Alternative Splicing of Fibroblast Growth Factor Receptor IgIII Loops in Cancer

Abstract

Alternative splicing of the IgIII loop of fibroblast growth factor receptors (FGFRs) 1-3 produces b- and c-variants of the receptors with distinctly different biological impact based on their distinct ligand-binding spectrum. Tissue-specific expression of these splice variants regulates interactions in embryonic development, tissue maintenance and repair, and cancer. Alterations in FGFR2 splicing are involved in epithelial mesenchymal transition that produces invasive, metastatic features during tumor progression. Recent research has elucidated regulatory factors that determine the splice choice both on the level of exogenous signaling events and on the RNA-protein interaction level. Moreover, methodology has been developed that will enable the in depth analysis of splicing events during tumorigenesis and provide further insight on the role of FGFR 1-3 IIIb and IIIc in the pathophysiology of various malignancies. This paper aims to summarize expression patterns in various tumor types and outlines possibilities for further analysis and application.

Figure 1

Schema splicing IIIb/IIIc. The extracellular domains of FGFRs consist of 3 Ig-like loops. The IgIII loops of FGFRs 1–3 are coded for by exons 7–9. Inclusion of exons 8 and 9 are mutually exclusive producing the IIIb and IIIc splice forms.

Figure 2

Tissue interactions mediated by IIIb/IIIc splice variants. Expression of FGFR2 variants is strictly tissue specific with the IIIb form found in the epithelium and the IIIc form in the mesenchyme. Together with expression of splice form-specific ligands in the respective complementary tissue, the splice variants mediate tissue interaction during embryonic development as well as in tissue maintenance and repair. The example FGFR2 with the IIIb-specific ligands FGF7 and 10 and the IIIc-specific ligands FGF4, 8 and 9, is involved in mouse limb development as well as in wound healing and reepithelialization in the skin [32].

Figure 3

Expression patterns of FGFR1–3 IIIb and IIIc exons in human and canine STS cells. Preliminary data with quantitative real-time RT-PCR indicate that FGFR1-IIIc demonstrate lowest cycle threshold (Ct) values and thus is expressed strongest in canine STS tumors (n = 13) and STS cell lines (n = 7) originated from human STS tumors [58]. Bars and error bars represent mean with SEM.

Figure 4

IIIb/IIIc splice reporter systems. Schematic of bichromatic fluorescence reporter constructs for IIIc (a) and IIIb/IIIc (b) analyses with adaptations from [90] and [92]. The reporter genes are positioned downstream of the splicing cassette (bold) in two different reading frames, each containing an individual stop codon. Inclusion of exon IIIc (and for b simultaneous skipping of IIIb) of FGFR2 results in a fusion protein in frame with EGFP (green) ending at stop codon 2. Skipping of this exon (and for b simultaneous inclusion of IIIb) results in DsRED (red) expression in another reading frame which contains stop codon 1. Possible adaptations of this splicing reporter system are indicated to exchange (1) species specific orthologous sequences, (2) sequences from IIIb splice variants, (3) sequences from paralogous FGFR 1 and 3, and (4) sequences for other fluorescence reporter proteins.