Fibroblast Growth Factor Receptor Kinase Research Articles

Fibroblast Growth Factor 2 Promotes Primitive Endoderm Development in Bovine Blastocyst Outgrowths1

Primitive endoderm (PE) is the second extraembryonic tissue to form during embryogenesis in mammals. The PE develops from pluripotent cells of the blastocyst inner cell mass. Experimental results described herein provide evidence that FGF2 stimulates PE development during bovine blastocyst development in vitro. Bovine blastocysts were cultured individually on a feeder layer-free, Matrigel-coated surface in the presence or absence of FGF2. A majority of blastocysts cultures formed outgrowths (76.8%) and the rate of outgrowth formation was not affected by FGF2 supplementation. However, supplementation with FGF2 increased the incidence of PE outgrowths on Days 13 and 15 after in vitro fertilization. Presumptive PE cultures contained cells with a phenotype distinct from trophectoderm (TE). Cell identity was validated by expression of GATA4 and GATA6 mRNA and transferrin protein, all markers of the PE lineage. Expression of GATA4 occurred coincident with blastocyst expansion and hatching. These cells did not express IFNT and CDX2 (TE lineage markers). Profiles of FGF receptor (FGFR) isoforms were distinct between PE and TE cultures. Specifically, FGFR1b and FGFR1c were the predominant FGFR transcripts in PE whereas FGFR2b transcripts were abundant in TE. Supplementation with FGF2 increased the mitotic index of PE but not TE. Moreover, FGF signaling appears important for initiation of PE formation in blastocysts, presumably by lineage committal from NANOG-positive epiblast cells, because chemical disruption of FGFR kinase activity with PD173074 reduces GATA4 expression and increases NANOG expression. Collectively, these results indicate that FGF2 and potentially other FGFs specify PE formation and mediate PE proliferation during early pregnancy in cattle.

Disruption of Fibroblast Growth Factor Receptor Signaling in Bovine Cumulus-Oocyte Complexes During In Vitro Maturation Impacts Subsequent Embryonic Development.

Several fibroblast growth factors (FGF) mediate folliculogenesis and oogenesis in cattle but it is unclear whether FGFs are required during the final stages of oocyte maturation. The objectives of this work were to determine whether blocking FGF receptor (FGFR) activity during in vitro maturation (IVM) affects oocyte fertilization and embryo development; examine changes in FGFR transcript profiles in cumulus cells and oocytes during IVM; and evaluate whether gonadotropins modulate FGFR transcript abundance during IVM. In the first set of studies, bovine cumulus-oocyte complexes (COCs) were matured in the presence of one of two FGFR kinase inhibitors (SU5402 or PD173074). After maturation, COCs were washed and cultured without inhibitors. Inhibitors did not affect cleavage rates but the percentage of 8-cell embryos at d 3 and blastocysts at d 7 and d 8 postfertilization were decreased when COCs were matured with either inhibitor. Profiles of FGFR mRNA variants were examined in cumulus cells and oocytes separated either immediately before (0 h) or at 6 or 21 h after beginning IVM. In cumulus cells, increases in R1b, R2b, and R2c abundance were detected when cultured in the absence of follicle-stimulating hormone (FSH). Supplementing FSH (1 or 25 M) increased the abundance of R1b, R1c, R2b, and R2c. In oocytes, no timeor SH-dependent changes in FGFR transcript abundance were detected. These observations implicate FGFs as crucial components f bovine oocyte competency and indicate that FSH augments FGFR mRNA abundance in cumulus cells during the final stages f oocyte maturation. 2012 Elsevier Inc. All rights reserved.

Functional interaction of fibroblast growth factor-8, bone morphogenetic protein and estrogen receptor in breast cancer cell proliferation

