Discovery of Potent and Selective Inhibitors of Wild-Type and Gatekeeper Mutant Fibroblast Growth Factor Receptor (FGFR) 2/3.

Upregulation of the fibroblast growth factor receptor (FGFR) signaling pathway has been implicated in multiple cancer types, including cholangiocarcinoma and bladder cancer. Consequently, small molecule inhibition of FGFR has emerged as a promising therapy for patients suffering from these diseases. First-generation pan-FGFR inhibitors, while highly effective, suffer from several drawbacks. These include treatment-related hyperphosphatemia and significant loss of potency for the mutant kinases. Herein, we present the discovery and optimization of novel FGFR2/3 inhibitors that largely maintain potency for the common gatekeeper mutants and have excellent selectivity over FGFR1. A combination of meticulous structure-activity relationship (SAR) analysis, structure-based drug design, and medicinal chemistry rationale ultimately led to compound 29, a potent and selective FGFR2/3 inhibitor with excellent in vitro absorption, distribution, metabolism, excretion (ADME), and pharmacokinetics in rat. A pharmacodynamic study of a closely related compound established that maximum inhibition of downstream ERK phosphorylation could be achieved with no significant effect on serum phosphate levels relative to vehicle.

Fibroblast growth factor (FGF), FGF receptor (FGFR), and cyclin D1 (CCND1) DNA methylation in head and neck squamous cell carcinomas is associated with transcriptional activity, gene amplification, human papillomavirus (HPV) status, and sensitivity to tyrosine kinase inhibitors.

BACKGROUND: Dysregulation of fibroblast growth factor receptor (FGFR) signaling pathway has been observed in head and neck squamous cell carcinoma (HNSCC) and is a promising therapeutic target for selective tyrosine kinase inhibitors (TKIs). Potential predictive biomarkers for response to FGFR-targeted therapies are urgently needed. Understanding the epigenetic regulation of FGF pathway related genes, i.e. FGFRs, FGFs, and CCND1, could enlighten the way towards biomarker-selected FGFR-targeted therapies. METHODS: We performed DNA methylation analysis of the encoding genes FGFR1, FGFR2, FGFR3, FGFR4, FGF1-14, FGF16-23, and CCND1 at single CpG site resolution (840 CpG sites) employing The Cancer Genome Research Atlas (TCGA) HNSCC cohort comprising N = 530 tumor tissue and N = 50 normal adjacent tissue samples. We correlated DNA methylation to mRNA expression with regard to human papilloma virus (HPV) and gene amplification status. Moreover, we investigated the correlation of methylation with sensitivity to the selective FGFR inhibitors PD 173074 and AZD4547 in N = 40 HPV(-) HNSCC cell lines. RESULTS: We found sequence-contextually nuanced CpG methylation patterns in concordance with epigenetically regulated genes. High methylation levels were predominantly found in the promoter flank and gene body region, while low methylation levels were present in the central promoter region for most of the analyzed CpG sites. FGFRs, FGFs, and CCND1 methylation differed significantly between tumor and normal adjacent tissue and was associated with HPV and gene amplification status. CCND1 promoter methylation correlated with CCND1 amplification. For most of the analyzed CpG sites, methylation levels correlated to mRNA expression in tumor tissue. Furthermore, we found significant correlations of DNA methylation of specific CpG sites with response to the FGFR1/3-selective inhibitors PD 173074 and AZD4547, predominantly within the transcription start site of CCND1. CONCLUSIONS: Our results suggest an epigenetic regulation of CCND1, FGFRs, and FGFs via DNA methylation in HNSCC and warrants further investigation of DNA methylation as a potential predictive biomarker for response to selective FGFR inhibitors in clinical trials.

Fibroblast growth factor receptor as a potential candidate for phosphate sensing.

