and a 319-base-pair product consistent with co-expression of exons IIIb and IIIc with the intervening intronic sequence removed (Figure 3). All of the keratinocyte cell lines expressed both amplification products but in H357, consistent expression of exon IIIc alone was obtained only with 40 PCR cycles. The presence of a product containing both exons spliced together was further supported by the amplification of a 268-base-pair product with primers 2 & 5. Sequence analysis of this product established that exons IIIb and IIIc were spliced together with the addition of a single base pair between the two exons (28). A product of ~240–245 base pairs was amplified in all of the malignant cell lines and normal keratinocytes with primers 1 & 6. The product was of the predicted size for FGFR 2 mRNA and contained either the IIIb exon or the IIIc exon, but it was not possible to resolve the difference between the size of the two products using gel separation. Sequencing of this product identified only FGFR 2 IIIb, which may reflect a greater proportion of the IIIb versus the IIIc isoform in the cDNA template. No other PCR products were amplified and, in particular, there was no band correlating to a full-length
FGF receptors in oral cancer
product that contained exons IIIb and IIIc spliced together. Amplification of normal oral fibroblast mRNA with primer pairs 1 & 5, 1 & 6, 3 & 5 and 3 & 6 revealed a single amplification product (Figure 4) of the predicted size for exon IIIc of FGFR 2. The fibroblasts did not express FGFR 2 exon IIIb and did not co-express the product of exons IIIb and IIIc. In order to examine whether sub-populations of cells within a heterogeneous malignant cell line expressed one or other of the IIIb or IIIc splice variant of FGFR 2, clonal sub-populations were derived from an undifferentiated (H314) and a differentiated (H413) malignant cell line. All of the clonal cell populations (73H314; 43H413) expressed the IIIb isoform of FGFR 2, the IIIc isoform of FGFR 2 and the variant containing exons IIIb and IIIc spliced together. The results of this study demonstrate that normal keratinocytes express exons IIIb and IIIc of FGFR 2 whereas only exon IIIc was expressed in fibroblasts. The data confirm the target cell specificity of KGF which binds to FGFR 2/IIIb and stimulates epithelial cells but has no effect on fibroblasts because of the lack of expression of FGFR 2/IIIb in this cell type (11–13). Somewhat surprisingly, the normal keratinocytes expressed exon IIIc of FGFR 2, a receptor isoform that gives rise to the bek variant of FGFR 2 and which is associated traditionally with the mesenchymal rather than the epithelial phenotype (11,13,14). It is possible that the amplification of exon IIIc in the normal keratinocytes was because of 3T3 or host fibroblast contamination and, whilst the fibroblasts were removed from the cultures of normal keratinocytes prior to RNA preparation, trace amounts may have remained. Interestingly, expression of FGFR 2/IIIc has also been found in normal epithelial cells from the cornea, breast and endometrium, but the possibility of fibroblast contamination in these studies was not eliminated (14,18,28,29). In a similar way to the normal keratinocytes, the malignant cell lines in this study also expressed both the IIIb and IIIc splice variant of FGFR 2. These cells were cultured in the absence of 3T3 fibroblasts from passage 3/4 and were used at passage ~25, which suggests that fibroblast contamination in these cultures was extremely unlikely. The expression of FGFR 2/IIIc in human malignant epithelial tumour cells is not unprecedented (14,15,18,30) and, invariably, exon IIIc is coexpressed with exon IIIb. In a minority of cell lines from the oral cavity, cervix, lung and endometrium, however, the expression of exon IIIc occurs in isolation (14,15,18,30). Taken together, the results suggest that the expression of FGFR 2/ IIIc may be more ubiquitous than was once thought, although it remains a possibility that the expression of both splice variants in keratinocytes occurs as a result of in vitro culture. Studies are in progress to examine whether the expression of the IIIc splice variant of FGFR 2 occurs in vivo and/or that exon IIIc expression is a specific characteristic of the malignant epithelial phenotype. In the present study, well-differentiated keratinocytes (H357 and H413) expressed proportionally more FGFR 2/IIIb than IIIc, whereas the poorly differentiated cells (H314) expressed more FGFR 2/IIIc than IIIb. Variable expression of FGFR 2/ IIIb and IIIc in head and neck carcinoma cell lines has been reported previously (22) but the significance remains unknown. Interestingly, a low level of FGFR 2/IIIb has been shown to be associated with a significantly poorer prognosis in transitional cell bladder carcinoma (21) and this possibly corresponds to the known lack of differentiation in H314 of the present study. The results are consistent with previous
observations in which the response of the cell lines to KGF (23) and FGF-2 (unpublished observations) have been examined. Well-differentiated keratinocytes, for example, are stimulated more by KGF than FGF-2 and undifferentiated cells show an increase in FGF-2-induced thymidine incorporation compared with that induced by KGF. In rodent tumours, there is a strong correlation between the expression of the different FGFR 2 exons and the degree of tumour cell differentiation. In these tumours, undifferentiated cells exclusively express FGFR 2 IIIc, which results in stromal independence, activation of genes encoding FGF-2, FGF-3, FGF-5 and FGFR 1 and the formation of multiple potential autocrine loops (31–33). The expression of FGFR 2 exon IIIb or IIIc is thought to arise by a mutually exclusive alternative splicing mechanism (11,34). All of the clonal cell populations in the present study, however, expressed both exons IIIb and IIIc. The results demonstrate that exon switching associated with the loss of cellular differentiation in human keratinocyte cell lines is not necessarily mutually exclusive. When cDNA from normal and tumour-derived keratinocytes, but not fibroblasts, was amplified with primers flanking IIIb/ IIIc, an additional PCR product was detected. This amplification product was sequenced and found to contain both the IIIb and IIIc splice variants of FGFR 2 joined by an additional base pair and with the intronic sequence between the exons removed. Interestingly, the cDNA that contained both exons could not be amplified using primer 6 with either primers 1 or 2, which suggests that either some sequences downstream of exon IIIc have been deleted or that the truncated receptor is encoded by a separate gene. The co-expression of exons IIIb and IIIc joined in this manner has been detected previously in normal and hyperplastic prostate epithelium and normal and malignant breast epithelium (27,28). The receptor expressed by breast epithelial cells also contained an additional base pair between the two exons and it was predicted that because of the frame shift caused by this additional base pair, the translated product would terminate at base pair 13 in the IIIc exon (28). This truncation would be predicted to produce a soluble protein with the ligand binding specificity of the KGF receptor (28). It is unknown at present whether this truncated receptor is secreted by keratinocytes and, if it is secreted, what its function might be. Other truncated soluble FGF receptors have been described (10,35) and it may be that these growth factors act to regulate the availability of the ligand to the cell. In conclusion, the results of this study demonstrate that FGFR 2/IIIb and IIIc are co-expressed in malignant human oral keratinocyte cell lines. With a loss of tumour cell differentiation, there appears to be a partial switch from the expression of FGFR2/IIIb to IIIc, and we suggest that this confers a selective growth advantage to the poorly differentiated cells by becoming more susceptible to the growth stimulatory effects of FGF 2. Acknowledgements C.S.Drugan was supported in part by the MRC (M17603). The authors also wish to acknowledge the support of Denman’s Charitable Trust.
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