Jørgen Wesche

Fibroblast growth factors and their receptors in cancer.

FGFs (fibroblast growth factors) and their receptors (FGFRs) play essential roles in tightly regulating cell proliferation, survival, migration and differentiation during development and adult life. Deregulation of FGFR signalling, on the other hand, has been associated with many developmental syndromes, and with human cancer. In cancer, FGFRs have been found to become overactivated by several mechanisms, including gene amplification, chromosomal translocation and mutations. FGFR alterations are detected in a variety of human cancers, such as breast, bladder, prostate, endometrial and lung cancers, as well as haematological malignancies. Accumulating evidence indicates that FGFs and FGFRs may act in an oncogenic fashion to promote multiple steps of cancer progression by inducing mitogenic and survival signals, as well as promoting epithelial-mesenchymal transition, invasion and tumour angiogenesis. Therapeutic strategies targeting FGFs and FGFRs in human cancer are therefore currently being explored. In the present review we will give an overview of FGF signalling, the main FGFR alterations found in human cancer to date, how they may contribute to specific cancer types and strategies for therapeutic intervention.

Dependence of Ricin Toxicity on Translocation of the Toxin A-chain from the Endoplasmic Reticulum to the Cytosol

Ricin acts by translocating to the cytosol the enzymatically active toxin A-chain, which inactivates ribosomes. Retrograde intracellular transport and translocation of ricin was studied under conditions that alter the sensitivity of cells to the toxin. For this purpose tyrosine sulfation of mutant A-chain in the Golgi apparatus, glycosylation in the endoplasmic reticulum (ER) and appearance of A-chain in the cytosolic fraction was monitored. Introduction of an ER retrieval signal, a C-terminal KDEL sequence, into the A-chain increased the toxicity and resulted in more efficient glycosylation, indicating enhanced transport from Golgi to ER. Calcium depletion inhibited neither sulfation nor glycosylation but inhibited translocation and toxicity, suggesting that the toxin is translocated to the cytosol by the pathway used by misfolded proteins that are targeted to the proteasomes for degradation. Slightly acidified medium had a similar effect. The proteasome inhibitor, lactacystin, sensitized cells to ricin and increased the amount of ricin A-chain in the cytosol. Anti-Sec61α precipitated sulfated and glycosylated ricin A-chain, suggesting that retrograde toxin translocation involves Sec61p. The data indicate that retrograde translocation across the ER membrane is required for intoxication.

Ubiquitination of α5β1 Integrin Controls Fibroblast Migration through Lysosomal Degradation of Fibronectin-Integrin Complexes

Cell migration requires endocytosis and recycling of integrins, but it is not known whether degradation of these membrane proteins is involved. Here we demonstrate that in migrating cells, a fraction of the endocytosed fibronectin receptor, alpha 5 beta 1 integrin, is sorted into multivesicular endosomes together with fibronectin and degraded in lysosomes. This sorting requires fibronectin-induced ubiquitination of the alpha 5 subunit, and the activity of the endosomal sorting complex required for transport (ESCRT) machinery, which interacts with alpha 5 beta 1 integrin. Importantly, we demonstrate that both alpha 5 ubiquitination and ESCRT functions are required for proper migration of fibroblasts. We propose that ligand-mediated degradation of alpha 5 beta 1 integrin via the ESCRT pathway is required in order to prevent endosomal accumulation of ligand-bound integrins that might otherwise form nonproductive adhesion sites. Fibronectin and alpha 5 beta 1 integrin therefore are trafficked to lysosomes in a similar way to growth factors and their receptors.

Roles of Fibroblast Growth Factor Receptors in Carcinogenesis

The fibroblast growth factor receptors (FGFR) play essential roles both during development and in the adult. Upon ligand binding, FGFRs induce intracellular signaling networks that tightly regulate key biological processes, such as cell proliferation, survival, migration, and differentiation. Deregulation of FGFR signaling can thus alter tissue homeostasis and has been associated with several developmental syndromes as well as with many types of cancer. In human cancer, FGFRs have been found to be deregulated by multiple mechanisms, including aberrant expression, mutations, chromosomal rearrangements, and amplifications. In this review, we will give an overview of the main FGFR alterations described in human cancer to date and discuss their contribution to cancer progression. Mol Cancer Res; 8(11); 1439–52. ©2010 AACR.