Estrogen is involved in the development and progression of breast cancer. Here we investigated the effect of fibroblast growth factor (FGF)-8 on breast cancer cell proliferation caused by estrogen using human breast cancer MCF-7 cells. MCF-7 cells express estrogen receptor (ER)α, ERβ, FGF receptors, and Smad signaling molecules. Estradiol stimulated MCF-7 cell proliferation in a concentration-responsive manner, whereas BSA-bound estradiol had a weak effect on MCF-7 cell mitosis compared with the effect of free estradiol. It is notable that estrogen-induced cell proliferation was enhanced in the presence of FGF-8 and that the combined effects were reversed in the presence of an FGF-receptor kinase inhibitor or an ER antagonist. It was also revealed that FGF-8 increased the expression levels of ERα, ERβ and aromatase mRNAs, while estradiol reduced the expression levels of ERs, aromatase and steroid sulfatase in MCF-7 cells. FGF-8-induced phosphorylation of FGF receptors was augmented by estradiol, which was reversed by an ER antagonist. FGF-8-induced activation of MAPKs and AKT signaling was also upregulated in the presence of estrogen. On the other hand, FGF-8 suppressed BMP-7 actions that are linked to mitotic inhibition by activating the cell cycle regulator cdc2. FGF-8 was revealed to inhibit BMP receptor actions including Id-1 promoter activity and Smad1/5/8 phosphorylation by suppressing expression of BMP type-II receptors and by increasing expression of inhibitory Smads. Collectively, the results indicate that FGF-8 acts to facilitate cell proliferation by upregulating endogenous estrogenic actions as well as by suppressing BMP receptor signaling in ER-expressing breast cancer cells.

Acenaphtho[1,2-b]pyrrole-Based Selective Fibroblast Growth Factor Receptors 1 (FGFR1) Inhibitors: Design, Synthesis, and Biological Activity

A novel series of acenaphtho[1,2-b]pyrrole derivatives as potent and selective inhibitors of fibroblast growth factor receptor 1 (FGFR1) were designed and synthesized. In silico target prediction revealed that tyrosine kinases might be the potential targets of the representative compound 2, which was subsequently validated by enzyme-linked immunosorbent assay (ELISA) for its selective and active FGFR1 inhibition of various tyrosine kinases. The structure-activity relationship (SAR) analysis aided by molecular docking simulation in the ATP-binding site demonstrated that acenaphtho[1,2-b]pyrrole carboxylic acid esters (2-5) are potent inhibitors of FGFR1 with IC(50) values ranging from 19 to 77 nM. Furthermore, these compounds exhibited favorable growth inhibition property against FGFR-expressing cancer cell lines with IC(50) values ranging from micromolar to submicromolar. Western blotting analysis showed that compounds 2, 3, and 2b inhibited activation of FGFR1 and extracellular-signal regulated kinase 1/2 (Erk1/2).

Abstract 4733: Activating FGFR2 kinase domain mutations provide resistance to dovitinib (TKI258)

Abstract Members of the fibroblast growth factor receptor (FGFR) family are amplified or mutationally activated in a variety of cancers, including breast, endometrial, ovarian, lung, gastric, and bladder cancers, glioblastoma and rhabdomyosarcoma. Consequently FGFRs are attractive therapeutic targets in cancer, with a number of FGFR inhibitors currently progressing through clinical trials. Dovitinib, a lead FGFR kinase inhibitor exhibits activity against FLT3, FGFRs, VEGFRs, and PDGFR, and has demonstrated considerable preclinical activity in cancer models with FGFR activation. Though targeted tyrosine kinase inhibitors (TKIs) have shown dramatic clinical responses, the long-term efficacy of these agents is frequently limited by development of resistance to the targeted agent, often due to mutation of the target kinase. Here we sought to identify the mutational mechanisms of resistance to Dovitinib using a BaF3 cell line screening strategy. The BaF3 cell line is an IL-3 dependent murine pro-B cell line that is commonly employed to model TKI resistant mutations. These cells do not express any FGF ligands or receptors and introduction and activation of FGFRs has been shown to substitute for IL-3 to promote cell proliferation. BaF3 cells transduced with FGFR2 were treated with TKI258 at 5x, 10x, and 15x the cellular IC50 for Dovitinib in these cells. Following clonal selection of Dovitinib resistant cells, the exons encoding the intracellular domain of FGFR2 were sequenced. Mutations in FGFR2 kinase domain were identified in 26 out of 63 (41.2%) resistant clones screened, with an increase in frequency of mutation with increasing selective pressure. Ten distinct Dovitinib-resistant mutations in FGFR2 were identified and subsequently confirmed to result in Dovitinib-resistance and kinase activation. The binding mode of Dovitinib and the mechanisms of action of the resistance mutations were studied using the crystal structures of unphosphorylated and phosphorylated FGFR2Ks. Mutations at N550 and E566 at the kinase hinge/interlobe region are expected to drive the kinase into the active state by disengaging the molecular brake that keeps the kinase in an autoinhibited state. Five additional mutations are also predicted to stabilize the active conformation of the kinase by strengthening a network of hydrophobic interactions between N- and C-terminal lobes of the kinase, termed the hydrophobic spine, that is a hallmark of the active state of the kinase. Hence our biochemical and structural data show that the drug predominantly binds the inactive state of the FGFR2 kinase. Our data have clinical ramifications as they suggest that cancer patients harboring these FGFR2 mutations may not respond to the anti-FGFR activity of Dovitinib. Taken together our study provides the first report of TKI-resistant mutations in FGFR2 and suggests that the active state of the FGFR2 kinase should be targeted for anti-cancer drug discovery. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4733. doi:10.1158/1538-7445.AM2011-4733

