Reciprocal priming between receptor tyrosine kinases at recycling endosomes orchestrates cellular signalling outputs

Integration of signalling downstream of individual receptor tyrosine kinases (RTKs) is crucial to fine‐tune cellular homeostasis during development and in pathological conditions, including breast cancer. However, how signalling integration is regulated and whether the endocytic fate of single receptors controls such signalling integration remains poorly elucidated. Combining quantitative phosphoproteomics and targeted assays, we generated a detailed picture of recycling‐dependent fibroblast growth factor (FGF) signalling in breast cancer cells, with a focus on distinct FGF receptors (FGFRs). We discovered reciprocal priming between FGFRs and epidermal growth factor (EGF) receptor (EGFR) that is coordinated at recycling endosomes. FGFR recycling ligands induce EGFR phosphorylation on threonine 693. This phosphorylation event alters both FGFR and EGFR trafficking and primes FGFR‐mediated proliferation but not cell invasion. In turn, FGFR signalling primes EGF‐mediated outputs via EGFR threonine 693 phosphorylation. This reciprocal priming between distinct families of RTKs from recycling endosomes exemplifies a novel signalling integration hub where recycling endosomes orchestrate cellular behaviour. Therefore, targeting reciprocal priming over individual receptors may improve personalized therapies in breast and other cancers.     

Cross-talk between FGF and other cytokine signalling pathways during endochondral bone development

FGF (fibroblast growth factor)/FGFR (FGF receptor) signalling plays an essential role in both endochondral and intramembranous bone development. FGF signalling pathways are important for the earliest stages of limb development and throughout skeletal development. The activity and the outcome of this signalling pathway during bone development are also influenced by many other intracellular and extracellular signals. In this review, we focus on the interplay between FGF signalling and other pathways, which is tightly regulated both spatially and temporally during endochondral skeletal development.     

In vitro assays using primary embryonic mouse lymphatic endothelial cells uncover key roles for FGFR1 signalling in lymphangiogenesis

Despite the importance of blood vessels and lymphatic vessels during development and disease, the signalling pathways underpinning vessel construction remain poorly characterised. Primary mouse endothelial cells have traditionally proven difficult to culture and as a consequence, few assays have been developed to dissect gene function and signal transduction pathways in these cells ex vivo. Having established methodology for the purification, short-term culture and transfection of primary blood (BEC) and lymphatic (LEC) vascular endothelial cells isolated from embryonic mouse skin, we sought to optimise robust assays able to measure embryonic LEC proliferation, migration and three-dimensional tube forming ability in vitro. In the course of developing these assays using the pro-lymphangiogenic growth factors FGF2 and VEGF-C, we identified previously unrecognised roles for FGFR1 signalling in lymphangiogenesis. The small molecule FGF receptor tyrosine kinase inhibitor SU5402, but not inhibitors of VEGFR-2 (SU5416) or VEGFR-3 (MAZ51), inhibited FGF2 mediated LEC proliferation, demonstrating that FGF2 promotes proliferation directly via FGF receptors and independently of VEGF receptors in primary embryonic LEC. Further investigation revealed that FGFR1 was by far the predominant FGF receptor expressed by primary embryonic LEC and correspondingly, siRNA-mediated FGFR1 knockdown abrogated FGF2 mediated LEC proliferation. While FGF2 potently promoted LEC proliferation and migration, three dimensional tube formation assays revealed that VEGF-C primarily promoted LEC sprouting and elongation, illustrating that FGF2 and VEGF-C play distinct, cooperative roles in lymphatic vascular morphogenesis. These assays therefore provide useful tools able to dissect gene function in cellular events important for lymphangiogenesis and implicate FGFR1 as a key player in developmental lymphangiogenesis in vivo.     

Role of heparan sulfate in fibroblast growth factor signalling: a structural view.

Fibroblast growth factors (FGFs) are among the best-studied heparin-binding proteins, and heparan sulfate proteoglycans regulate FGF signalling by direct molecular association with FGF and its tyrosine kinase receptor, FGFR. Two recently determined crystal structures of FGF-FGFR-heparin complexes have provided new structural information on how heparin binds to FGF and FGFR, and lead to different models for receptor dimerisation.     

