FGFR3 signaling and function in triple negative breast cancer

BACKGROUND: Triple negative breast cancer (TNBC) accounts for 16% of breast cancers and represents an aggressive subtype that lacks targeted therapeutic options. In this study, mass spectrometry (MS)-based tyrosine phosphorylation profiling identified aberrant FGFR3 activation in a subset of TNBC cell lines. This kinase was therefore evaluated as a potential therapeutic target. METHODS: MS-based tyrosine phosphorylation profiling was undertaken across a panel of 24 TNBC cell lines. Immunoprecipitation and Western blot were used to further characterize FGFR3 phosphorylation. Indirect immunofluorescence and confocal microscopy were used to determine FGFR3 localization. The selective FGFR1-3 inhibitor, PD173074 and siRNA knockdowns were used to characterize the functional role of FGFR3 in vitro. The TCGA and Metabric breast cancer datasets were interrogated to identify FGFR3 alterations and how they relate to breast cancer subtype and overall patient survival. RESULTS: High FGFR3 expression and phosphorylation were detected in SUM185PE cells, which harbor a FGFR3-TACC3 gene fusion. Low FGFR3 phosphorylation was detected in CAL51, MFM-223 and MDA-MB-231 cells. In SUM185PE cells, the FGFR3-TACC3 fusion protein contributed the majority of phosphorylated FGFR3, and largely localized to the cytoplasm and plasma membrane, with staining at the mitotic spindle in a small subset of cells. Knockdown of the FGFR3-TACC3 fusion and wildtype FGFR3 in SUM185PE cells decreased FRS2, AKT and ERK phosphorylation, and induced cell death. Knockdown of wildtype FGFR3 resulted in only a trend for decreased proliferation. PD173074 significantly decreased FRS2, AKT and ERK activation, and reduced SUM185PE cell proliferation. Cyclin A and pRb were also decreased in the presence of PD173074, while cleaved PARP was increased, indicating cell cycle arrest in G1 phase and apoptosis. Knockdown of FGFR3 in CAL51, MFM-223 and MDA-MB-231 cells had no significant effect on cell proliferation. Interrogation of public datasets revealed that increased FGFR3 expression in breast cancer was significantly associated with reduced overall survival, and that potentially oncogenic FGFR3 alterations (eg mutation and amplification) occur in the TNBC/basal, luminal A and luminal B subtypes, but are rare. CONCLUSIONS: These results indicate that targeting FGFR3 may represent a therapeutic option for TNBC, but only for patients with oncogenic FGFR3 alterations, such as the FGFR3-TACC3 fusion. Video abstract