Fibroblast growth factor requirements for in vitro development of bovine embryos

The overall goal was to describe the importance of fibroblast growth factors (FGFs) during development of the bovine embryo. An inhibitor of FGF receptor kinase activity (SU5402) was used to examine whether FGF signaling is required for embryo development. Addition of 20 μM SU5402 on Day 0 (Day of IVF) reduced (P = 0.04) the percentage of oocytes becoming blastocysts on Day 7 compared to controls (5.9 ± 2.1 vs 16.9 ± 2.4; average ± SEM). Also, Day-8 blastocysts placed into individual culture drops of medium containing SU5402 tended to have decreased (P = 0.08) blastomere numbers at Day 11 (211.1 ± 27.5 vs 297.8 ± 25.0). A second series of studies determined if supplemental FGF2 enhances development in vitro. There was no effect of FGF2 on cleavage or blastocyst development rates when 5 or 100 ng/mL FGF2 was provided immediately after fertilization. Also, FGF2 supplementation beginning on Day 5 post-fertilization did not significantly affect blastocyst rates or the number of trophoblast and inner cell mass cells. However, addition of 500 ng/mL FGF2 at both Day 0 and Day 4 increased (P = 0.03) the percentage of oocytes that became blastocysts on Day 7 compared with controls (27.4 ± 1.3 vs 19.7 ± 1.3). In a final study, the thermal-protective ability of FGF2 was examined by adding FGF2 1 h before exposing Day 5 embryos to heat shock. Addition of FGF2 did not significantly influence embryo thermal-tolerance. In conclusion, FGF receptor activation was important for optimal blastocyst formation and FGF2 supplementation increased bovine blastocyst formation when provided at high concentrations.

N-Phenyl- N′-[4-(5 H-pyrrolo[3,2- d]pyrimidin-4-yloxy)phenyl]ureas as novel inhibitors of VEGFR and FGFR kinases

We have recently reported the discovery of pyrrolo[3,2- d]pyrimidine derivatives 1a and 1b as potent triple inhibitors of vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and Tie-2 kinases. To identify compounds having strong inhibitory activity against fibroblast growth factor receptor (FGFR) kinase, further modification was conducted using the co-crystal structure analysis of VEGFR2 and 1b. Among the compounds synthesized, urea derivative 11l having a piperazine moiety on the terminal benzene ring showed strong inhibitory activity against FGFR1 kinase as well as VEGFR2 kinase. A binding model of 11l complexed with VEGFR2 suggested that the piperazine moiety forms additional interactions with Ile1025 and His1026.

The Heterohexameric Complex Structure, a Component in the Non-classical Pathway for Fibroblast Growth Factor 1 (FGF1) Secretion

Fibroblast growth factors (FGFs) are key regulators of cell proliferation, tumor-induced angiogenesis, and migration. FGFs are essential for early embryonic development, organ formation, and angiogenesis. FGF1 also plays an important role in inflammation, wound healing, and restenosis. The biological effects of FGF1 are mediated through the activation of the four transmembrane phosphotyrosine kinase fibroblast growth factor receptors in the presence of heparin sulfate proteoglycans and, therefore, require the release of the protein into the extracellular space. FGF1 is exported through a non-classical release pathway involving the formation of a specific multiprotein complex. The protein constituents of this complex include FGF1, S100A13, and the p40 form of synaptotagmin 1 (Syt1). Because FGF1 plays an important role in tumor formation, it is clear that preventing the formation of the multiprotein complex would be an effective strategy to inhibit a wide range of cancers. To understand the molecular events in the FGF1 release pathway, we studied the FGF1-S100A13 tetrameric and FGF1-S100A13-C2A hexameric complex structures, which are both complexes possibly formed during the non-classical pathway of FGF1 release.

