Ellen Margrethe Haugsten

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.

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.

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.

Production of phosphatidylinositol 5-phosphate via PIKfyve and MTMR3 regulates cell migration

Although phosphatidylinositol 5‐phosphate (PtdIns5P) is present in many cell types and its biogenesis is increased by diverse stimuli, its precise cellular function remains elusive. Here we show that PtdIns5P levels increase when cells are stimulated to move and we find PtdIns5P to promote cell migration in tissue culture and in a Drosophila in vivo model. First, class III phosphatidylinositol 3‐kinase, which produces PtdIns3P, was shown to be involved in migration of fibroblasts. In a cell migration screen for proteins containing PtdIns3P‐binding motifs, we identified the phosphoinositide 5‐kinase PIKfyve and the phosphoinositide 3‐phosphatase MTMR3, which together constitute a phosphoinositide loop that produces PtdIns5P via PtdIns(3,5)P2. The ability of PtdIns5P to stimulate cell migration was demonstrated directly with exogenous PtdIns5P and a PtdIns5P‐producing bacterial enzyme. Thus, the identified phosphoinositide loop defines a new role for PtdIns5P in cell migration.

Phosphorylation of Fibroblast Growth Factor (FGF) Receptor 1 at Ser777 by p38 Mitogen-Activated Protein Kinase Regulates Translocation of Exogenous FGF1 to the Cytosol and Nucleus▿

ABSTRACT Exogenous fibroblast growth factor 1 (FGF1) signals through activation of transmembrane FGF receptors (FGFRs) but may also regulate cellular processes after translocation to the cytosol and nucleus of target cells. Translocation of FGF1 occurs across the limiting membrane of intracellular vesicles and is a regulated process that depends on the C-terminal tail of the FGFR. Here, we report that translocation of FGF1 requires activity of the α isoform of p38 mitogen-activated protein kinase (MAPK). FGF1 translocation was inhibited after chemical inhibition of p38 MAPK or after small interfering RNA knockdown of p38α. Translocation was increased after stimulation of p38 MAPK with anisomycin, mannitol, or H2O2. The activity level of p38 MAPK was not found to affect endocytosis or intracellular sorting of FGF1/FGFR1. Instead, we found that p38 MAPK regulates FGF1 translocation by phosphorylation of FGFR1 at Ser777. The FGFR1 mutation S777A abolished FGF1 translocation, while phospho-mimetic mutations of Ser777 to Asp or Glu allowed translocation to take place and bypassed the requirement for active p38 MAPK. Ser777 in FGFR1 was directly phosphorylated by p38α in a cell-free system. These data demonstrate a crucial role for p38α MAPK in the regulated translocation of exogenous FGF1 into the cytosol/nucleus, and they reveal a specific role for p38α MAPK-mediated serine phosphorylation of FGFR1.

Different abilities of the four FGFRs to mediate FGF-1 translocation are linked to differences in the receptor C-terminal tail.

Members of the fibroblast growth factor family bind to one or more of the four closely related membrane-spanning FGF receptors. In addition to signaling through the receptors, exogenous FGF-1 and FGF-2 are endocytosed and translocated to the cytosol and nucleus where they stimulate RNA and DNA synthesis. Here we have studied the ability of the four FGF receptors to facilitate translocation of exogenous FGF-1 to the cytosol and nucleus. FGFR1 and FGFR4 were able to mediate translocation, whereas FGFR2 and FGFR3 completely lacked this ability. By analyzing mutant FGFRs we found that the tyrosine kinase domain could be deleted from FGFR1 without abolishing translocation, whereas the C-terminal tail of the FGFRs, constituted by approximately 50 amino acids downstream of the kinase domain, plays a crucial role in FGF-1 translocation. Three amino acids residues within the C-terminal tail were found to be of particular importance for translocation. For FGFR2, the two amino acid substitutions Q774M and P800H were sufficient to enable the receptor to support FGF-1 translocation. The results demonstrate a striking diversity in function of the four FGFRs determined by their C-terminal domain.