PURPOSE OF REVIEW: Phosphate plays essential roles in many biological processes. Serum phosphate level needs to be regulated because hypophosphatemia and hyperphosphatemia cause rickets/osteomalacia and ectopic calcification, respectively. Fibroblast growth factor (FGF) 23 is the principal hormone to regulate serum phosphate level. FGF23 is produced by the bone and works to reduce serum phosphate level by binding to FGF receptor (FGFR) 1c and α-Klotho complex in the kidney. It has been unclear how the bone senses the changes of serum phosphate level and how the bone regulates the production of FGF23. RECENT FINDINGS: Our recent results indicate that high extracellular phosphate activates FGFR1c. Its downstream intracellular signalling pathway regulates the expression of GALNT3 encoding a protein involved in the regulation of the posttranslational modification of FGF23 protein. This FGFR1c-GALNT3 axis is considered to be the main regulatory mechanism of enhanced FGF23 production in response to high phosphate. SUMMARY: We propose that FGFR1c works as a phosphate-sensing molecule in the regulation of FGF23 production and serum phosphate level. Feedback system is present in the regulation of serum phosphate involving FGFR1c and FGF23. These findings uncover so far unrecognized function of FGFR and molecular basis of phosphate sensing.

Blockade of Pan-Fibroblast Growth Factor Receptors Mediates Bidirectional Effects in Lung Fibrosis.

FGFs (fibroblast growth factors) are major factors associated with the pathogenesis of pulmonary fibrosis. On the one hand, nintedanib, a tyrosine kinase inhibitor targeting several growth factor receptors, including the FGF receptor (FGFR), has been approved for the treatment of idiopathic pulmonary fibrosis. On the other hand, recent reports suggest that FGFs are required for epithelial recovery. In this study, we focused on FGF signaling to both fibroblasts and alveolar epithelial cells (AECs), and we examined the effect of a pan-FGFR blocker on experimental pulmonary fibrosis in mice. The effects of BGJ398, a pan-FGFR inhibitor, on the migration and proliferation of fibroblasts and AECs were assessed using Transwell migration or [3H]thymidine incorporation assays. The expression of FGFR was analyzed using IB or flow cytometry. We also investigated the effect of BGJ398 on pulmonary fibrosis induced by bleomycin in mice. Both lung fibroblasts and AECs expressed FGFRs. BGJ398 significantly inhibited the proliferation and migration of lung fibroblasts stimulated with FGF2. BGJ398 also reduced the proliferation of AECs in response to FGF2. Although the administration of BGJ398 ameliorated pulmonary fibrosis in bleomycin-treated mice, it increased mortality resulting from alveolar injury and inhibition of AEC regeneration. These data suggest that the total inhibition of FGFR signaling can suppress lung fibrosis by inhibiting fibroblast activities, although alveolar injury is simultaneously caused.

Nintedanib-induced glomerular microangiopathy: a case report.

Nintedanib, a triple tyrosine kinase inhibitor of vascular endothelial growth factor receptor, platelet-derived growth factor receptor, and fibroblast growth factor receptor, has been used in idiopathic pulmonary fibrosis and adenocarcinoma in advanced non-small cell lung cancer. Although vascular endothelial growth factor inhibitors have been reported to cause endothelial injury and glomerular microangiopathy, nintedanib-induced glomerular microangiopathy has not been reported. A 68-year-old man with a history of primary aldosteronism, idiopathic pulmonary fibrosis, and pleomorphic carcinoma of the lung developed proteinuria and leg edema after nintedanib initiation. Kidney biopsy revealed prominent endothelial and mesangial injury. Proteinuria improved after nintedanib withdrawal. To the best of our knowledge, this is the second case report of nintedanib-induced glomerular microangiopathy. Although the incidence of nephropathy among patients receiving nintedanib is unknown at this moment, we recommend monitoring urinary protein excretion and blood pressure in patients receiving nintedanib and performing kidney biopsy to determine any histopathological change.

Evaluation of fibroblast growth factor activity exerted by placental extract used as a cosmetic ingredient.