Abstract 3380: The serine protease inhibitor PN-1 induces activation of FGFR-mediated signaling

Abstract We are studying the role of PN-1 (Protease Nexin-1) in breast cancer. PN-1 is a serine protease inhibitor (serpin) that blocks a broad spectrum of proteases including among others tPA, uPA and thrombin. Once bound to a target protease, the PN-1/protease complexes bind with high affinity to Low Density Lipoprotein Receptor related Protein (LRP-1). A bioinformatic analysis revealed that PN-1 is overexpressed in a significant proportion of human tumors from different origins. Moreover, PN-1 expression levels correlate with markers of poor prognosis in breast cancer. PN-1 is also expressed in breast tumor cell lines and we recently showed that PN-1 is required by the malignant 4T1 mammary cancer cell line to metastasize to the lungs. Indeed, when mammary fat pads of Balb/c mice were injected with PN-1-knock down (KD)-4T1 cells, primary mammary tumors developed and these grew similarly as control 4T1 cells. However, the PN-1 KD tumors displayed a significant decrease in their potential to form lung metastasis, in comparison to control tumors. The mechanism by which complexes of serpin/protease mediate their effects in cancer cells is still largely unknown. Thus, our current studies are aimed at deciphering how the protease/PN-1 complex impacts on intracellular signaling in order to promote metastasis. Using the PN-1 negative 168FARN mammary tumor cell line, we have found that PN-1/protease complexes bind LRP-1 receptor and induce MMP-9 expression via Erk1/2 pathway activation. Importantly, the PN-1-driven regulation of MMP-9 expression was also demonstrated to be central to PN-1's prometastatic effect in 4T1 cells. Here by the use of Western Blot and specific inhibitors, we show that PN-1/protease complex treatment of fibroblast growth factor receptor (FGFR)-expressing tumor cells induces Erk signaling. Furthermore, fibroblast growth factor receptor substrate 2 (FRS2), a lipid anchored docking protein that plays a crucial role in mediating FGFR and tropomyosin receptor kinase (Trk)-induced signaling, was also found to be phosphorylated upon PN-1/protease complex addition. Preincubation of the cells with the FGFR inhibitor, TKI 258, abrogated phosphorylation of FRS2, Erk and Shc upon PN-1/protease complex treatment. In contrast, inhibitors of EGFR or PDGR did not have any effect on PN-1-induced phosphorylation of these proteins. This suggests that the PN-1/protease complex via binding to LRP-1 may transactivate FGFR and potentially other receptors known to activate mitogenic signaling in cancer cells. Our results are in agreement with a recent study showing that other ligands for LRP-1, (e.g, tPA and α2-macroglobulin), could transactivate TrkA receptor. Thus, PN-1 may also transactivate tyrosine kinase receptors. Hence, these results suggest that PN-1/protease complexes might be considered as a new target in cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3380.

Amplification of fibroblast growth factor receptor-1 in breast cancer and the effects of brivanib alaninate

Fibroblast growth factor receptor-1 (FGFR-1) is amplified in 10% of human breast cancers. The goal of this study was to test the correlation between FGFR-1 amplification and expression and sensitivity to brivanib, an FGFR-1 small molecule inhibitor, in breast cancer cell lines in vitro. Using CGH array and gene expression profiling, FGFR-1 DNA copy number, mRNA, and protein expression were measured in 21 cell lines and correlated with growth inhibition by brivanib. We examined FGFR-1 autophosphorylation and kinase activity, as well as phosphorylation of downstream signaling molecules in response to bFGF and brivanib exposure. CAMA, MDA-MB-361, and HCC38 cells had FGFR-1 amplification and protein overexpression. Brivanib GI(50) values were significantly lower in the gene amplified (15.17 μM, n = 3) compared to normal copy number (69.09 μM, n = 11) or FGFR-1 deleted (76.14 μM, n = 7) cells (P = 0.0107). Among nonamplified cells, there was no correlation between FGFR-1 mRNA or protein expression levels and brivanib sensitivity. Two of three FGFR-1 amplified cells were sensitive to bFGF-induced growth stimulation, which was blocked by brivanib. In cells with amplified FGFR-1, brivanib decreased receptor autophosphorylation, inhibited bFGF-induced tyrosine kinase activity, and reduced phosphorylation of ERK and AKT. Breast cancer cell lines with FGFR-1 gene amplification and protein overexpression are more sensitive to growth inhibition by brivanib than nonamplified cells. These findings suggest that FGFR-1 amplification or protein overexpression in breast cancers may be an indicator for brivanib treatment, where it may have direct anti-proliferative effects in addition to its' anti-angiogenic effects.

