[Effect of Juanbi Qianggu Formula on biological behaviors of fibroblast-like synoviocytes in rheumatoid arthritis by regulating FGFR1 signaling pathway based on network pharmacology and cell function experiments].

This study aimed to explore the molecular mechanism of Juanbi Qianggu Formula(JBQGF), an empirical formula formulated by the prestigious doctor in traditional Chinese medicine, in the treatment of rheumatoid arthritis based on network pharmacology and cell function experiments. The main active components and targets of JBQGF were obtained through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and Encyclopedia of Traditional Chinese Medicine(ETCM), and the core targets underwent functional enrichment analysis and signaling pathway analysis. Cytoscape 3.6.0 was used to construct a visualized "active component-target-signaling pathway" network of JBQGF. After screening, nine potential pathways of JBQGF were obtained, mainly including G protein-coupled receptor signaling pathway and tyrosine kinase receptor signaling pathway. As previously indicated, the fibroblast growth factor receptor 1(FGFR1) signaling pathway was highly activated in active fibroblast-like synoviocytes(FLS) in rheumatoid arthritis, and cell and animal experiments demonstrated that inhibition of the FGFR1 signaling pathway could significantly reduce joint inflammation and joint destruction in collagen-induced arthritis(CIA) rats. In terms of the tyrosine kinase receptor signal transduction pathway, the analysis of its target genes revealed that FGFR1 might be a potential target of JBQGF for rheumatoid arthritis treatment. The biological effect of JBQGF by inhibiting FGFR1 phosphorylation was preliminarily verified by Western blot, Transwell invasion assay, and pannus erosion assay, thereby inhibiting matrix metalloproteinase 2(MMP2) and receptor activator of nuclear factor-κB ligand(RANKL) and suppressing the invasion of fibroblasts in rheumatoid arthritis and erosive effect of pannus bone. This study provides ideas for searching potential targets of rheumatoid arthritis treatment and TCM drugs through network pharmacology.

Hypocrellin A exerts antitumor effects by inhibiting the FGFR1 signaling pathway in non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer, which is the deadliest form of cancer worldwide. Recent studies have shown that genes in the fibroblast growth factor (FGF) family are highly mutated in lung cancer, and fibroblast growth factor receptor 1 (FGFR1) has been found to be involved in various cancers, including lung cancer, suggesting that FGFR1 is a valid therapeutic target. Hypocrellin A (HA), a molecule with multiple biological activities, has been shown to influence cancer growth, but the specific mechanisms of its antitumor action have not been fully explored. METHODS: MTT, colony formation, wound healing, transwell cell invasion and EdU cell proliferation assays were performed upon HA treatment of three NSCLC cell lines, H460, PC-9 and H1975. Hoechst 33258 staining and caspase 3 activity assays were carried out to investigate the impact of HA on apoptosis in these cells. Molecular docking and surface plasmon resonance were conducted to assess binding of HA to FGFR1. A mouse tumor model was used to detect the NSCLC-inhibitory ability of HA in vivo. RESULTS: Through in vitro assays, HA was shown to negatively impact cell viability, migration, invasion and promote apoptosis in three human NSCLC cell line models. HA was shown to bind to FGFR1 and to inhibit its autophosphorylation and the phosphorylation of downstream signaling molecules. Inhibition of tumor growth was also demonstrated in a mouse xenograft tumor model, and no toxic effects of HA treatment were observed. CONCLUSIONS: HA inhibits the activity of the FGFR1 and STAT3 signaling pathways. HA thus represents a potential new FGFR1-targeted treatment for NSCLC.

FGF21 in obesity and cancer: New insights.