BACKGROUND: The biological activities claimed for placental extract (PE) in its medical and cosmetic applications are largely assumed to be the combined effects of its various signaling molecules and nutritional constituents. But there are considerable uncertainties about this assumption. AIMS: To determine the specific biological activity of PE at a molecular level. METHODS: Fibroblast growth factor (FGF) activity was assessed based on the ability to induce proliferation of FGF receptor (FGFR)-overexpressing BaF3 cells. RESULTS: Porcine PE (PPE), an ingredient in numerous cosmetics, activated proliferation of BaF3 cells overexpressing FGFR subtypes 1c, 2c, 2b, 3c, or 4, that is, all the major FGFR subtypes. The effect was suppressed largely or partially when the cells were treated with a FGFR inhibitor PD173074, and the FGFR-negative BaF3 parent cells exhibited minimal growth promotion as compared to the FGFR-expressing BaF3 cells. The high (>10 kDa) and low (<3 kDa) molecular weight fractions of PPE were effective activators of FGFR signaling. PPE was found to contain sulfated glycosaminoglycans, including heparin/heparan sulfate and chondroitin sulfate, which serve as both structural stabilizers of FGFs and indispensable cofactors for FGF-FGFR signaling. CONCLUSIONS: These results indicate that PPE is capable of evoking FGF signaling in cells via FGFRs. Given that recombinant FGFs have proven useful for medical/cosmetic purposes, our results suggest that the medical/cosmetic utility of PPE is provided at least partly through the activation of FGF signaling in epidermal, dermal, and subdermal tissues.

[Regulation of airway stem cell proliferation in idiopathic pulmonary fibrosis].

OBJECTIVE: To investigate the effect of fibroblasts on regulating airway stem cell proliferation in idiopathic pulmonary fibrosis. METHODS: Lung cell suspension was prepared from ß-actin-GFP mice. Airway stem cells were obtained by fluorescence activated cell sorting and co-cultured with lung fibroblasts. The fibroblasts were treated with TGF-ß inhibitor SB43142. The expression of growth factors FGF1/2 and the effect of FGF1/2 on stem cell proliferation were observed. RESULTS: The cloning efficiency of airway stem cells, when co-cultured with normal lung fibroblast cells for 8 days, was (3.5±1.1)%, while the cloning efficiency was reduced to (0.04±0.04)% when co-cultured with lung fibroblasts from idiopathic pulmonary fibrosis patients. The difference between the 2 groups was statistically significant(P=0.002 5). TGF-ß receptor inhibitor SB431542 increased lung fibroblast growth factors FGF1/2 expression.FGF1 mRNA expression was increased to the experimental group 0.005 5 from 0.000 2 in the control group.FGF2 mRNA expression of the amount raised to the experimental group 0.000 15 from 0.000 8 in the control group.FGF1/2 promoted the growth of airway stem cells. After FGF1/2 was co-cultured with normal lung fibroblast cells for 8 days, the cloning efficiency of airway stem cells was (0.3±0.1)%. CONCLUSION: During the development of idiopathic pulmonary fibrosis, fibroblast secreted FGF1/2 regulate airway stem cell proliferation.

The effects of mycophenolate mofetil on cytokines and their receptors in pulmonary arterial hypertension in rats.

OBJECTIVES: To analyse the effects of mycophenolate mofetil (MMF) on pulmonary artery pressure (PAP) and the expression of cytokines and their receptors in a rat model of pulmonary arterial hypertension (PAH). METHOD: Thirty-eight healthy male inbred Sprague-Dawley rats were divided randomly into four groups: a control group, a monocrotaline (MCT) group, an MMF20 group (MCT+20 mg/kg/day MMF), and an MMF40 group (MCT+40 mg/kg/day MMF). Systolic PAP (SPAP), the right ventricular hypertrophy index (RVI), and the levels of expression of cytokines and their receptors were measured and analysed. RESULTS: SPAP, RVI, levels of expression of basic fibroblast growth factor (bFGF) in serum and lung homogenates, alveolar arterial wall thickness, and the number of muscular arteries in the MMF20 and MMF40 groups were decreased in comparison with the MCT group. CONCLUSIONS: MMF inhibits the formation of vascular muscle and decreases SPAP and RVI by inhibition of the expression of bFGF, endothelin-1 (ET-1), and their receptors, resulting in the inhibition of smooth muscle proliferation and amelioration of PAH.

Promotion of Dental Pulp Cell Migration and Pulp Repair by a Bioceramic Putty Involving FGFR-mediated Signaling Pathways.

