We have reported that normal human salivary gland-derived epithelial cells exclusively express keratinocyte growth factor receptor (KGFR). In the process of malignant transformation of human salivary gland tumors, KGFR gene expression disappeared concomitantly with the de novo expression of the fibroblast growth factor receptor 1 (FGFR1) and FGFR4 genes. In the present study, we introduced wild-type KGFR cDNA or chimeric KGFR͞ FGFR1 cDNA, which encoded the extracellular domain of KGFR and the intracellular domain of FGFR1, into the HSY human salivary adenocarcinoma cell line. The KGFR tyrosine kinase suppressed the activity of FGF receptor substrate 2 (FRS2) and inhibited the growth of HSY by inducing differentiation and apoptosis in vitro and in vivo. Our results provided significant insight into the mechanism of KGFR tumor suppression and suggest that KGFR gene therapy might be a viable method of inhibiting human salivary adenocarcinoma growth. K eratinocyte growth factor (KGF͞FGF7) is a member of the fibroblast growth factor (FGF) family (1). Its activity is largely restricted to epithelial cells, which express the KGF receptor (KGFR), a transmembrane tyrosine kinase encoded by the IIIb splice variant of FGFR2 (FGFR2-IIIb) (2, 3). KGFR binds KGF and FGF-1 with high affinity. In contrast, FGFR2-IIIc, another splice variant of FGFR2, binds FGF-1 and FGF-2, but not KGF (3, 4). Analysis of KGF and KGFR expression during embryonic development, including that of mammary glands, has provided evidence that KGF is an important mesenchymal mediator of epithelial growth and differentiation. In normal human skin, KGFR immunostaining localizes to the suprabasal layers (5, 6). The lack of KGFR in basal cell progenitors in skin suggests that KGFR might regulate keratinocyte differentiation (7).Malignant salivary tumors are highly aggressive neoplasms that readily invade adjacent tissues and metastasize to distant organs at early stages of the disease. Malignant salivary tumors exhibit enhanced expression of both FGF-1 and FGF-2 compared with normal salivary gland (8). FGF-1 and FGF-2 can act in an autocrine manner to stimulate the proliferation of salivary adenocarcinoma cells (8,9). Normal salivary gland epithelial cells and benign salivary gland tumors exclusively express the KGFR gene. During the malignant transformation of salivary gland epithelial cells, the expression of the KGFR gene is abolished, whereas the FGFR1-IIIc and FGFR4 genes are activated (10). The exclusive expression of the KGFR gene in both normal and premalignant salivary epithelial cells correlates well with the slow-growing and differentiated phenotype of premalignant tumors. In contrast, the loss of the KGFR gene and the expression of both the FGFR1 gene and its specific ligand FGF-2 in malignant tumor cells are associated with the emergence of the malignant phenotype. Thus, the loss of normal KGFR gene function may promote tumorigenicity, whereas KGFR may function to suppress human salivary adenocarcinomas.The Ras-MAP kinase signaling pathway plays...

A G to T mutation at nucleotide position 2128 in the human FGFR3b coding region resulting in a Cys for Gly substitution (G697C) in the tyrosine kinase domain was observed in 62% (44/ 71) of oral squamous cell carcinomas (OSCC) examined. Immunostained FGFR3b was found in the cytoplasm of prickle cells in normal epithelia, and FGFR3b was localized in the cytoplasm and nucleus in non-FGFR3b mutant OSCC. Overexpressed FGFR3b protein on plasma membranes was noted in OSCC bearing the FGFR3b mutation. Enhanced tyrosine kinase activity of G697C FGFR3b was confirmed. Our results indicate that G697C is an activating mutation causing constitutive ligand-independent FGFR3b signaling. This mutation may be involved in the progression of OSCC and thus the FGFR3b coding sequence may have diagnostic or prognostic value for OSCC. ' 2005 Wiley-Liss, Inc.Key words: FGFR3b; oral squamous cell carcinomas; mutationThe 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 immunoglobulinlike 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-tocysteine s...

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BackgroundVascular endothelial growth factor (VEGF) is a highly specific signaling protein for vascular endothelial cells that plays a critical role in tumor growth and invasion through angiogenesis, and may contribute to cell migration and activation of pre-osteoclasts, osteoclasts and some tumor cells.ObjectivesWe aimed to clarify the detailed roles of VEGF-Flt-1 signaling in bone invasion of oral squamous cell carcinoma (OSCC) cells.ResultsForty-two (42) of 54 cases with gingival SCC (77.8%) strongly expressed VEGF, and had a significantly increased number of Flt-1+ osteoclasts (p<0.01) and more aggressive bone invasion (p<0.05). PlGF, a ligand of Flt-1, induced osteoclastogenesis in single culture of bone marrow cells (BMCs), and inhibition of Flt-1-signaling by VEGF tyrosine kinase inhibitor and It’s down stream (Akt and ERK1/2) inhibitos reduced osteoclastogenesis in PlGF-stimulated BMCs (p<0.01). In molecular level, PlGF stimulation significantly upregulated RANKL expression in Flt-1-expressing HSC2 cells via phosphorylation of Akt and ERK1/2. In the co-culture of VEGF-producing HSC2 cells and BMCs, number of TRAP-positive osteoclasts markedly increased (p<0.01). The osteoclastogenesis was significantly inhibited by RANKL-neutralizing antibody (p<0.01) as well as by VEGF tyrosine kinase inhibitor (p<0.01) and it’s downstream (Akt and ERK1/2) inhibitors (p<0.01, p<0.05, respectively).ConclusionVEGF-Flt-1 signaling induces osteoclastogenesis in OSCC through two possible ways: 1) VEGF produced from OSCC cells can directly stimulate the Flt-1 pathway in preosteoclasts to induce migration to future bone resorbing area and differentiation into osteoclasts, and 2) VEGF-Flt-1 signaling upregulates RANKL expression in OSCC cells, which indirectly leads to osteoclast differentiation. Therefore, blocking of the VEGF-Flt-1 signaling may help inhibit bone invasion of OSCC.