Flat clathrin coats on endosomes mediate degradative protein sorting by scaffolding Hrs in dynamic microdomains

Endocytosed membrane proteins that are destined for degradation in lysosomes are ubiquitylated and recognised by sorting complexes on endosome membranes. The ubiquitin-binding sorting component Hrs as well as ubiquitylated cargo are enriched in a characteristic flat clathrin coat on the endosome membrane. The function of clathrin within this coat has not been investigated. Here, we show that both clathrin and the clathrin-box motif of Hrs are required for the clustering of Hrs into restricted microdomains. The C-terminus of Hrs, which contains the clathrin-box, is sufficient to redirect a phosphatidylinositol(3)-phosphate-binding protein into the Hrs- and clathrin-containing microdomains. Although these microdomains show little lateral diffusion in the membrane, they are dynamic structures that exchange Hrs and clathrin with similar kinetics, and acquire the downstream sorting component Tsg101. The clathrin-mediated clustering is essential for the function of Hrs in degradative protein sorting. We conclude that clathrin is responsible for concentrating Hrs in endosomal microdomains specialised for recognition of ubiquitylated membrane proteins, thus enabling efficient sorting of cargo into the degradative pathway.

Ubiquitination of Fibroblast Growth Factor Receptor 1 Is Required for Its Intracellular Sorting but Not for Its Endocytosis

Endocytosis and targeting of growth factor receptors for lysosomal degradation have been associated with ubiquitination of the intracellular part of the receptors. To elucidate the role of receptor ubiquitination in internalization and sorting of fibroblast growth factor receptor (FGFR), we constructed several mutants of FGFR1 in which lysines, potential ubiquitination sites, were substituted for arginines. Substitution of all lysine residues in the intracellular part of FGFR1 resulted in inactivation of the tyrosine kinase domain of the receptor. However, several multilysine FGFR1 mutants, where up to 26 of 29 lysines in the intracellular part of the receptor were mutated, retained tyrosine kinase activity. The active multilysine mutants were poorly ubiquitinated, but internalized normally, indicating that ubiquitination of the receptor is not required for endocytosis. In contrast, degradation of the multilysine mutants was dramatically reduced as the mutants were inefficiently transported to lysosomes but rather sorted to recycling endosomes. The altered sorting resulted in sustained signaling. The duration of FGFR1 signaling seems to be tightly regulated by receptor ubiquitination and subsequent sorting to the lysosomes for degradation.

Different intracellular trafficking of FGF1 endocytosed by the four homologous FGF receptors.

Many growth factors and cytokines bind to more than one receptor, but in many cases the different roles of the separate receptors in signal transduction are unclear. Intracellular sorting of ligand-receptor complexes may modulate the signalling, and we have here studied the intracellular trafficking of ligand bound to receptors for fibroblast growth factors (FGFs). For this purpose, we transfected HeLa cells with any one of the four tyrosine kinase FGF receptors (FGFR1-4). In cells expressing any one of these receptors, externally added FGF1 was localized to sorting/early endosomes after 15 minutes at 37°C. After longer incubation times, FGF1 internalized in cells expressing FGFR1 was localized mainly to late endosomes/lysosomes, similarly to EGF. By contrast, FGF1 internalized in cells expressing FGFR4 followed largely the same intracellular pathway as the recycling ligand, transferrin. In cells expressing FGFR2 or FGFR3, sorting of FGF1 to lysosomes was somewhat less efficient than that observed for FGFR1. Furthermore, FGF1 was more slowly degraded in cells expressing FGFR4 than in cells expressing FGFR1-3 and in addition, internalized FGFR4 as such was more slowly degraded than the other receptors. The data indicate that after endocytosis, FGFR4 and its bound ligand are sorted mainly to the recycling compartment, whereas FGFR1-3 with ligand are sorted mainly to degradation in the lysosomes. Alignment of the amino acid sequence of the intracellular part of the four FGFRs revealed several lysines conserved in FGFR1-3 but absent in FGFR4. Lysines are potential ubiquitylation sites and could thus target a receptor to lysosomes for degradation. Indeed, we found that FGFR4 is less ubiquitylated than FGFR1, which could be the reason for the different sorting of the receptors.