Abstract A139: Discovery and biological profiling of potent fibroblast growth factor receptor (FGFR) kinase inhibitors derived from in silico design with in vivo antitumor activity against FGFR2-dependent human tumors

Abstract Dysregulation of members of the FGFR tyrosine kinase family has been increasingly implicated in a number of human cancers, including gastric, breast, endometrial, and bladder carcinomas. A proprietary structure-based design paradigm was employed to identify inhibitors which favor a mode of binding that is distinct from the commonly described ATP competitive inhibitors. A molecular template was identified (ARQ 523) which, upon further modification, provided molecules that were shown to inhibit FGFR kinases in the low micromolar range, to bind to FGFR2 in an enantiomeric-specific fashion, to bind to unphosphorylated FGFR2 with a KD of 5 µM; and, after pre-incubation with inactive FGFR2, to prevent a fluorescent analogue of ATP from binding to the enzyme. We have designated this novel type of kinase inhibitor as “ATP-exclusionary” or “Type IV” to differentiate these inhibitors from extant molecules. Following a lead optimization campaign, biochemical potencies of 1 nM or less against FGFR kinases with corresponding increases in binding affinities to FGFR2 in biophysical assays were documented in several compounds in two distinct series. These compounds showed sub-micromolar activity in both FGFR2-dependent pharmacodynamic and cytotoxicity assays, and demonstrated a moderately high degree of selectivity across the human kinome. A representative compound showed marked FGFR2 pharmacodynamic suppression and corresponding growth inhibition in KATO III and SNU-16 human gastric carcinoma cells. In addition, growth of SNU-16 tumor xenografts in athymic mice was markedly suppressed (58% regression as compared to vehicle-treated controls) after daily intraperitoneal administration for 9 days. A paired Ba/F3 xenograft model was also employed, using both Ba/F3 cells transfected with either FGFR2 or the unrelated insulin receptor. Significant tumor growth inhibition (77% inhibition as compared to vehicle-treated controls) was observed in treated mice bearing FGFR2-transfected Ba/F3 tumors but not in the insulin receptor transfectants. In summary, we have achieved proof-of-principle of a structure-based kinase inhibitor design paradigm for the identification of a series of FGFR kinase inhibitors that exhibits a preference for the inactive form of the kinase and excludes ATP upon binding. One advanced lead molecule demonstrated the ability to exert profound anti-tumor effects against cancer cell lines in which FGFR2 is the dominant oncogenic driver. The opportunity now exists to significantly expand the number of new chemotypes available for drug discovery against disease-relevant kinases in oncology and in other therapeutic areas, since it is predicted that approximately half of the human kinome is amenable to this novel mode of inhibition. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A139.

Design, Synthesis, and Evaluation of Indolinones as Triple Angiokinase Inhibitors and the Discovery of a Highly Specific 6-Methoxycarbonyl-Substituted Indolinone (BIBF 1120)

Inhibition of tumor angiogenesis through blockade of the vascular endothelial growth factor (VEGF) signaling pathway is a new treatment modality in oncology. Preclinical findings suggest that blockade of additional pro-angiogenic kinases, such as fibroblast and platelet-derived growth factor receptors (FGFR and PDGFR), may improve the efficacy of pharmacological cancer treatment. Indolinones substituted in position 6 were identified as selective inhibitors of VEGF-, PDGF-, and FGF-receptor kinases. In particular, 6-methoxycarbonyl-substituted indolinones showed a highly favorable selectivity profile. Optimization identified potent inhibitors of VEGF-related endothelial cell proliferation with additional efficacy on pericyctes and smooth muscle cells. In contrast, no direct inhibition of tumor cell proliferation was observed. Compounds 2 (BIBF 1000) and 3 (BIBF 1120) are orally available and display encouraging efficacy in in vivo tumor models while being well tolerated. The triple angiokinase inhibitor 3 is currently in phase III clinical trials for the treatment of nonsmall cell lung cancer.

