Intracellular trafficking of FGF1 endocytosed by its four tyrosine kinase receptors

Fibroblast growth factors and the four related high-affinity, tyrosine kinase fibroblast growth factor receptors are involved in the regulation of many key cellular responses in developmental and physiological processes. Irregularities in FGF-mediated signalling are implicated in several serious disorders such as cancer and various forms of dwarfism. Little is known about the fate of endocytosed fibroblast growth factors and their receptors and the main purpose of this project is to study and compare the intracellular trafficking of the fibroblast growth factor 1 and the four related tyrosine kinase fibroblast growth factor receptors upon internalization

Fibroblast growth factor 2 conjugated with monomethyl auristatin E inhibits tumor growth in a mouse model

Worldwide, cancer is the second leading cause of death. Regardless of the continuous progress in medicine, we still do not have a fully effective anti-cancer therapy. Therefore, the search for new targeted anti-cancer drugs is still an unmet need. Here, we present novel protein–drug conjugates that inhibit tumor growth in a mouse model of human breast cancer. We developed conjugates based on fibroblast growth factor (FGF2) with improved biophysical and biological properties for the efficient killing of cancer cells overproducing fibroblast growth factor receptor 1 (FGFR1). We used hydrophilic and biocompatible PEG4 or PEG27 molecules as a spacer between FGF2 and the toxic agent monomethyl auristatin E. All conjugates exhibited a cytotoxic effect on FGFR1-positive cancer cell lines. The conjugate with the highest hydrodynamic size (42 kDa) and cytotoxicity was found to efficiently inhibit tumor growth in a mouse model of human breast cancer

Strategies to inhibit FGFR4 V550L-driven rhabdomyosarcoma

Background: Rhabdomyosarcoma (RMS) is a paediatric cancer driven either by fusion proteins (e.g., PAX3-FOXO1) or by mutations in key signalling molecules (e.g., RAS or FGFR4). Despite the latter providing opportunities for precision medicine approaches in RMS, there are currently no such treatments implemented in the clinic. Methods: We evaluated biologic properties and targeting strategies for the FGFR4 V550L activating mutation in RMS559 cells, which have a high allelic fraction of this mutation and are oncogenically dependent on FGFR4 signalling. Signalling and trafficking of FGFR4 V550L were characterised by confocal microscopy and proteomics. Drug effects were determined by live-cell imaging, MTS assay, and in a mouse model. Results: Among recently developed FGFR4-specific inhibitors, FGF401 inhibited FGFR4 V550L-dependent signalling and cell proliferation at low nanomolar concentrations. Two other FGFR4 inhibitors, BLU9931 and H3B6527, lacked potent activity against FGFR4 V550L. Alternate targeting strategies were identified by RMS559 phosphoproteomic analyses, demonstrating that RAS/MAPK and PI3K/AKT are essential druggable pathways downstream of FGFR4 V550L. Furthermore, we found that FGFR4 V550L is HSP90- dependent, and HSP90 inhibitors efficiently impeded RMS559 proliferation. In a RMS559 mouse xenograft model, the pan-FGFR inhibitor, LY2874455, did not efficiently inhibit growth, whereas FGF401 potently abrogated growth. Conclusions: Our results pave the way for precision medicine approaches against FGFR4 V550L-driven RMS

Clathrin- and Dynamin-Independent Endocytosis of FGFR3 – Implications for Signalling

Endocytosis of tyrosine kinase receptors can influence both the duration and the specificity of the signal emitted. We have investigated the mechanisms of internalization of fibroblast growth factor receptor 3 (FGFR3) and compared it to that of FGFR1 which is internalized predominantly through clathrin-mediated endocytosis. Interestingly, we observed that FGFR3 was internalized at a slower rate than FGFR1 indicating that it may use a different endocytic mechanism than FGFR1. Indeed, after depletion of cells for clathrin, internalization of FGFR3 was only partly inhibited while endocytosis of FGFR1 was almost completely abolished. Similarly, expression of dominant negative mutants of dynamin resulted in partial inhibition of the endocytosis of FGFR3 whereas internalization of FGFR1 was blocked. Interfering with proposed regulators of clathrin-independent endocytosis such as Arf6, flotillin 1 and 2 and Cdc42 did not affect the endocytosis of FGFR1 or FGFR3. Furthermore, depletion of clathrin decreased the degradation of FGFR1 resulting in sustained signalling. In the case of FGFR3, both the degradation and the signalling were only slightly affected by clathrin depletion. The data indicate that clathrin-mediated endocytosis is required for efficient internalization and downregulation of FGFR1 while FGFR3, however, is internalized by both clathrin-dependent and clathrin-independent mechanisms