Binding of FGF‐1 variants to protein kinase CK2 correlates with mitogenicity

Fibroblast growth factor‐1 (FGF‐1) has both extra‐ and intracellular functions. To identify intracellular binding partners for FGF‐1, we isolated proteins from U2OS human osteosarcoma cells interacting specifically with FGF‐1. One of the isolated proteins was identified as protein kinase CK2 (CK2). We here provide evidence that FGF‐1 binds to both the catalytic α‐subunit and to the regulatory β‐subunit of CK2. The interaction between FGF‐1 and CK2α and β was characterized by surface plasmon resonance, giving KD values of 0.4 ± 0.3 and 1.2 ± 0.2 μM, respectively. By using a novel assay for intracellular protein interaction, FGF‐1 and CK2α are shown to interact in vivo. In vitro, FGF‐1 and FGF‐2 are phosphorylated by CK2, and the presence of FGF‐1 or FGF‐2 was found to enhance the autophosphorylation of CK2β. A correlation between the mitogenic potential of FGF‐1 mutants and their ability to bind to CK2α was observed. The possible involvement of CK2 in the FGF‐induced stimulation of DNA synthesis is discussed.

Vesicle transmembrane potential is required for translocation to the cytosol of externally added FGF-1

Externally added fibroblast growth factor‐1 (FGF‐1) is capable of crossing cellular membranes to reach the cytosol and the nucleus in a number of cell types. We have monitored the translocation of the growth factor by two methods: phosphorylation of FGF‐1, and prenylation of an FGF‐1 mutant that contains a C‐terminal prenylation signal. Inhibition of endosomal acidification by ammonium chloride or monensin did not block the translocation of FGF‐1, whereas bafilomycin A1, a specific inhibitor of vacuolar proton pumps, blocked translocation completely. A combination of ionophores expected to dissipate the vesicular membrane potential (valinomycin plus monensin) also fully inhibited the translocation. The inhibition of translocation by bafilomycin A1 was overcome in the presence of monensin or nigericin, while ouabain blocked translocation under these conditions. The data indicate that translocation of FGF‐1 to cytosol occurs from the lumen of intracellular vesicles possessing vacuolar proton pumps, and that a vesicular membrane potential is required. Apparently, activation of vesicular Na+/K+‐ATPase by monensin or nigericin generates a membrane potential that can support translocation when the proton pump is blocked.

FGF-1 and FGF-2 Require the Cytosolic Chaperone Hsp90 for Translocation into the Cytosol and the Cell Nucleus

Similarly to many protein toxins, the growth factors fibroblast growth factor 1 (FGF-1) and FGF-2 translocate from endosomes into the cytosol. It was recently found that certain toxins are dependent on cytosolic Hsp90 for efficient translocation across the endosomal membrane. We therefore investigated the requirement for Hsp90 in FGF translocation. We found that low concentrations of the specific Hsp90 inhibitors, geldanamycin and radicicol, completely blocked the translocation of FGF-1 and FGF-2 to the cytosol and the nucleus. The drugs did not interfere with the initial binding of FGF-1 to the growth factor receptors at the cell-surface or with the subsequent internalization of the growth factors into endosomes. The activation of known signaling cascades downstream of the growth factor receptors was also not affected by the drugs. The data indicate that the drugs block translocation from endosomes to the cytosol implying that Hsp90 is required for translocation of FGF-1 and FGF-2 across the endosomal membrane.