Deciphering role of FGFR signalling pathway in pancreatic cancer

Recently, fibroblast growth factors are identified to play a vital role in the development and progression of human pancreatic cancer. FGF pathway is critical involved in numerous cellular processes through regulation of its downstream targets, including proliferation, apoptosis, migration, invasion, angiogenesis and metastasis. In this review article, we describe recent advances of FGFR signalling pathway in pancreatic carcinogenesis and progression. Moreover, we highlight the available chemical inhibitors of FGFR pathway for potential treatment of pancreatic cancer. Furthermore, we discuss whether targeting FGFR pathway is a novel therapeutic strategy for pancreatic cancer clinical management.     

Phosphorylation of RSK2 at Tyr529 by FGFR2-p38 enhances human mammary epithelial cells migration

The members of p90 ribosomal S6 kinase (RSK) family of Ser/Thr kinases are downstream effectors of MAPK/ERK pathway that regulate diverse cellular processes including cell growth, proliferation and survival. In carcinogenesis, RSKs are thought to modulate cell motility, invasion and metastasis. Herein, we have studied an involvement of RSKs in FGF2/FGFR2-driven behaviours of mammary epithelial and breast cancer cells. We found that both silencing and inhibiting of FGFR2 attenuated phosphorylation of RSKs, whereas FGFR2 overexpression and/or its stimulation with FGF2 enhanced RSKs activity. Moreover, treatment with ERK, Src and p38 inhibitors revealed that p38 kinase acts as an upstream RSK2 regulator. We demonstrate for the first time that in FGF2/FGFR2 signalling, p38 but not MEK/ERK, indirectly activated RSK2 at Tyr529, which facilitated phosphorylation of its other residues (Thr359/Ser363, Thr573 and Ser380). In contrast to FGF2-triggered signalling, inhibition of p38 in the EGF pathway affected only RSK2-Tyr529, without any impact on the remaining RSK phosphorylation sites. p38-mediated phosphorylation of RSK2-Tyr529 was crucial for the transactivation of residues located at kinase C-terminal domain and linker-region, specifically, in the FGF2/FGFR2 signalling pathway. Furthermore, we show that FGF2 promoted anchorage-independent cell proliferation, formation of focal adhesions and cell migration, which was effectively abolished by treatment with RSKs inhibitor (FMK). These indicate that RSK2 activity is indispensable for FGF2/FGFR2-mediated cellular effects. Our findings identified a new FGF2/FGFR2-p38-RSK2 pathway, which may play a significant role in the pathogenesis and progression of breast cancer and, hence, may present a novel therapeutic target in the treatment of FGFR2-expressing tumours.     

Role of fibroblast growth factors in elicitation of cell responses

Fibroblast growth factors (FGFs) are signalling peptides that control important cell processes such as proliferation, differentiation, migration, adhesion and survival. Through binding to different types of receptor on the cell surface, these peptides can have different effects on a target cell, the effect achieved depending on many features. Thus, each of the known FGFs elicits specific biological responses. FGF receptors (FGFR 1–5) initiate diverse intracellular pathways, which in turn lead to a variety of results. FGFs also bind the range of FGFRs with a series of affinities and each type of cells expresses FGFRs in different qualitative and quantitative patterns, which also affect responses. To summarize, cell response to binding of an FGF ligand depends on type of FGF, FGF receptor and target cell, all interacting in concert. This review aims to examine properties of the FGF family and its members receptors. It also aims to summarize features of intracellular signalling and highlight differential effects of the various FGFs in different circumstances.     

Fibroblast growth factor receptor signalling is dictated by specific heparan sulphate saccharides

Signalling by fibroblast growth factors (FGFs) through FGF receptors (FGFRs) depends on the cell-surface polysaccharide heparan sulphate (HS) [1] [2]. HS has an ordered domain structure of highly diverse saccharide motifs that present unique displays of sulphate, carboxyl and hydroxyl groups [3]. These motifs interact with many proteins, particularly growth factors. HS binds both to FGFs [4] [5] [6] and FGFRs [7], and probably activates signalling by facilitating ligand-induced receptor dimerisation [8] [9]. Nevertheless, the extent to which specific HS saccharide sequences play a regulatory role has not been established. By screening a library of structurally diverse HS decasaccharides in bioassays of FGF signalling mediated by three different FGFR isoforms, we found that saccharides showed specificity for both ligands and receptors; some saccharides selectively activated FGF signalling through different FGFR isoforms, others acted as negative regulators. We conclude that HS saccharides play critical roles in dictating the specificity of ligand-receptor interactions in FGFR signalling. Controlled alterations in HS structures [10] would provide a mechanism for regulation of cellular responsiveness to growth factors that bind HS.     