Roles of the FGF-FGFR Signaling System in Cancer Development and Inflammation

For multi-cellular organisms to organize tissues, their cells must communicate with each other [...

Cancer Mutations in FGFR2 Prevent a Negative Feedback Loop Mediated by the ERK1/2 Pathway

Tight regulation of signaling from receptor tyrosine kinases is required for normal cellular functions and uncontrolled signaling can lead to cancer. Fibroblast growth factor receptor 2 (FGFR2) is a receptor tyrosine kinase that induces proliferation and migration. Deregulation of FGFR2 contributes to tumor progression and activating mutations in FGFR2 are found in several types of cancer. Here, we identified a negative feedback loop regulating FGFR2 signaling. FGFR2 stimulates the Ras/MAPK signaling pathway consisting of Ras-Raf-MEK1/2-ERK1/2. Inhibition of this pathway using a MEK1/2 inhibitor increased FGFR2 signaling. The putative ERK1/2 phosphorylation site at serine 780 (S780) in FGFR2 corresponds to serine 777 in FGFR1 which is directly phosphorylated by ERK1/2. Substitution of S780 in FGFR2 to an alanine also increased signaling. Truncated forms of FGFR2 lacking the C-terminal tail, including S780, have been identified in cancer and S780 has been found mutated to leucine in bladder cancer. Substituting S780 in FGFR2 with leucine increased FGFR2 signaling. Importantly, cells expressing these mutated versions of S780 migrated faster than cells expressing wild-type FGFR2. Thus, ERK1/2-mediated phosphorylation of S780 in FGFR2 constitutes a negative feedback loop and inactivation of this feedback loop in cancer cells causes hyperactivation of FGFR2 signaling, which may result in increased invasive properties

Endocytosis and Transport of Growth Factor Receptors in Peripheral Axon Regeneration: Novel Lessons from Neurons Expressing Lysine‐Deficient FGF Receptor Type 1 in vitro

In the course of peripheral nerve regeneration, axons encounter different extracellular growth factors secreted by non‐neuronal cells at the injury site and retrogradely transported after binding to neuronal membrane receptor tyrosine kinases. The present study reviews the role of receptor transport in peripheral axon outgrowth and provides novel data on trafficking of fibroblast growth factor receptor type 1 (FGFR1). Differences in receptor transport are determined by different numbers of lysine residues acting as ubiquitination sites in the intracellular receptor domain. We previously demonstrated that overexpression of mutant FGFR1‐25R (25 out of 29 intracellular lysines replaced with arginine) results in enhanced receptor recycling as compared to wild‐type FGFR1 followed by strong stimulation of elongative axon growth in vitro . Here, the effects of lysine‐deficient FGFR1 (FGFR1‐29R lacking all 29 cytoplasmic lysine residues) or of only 15 lysine mutations (FGFR1‐15R) on axon outgrowth and concomitant changes in signal pathway activation were investigated by immunocytochemistry and morphometry of cultured primary neurons. Overexpression of FGFR1‐15R in adult sensory neurons resulted in enhanced receptor recycling, which was accompanied by increased axon elongation without stimulating axon branching. By contrast, FGFR1‐29R was neither endocytosed nor axon outgrowth affected. Although overexpression of FGFR1‐15R or FGFR1‐25Ra strongly promoted elongation, we did not detect increased signal pathway activation (ERK, AKT, PLC, or STAT3) in neurons expressing mutant FGFR1 as compared with wild‐type neurons raising the possibility that other signaling pathways or signaling independent mechanisms may be involved in the axon outgrowth effects of recycled FGF receptors. Anat Rec, 302:1268–1275, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists

Efficacy and selectivity of fgf2-saporin cytosolically delivered by pci in cells overexpressing fgfr1

Fibroblast growth factor receptors (FGFRs) have become an attractive target in cancer research and therapy due to their implication in several cancers. Limitations of current treatment options require a need for additional, more specific and potent strategies to overcome cancers driven by FGFRs. Photochemical internalization (PCI) is a light-controlled method for cytosolic delivery of drugs that are entrapped in endosomes and lysosomes. We here evaluated the efficacy and selectivity of PCI of FGF2-saporin (FGF-SAP) in cells overexpressing FGFR1. FGF-SAP is a conjugate of FGF2 and the highly cytotoxic ribosome-inactivating protein (RIP) saporin, which is used as payload to eliminate cancer cells. Evaluation of the targeting effect of PCI of FGF-SAP was done by comparing the cytotoxic response in osteosarcoma cells with very low levels of FGFR1 (U2OS) to cells overexpressing FGFR1 (U2OS-R1). We demonstrate that PCI greatly enhances cytotoxicity of the drug showing efficient cell killing at pM concentrations of the drug in U2OS-R1 cells. However, U2OS cells were also sensitive to the toxin after PCI. Binding experiments using confocal microscopy and Western blotting techniques indicate that FGF-SAP is taken up by cells through heparan sulfate proteoglycans (HSPGs) in U2OS cells. We further show that the cytotoxicity of FGF-SAP in U2OS cells was reduced when cells were co-treated with heparin to compete out binding to HSPG, demonstrating that the cytotoxic effect was due to internalization by HSPGs. We conclude that to prevent off-target effects of FGF-based toxins, it will be necessary to circumvent binding to HSPGs, for example by mutating the binding site of FGF2 to HSPGs

Negative Regulation of FGFR (Fibroblast Growth Factor Receptor) Signaling

FGFR (fibroblast growth factor receptor) signaling controls fundamental processes in embryonic, fetal and adult human life. The magnitude, duration, and location of FGFR signaling must be strictly controlled in order to induce the correct biological response. Uncontrolled receptor signaling has been shown to lead to a variety of diseases, such as skeletal disorders and cancer. Here we review the numerous cellular mechanisms that regulate and turn off FGFR signaling, once the receptor is activated. These mechanisms include endocytosis and endocytic sorting, phosphatase activity, negative regulatory proteins and negative feedback phosphorylation events. The mechanisms act together simultaneously or sequentially, controlling the same or different steps in FGFR signaling. Although more work is needed to fully understand the regulation of FGFR signaling, it is clear that the cells in our body have evolved an extensive repertoire of mechanisms that together keep FGFR signaling tightly controlled and prevent excess FGFR signaling

Proximity Labeling by a Recombinant APEX2–FGF1 Fusion Protein Reveals Interaction of FGF1 with the Proteoglycans CD44 and CSPG4

Fibroblast growth factor 1 (FGF1) binds to specific FGF receptors (FGFRs) at the surface of target cells to initiate intracellular signaling. While heparan sulfate proteoglycans (HSPGs) are well-described coreceptors, it is uncertain whether there are additional binding sites for FGF1 at the cell surface. To address this, we devised and tested a method to identify novel binding sites for FGF1 at the cell surface, which may also be applicable for other protein ligands. We constructed an APEX2–FGF1 fusion protein to perform proximal biotin labeling of proteins following binding of the fusion protein to the cell surface. After functional validation of the fusion protein by a signaling assay, we used this method to identify binding sites for FGF1 on cell surfaces of living cells. We confirmed the feasibility of our approach by detection of FGFR4, a well-known and specific receptor for FGF1. We subsequently screened for novel interactors using RPE1 cells and identified the proteoglycans CSPG4 (NG2) and CD44. We found that FGF1 binds CD44 through its heparin-binding moiety. Moreover, we found that FGF1 was colocalized with both CSPG4 and CD44 at the cell surface, suggesting that these receptors act as storage molecules that create a reservoir of FGF1. Importantly, our data demonstrate that recombinant ligand–APEX2 fusion proteins can be used to identify novel receptor interactions on the cell surface