B69
Regulation of Fgf-10 and its implication in breast cancer
Athina-myrto Chioni, Richard Grose
Barts and The London, QMUL, Institute of Cancer, Tumour Biology, London, UK
Introduction
Fgf-10 plays an important role in tissue development and disease, signalling primarily through Fgfr2b to regulate cell proliferation, migration and differentiation. Aberrant Fgf signalling is implicated in tumorigenesis, with studies of several cancers either reporting Fgf-10 upregulation or identifying Fgfr2b mutations.
Aims
(1) Identify the fgf-10 transcriptional start site and analyse the upstream promoter region to gain insight into the transcriptional regulation of FGF-10 expression. (2) Investigate whether Fgf10 signalling is involved in breast cancer (BCa) cell behaviour and, if so, elucidate the signalling mechanisms.
Method
RNA from E18.5 mouse lung was used for 5’ RLM-RACE, to clone the full length Fgf-10 5’ cDNA sequence, and the new promoter region of fgf-10 analysed using TESS and ChIP. Having established the basal levels of FGF-10 and FGFR2 mRNA expression in breast cancer cell lines by RT-PCR, the role of the potential regulatory transcription factor on FGF-10 expression was investigated by RNAi. The effect of FGF-10 on BCa progression was studied using strongly (MDA-MB-231) and weakly (MCF-7) metastatic BCa cell lines in biochemical analysis, cell-based proliferation and migration assays, and an organotypic invasion model.
Results
We have identified a novel transcription start site upstream of the published site. In silico analysis predicted multiple binding sites for the transcription factor Pea3 -230 to -130 bp upstream of this site. Binding was confirmed by ChIP and functional significance confirmed by RNAi knockdown of Pea3 leading to 3.5 fold increased FGF-10 mRNA expression.
MCF-7 cells expressed FGF-10, FGFR1 and FGFR2 (IIIb and IIIc) mRNA, however MDA-MB-231 cells expressed FGF-10, FGFR1 and FGFR2IIIc only. Both cell lines showed rapid ERK phosphorylation upon FGF-10 treatment. This was abrogated by pre-treatment with a specific inhibitor for FGFR (PD173074). Interestingly, the signalling pathways downstream of FGF-10 stimulation differed between the two cell lines. In MCF-7 cells, FGF-10 stimulation activated P-FRS2 whereas in MDA-MB-231 cells the PLC-γ gamma pathway was activated. FGF-10 treatment stimulated cell proliferation only in MCF-7 cells, suggesting that its mitogenic effect acts via P-FRS2. Nevertheless, Transwell assays showed that FGF10 stimulation increased cell migration both in MCF-7 and MDA-MB-231 cells, while co-treatment with PD173074 blocked this effect. Interestingly, treatment with PD173074, in the absence of FGF-10, decreased basal levels of cell migration in MDA-MB-231, but not MCF-7, cells.
Conclusion
We have identified a novel fgf-10 transcription start site and show that Fgf-10 is regulated transcriptionally by Pea3, a target of Fgf signalling not known to regulate Fgf expression. PEA3, in addition with FGF-10, have been implicated in several cancers, including breast, so regulation of FGF-10 by PEA3 might prove important in tumourigenesis.
Biochemical studies revealed that signalling downstream of FGF-10 stimulation differs between MCF-7 and MDA-MB-231 cells, although both show a significant role for FGF-10 in cell invasion. In strongly metastatic MDA-MB-231 cells, basal levels of FGF-10 expression contribute to driving cell invasion but exogenous FGF-10 stimulation increased this both in strongly and weakly metastatic cells, highlighting a potential importance of FGF-10 regulation in breast cancer. Our study suggests that transcriptional (i.e. Pea3) or post-transcriptional (i.e. use of FGFR inhibitors) regulation of FGF-10 might be useful target for controlling cancer migration/invasion.