Fibroblast Growth Factor Receptor 3 Associates with and Tyrosine Phosphorylates p90 RSK2, Leading to RSK2 Activation That Mediates Hematopoietic Transformation

Dysregulation of the receptor tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) plays a pathogenic role in a number of human hematopoietic malignancies and solid tumors. These include t(4;14) multiple myeloma associated with ectopic expression of FGFR3 and t(4;12)(p16;p13) acute myeloid leukemia associated with expression of a constitutively activated fusion tyrosine kinase, TEL-FGFR3. We recently reported that FGFR3 directly tyrosine phosphorylates RSK2 at Y529, which consequently regulates RSK2 activation. Here we identified Y707 as an additional tyrosine in RSK2 that is phosphorylated by FGFR3. Phosphorylation at Y707 contributes to RSK2 activation, through a putative disruption of the autoinhibitory alphaL-helix on the C terminus of RSK2, unlike Y529 phosphorylation, which facilitates ERK binding. Moreover, we found that FGFR3 interacts with RSK2 through residue W332 in the linker region of RSK2 and that this association is required for FGFR3-dependent phosphorylation of RSK2 at Y529 and Y707, as well as the subsequent RSK2 activation. Furthermore, in a murine bone marrow transplant assay, genetic deficiency in RSK2 resulted in a significantly delayed and attenuated myeloproliferative syndrome induced by TEL-FGFR3 as compared with wild-type cells, suggesting a critical role of RSK2 in FGFR3-induced hematopoietic transformation. Our current and previous findings represent a paradigm for tyrosine phosphorylation-dependent regulation of serine-threonine kinases.

The FGF family: biology, pathophysiology and therapy

The family of fibroblast growth factors (FGFs) regulates a plethora of developmental processes, including brain patterning, branching morphogenesis and limb development. Several mitogenic, cytoprotective and angiogenic therapeutic applications of FGFs are already being explored, and the recent discovery of the crucial roles of the endocrine-acting FGF19 subfamily in bile acid, glucose and phosphate homeostasis has sparked renewed interest in the pharmacological potential of this family. This Review discusses traditional applications of recombinant FGFs and small-molecule FGF receptor kinase inhibitors in the treatment of cancer and cardiovascular disease and their emerging potential in the treatment of metabolic syndrome and hypophosphataemic diseases.

FGF acts as a co-transmitter through adenosine A2A receptor to regulate synaptic plasticity

Abnormalities of striatal function have been implicated in several major neurological and psychiatric disorders, including Parkinson's disease, schizophrenia and depression. Adenosine, via activation of A(2A) receptors, antagonizes dopamine signaling at D2 receptors and A(2A) receptor antagonists have been tested as therapeutic agents for Parkinson's disease. We found a direct physical interaction between the G protein-coupled A(2A) receptor (A(2A)R) and the receptor tyrosine kinase fibroblast growth factor receptor (FGFR). Concomitant activation of these two classes of receptors, but not individual activation of either one alone, caused a robust activation of the MAPK/ERK pathway, differentiation and neurite extension of PC12 cells, spine morphogenesis in primary neuronal cultures, and cortico-striatal plasticity that was induced by a previously unknown A(2A)R/FGFR-dependent mechanism. The discovery of a direct physical interaction between the A(2A) and FGF receptors and the robust physiological consequences of this association shed light on the mechanism underlying FGF functions as a co-transmitter and open new avenues for therapeutic interventions.

