BACKGROUND: Fibroblast growth factor receptor 3 (FGFR3) point mutations have been recorded in 40-60% superficial bladder tumors resulting in constitutive activation of the receptor. Such an event has been shown to play a role in neoplastic progression. In this study we have assessed the in vitro efficacy of the FGFR selective inhibitor PD173074 to target bladder carcinoma cells harboring FGFR3 mutations displaying an activated receptor status.
METHODS: Within a panel of 17 bladder carcinoma cell lines the FGFR3 mutation status in exons 7, 10 and 15 was determined using single strand conformation polymorphism (SSCP) followed by the TA-cloning and sequencing of aberrantly migrating bands. The constitutive activation status of FGFR3 in cells was established in immunoprecipitated receptor complexes probed in western blot with phospho-FGFR3 antibody. Clonogenic assays were performed following exposure of cell lines to different drug concentrations over a 24-48 hour period with subsequent maintenance in culture for 7-10 days. PTEN expression was restored to the J82 cell line, in which the gene is deleted, using lipofection. The assessment of the activation status of the MAP and Akt signaling pathways was performed in western blot analysis probing with antibodies to the phosphorylated form of the proteins.
RESULTS: Sequencing of aberrantly migrating bands in SSCP revealed FGFR3 mutations in 2/17 bladder carcinoma cell lines, PSI (S249C) and J82 (K652E). These cell lines showed constitutive activation of FGFR3 in western blot analysis in contrast to cell lines harboring a wild-type FGFR3 sequence. Introduction of mutated FGFR3 into NIH/3T3 cells resulted in an increased migration potential over the wild-type FGFR3 transfectants. Incubation of cell lines with PD173074 showed a reduction in the activation status of constitutively activated FGFR3. Clonogenic assays in human bladder carcinoma cell panel revealed suppression of growth at micromolar concentrations in an IC₅₀ range of 5-15μM inducing concentration-dependent cell death. A range of sensitivity to the action of PD173074 was recorded where PSI and J82 were among the most sensitive along with alternative cell lines harboring a wild-type FGFR3 gene. Analysis of signaling events following exposure to the inhibitor revealed modulations in the MAP and Akt signaling pathways. However, restoration of PTEN in cell line J82, resulting in reduced activation of Akt, did not alter the response of the cells to the PD173074.
CONCLUSIONS: Although mutations in the FGFR3 gene are common in superficial bladder tumors the activation status of the receptor does not define sensitivity to the action of PD173074. Differential sensitivity to PD173074 was recorded within a panel of bladder carcinoma cell lines where deactivation events were identified in downstream signaling. Presently, the mechanism defining sensitivity to PD173074 in bladder carcinoma cells is ill-defined. However, even in the absence of FGFR3 mutations PD173074 has promise as a new therapeutic agent in bladder cancer.