Nedd4‐1 binds and ubiquitylates activated FGFR1 to control its endocytosis and function

Fibroblast growth factor receptor 1 (FGFR1) has critical roles in cellular proliferation and differentiation during animal development and adult homeostasis. Here, we show that human Nedd4 (Nedd4‐1), an E3 ubiquitin ligase comprised of a C2 domain, 4 WW domains, and a Hect domain, regulates endocytosis and signalling of FGFR1. Nedd4‐1 binds directly to and ubiquitylates activated FGFR1, by interacting primarily via its WW3 domain with a novel non‐canonical sequence (non‐PY motif) on FGFR1. Deletion of this recognition motif (FGFR1‐Δ6) abolishes Nedd4‐1 binding and receptor ubiquitylation, and impairs endocytosis of activated receptor, as also observed upon Nedd4‐1 knockdown. Accordingly, FGFR1‐Δ6, or Nedd4‐1 knockdown, exhibits sustained FGF‐dependent receptor Tyr phosphorylation and downstream signalling (activation of FRS2α, Akt, Erk1/2, and PLCγ). Expression of FGFR1‐Δ6 in human embryonic neural stem cells strongly promotes FGF2‐dependent neuronal differentiation. Furthermore, expression of this FGFR1‐Δ6 mutant in zebrafish embryos disrupts anterior neuronal patterning (head development), consistent with excessive FGFR1 signalling. These results identify Nedd4‐1 as a key regulator of FGFR1 endocytosis and signalling during neuronal differentiation and embryonic development.     

FGF/FGFR signaling: From lung development to respiratory diseases

The fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling system regulates a variety of biological processes, including embryogenesis, angiogenesis, wound repair, tissue homeostasis, and cancer. It exerts these regulatory functions by controlling proliferation, differentiation, migration, survival, and metabolism of target cells. The morphological structure of the lung is a complex tree-like network for effective oxygen exchange, and the airway terminates in the middle and distal ends of many alveoli. FGF/FGFR signaling plays an important role in the pathophysiology of lung development and pathogenesis of various human respiratory diseases. Here, we mainly review recent advances in FGF/FGFR signaling during human lung development and respiratory diseases, including lung cancer, acute lung injury (ALI), pulmonary arterial hypertension (PAH), chronic obstructive pulmonary disease (COPD), asthma, and pulmonary fibrosis.     

Mouse genetics identifies unique and overlapping functions of fibroblast growth factor receptors in keratinocytes

Fibroblast growth factors (FGFs) are key regulators of tissue development, homeostasis and repair, and abnormal FGF signalling is associated with various human diseases. In human and murine epidermis, FGF receptor 3 (FGFR3) activation causes benign skin tumours, but the consequences of FGFR3 deficiency in this tissue have not been determined. Here, we show that FGFR3 in keratinocytes is dispensable for mouse skin development, homeostasis and wound repair. However, the defect in the epidermal barrier and the resulting inflammatory skin disease that develops in mice lacking FGFR1 and FGFR2 in keratinocytes were further aggravated upon additional loss of FGFR3. This caused fibroblast activation and fibrosis in the FGFR1/FGFR2 double‐knockout mice and even more in mice lacking all three FGFRs, revealing functional redundancy of FGFR3 with FGFR1 and FGFR2 for maintaining the epidermal barrier. Taken together, our study demonstrates that FGFR1, FGFR2 and FGFR3 act together to maintain epidermal integrity and cutaneous homeostasis, with FGFR2 being the dominant receptor.     