Frs2α-deficiency in cardiac progenitors disrupts a subset of FGF signals required for outflow tract morphogenesis

The cardiac outflow tract (OFT) is a developmentally complex structure derived from multiple lineages and is often defective in human congenital anomalies. Although emerging evidence shows that fibroblast growth factor (FGF) is essential for OFT development, the downstream pathways mediating FGF signaling in cardiac progenitors remain poorly understood. Here, we report that FRS2alpha (FRS2), an adaptor protein that links FGF receptor kinases to multiple signaling pathways, mediates crucial aspects of FGF-dependent OFT development in mouse. Ablation of Frs2alpha in mesodermal OFT progenitor cells that originate in the second heart field (SHF) affects their expansion into the OFT myocardium, resulting in OFT misalignment and hypoplasia. Moreover, Frs2alpha mutants have defective endothelial-to-mesenchymal transition and neural crest cell recruitment into the OFT cushions, resulting in OFT septation defects. These results provide new insight into the signaling molecules downstream of FGF receptor tyrosine kinases in cardiac progenitors.

Relative role of upstream regulators of Akt, ERK and CREB in NCAM- and FGF2-mediated signalling

Although a large number of signalling cascades are known to be activated downstream of NCAM, only little is known regarding the hierarchical relationship between the involved molecules in the individual cascades and the level of cross talk between the cascades. Here, we evaluated the requirement of putative upstream signalling cascades for the phosphorylation of the kinases extracellular signal-regulated kinase (ERK) and Akt and the transcription factor cyclic adenosine monophosphate (cAMP) response-element binding protein (CREB) following stimulation of NCAM in rat cerebellar granule neurons with an NCAM ligand, the C3d peptide. NCAM-mediated ERK phosphorylation depended on activation of the fibroblast growth factor receptor (FGFR), Src-family kinases, MEK (MAP and ERK kinase) and G 0/G i-proteins, whereas NCAM-mediated CREB phosphorylation depended on the activity of Src-family kinases and MEK. NCAM-specific Akt phosphorylation depended on cyclic guanosine monophosphate (cGMP) and phosphatidylinositide 3-kinase (PI3K). All three phosphorylation events were independent of activation of the signalling molecules phospholipase C, protein kinase C, protein kinase A, and CamKII, which all have been demonstrated previously to be involved in NCAM signalling. For comparison, we also evaluated the role of upstream signalling cascades on fibroblast growth factor 2 (FGF2)-mediated phosphorylation of ERK, Akt, and CREB and found that FGF2 required the activity of both FGFR and Src-family kinases for phosphorylation of ERK, Akt, and CREB. MEK was required for phosphorylation of ERK and CREB, but not Akt, whereas G 0/G i-proteins were necessary for phosphorylation of Akt and CREB, and cGMP was necessary for Akt phosphorylation. We thus demonstrate that even though NCAM and FGF2 have many signalling features in common, and even though both are known to activate FGFR, there are a number of differences in the intracellular signalling network activated by the NCAM ligand C3d and the FGFR ligand FGF2.

BIBF 1120: Triple Angiokinase Inhibitor with Sustained Receptor Blockade and Good Antitumor Efficacy

Inhibition of tumor angiogenesis through blockade of the vascular endothelial growth factor (VEGF) signaling pathway is a novel treatment modality in oncology. Preclinical findings suggest that long-term clinical outcomes may improve with blockade of additional proangiogenic receptor tyrosine kinases: platelet-derived growth factor receptors (PDGFR) and fibroblast growth factor receptors (FGFR). BIBF 1120 is an indolinone derivative potently blocking VEGF receptor (VEGFR), PDGFR and FGFR kinase activity in enzymatic assays (IC(50), 20-100 nmol/L). BIBF 1120 inhibits mitogen-activated protein kinase and Akt signaling pathways in three cell types contributing to angiogenesis, endothelial cells, pericytes, and smooth muscle cells, resulting in inhibition of cell proliferation (EC(50), 10-80 nmol/L) and apoptosis. In all tumor models tested thus far, including human tumor xenografts growing in nude mice and a syngeneic rat tumor model, BIBF 1120 is highly active at well-tolerated doses (25-100 mg/kg daily p.o.), as measured by magnetic resonance imaging of tumor perfusion after 3 days, reducing vessel density and vessel integrity after 5 days, and inducing profound growth inhibition. A distinct pharmacodynamic feature of BIBF 1120 in cell culture is sustained pathway inhibition (up to 32 hours after 1-hour treatment), suggesting slow receptor off-kinetics. Although BIBF 1120 is rapidly metabolized in vivo by methylester cleavage, resulting in a short mean residence time, once daily oral dosing is fully efficacious in xenograft models. These distinctive pharmacokinetic and pharmacodynamic properties may help explain clinical observations with BIBF 1120, currently entering phase III clinical development.