The endocrine FGF21 was discovered as a novel metabolic regulator in 2005 with new functions bifurcating from the canonic heparin-binding FGFs that directly promote cell proliferation and growth independent of a co-receptor. Early studies have demonstrated that FGF21 is a stress sensor in the liver and possibly, several other endocrine and metabolic tissues. Hepatic FGF21 signals via endocrine routes to quench episodes of metabolic derangements, promoting metabolic homeostasis. The convergence of mouse and human studies shows that FGF21 promotes lipid catabolism, including lipolysis, fatty acid oxidation, mitochondrial oxidative activity, and thermogenic energy dissipation, rather than directly regulating insulin and appetite. The white and brown adipose tissues and, to some extent, the hypothalamus, all of which host a transmembrane receptor binary complex of FGFR1 and co-receptor KLB, are considered the essential tissue and molecular targets of hepatic or pharmacological FGF21. On the other hand, a growing body of work has revealed that pancreatic acinar cells form a constitutive high-production site for FGF21, which then acts in an autocrine or paracrine mode. Beyond regulation of macronutrient metabolism and physiological energy expenditure, FGF21 appears to function in forestalling the development of fatty pancreas, steato-pancreatitis, fatty liver, and steato-hepatitis, thereby preventing the development of advanced pathologies such as pancreatic ductal adenocarcinoma or hepatocellular carcinoma. This review is intended to provide updates on these new discoveries that illuminate the protective roles of FGF21-FGFR1-KLB signal pathway in metabolic anomalies-associated severe tissue damage and malignancy, and to inform potential new preventive or therapeutic strategies for obesity-inflicted cancer patients via reducing metabolic risks and inflammation.

Involvement of estrogen in phosphorus-induced nephrocalcinosis through fibroblast growth factor 23.

Excessive phosphorus intake adversely affects bone and mineral metabolism. Estrogen is one of the factors affecting fibroblast growth factor 23 (FGF23), a phosphorus-regulating hormone. However, the interaction between excess phosphorus and estrogen status has not been fully elucidated. This study investigated the involvement of estrogen in the effects of high phosphorus intake on bone metabolism and ectopic calcification in ovariectomized (OVX) rats. The interaction between high phosphorus diet and OVX was not observed in bone mineral density and aortic calcium. In contrast, high phosphorus intake markedly increased renal calcium concentration in sham rats, whereas the effect was attenuated in OVX rats, which was reversed by a selective estrogen-receptor modulator treatment. A strong positive correlation between renal calcium and serum FGF23 was observed. In addition, fibroblast growth factor receptor 1 (FGFR1: a predominant receptor of FGF23) inhibitor treatment partially decreased renal calcium concentrations in rats with high phosphorus intake. In conclusion, the effect of high phosphorus intake on bone metabolism and aortic calcification did not depend on the estrogen status; in contrast, high phosphorus intake synergistically induced nephrocalcinosis in the presence of estrogenic action on the bone. Furthermore, FGF23 was involved in the nephrocalcinosis induced by high phosphorus intake partially through FGFR1 signaling.

Effect of anti-fibroblast growth factor receptor 1 antibodies on phosphorus metabolism in laying hens and their progeny chicks.

Targeting fibroblast growth factor 23 (FGF-23) signaling pathway is of interest in controlling body phosphate metabolism. This study investigated the effect of anti-fibroblast growth factor receptor 1 (FGFR1, major FGF-23 receptor in the kidney) antibodies on phosphate metabolism. White Leghorn laying hens (65-wk-old) were vaccinated with either a FGFR1 peptide vaccine (five 8-amino-acid peptides were selected, CrZ-1:LPEDPRWE, CrZ-2:LDKDKPNR, CrZ-3:RRPPGMEY, CrZ-4:GSPYPGVP, and CrZ-5:RMDKPSNC) or adjuvant control. At peak antibody titer, hens were artificially inseminated. Chicks from control-vaccinated hens were fed either a non-phytate phosphorus (nPP) sufficient (nPP = 0.45%, positive control) or deficient (nPP = 0.20%, negative control) diet, while chicks from each of the FGFR1 peptide vaccinated hens were fed with the above nPP-deficient diet, for 14 D. When compared to control hens, plasma phosphate in CrZ-1, CrZ-2, CrZ-3, CrZ-4, and CrZ-5 vaccinated hens were decreased by 33, 30, 24, 20, and 26%, respectively (P < 0.05); egg weight in CrZ-2 and CrZ-5 vaccinated hens were increased by 6 and 7%, respectively (P < 0.05); egg production in CrZ-3, CrZ-4, and CrZ-5 vaccinated hens tended to decrease (P = 0.085; decreased by 14, 15, and 13%, respectively). When compared to positive control, chicks from all other groups had decreased body weight gain (BWG) and feed intake (FI) during 1 to 14 D, and had decreased plasma phosphate, tibiotarsus ash, and 24-h phosphorus excretion on day 14. When compared to negative control, BWG of CrZ-1, CrZ-2, CrZ-3, and CrZ-4 antibody chicks were decreased by 23, 28, 26, and 20%, respectively (P < 0.05); FI of CrZ-1, CrZ-2, and CrZ-3 antibody chicks were decreased by 15, 15, and 18%, respectively (P < 0.05); plasma phosphate of CrZ-5 antibody chicks were decreased by 26% (P < 0.05); plasma FGF-23 levels of CrZ-4 antibody chicks were increased by 18% (P < 0.05); tibiotarsus ash content of CrZ-2, CrZ-3, and CrZ-4 antibody chicks were decreased by 20, 20, and 21%, respectively (P < 0.05). In conclusion, anti-FGFR1 peptide antibodies decreased egg production of hens and growth performance of their progeny chicks probably by activating FGF-23 signaling and stimulating FGF-23 production.