Mineral trioxide aggregate is the currently recommended material of choice for clinical pulp repair despite several disadvantages, including handling inconvenience. Little is known about the signaling mechanisms involved in bioceramic-mediated dental pulp repair-particularly, dental pulp cell (DPC) migration. This study evaluated the effects of iRoot BP Plus, a novel ready-to-use nanoparticulate bioceramic putty, on DPC migration in vitro and pulp repair in vivo, focusing on possible involvement of fibroblast growth factor receptor (FGFR)-related signaling, including mitogen-activated protein kinase and Akt pathways. Treatment with iRoot BP Plus extracts enhanced horizontal and vertical migration of DPCs, which was comparable with the effects induced by mineral trioxide aggregate extracts. The DPCs exposed to iRoot BP Plus extracts demonstrated no evident apoptosis. Importantly, treatment with iRoot BP Plus extracts resulted in rapid activation of FGFR, p38 mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK) 1/2, c-Jun-N-terminal kinase (JNK), and Akt signaling in DPCs. Confocal immunofluorescence staining revealed that iRoot BP Plus stimulated focal adhesion formation and stress fiber assembly in DPCs, in addition to upregulating the expression of focal adhesion molecules, including p-focal adhesion kinase, p-paxillin, and vinculin. Moreover, activation of FGFR, ERK, JNK, and Akt were found to mediate the upregulated expression of focal adhesion molecules, stress fiber assembly, and enhanced DPC migration induced by iRoot BP Plus. Consistent with the in vitro results, we observed induction of homogeneous dentin bridge formation and expression of p-focal adhesion kinase, p-FGFR, p-ERK 1/2, p-JNK, and p-Akt near injury sites by iRoot BP Plus in an in vivo pulp repair model. These data demonstrate that iRoot BP Plus can promote DPC migration and pulp repair involving the FGFR-mediated ERK 1/2, JNK, and Akt pathways. These findings provide valuable insights into the signaling mechanisms underlying nanoparticulate bioceramic-mediated pulp repair.

Anti-fibrotic effects of nintedanib in lung fibroblasts derived from patients with idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. The kinase inhibitor nintedanib specific for vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR) and fibroblast growth factor receptor (FGFR) significantly reduced the rate of decline of forced vital capacity versus placebo. AIM: To determine the in vitro effect of nintedanib on primary human lung fibroblasts. METHODS: Fibroblasts were isolated from lungs of IPF patients and from non-fibrotic controls. We assessed the effect of VEGF, PDGF-BB and basic FGF (bFGF) ± nintedanib on: (i) expression/activation of VEGFR, PDGFR, and FGFR, (ii) cell proliferation, secretion of (iii) matrix metalloproteinases (MMP), (iv) tissue inhibitor of metalloproteinase (TIMP), and (v) collagen. RESULTS: IPF fibroblasts expressed higher levels of PDGFR and FGFR than controls. PDGF-BB, bFGF, and VEGF caused a pro-proliferative effect which was prevented by nintedanib. Nintedanib enhanced the expression of pro-MMP-2, and inhibited the expression of TIMP-2. Transforming growth factor-beta-induced secretion of collagens was inhibited by nintedanib. CONCLUSION: Our data demonstrate a significant anti-fibrotic effect of nintedanib in IPF fibroblasts. This effect consists of the drug's anti-proliferative capacity, and on its effect on the extracellular matrix, the degradation of which seems to be enhanced.

Fibroblast growth factor receptor 2 tyrosine kinase fusions define a unique molecular subtype of cholangiocarcinoma.

UNLABELLED: Cholangiocarcinoma is an intractable cancer, with limited therapeutic options, in which the molecular mechanisms underlying tumor development remain poorly understood. Identification of a novel driver oncogene and applying it to targeted therapies for molecularly defined cancers might lead to improvements in the outcome of patients. We performed massively parallel whole transcriptome sequencing in eight specimens from cholangiocarcinoma patients without KRAS/BRAF/ROS1 alterations and identified two fusion kinase genes, FGFR2-AHCYL1 and FGFR2-BICC1. In reverse-transcriptase polymerase chain reaction (RT-PCR) screening, the FGFR2 fusion was detected in nine patients with cholangiocarcinoma (9/102), exclusively in the intrahepatic subtype (9/66, 13.6%), rarely in colorectal (1/149) and hepatocellular carcinoma (1/96), and none in gastric cancer (0/212). The rearrangements were mutually exclusive with KRAS/BRAF mutations. Expression of the fusion kinases in NIH3T3 cells activated MAPK and conferred anchorage-independent growth and in vivo tumorigenesis of subcutaneous transplanted cells in immune-compromised mice. This transforming ability was attributable to its kinase activity. Treatment with the fibroblast growth factor receptor (FGFR) kinase inhibitors BGJ398 and PD173074 effectively suppressed transformation. CONCLUSION: FGFR2 fusions occur in 13.6% of intrahepatic cholangiocarcinoma. The expression pattern of these fusions in association with sensitivity to FGFR inhibitors warrant a new molecular classification of cholangiocarcinoma and suggest a new therapeutic approach to the disease.