Photoactivation Approaches Reveal a Role for Rab11 in FGFR4 Recycling and Signalling

Fibroblast growth factor receptor 4 (FGFR4) plays important roles during development and in the adult to maintain tissue homeostasis. Moreover, overexpression of FGFR4 or activating mutations in FGFR4 has been identified as tumour‐promoting events in several forms of cancer. Endocytosis is important for regulation of signalling receptors and we have previously shown that FGFR4 is mainly localized to transferrin‐positive structures after ligand‐induced endocytosis. Here, using a cell line with a defined pericentriolar endocytic recycling compartment, we show that FGFR4 accumulates in this compartment after endocytosis. Furthermore, using classical recycling assays and a new, photoactivatable FGFR4‐PA‐GFP fusion protein combined with live‐cell imaging, we demonstrate that recycling of FGFR4 is dependent on Rab11. Upon Rab11b depletion, FGFR4 is trapped in the pericentriolar recycling compartment and the total levels of FGFR4 in cells are increased. Moreover, fibroblast growth factor 1 (FGF1)‐induced autophosphorylation of FGFR4 as well as phosphorylation of phospholipase C (PLC)‐γ is prolonged in cells depleted of Rab11. Interestingly, the activation of mitogen‐activated protein kinase and AKT pathways were not prolonged but rather reduced in Rab11‐depleted cells, indicating that recycling of FGFR4 is important for the nature of its signalling output. Thus, Rab11‐dependent recycling of FGFR4 maintains proper levels of FGFR4 in cells and regulates FGF1‐induced FGFR4 signalling.      

Cell responses to FGFR3 signalling: growth, differentiation and apoptosis

FGFR3 is a receptor tyrosine kinase (RTK) of the FGF receptor family, known to have a negative regulatory effect on long bone growth. Fgfr3 knockout mice display longer bones and, accordingly, most germline-activating mutations in man are associated with dwarfism. Somatically, some of the same activating mutations are associated with the human cancers multiple myeloma, cervical carcinoma and carcinoma of the bladder. How signalling through FGFR3 can lead to either chondrocyte apoptosis or cancer cell proliferation is not fully understood. Although FGFR3 can be expressed as two main splice isoforms (IIIb or IIIc), there is no apparent link with specific cell responses, which may rather be associated with the cell type or its differentiation status. Depending on cell type, differential activation of STAT proteins has been observed. STAT1 phosphorylation seems to be involved in inhibition of chondrocyte proliferation while activation of the ERK pathway inhibits chondrocyte differentiation and B-cell proliferation (as in multiple myeloma). The role of FGFR3 in epithelial cancers (bladder and cervix) is not known. Some of the cell specificity may arise via modulation of signalling by crosstalk with other signalling pathways. Recently, inhibition of the ERK pathway in achondroplastic mice has provided hope for an approach to the treatment of dwarfism. Further understanding of the ability of FGFR3 to trigger different responses depending on cell type and cellular context may lead to treatments for both skeletal dysplasias and cancer.     

The distribution of the growth factors FGF-2 and VEGF, and their receptors, in growing red deer antler

The cellular distributions of the growth factors FGF-2 and VEGF, and their receptors FGFR1, FGFR2 and FGFR3, and VEGFR-2 respectively, were visualized by immunohistochemistry and light microscopy in sections of growing red deer antler. Both of these signalling systems were widely expressed in the integument and osteocartilaginous compartments. FGF-2 was found in the same cells as all three FGFRs, indicating that FGF signalling may be principally autocrine. The patterns of labelling for VEGF and its receptor were similar to those seen for FGF-2 and FGFR-3, in both compartments. Our data are consistent with the findings of others in suggesting that FGF-2 induces expression of VEGF, to stimulate and maintain high rates of neovascularisation and angiogenesis, thereby providing nutrients to both velvet and bone as they rapidly grow and develop. The presence of FGF and VEGF and their receptors in epithelial cells suggests that these signalling systems play a role in skin development, raising the possibility that one or both may be involved in the close coupling of the coordinated growth of the integument and osteocartilage of antler, a process which is poorly understood at present.     