Voltage-Gated Na+Channel β1 Subunit-Mediated Neurite Outgrowth Requires Fyn Kinase and Contributes to Postnatal CNS DevelopmentIn Vivo

Voltage-gated Na(+) channel beta1 subunits are multifunctional, participating in channel modulation and cell adhesion in vitro. We previously demonstrated that beta1 promotes neurite outgrowth of cultured cerebellar granule neurons (CGNs) via homophilic adhesion. Both lipid raft-associated kinases and nonraft fibroblast growth factor (FGF) receptors are implicated in cell adhesion molecule-mediated neurite extension. In the present study, we reveal that beta1-mediated neurite outgrowth is abrogated in Fyn and contactin (Cntn) null CGNs. beta1 protein levels are unchanged in Fyn null brains, whereas levels are significantly reduced in Cntn null brain lysates. FGF or EGF (epidermal growth factor) receptor kinase inhibitors have no effect on beta1-mediated neurite extension. These results suggest that beta1-mediated neurite outgrowth occurs through a lipid raft signaling mechanism that requires the presence of both fyn kinase and contactin. In vivo, Scn1b null mice show defective CGN axon extension and fasciculation indicating that beta1 plays a role in cerebellar microorganization. In addition, we find that axonal pathfinding and fasciculation are abnormal in corticospinal tracts of Scn1b null mice consistent with the suggestion that beta1 may have widespread effects on postnatal neuronal development. These data are the first to demonstrate a cell-adhesive role for beta1 in vivo. We conclude that voltage-gated Na(+) channel beta1 subunits signal via multiple pathways on multiple timescales and play important roles in the postnatal development of the CNS.

Epidermal Growth Factor Stimulates RSK2 Activation through Activation of the MEK/ERK Pathway and Src-dependent Tyrosine Phosphorylation of RSK2 at Tyr-529

The Ser/Thr kinase ribosomal S6 kinase 2 (RSK2) has been demonstrated to phosphorylate transcription factor CREB (cyclic AMP-responsive-binding protein) and histone H3 in response to mitogenic stimulation by epidermal growth factor (EGF). EGF activates the MEK/ERK pathway to activate RSK2. We recently reported that receptor tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) directly tyrosine phosphorylates RSK2 at Tyr-529, which consequently regulates RSK2 activation by facilitating inactive ERK binding to RSK2 that is required for ERK-dependent phosphorylation and activation of RSK2 (Kang, S., Dong, S., Gu, T. L., Guo, A., Cohen, M. S., Lonial, S., Khoury, H. J., Fabbro, D., Gilliland, D. G., Bergsagel, P. L., Taunton, J., Polakiewicz, R. D., and Chen, J. (2007) Cancer Cell 12, 201-214). Here we report that upon treatment of EGF, RSK2 was tyrosine-phosphorylated at Tyr-529 and activated in 293T and COS7 cells that do not express FGFR3. In contrast to FGFR3, the receptor tyrosine kinase EGF receptor did not directly phosphorylate RSK2 at Tyr-529 in an in vitro kinase assay using recombinant RSK2 and active EGF receptor or FGFR3. By mass spectroscopy-based studies, we identified Src tyrosine kinase family members Src and Fyn as upstream kinases of RSK2 Tyr-529. Treatment of Src inhibitor PP2 effectively attenuated EGF-dependent activation and Tyr-529 phosphorylation of RSK2, suggesting that Src family members are the kinases that phosphorylate RSK2 at Tyr-529 in response to EGF. Src and Fyn were able to directly phosphorylate RSK2 at Tyr-529 in the in vitro kinase assay. In vitro reconstitution of Tyr-529 phosphorylation by Src in glutathione S-transferase-tagged RSK2 enhanced inactive ERK binding to RSK2 wild type, but not the Y529F mutant. Together, our findings suggest that Src-dependent phosphorylation at Tyr-529 facilitates inactive ERK binding to RSK2, which might be a general requirement for RSK2 activation by EGF through the MEK/ERK pathway.