A RTK-based functional RNAi screen reveals determinants of PTX-3 expression.

AIM: The aim of the present study was to explore the role of receptor tyrosine kinases (RTKs) in the regulation of expression of PTX-3, a protector in atherosclerosis. METHODS: Human monocytic U937 cells were infected with a shRNA lentiviral vector library targeting human RTKs upon LPS stimuli and PTX-3 expression was determined by ELISA analysis. The involvement of downstream signaling in the regulation of PTX-3 expression was analyzed by both Western blotting and ELISA assay. RESULTS: We found that knocking down of ERBB2/3, EPHA7, FGFR3 and RET impaired PTX-3 expression without effects on cell growth or viability. Moreover, inhibition of AKT, the downstream effector of ERBB2/3, also reduced PTX-3 expression. Furthermore, we showed that FGFR3 inhibition by anti-cancer drugs attenuated p38 activity, in turn induced a reduction of PTX-3 expression. CONCLUSION: Altogether, our study demonstrates the role of RTKs in the regulation of PTX-3 expression and uncovers a potential cardiotoxicity effect of RTK inhibitor treatments in cancer patients who have symptoms of atherosclerosis or are at the risk of atherosclerosis.

Structural stability of human fibroblast growth factor-1 is essential for protective effects against radiation-induced intestinal damage.

PURPOSE: Human fibroblast growth factor-1 (FGF1) has radioprotective effects on the intestine, although its structural instability limits its potential for practical use. Several stable FGF1 mutants were created increasing stability in the order, wild-type FGF1, single mutants (Q40P, S47I, and H93G), Q40P/S47I, and Q40P/S47I/H93G. This study evaluated the contribution of the structural stability of FGF1 to its radioprotective effect. METHODS AND MATERIALS: Each FGF1 mutant was administered intraperitoneally to BALB/c mice in the absence of heparin 24 h before or after total body irradiation (TBI) with γ-rays at 8-12 Gy. Several radioprotective effects were examined in the jejunum. RESULTS: Q40P/S47I/H93G could activate all subtypes of FGF receptors in vitro much more strongly than the wild-type without endogenous or exogenous heparin. Preirradiation treatment with Q40P/S47I/H93G significantly increased crypt survival more than wild-type FGF1 after TBI at 10 or 12 Gy, and postirradiation treatment with Q40P/S47I/H93G was effective in promoting crypt survival after TBI at 10, 11, or 12 Gy. In addition, crypt cell proliferation, crypt depth, and epithelial differentiation were significantly promoted by postirradiation treatment with Q40P/S47I/H93G. The level of stability of FGF1 mutants correlated with their mitogenic activities in vitro in the absence of heparin; however, preirradiation treatment with the mutants increased the crypt number to almost the same level as Q40P/S47I/H93G. When given 24 h after TBI at 10 Gy, all FGF1 mutants increased crypt survival more than wild-type FGF1, and Q40P/S47I/H93G had the strongest mitogenic effects in intestinal epithelial cells after radiation damage. Moreover, Q40P/S47I/H93G prolonged mouse survival after TBI because of the repair of intestinal damage. CONCLUSION: These findings suggest that the structural stability of FGF1 can contribute to the enhancement of protective effects against radiation-induced intestinal damage. Therefore, Q40P/S47I/H93G is pharmacologically one of the most promising candidates for clinical applications for radiation-induced gastrointestinal syndrome.

[Possibilities for inhibiting tumor-induced angiogenesis: results with multi-target tyrosine kinase inhibitors]. / A tumorindukált angiogenezis gátlásának lehetõségei: eredmények multi-target tirozinkináz-gátlókkal.

