Fig 2: Therapeutic potential of FGFR inhibition in a specific subgroup of UPS (A) Assessment of cell viability with pan-FGFR inhibitor JNJ-42756493 in 4 UPS cell lines; Growth curves indicate growth inhibition and IC50 of the 4 UPS cell lines after JNJ-42756493 treatment for 72 h (n = 6) (****p < 0.0001, one-way ANOVA). (B) FGFR-inhibitor induces MAPK pathway inhibition in FGFR2 overexpressing cell lines; Phospho-FGFR2/FGFR2 ratio and Phospho-Erk /Erk ratio decrease in FGFR2 overexpressing cell lines after 24 h of treatment with JNJ-42756493 at 1 µM (n = 3) (*p < 0.05 and **p < 0.01, two-way ANOVA) (Western-blot). (C) Activity of pan-FGFR inhibitor (JNJ-42756493) on cell cycle of 4 UPS cell lines; Top: cell-cycle profiles after 24 h of treatment with or without JNJ-42756493 at the IC50 analyzed by Propidium Iodide incorporation and flow cytometry; Bottom: cell cycle phase distributions were analyzed with FlowJo software and presented as mean ± SEM of 3 independent experiments (n = 3) (**p < 0.01 and ***p < 0.001, ns: not significant, two-way ANOVA). (D) Antitumoral effect of a pan-FGFR inhibitor (JNJ-42756493) in two Patient-Derived Xenograft (PDX) models of UPS; Mice were randomly assigned to receive 30 mg/kg of drug or vehicle; Tumor volume progression curves were drawn over 3 to 4 weeks of JNJ-42756493 treatment, arrows represent treatment beginning; The data points represent an average of 11 mice for the JR588 PDX (top) and 8 mice for the KN473 PDX (bottom) per condition (bars, SEM) (***p < 0.001, two-way ANOVA).

Fig 3: FGF7/FGFR2 signaling regulates proteasomal degradation of ER in BCa cells. (A) T47D and MCF7 cells were grown in the presence of FGF7 and/or tamoxifen for 24, 48, and 72 h. ER expression was evaluated by western blotting. Densitometry was done with ImageJ software. (B) FGF7-dependent effect on ER expression in T47D cells is mediated by FGFR2 and (C) involves proteasomal activities (48 h incubation with MG132). D) FGF7/FGFR2-signaling triggers ER ubiquitination. T47D vs T47D shFGFR2 cells were serum starved and incubated with FGF7 (10 ng/ml) and/or or tamoxifen (1 μM) for 24 h. Cell lysis and immunoprecipitation of ER was done in 1% Triton-X. Ubiquitination level was evaluated by western blot analyses. Protein amount in both lysate and immunoprecipitated fraction was normalized. E) qPCR analyses of ESR1 (coding ER) expression upon FGF7 and/or tamoxifen exposure. A, D, E experiments were done in triplicates; B and C experiments were done in duplicates.

Fig 4: FGFR2 S780 is mutated in cancer. (a) The sequence of the C-terminal tail of FGFR2. Variations that might influence S780 phosphorylation and have been identified in cancer patients are indicated. An asterisk indicates a stop codon. Numbers refer to the amino acid numbering used for human FGFR2 (NCBI: NM_000141). The variations are reported in cBioPortal and COSMIC. (b) U2OS-R2 cells or U2OS-R2 S780L cells were kept in serum-free media for two hours and then treated or not with 100 ng/mL FGF1 for 15 min in the presence of heparin (20 U/mL). After lysis, the cellular material was analyzed by immunoblotting using the indicated antibodies. A p in front of the name of the antibody indicates that it recognizes the phosphorylated form of the protein. One representative experiment is shown. (c) U2OS-R2 S780L cells were kept at 4 °C with DL550-FGF1 for one hour. Next, the cells were either fixed directly (upper panel) or incubated for 30 min at 37 °C before fixation (lower panel). The cells were then stained with anti-EEA1 antibodies and Hoechst and analyzed by confocal microscopy. Representative images are shown. Scale bar: 5 µM. (d) U2OS-R2 S780L cells were treated with 200 ng/mL DL550-FGF1 in the presence of heparin (50 U/mL) for 30 min. The cells were then fixed, stained with anti-FGFR2 antibodies and Hoechst, and analyzed by confocal microscopy. Representative images are shown. Scale bar: 5 µM.

Fig 5: FGFR2 functions in MKN-45 cells. MKN-45 cells were transfected with FGFR2 overexpressed plasmid and (A) Cell viability was measured by MTT assay at the points of 24h, 48h and 72h; (B, C) Cell proliferation was measured by colony formation assay; (D–G) Cell migration was measured by transwell assay and scratch assay, and migration associated proteins were measured by western blot. (H, I) Cell apoptosis rates were measured by flow cytometry and apoptosis associated proteins were measured by Western blot. MKN-45 cells were transfected with FGFR2 overexpressed plasmid or its control, at the same time were infected with miR-5701 or its control, and (J) Cell viability were measured by MTT assay at the points of 24h, 48h and 72h; (K) Cell apoptosis rates were measured by flow cytometry at 48h.