Unique signalling connectivity of FGFR3-TACC3 oncoprotein revealed by quantitative phosphoproteomics and differential network analysis

Abstract

The FGFR3-TACC3 fusion is an oncogenic driver in diverse malignancies, including bladder cancer, characterized by upregulated tyrosine kinase activity. To gain insights into distinct properties of FGFR3-TACC3 down-stream signalling, we utilised telomerase-immortalised normal human urothelial cell lines expressing either the fusion or wild-type FGFR3 (isoform IIIb) for subsequent quantitative proteomics and network analysis. Cellular lysates were chemically labelled with isobaric tandem mass tag reagents and, after phosphopeptide enrichment, liquid chromatography-high mass accuracy tandem mass spectrometry (LC-MS/MS) was used for peptide identification and quantification. Comparison of data from the two cell lines under non-stimulated and FGF1 stimulated conditions and of data representing physiological stimulation of FGFR3 identified about 200 regulated phosphosites. The identified phosphoproteins and quantified phosphosites were further analysed in the context of functional biological networks by inferring kinase-substrate interactions, mapping these to a comprehensive human signalling interaction network, filtering based on tissue-expression profiles and applying disease module detection and pathway enrichment methods. Analysis of our phosphoproteomics data using these bioinformatics methods combined into a new protocol—Disease Relevant Analysis of Genes On Networks (DRAGON)—allowed us to tease apart pathways differentially involved in FGFR3-TACC3 signalling in comparison to wild-type FGFR3 and to investigate their local phospho-signalling context. We highlight 9 pathways significantly regulated only in the cell line expressing FGFR3-TACC3 fusion and 5 pathways regulated only by stimulation of the wild-type FGFR3. Pathways differentially linked to FGFR3-TACC3 fusion include those related to chaperone activation and stress response and to regulation of TP53 expression and degradation that could contribute to development and maintenance of the cancer phenotype.

Strong anion exchange‐mediated phosphoproteomics reveals extensive human non‐canonical phosphorylation

“…TiO 2 or IMAC, we hypothesise that this is because UPAX separates, rather than specifically enriches, phosphopeptides from non-phosphopeptides. Based on a careful comparison of the number of phosphosites identified using our "all-pX" search strategy with the number of phosphorylation sites identified for the theoretical non-phosphorylatable residue Ala, we show that the commonly applied ptmRS score of 0.75 for "class I" phosphosite localisation (Taus et al, 2011;Meijer et al, 2013;Giansanti et al, 2015;Roitinger et al, 2015;Lombardi et al, 2017) is not acceptable for broad-scale analysis of canonical and non-canonical phosphorylation, since it yields an unacceptably high FLR (Fig 2 and Appendix Tables S5 and S7). To improve confidence in our phosphopeptide datasets, facilitating motif and functional characterisation of the sites and proteins identified, we increased the acceptable site localisation stringency to ptmRS ≥ 0.90, further filtering the data to a ptmRS value ≥ 0.99 for improved confidence when interrogating pX consensus motifs.…”

Exploiting protein family and protein network data to identify novel drug targets for bladder cancer

“…Some approaches search for mutations clustering in the protein structure, since clusters lying close to protein functional sites are particularly indicative of driver mutations likely to be causing gain or loss of function [11]. Other strategies for detecting putative drivers use network modules enriched in mutated or highly expressed genes [12][13][14][15][16].…”

Oncogenic driver FGFR3-TACC3 is dependent on membrane trafficking and ERK signaling

“…Although altered cellular localization and effects on mitotic defects have been well explored, it is unclear if these effects represent the initial drivers of oncogenic proliferation by FGFR3-TACC3. It has also been demonstrated that the fusion protein FGFR3-TACC3 leads to an upregulation of PI3K/AKT, STAT and MAPK pathways and pathways related to stress response and chaperone activation [ 2 , 20 , 21 ]. Here, we demonstrate that the oncogenic mechanism initiated by FGFR3-TACC3 is through the overactivation of canonical FGFR pathways which requires the localization of FGFR3-TACC3 to the secretory pathway and plasma membrane.…”