Translocation of exogenous FGF1 into cytosol and nucleus is a periodic event independent of receptor kinase activity

Fibroblast growth factor 1 (FGF1) has the property to become translocated from the extracellular space into the cell cytosol and nucleus. Membrane translocation of FGF1 occurs subsequent to endocytic uptake and is strictly FGF-receptor (FGFR) dependent. Here we have investigated the timing of FGF1 translocation in relation to FGFR1 signalling. We found that the translocation of FGF1 is a periodic event that occurs with 24 h intervals. Serum-starved cells translocated the growth factor with peak occurrences ~ 6 h, ~ 30 h, and ~ 54 h after the addition of FGF1. The periodic FGF1 translocation was totally independent of the FGFR1 tyrosine kinase activity as it proceeded unchanged when the kinase activity was chemically inhibited or the kinase domain was deleted. Furthermore, FGF1 translocation was not restricted to a particular phase of the cell cycle or dependent on cell cycle progression. The results demonstrate that the FGF1/FGFR1 complex constitutes a signalling module that independently of the receptor tyrosine kinase can convey a signal that initiates a strictly timed and periodic release of endocytosed FGF1 into the cytosol/nucleus.     

Small molecule inhibition of fibroblast growth factor receptors in cancer

Fibroblast growth factors (FGFs) signal through FGF receptors (FGFRs), which are a sub-family of the superfamily of receptor tyrosine kinases, to regulate human development and metabolism. Uncontrolled FGF signaling is responsible for diverse array of developmental disorders, most notably skeletal syndromes due to FGFR gain-of-function mutations. Studies in the last few years have provided significant evidence for the importance of FGF signaling in the pathogenesis of diverse cancers, including endometrial and bladder cancers. FGFs are both potent mitogenic and angiogenic factors and can contribute to carcinogenesis by stimulating cell proliferation and tumor angiogenesis. Gene knockout and pharmacological inhibition of FGFRs in in vivo and in vitro models validate FGFRs as a target for cancer treatment. Considerable efforts are being expended to develop specific, small-molecule inhibitors for treating FGFR-driven cancers. Recent reviews on the FGF/FGFR system have focused primarily on signaling, pathophysiology, and functions in cancer. In this article, we review the key roles of FGFR in cancer, provide an update on the status of clinical trials with small-molecule FGFR inhibitors, and discuss how the current structural data on FGFR kinases guide the design and characterization of new FGFR inhibitors.     

Activating Somatic FGFR2 Mutations in Breast Cancer

It is known that FGFR2 gene variations confer a risk for breast cancer. FGFR2 and FGF10, the main ligand of FGFR2, are both overexpressed in 5–10% of breast tumors. In our study, we sequenced the most important coding regions of FGFR2 in somatic tumor tissue of 140 sporadic breast cancer patients and performed MLPA analysis to detect copy number variations in FGFR2 and FGF10. We identified one somatic heterozygous missense mutation, p.K660N (c.1980G>C), within the tyrosine kinase domain of FGFR2 in tumor tissue of a sporadic breast cancer patient, which is likely mediated by the FGFR2-IIIb isoform. The presence of wild type and mutated alleles in equal quantities suggests that the mutation has driven clonal amplification of mutant cells. We have analyzed the tyrosine kinase activity of p.K660N and another recently described somatic breast cancer mutation in FGFR2, p.R203C, after expression in HEK293 cells and demonstrated that the intrinsic tyrosine kinase activity of both mutant proteins is strongly increased resulting in elevated phosphorylation and activity of downstream effectors. To our knowledge, this is the first report of functional analysis of somatic breast cancer mutations in FGFR2 providing evidence for the activating nature of FGFR2-mediated signalling in the pathogenesis of breast cancer.     

FGF signals specifically regulate the structure of hair shaft medulla via IGF-binding protein 5

Reciprocal interactions between the dermal papilla and the hair matrix control proliferation and differentiation in the mature hair follicle. Analysis of expression suggests an important role for FGF7 and FGF10, as well as their cognate receptor FGFR2-IIIb, in these processes. Transgenic mice that express a soluble dominant-negative version of this receptor in differentiating hair keratinocytes were generated to interfere with endogenous FGF signalling. Transgenic mice develop abnormally thin but otherwise normal hairs, characterised by single columns of medulla cells in all hair types. All structural defects and the accompanying changes of global gene expression patterns are restricted to the hair medulla. Forced transgenic expression of IGF-binding protein 5, whose expression level is elevated upon suppression of FGFR2-IIIb-mediated signalling largely phenocopies the defect of dnFgfr2-IIIb-expressing hairs. Thus, the results identify Igfbp5-mediated FGFR2-IIIb signals as a key regulator of the genetic program that controls the structure of the hair shaft medulla.