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. 2009 Dec 3;28(48):4306-16.
doi: 10.1038/onc.2009.280. Epub 2009 Sep 14.

Mutant fibroblast growth factor receptor 3 induces intracellular signaling and cellular transformation in a cell type- and mutation-specific manner

E di Martino  1 , C G L'Hôte, W Kennedy, D C Tomlinson, M A Knowles
Affiliations

Mutant fibroblast growth factor receptor 3 induces intracellular signaling and cellular transformation in a cell type- and mutation-specific manner

E di Martino et al. Oncogene. .

Abstract

Although activating mutations of fibroblast growth factor receptor 3 (FGFR3) are frequent in bladder tumors, little information is available on their specific effects in urothelial cells or the basis for the observed mutation spectrum. We investigated the phenotypic and signaling consequences of three FGFR3 mutations (S249C, Y375C, and K652E) in immortalized normal human urothelial cells (TERT-NHUC) and mouse fibroblasts (NIH-3T3). In TERT-NHUC, all mutant forms of FGFR3 induced phosphorylation of FRS2alpha and ERK1/2, but not AKT or SRC. PLCgamma1 phosphorylation was only observed in TERT-NHUC expressing the common S249C and Y375C mutations, and not the rare K652E mutation. Cells expressing S249C and Y375C FGFR3 displayed an increased saturation density, related to increased proliferation and viability. This effect was significantly dependent on PLCgamma1 signaling and undetectable in cells expressing K652E FGFR3, which failed to phosphorylate PLCgamma1. In contrast to TERT-NHUC, expression of mutant FGFR3 in NIH-3T3 resulted in phosphorylation of Src and Akt. In addition, all forms of mutant FGFR3 were able to phosphorylate Plcgamma1 and induce morphological transformation, cell proliferation, and anchorage-independent growth. Our results indicate that the effects of mutant FGFR3 are both cell type specific and mutation specific. Mutant FGFR3 may confer a selective advantage in the urothelium by overcoming normal contact inhibition of proliferation.

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Figures

Figure 1
Figure 1
Effects of mutant FGFR3 in NIH-3T3 cells. (a) Morphology of cells expressing wildtype (WT), S249C, Y375C, and K652E FGFR3. Line bars indicate 100 μm. (b) Representative experiment showing the total cell numbers reached on day 7 by cells expressing WT or mutant FGFR3 and by control cells. (c) Fold difference in the number of colonies formed in soft agar by cells expressing WT or mutant FGFR3 and control cells. (d) Constitutive phosphorylation of mutant FGFR3 and ligand-independent activation of signaling cascades. Significant differences to control cells (transduced with an empty pFB vector) are indicated as ‘*’ (p<0.05) or ‘**’ (p<0.01)
Figure 2
Figure 2
Effects of mutant FGFR3 in TERT-NHUC. (a) Saturation density of confluent cells. (b) Flow cytometric analysis of cell cycle profiles of confluent cells (day 12). (c) Representative experiment showing the number of viable cells expressing WT or mutant FGFR3, compared to control cells, at subconfluence (day 4) and confluence (day 12). (d) Expression levels of proteins involved in cell cycle and cell survival in confluent cultures (day 12); proteins were extracted from cells grown in full medium unless otherwise specified (DM, depleted medium). Significant differences to control cells (transduced with an empty pFB vector) are indicated as ‘*’ (p<0.05) or ‘**’ (p<0.01)
Figure 3
Figure 3
Constitutive activation of mutant FGFR3 and downstream signaling cascades in TERT-NHUC. (a) Constitutive phosphorylation of mutant FGFR3. (b) Western blot of cell lysates run under non-denaturing conditions showing ligand-independent dimerization of S249C and Y375C FGFR3. (c) Downstream signaling activated by mutant FGFR3 in confluent (day 12) and subconfluent (day 4) cells in full medium, and in subconfluent cells after 1 h in depleted medium.
Figure 4
Figure 4
Ligand independence of different forms of mutant FGFR3 expressed in TERT-NHUC. Receptor phosphorylation (a) and signaling activation (b) in starved subconfluent cells, before and after stimulation with FGF1.
Figure 5
Figure 5
Contribution of Plcγ1 phosphorylation to FGFR3-mediated transformation in NIH-3T3. (a) Activation of signaling cascades in cells expressing S249C FGFR3 or S249C with a mutation in the PLCγ1 binding site (S249C+Y762F). (b) Morphology of NIH-3T3 cells expressing S249C+Y762F FGFR3 and control cells. Line bars indicate 100 μm. (c) Representative experiment showing total cell numbers reached on day 7 by NIH-3T3 cells expressing S249C and S249C+Y762F FGFR3, compared with controls (d) Fold difference in the number of colonies >200 μm formed in soft agar by NIH-3T3 cells expressing S249C and S249C+Y762F FGFR3, compared with control cells. Significant differences to control cells (transduced with an empty pFB vector) are indicated as ‘*’ (p<0.05) or ‘**’ (p<0.01)
Figure 6
Figure 6
Contribution of PLCγ1 phosphorylation to saturation density in TERT-NHUC. (a) Growth curves showing saturation density achieved by cells expressing S249C+Y762F FGFR3, compared with cells expressing S249C FGFR3 and control cells. (b) Flow cytometric analysis of cell cycle profiles of confluent cells (day 12) expressing S249C+Y762F FGFR3. (c) Representative experiment showing the number of viable cells at confluence (day 12) in cultures expressing S249C+Y762F FGFR3 compared with cells expressing S249C FGFR3. Significant differences to S249C cells are indicated as ‘*’ (p<0.05) or ‘**’ (p<0.01)
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