The fibroblast growth factor (FGF) family currently includes 22 different gene products that bind to 4 distinct FGF receptors (FGFR1-FGFR4).1, 2 The activities of FGF are mediated by high-affinity FGFR located on the cell surface, which share a common structure of 2 or 3 extracellular immunoglobulin-like loops, a transmembrane domain, and an intracellular split tyrosine kinase domain.3, 4 It is believed that FGFR, when activated by bound FGF in the presence of heparin or heparan sulfate proteoglycan, forms a dimer, which undergoes phosphorylation on tyrosine residues, and transduces signals of cell growth and differentiation.5, 6 It was shown recently that mutations in FGFR cause various congenital skeletal and chondral dysplasia.7 In these congenital diseases, constitutively active tyrosine kinase activity due to ligand-independent receptor dimerization was observed, and the degree of receptor tyrosine kinase activity correlated with the severity of disease. Squamous cell carcinoma (SCC) is a cancer originating from stratified squamous epithelia that mainly cover skin, oral cavity, esophagus, vagina and bronchus. We have reported previously that all 4 FGFR, including FGFR1 (c isoform), FGFR2 (b isoform), FGFR3 (b isoform) and FGFR4 were expressed exclusively in cells derived from normal oral epithelia and oral squamous cell carcinomas (OSCC).8 The growth of normal epithelial cells was stimulated by FGF whereas OSCC cell proliferation was not.9 Mutations in FGFR genes have been reported in human bladder, cervical, gastric and colorectal carcinomas,10, 11 however no such mutations have been reported in OSCC.
We examined the mutational status of FGFR3b in a series of human OSCC to determine whether FGFR3b is involved in oral squamous tumorigenesis. We found that 44 OSCC DNAs showed band shifts in exon 17 of the FGFR3b gene by SSCP analysis (Fig. 1a). The DNA sequence of exon 17 in each sample was investigated by direct nucleotide sequencing. A heterozygous missense mutation (G2128T) was observed in exon 17 in all cases (44/71) that exhibited a band shift in PCR-SSCP (Fig. 1b). This mutation resulted in a glycine-to-cysteine substitution at position 697 (G697C) in the second part of kinase domain of FGFR3b. Sequencing analysis of available blood genomic DNAs obtained from 12 OSCC patients with G2128T mutation showed that all of them exhibited normal sequence indicating G2128T mutation is somatic.
Mutations in several oncogenes and tumor suppressor genes have been found in SCC. According to previous reports, the frequencies of these mutations were 10% in H-ras,13 20% in MTS1,14 10% in ING115 and 55% in p53.16 The G2128T mutation of FGFR3b in OSCC was observed at a high frequency (62%), which strongly suggested that the mutation was closely related to the pathogenesis of OSCC.
It was reported that cysteine is an important amino acid for maintaining biological activity of proteins because it affects protein conformation through the formation of intra- and inter-molecular disulfide bonds.17 The substitution of glycine with cysteine might cause ligand-independent dimerization of FGFR molecules, which results in alterations in receptor function including increased levels of membrane-localized FGFR3b and enhanced autophosphorylation activity. In our present study, the expression of FGFR3b protein was observed in the cytoplasm of prickle cells in normal epithelial tissue (Fig. 2a). In the OSCC having no G2128T mutation, the expression of FGFR3b protein was observed in the cytoplasm and nucleus of the cancer cells (Fig. 2b). By contrast, in OSCC bearing the G2128T mutation strong expression of FGFR3b protein was observed on the cell membrane of cancer cells (Fig. 2c). The difference in localization of receptor proteins is thought to indicate differential receptor expression or differential receptor activity. It was reported that in COS cells FGFR2 and FGFR3 were localized in the cytoplasm and nucleus, whereas when the cells over-expressed wtFGFR1, wtFGFR2, wtFGFR3 and wtFGFR4, all 4 FGFR protein were detected on the cell surface.18 In OSCC bearing the G2128T mutation, the overexpressed G697CFGFR3b protein is similarly localized to the cell surface. In non-malignant cells FGFR molecules on the cell surface are difficult to detect immunohistochemically because the expression levels of these receptors is normally low.
To investigate the functional significance of G697C amino acid substitution of FGFR3b in OSCC, tyrosine kinase activity was compared between cells expressing the wtFGFR3b and G697CFGFR3b. When a Western blot of Sf9 cell lysate was probed with 4G10 anti-phosphotyrosine antibody, a weak band of 65 kDa corresponding to the FGFR3b band was detected in the wtFGFR3b sample. The equivalent band in the G697CFGFR3b sample was highly autophosphorylated. To control for sample loading the blot was probed with an anti-FGFR3 antibody, which yielded bands of similar intensities in the 2 samples (Fig. 3a). Densitometric analysis was carried out by using NIH image to quantitate the phosphorylation activity of wtFGFR3b and G697CFGFR3b. The band intensities of phosphorylation were normalized to the intensity of FGFR3b protein band, and relative ratio of phosphorylated-FGFR3b protein was calculated as increase of phosphorylation in G697CFGFR3b over that in wtFGFR3b. The densitometric analysis confirmed that G697CFGFR3b had 23 times as much activity as wtFGFR3b (Fig. 3b). These results indicated that the G697C amino acid substitution in the kinase domain of FGFR3b resulted in an enhanced tyrosine kinase activity, which would promote FGFR3b signaling. Su et al.19 expressed a K650E mutant FGFR3 in 293T cells and demonstrated that the mutant FGFR3 exhibited constitutive tyrosine kinase activity. Onose et al.20 found that thyroid cancer cells overexpressing FGFR3 did not differ from control cells in growth rate, however, they continued to proliferate even after the control cells ceased growing. This experiment suggests that FGFR3 is involved in the control of contact inhibition.
In our present study, the G697C substitution caused constitutively active ligand-independent FGFR3b kinase activity. The apparent over-expression of G697CFGFR3b in 62% of the OSCC cases investigated strongly suggests that this particular mutation is involved in the progression of OSCC. FGFR3b may therefore be an important diagnostic and prognostic marker for OSSC and a molecular target for future therapies.
This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and Smoking Research Foundation to T.O. and J.D.S was supported by grant P20 RR16463 from the National Center for Research Resources, National Institutes of Health.