Functional blood vasculature is essential for tumor progression. The main signalization pathways that play a key role in the survival and growth of tumor vessels originate from the VEGF-, PDGF- and FGF tyrosine kinase receptors. In the past decade, significant results have been published on receptor tyrosine kinase inhibitors (RTKIs). In this paper, the mechanisms of action and the results so far available of experimental and clinical studies on multi-target antiangiogenic TKIs are discussed. On the one hand, notable achievements have been made recently and these drugs are already used in clinical practice in some patient populations. On the other hand, the optimal combination and dosage of these drugs, selection of the apropriate biomarker and better understanding of the conflicting role of PDGFR and FGFR signaling in angiogenesis remain future challenges.

Adding to the mix: fibroblast growth factor and platelet-derived growth factor receptor pathways as targets in non-small cell lung cancer.

The treatment of advanced non � small cell lung cancer (NSCLC) increasingly involves the use of molecularly targeted therapy with activity against either the tumor directly, or indirectly, through activity against host-derived mechanisms of tumor support such as angiogenesis. The most well studied signaling pathway associated with angiogenesis is the vascular endothelial growth factor (VEGF) pathway, and the only antiangiogenic agent currently approved for the treatment of NSCLC is bevacizumab, an antibody targeted against VEGF. More recently, preclinical data supporting the role of fibroblast growth factor receptor (FGFR) and platelet-derived growth factor receptor (PDGFR) signaling in angiogenesis have been reported. The platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) pathways may also stimulate tumor growth directly through activation of downstream mitogenic signaling cascades. In addition, 1 or both of these pathways have been associated with resistance to agents targeting the epidermal growth factor receptor (EGFR) and VEGF. A number of agents that target FGF and/or PDGF signaling are now in development for the treatment of NSCLC. This review will summarize the potential molecular roles of PDGFR and FGFR in tumor growth and angiogenesis, as well as discuss the current clinical status of PDGFR and FGFR inhibitors in clinical development.

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.

TSU68, an antiangiogenic receptor tyrosine kinase inhibitor, induces tumor vascular normalization in a human cancer xenograft nude mouse model.

PURPOSE: Combination therapy using antiangiogenic and cytotoxic agents is a useful strategy for advanced cancer, but the mechanism has not yet been elucidated. Moreover, there is a persistent paradox that destroying tumor vasculature with antiangiogenic agents disturbs the delivery of cytotoxic agents. It has been hypothesized that antiangiogenic agents can lead to normalization of tumor vessels that are structurally and functionally abnormal. The normalization means enhancing the deliver of cytotoxic agents. Our purpose was to investigate whether TSU68, a multiple receptor tyrosine kinase inhibitor that targets vascular endothelial growth factor receptor-2 (VEGFR2), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR), would induce the normalization of tumor vessels. METHODS: TSU68 was administered for 7 days to mice with xenografted tumors. Tumors of interstitial fluid pressure (IFP) were measured before and after administration of agents. Immunofluorescence double staining for CD31 and alpha-SMA was performed, and a medical video endoscopy system with narrowband illumination (NBI) was used to visualize the vascular pattern. RESULTS: TSU68 treatment decreased IFP significantly. Immunofluorescence double staining showed a significant increase in the fraction of pericyte coverage in the TSU68-treated group. NBI endoscopy showed that many tumor vessels in TSU68-treated mice were pruned and the diameters of remaining vessels were reduced. CONCLUSION: The data supported our hypothesis of tumor vascular normalization by the antiangiogenic agent TSU68.

The fibroblast growth factor receptor inhibitor PD173074 blocks small cell lung cancer growth in vitro and in vivo.