Bufalin suppresses the migration and invasion of prostate cancer cells through HOTAIR, the sponge of miR-520b.

Bufalin, the major active component of the traditional Chinese medicine ChanSu obtained from the skin and parotid venom glands of toads, has long been known as an anticancer agent. Recent studies show that microRNAs (miRs) are involved in the anticancer activities of bufalin, while long non-coding RNAs (lncRNAs) are known to interact with miRNAs to regulate various biological functions. In this paper, we investigated the possible network related to the antimetastatic effect of bufalin in prostate cancer (PCa) cells. We demonstrated that bufalin (0.05-10 µM) dose-dependently suppressed the proliferation of prostate cancer DU145 and PC3 cells with IC50 values of 0.89 and 1.28 µM, respectively. Furthermore, bufalin treatment significantly suppressed the cell migration and invasion. To explore the role of lncRNAs in the antimetastatic activity of bufalin, we used an lncRNA microarray and found that HOX transcript antisense RNA (HOTAIR) was the most markedly downregulated lncRNA in bufalin-treated PCa cells. Overexpression of HOTAIR counteracted the suppressing effects of bufalin on DU145 and PC3 cells. We then predicted and verified that HOTAIR upregulated FGFR1 expression by sponging miR-520b in PCa cells. In 40 patients with PCa bone metastasis, we used in situ hybridization or immunohistochemical assay to assess the HOTAIR and FGFR1 expression, which revealed that both HOTAIR and FGFR1 expression were significantly higher in bone metastasis tissues than in the primary PCa tissues. In addition, the level of serum HOTAIR was positively associated with the levels of serum bone metabolic markers (CTx, OST, B-ALP and PINP) and may serve as a reasonable biomarker for PCa bone metastasis. Taken together, this is the first study revealing that HOTAIR promotes PCa bone metastasis, and bufalin may be a promising candidate for the treatment of this disease.

FGF19 subfamily members: FGF19 and FGF21.

Fibroblast growth factors (FGF) constitute a large family of proteins with pleiotropic effects on development, organogenesis, and metabolism. The FGF19 subclass includes growth factors circulating with the blood referred to as endocrine FGF. Representatives of the FGF19 subclass, including FGF19, FGF21, and FGF23, act via FGFR receptors. The proteins of FGF19 subfamily influence the enterohepatic circulation of bile, participate in glucose and lipid metabolism regulation, and maintenance of phosphorus and vitamin D3 homeostasis. FGF19 and FGF21 are activated under different physiological and pathological conditions.

A novel FGFR1-binding peptide attenuates the degeneration of articular cartilage in adult mice.