Nuclear Import of Exogenous FGF1 Requires the ER‐Protein LRRC59 and the Importins Kpnα1 and Kpnβ1

Fibroblast growth factor 1 (FGF1) taken up by cells into endocytic vesicles can be translocated across vesicular membranes into the cytosol and the nucleus where it has a growth regulatory activity. Previously, leucine‐rich repeat containing 59 (LRRC59) was identified as an intracellular binding partner of FGF1, but its biological role remained unknown. Here, we show that LRRC59 is strictly required for nuclear import of exogenous FGF1. siRNA‐mediated depletion of LRRC59 did not inhibit the translocation of FGF1 into cytosol, but blocked the nuclear import of FGF1. We also found that an nuclear localization sequence (NLS) in FGF1, Ran GTPase, karyopherin‐α1 (Kpnα1), and Kpnβ1 were required for nuclear import of FGF1. Nuclear import of exogenous FGF2, which depends on CEP57/Translokin, was independent of LRRC59, but was dependent on Kpnα1 and Kpnβ1, while the nuclear import of FGF1 was independent of CEP57. LRRC59 is a membrane‐anchored protein that localizes to the endoplasmic reticulum (ER) and the nuclear envelope (NE). We found that LRRC59 possesses NLS‐like sequences in its cytosolic part that can mediate nuclear import of soluble LRRC59 variants, and that the localization of LRRC59 to the NE depends on Kpnβ1. We propose that LRRC59 facilitates transport of cytosolic FGF1 through nuclear pores by interaction with Kpns and movement of LRRC59 along the ER and NE membranes.

ERK-Mediated Phosphorylation of Fibroblast Growth Factor Receptor 1 on Ser777 Inhibits Signaling

Serine phosphorylation limits signaling by a fibroblast growth factor receptor. Limiting Growth Factor Signaling Binding of fibroblast growth factors (FGFs) to receptor tyrosine kinases in the FGFR family triggers activation of these receptors through phosphorylation of tyrosine residues, thereby initiating signaling that stimulates cell proliferation and differentiation in various developmental processes, such as axonal growth. Zakrzewska et al. investigated the effect of phosphorylation of serine residues in FGFR1 on receptor activity. FGF-dependent activation of the mitogen-activated protein kinases ERK1 and ERK2 (ERK1/2) resulted in phosphorylation of Ser777 in the C-terminal region of the receptor, and this phosphorylation event was associated with decreased activation of FGFR1 and attenuated signaling. Dorsal root ganglion neurons expressing a S777A mutant FGFR1 exhibited enhanced and sustained receptor activation and extended longer axons than those expressing the wild-type receptor. In addition, previous stimulation of cells with epidermal growth factor, a ligand for a distinct receptor tyrosine kinase, resulted in the ERK1/2-mediated serine phosphorylation of FGFR1 and a reduction in subsequent FGF-dependent signaling. Together, these data suggest that an ERK-dependent mechanism initiated by activation of FGFR1 or other growth factor receptors prevents excessive FGFR1 signaling. Fibroblast growth factor 1 (FGF1) controls cellular activities through the activation of specific cell-surface FGF receptors (FGFRs). Transphosphorylation of tyrosine residues in the kinase domain of FGFRs leads to activation of intracellular signaling cascades, including those mediated by mitogen-activated protein kinases (MAPKs). FGFRs also contain a serine-rich C-terminal tail. We identified a regulatory mechanism of FGFR signaling involving phosphorylation of Ser777 in the C-terminal region of FGFR1 by the MAPKs extracellular signal–regulated kinase 1 (ERK1) and ERK2. Prevention of the phosphorylation of Ser777 in FGFR1 or mutation of Ser777 to alanine enhanced FGF-stimulated receptor tyrosine phosphorylation and increased cell proliferation, cell migration, and axonal growth. A form of FGFR1 with a phosphomimetic mutation at Ser777 exhibited reduced signaling. Activation of MAPKs by other receptor tyrosine kinases also resulted in phosphorylation of Ser777 in FGFR1, thereby enabling crosstalk regulation of FGFR activity by other signaling pathways. Our data reveal a negative feedback mechanism that controls FGF signaling and thereby protects the cell from excessive activation of FGFR.

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.