Lung cancer is the commonest cancer killer. Small cell lung cancer (SCLC) is initially chemosensitive, but rapidly relapses in a chemoresistant form with an overall survival of <5%. Consequently, novel therapies are urgently required and will likely arise from an improved understanding of the disease biology. Our previous work showed that fibroblast growth factor-2 induces proliferation and chemoresistance in SCLC cells. Here, we show that the selective fibroblast growth factor receptor (FGFR) inhibitor PD173074 blocks H-510 and H-69 SCLC proliferation and clonogenic growth in a dose-dependent fashion and prevents FGF-2-induced chemoresistance. These effects correlate with the inhibition of both FGFR1 and FGFR2 transphosphorylation. We then determined the efficacy of daily oral administration of PD173074 for 28 days in two human SCLC models. In the H-510 xenograft, tumor growth was impaired similar to that seen with single-agent cisplatin administration, increasing median survival compared with control sham-treated animals. Crucially, the effect of cisplatin was significantly potentiated by coadministration of PD173074. More dramatically, in H-69 xenografts, PD173074 induced complete responses lasting >6 months in 50% of mice. These effects were not a consequence of disrupted tumor vasculature but instead correlated with increased apoptosis (caspase 3 and cytokeratin 18 cleavage) in excised tumors. Moreover, in vivo imaging with 3'-deoxy-3'-[(18)F]fluorothymidine-positron emission tomography ([(18)F]FLT-PET) showed decreased intratumoral proliferation in live animals treated with the compound at 7 to 14 days. Our results suggest that clinical trials of FGFR inhibitors should be undertaken in patients with SCLC and that [(18)F]FLT-PET imaging could provide early in vivo evidence of response.

Neural cell adhesion molecule stimulates survival of premyelinating oligodendrocytes via the fibroblast growth factor receptor.

Axonal signals are critical in promoting the survival and maturation of oligodendrocytes during myelination, with contact-dependent signals thought to play a key role. However, the exact nature of these signals remains unclear. Neural cell adhesion molecule (NCAM) is expressed by both axons and oligodendrocytes and is ideally localized to transduce signals from the axon. This study sought to investigate the influence of NCAM on premyelinating oligodendrocytes in vitro. Both a soluble molecule comprising the extracellular domain of NCAM and a peptide derived from the fibroblast growth factor receptor (FGFR) binding motif within the first fibronectin domain stimulated a dose-dependent increase in survival of premyelinating oligodendrocytes in vitro. The survival effect was blocked by a mitogen-activated protein kinase (MAPK) inhibitor and an FGFR inhibitor, suggesting that activation of MAPK signalling pathways following interaction with the FGFR is involved in the survival effect of NCAM. Furthermore, NCAM presented in a cellular monolayer induced an increase in radial process outgrowth of oligodendrocyte progenitor cells. These data suggest that NCAM may play a role in axon-oligodendrocyte signalling during myelination, leading to an increase in oligodendrocyte survival and process outgrowth following axonal contact.

Fibroblast growth factor-derived peptides: functional agonists of the fibroblast growth factor receptor.

A series of peptides, termed dekafins, were derived from the beta10-beta11 loop regions of fibroblast growth factors (FGFs) 1, 2, 3, 5, 6, 8, 9, 10, and 17. The dekafins share a homologous amino acid sequence similar to a sequence in the first fibronectin type III module of the neural cell adhesion molecule. All dekafins were shown by surface plasmon resonance analysis to bind fibroblast growth factor receptor (FGFR)1-IIIc-Ig2-3 and FGFR2-IIIb-Ig2-3, respectively, with K(d) values of approximately 10(-7) to 10(-8) mol/L. Binding of dekafin1 to FGFR1-IIIc-Ig2-3 was inhibited by a heparin analog, sucrose octasulfate, indicating that heparin sulfate moiety can modulate dekafin binding to FGFRs. Treatment of transcription and mRNA export (TREX) cells permanently expressing Strep-tag-labeled FGFR1-IIIc with dekafins resulted in receptor phosphorylation. FGF1-induced FGFR1-IIIc phosphorylation was inhibited by dekafin1 and 10 in high concentrations, indicating that dekafins are FGFR partial agonists. The dekafins induced neuronal differentiation as reflected by neurite outgrowth from cerebellar granule neurons, an effect that was abolished by SU5402, a specific inhibitor of the FGFR tyrosine kinase, and by inositolhexaphosphate, an extracellularly acting FGFR antagonist. Some, but not all, dekafins were capable of promoting survival of cerebellar granule neurons induced to undergo apoptosis. Thus, the dekafins are functional FGFR agonists with apparent therapeutic potential.