OBJECTIVE: We previously reported that genetic ablation of (Fibroblast Growth Factors Receptors) FGFR1 in knee cartilage attenuates the degeneration of articular cartilage in adult mice, which suggests that FGFR1 is a potential targeting molecule for osteoarthritis (OA). Here, we identified R1-P1, an inhibitory peptide for FGFR1 and investigated its effect on the pathogenesis of OA in mice induced by destabilization of medial meniscus (DMM). DESIGN: Binding ability between R1-P1 and FGFR1 protein was evaluated by enzyme-linked immuno sorbent assay (ELISA) and molecular docking. Alterations in cartilage were evaluated histologically. The expression levels of molecules associated with articular cartilage homeostasis and FGFR1 signaling were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting and immunohistochemistry (IHC). The chondrocyte apoptosis was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay. RESULTS: R1-P1 had highly binding affinities to human FGFR1 protein, and efficiently inhibited extracellular signal-regulated kinase (ERK)1/2 pathway in mouse primary chondrocytes. In addition, R1-P1 attenuated the IL-1ß induced significant loss of proteoglycan in full-thickness cartilage tissue from human femur head. Moreover, this peptide can significantly restore the IL-1ß mediated loss of proteoglycan and type II collagen (Col II) and attenuate the expression of matrix metalloproteinase-13 (MMP13) in mouse primary chondrocytes. Finally, intra-articular injection of R1-P1 remarkably attenuated the loss of proteoglycan and the destruction of articular cartilage and decreased the expressions of extracellular matrix (ECM) degrading enzymes and apoptosis in articular chondrocytes of mice underwent DMM surgery. CONCLUSIONS: R1-P1, a novel inhibitory peptide for FGFR1, attenuates the degeneration of articular cartilage in adult mice, which is a potential leading molecule for the treatment of OA.

Investigation of the effects of Achillea millefolium extract in diabetic rats with second-degree burns.

Our aim was to investigate the effects of Achillea millefolium on wound healing in second-degree burns formed in diabetic rats. This study was conducted at the Kafkas University Experimental Research Center. 20 Sprague Dawley rats were divided into 2 groups. Group I (n=10 rats) was the Control group and contained the diabetic rats with burn injury. Group II (n=10 rats) was the group where burn injury was created and Achillea millefolium was administered to diabetic rats. The backs of the rats were shaved so as to include 30% of the body area. A 10% lanolin extract was administered for 14 days after the burn injury was created. Tissue was obtained from the burn area of the rats sacrificed. No significant difference was found in Group II in terms of the severity of the dermatitis and inflammatory cell reactions when compared to Group I. No significant difference was observed between FGFR1immunoreactivity in the epidermis and dermis in Group I. While FGFR1immunoreactivity in 3 rats in Group II was similar to Group I, strong immunore activity that was more prominent in the epidermis was found in 7 rats in Group II. We believe that Achillea millefolium contributes to wound healing in burn injury due to its antioxidant and anti-inflammatory properties.

A gut-brain axis regulating glucose metabolism mediated by bile acids and competitive fibroblast growth factor actions at the hypothalamus.

OBJECTIVE: Bile acids have been implicated as important regulators of glucose metabolism via activation of FXR and GPBAR1. We have previously shown that FGF19 can modulate glucose handling by suppressing the activity of hypothalamic AGRP/NPY neurons. As bile acids stimulate the release of FGF19/FGF15 into the circulation, we pursued the potential of bile acids to improve glucose tolerance via a gut-brain axis involving FXR and FGF15/FGF19 within enterocytes and FGF receptors on hypothalamic AGRP/NPY neurons. METHODS: A 5-day gavage of taurocholic acid, mirroring our previous protocol of a 5-day FGF19 treatment, was performed. Oral glucose tolerance tests in mice with genetic manipulations of FGF signaling and melanocortin signaling were used to define a gut-brain axis responsive to bile acids. RESULTS: The taurocholic acid gavage led to increased serum concentrations of taurocholic acid as well as increases of FGF15 mRNA in the ileum and improved oral glucose tolerance in obese (ob/ob) mice. In contrast, lithocholic acid, an FXR antagonist but a potent agonist for GPBAR1, did not improve glucose tolerance. The positive response to taurocholic acid is dependent upon an intact melanocortinergic system as obese MC4R-null mice or ob/ob mice without AGRP did not show improvements in glucose tolerance after taurocholate gavage. We also tested the FGF receptor isoform necessary for the bile acid response, using AGRP:Fgfr1-/- and AGRP:Fgfr2-/- mice. While the absence of FGFR1 in AGRP/NPY neurons did not alter glucose tolerance after taurocholate gavage, manipulations of Fgfr2 caused bidirectional changes depending upon the experimental model. We hypothesized the existence of an endogenous hypothalamic FGF, most likely FGF17, that acted as a chronic activator of AGRP/NPY neurons. We developed two short peptides based on FGF8 and FGF17 that should antagonize FGF17 action. Both of these peptides improved glucose homeostasis after a 4-day course of central and peripheral injections. Significantly, daily average blood glucose from continuous glucose monitoring was reduced in all tested animals but glucose concentrations remained in the euglycemia range. CONCLUSIONS: We have defined a gut-brain axis that regulates glucose metabolism mediated by antagonistic fibroblast growth factors. From the intestine, bile acids stimulate FGF15 secretion, leading to activation of the FGF receptors in hypothalamic AGRP/NPY neurons. FGF receptor intracellular signaling subsequently silences AGRP/NPY neurons, leading to improvements of glucose tolerance that are likely mediated by the autonomic nervous system. Finally, short peptides that antagonize homodimeric FGF receptor signaling within the hypothalamus have beneficial effects on glucose homeostasis without inducing hypoglycemia. These peptides could provide a new mode of regulating glucose metabolism.

Preclinical pharmacokinetic characterization of an adipose tissue-targeting monoclonal antibody in obese and non-obese animals.

Target receptor levels can influence pharmacokinetics (PK) or pharmacodynamics (PD) of monoclonal antibodies (mAbs), and can affect drug development of this class of molecules. We generated an effector-less humanized bispecific antibody that selectively activates fibroblast growth factor receptor (FGFR)1 and ßKlotho receptor, a FGF21 receptor complex highly expressed in both white and brown adipocytes. The molecule shows cross-species binding with comparable equilibrium binding affinity (Kd) for human, cynomolgus monkey, and mouse FGFR1/ßKlotho. To understand the PK/PD relationship in non-obese and obese animals, we evaluated the adipose tissue distribution of the antibody, serum exposures, and an associated PD marker (high-molecular-weight adiponectin), in both non-obese and obese mice and monkeys. Antibody uptake into fat tissue was found to be higher on a per gram basis in non-obese animals compared to obese animals. Since obesity has been reported to be associated with reduced expression of FGFR1 and ßKlotho receptor in white adipose tissues in mice, our results suggest that the distribution in adipose tissues was influenced by target expression levels. Even so, the overall dose-normalized serum exposures were comparable between non-obese and obese mice and monkeys, suggesting that adipose tissue uptake plays a limited role in overall systemic PK determination. It remains to be determined if and how obesity and receptor expression in humans influence the PK and PD profile of this novel therapeutic candidate.

Antiangiogenic Effects of Ziyuglycoside II, a Major Active Compound of Sanguisorba officinalis L.

Ziyuglycoside II, a major bioactive compound of Sanguisorba officinalis L., displays anticancer potential against several human cancer cells. However, little information concerning its antiangiogenic properties and possible mechanisms is available. The aim of this study was to investigate the inhibitory effects of ziyuglycoside II on angiogenesis. Ziyuglycoside II inhibited the proliferation, migration, and tubule formation of human umbilical vein endothelial cells, as well as the number of microvessels growing from the aortic rings. The underlying antiangiogenic mechanism of ziyuglycoside II correlated with blocking vascular endothelial growth factor receptor-2 and the fibroblast growth factor receptor-1 mediated signaling pathway. Moreover, an in vivo Matrigel plug assay in mice showed a significant decrease in vascularization and hemoglobin content in the plugs from ziyuglycoside II-treated mice compared with control mice. Overall, these results suggest that ziyuglycoside II inhibits various attributes of angiogenesis, which might contribute to its reported antitumor effects. Copyright © 2017 John Wiley & Sons, Ltd.

Thermal Preconditioning May Prevent Tendon Adhesion by Up-Regulating HSP72 in Rats.

BACKGROUND/AIMS: The study aims to determine the effects of thermal preconditioning on tendon adhesion by regulating the expression of heat shock protein 72 (HSP72) in rat models. METHODS: Sixty male Wistar rats were collected and randomly assigned into the thermal preconditioning and control groups. During the 4th and 8th weeks following surgery, 15 rats were sacrificed in each period respectively, and their tendon adhesion was observed and evaluated. Biomechanical testing was performed to measure the tensile strength and gliding distance of tendons. Hematoxylin-eosin (HE) was used to observe the morphological structure of the tendons. Immunohistochemical staining, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the HSP72, fibroblast growth factor-2 (FGF-2), fibroblast growth factor receptor-1 (FGFR-1), ß-catenin, epithelial cell adhesion molecule (EPCAM), Tenomodulin and scleraxis protein expressions. Pearson correlation analysis was applied to analyze the correlation between HSP72 expression and tendon adhesion. RESULTS: At the 4th week after surgery, we found no differences in the tendon adhesion scores or mRNA and protein expressions of HSP72 between the thermal preconditioning and control groups. However, after the 8th week after surgery, the thermal preconditioning group had a lower tendon adhesion score and higher mRNA and protein expressions of HSP72 than the control group. During the same period, we found longer gliding distance and higher expression levels of FGF-2, FGFR-1, ß-catenin, Tenomodulin and scleraxis, but lower EPCAM expression in the thermal preconditioning group. Pearson correlation analysis indicated that HSP72 mRNA and protein expression levels were negatively correlated with tendon adhesion. CONCLUSIONS: These findings provide evidence that thermal preconditioning may alleviate tendon adhesions via upregulation of HSP72 expression.

KLB, encoding ß-Klotho, is mutated in patients with congenital hypogonadotropic hypogonadism.

Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic form of isolated gonadotropin-releasing hormone (GnRH) deficiency caused by mutations in > 30 genes. Fibroblast growth factor receptor 1 (FGFR1) is the most frequently mutated gene in CHH and is implicated in GnRH neuron development and maintenance. We note that a CHH FGFR1 mutation (p.L342S) decreases signaling of the metabolic regulator FGF21 by impairing the association of FGFR1 with ß-Klotho (KLB), the obligate co-receptor for FGF21. We thus hypothesized that the metabolic FGF21/KLB/FGFR1 pathway is involved in CHH Genetic screening of 334 CHH patients identified seven heterozygous loss-of-function KLB mutations in 13 patients (4%). Most patients with KLB mutations (9/13) exhibited metabolic defects. In mice, lack of Klb led to delayed puberty, altered estrous cyclicity, and subfertility due to a hypothalamic defect associated with inability of GnRH neurons to release GnRH in response to FGF21. Peripheral FGF21 administration could indeed reach GnRH neurons through circumventricular organs in the hypothalamus. We conclude that FGF21/KLB/FGFR1 signaling plays an essential role in GnRH biology, potentially linking metabolism with reproduction.

Nintedanib, a triple tyrosine kinase inhibitor, attenuates renal fibrosis in chronic kidney disease.

Nintedanib (BIBF1120) is a triple kinase inhibitor of platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptors (FGFR), vascular endothelial growth factor receptor (VEGFR), and Src family kinase, which has recently been approved by FDA to treat idiopathic pulmonary fibrosis. Whether it affects renal fibrosis remains unknown. Here, we demonstrated that administration of nintedanib immediately or 3 days after unilateral ureteral obstruction (UUO) injury and with folic acid (FA) injection attenuated renal fibrosis and inhibited activation of renal interstitial fibroblasts. Delayed administration of nintedanib also partially reversed established renal fibrosis. Treatment with nintedanib blocked UUO-induced phosphorylation of PDGFRß, FGFR1, FGFR2, VEGFR2, and several Src family kinases including Src, Lck, Lyn as well as activation of signal transducer and activator of transcription-3 (STAT3), nuclear factor-κB (NF-κB), and Smad-3 in the kidney. Furthermore, nintedanib inhibited UUO-elicited renal proinflammatory cytokine expression and macrophage infiltration. These data indicate that nintedanib is a potent anti-fibrotic agent in the kidney and may hold therapeutic potential as a treatment of chronic fibrotic kidney disease.

The inhibition of FGF receptor 1 activity mediates sorafenib antiproliferative effects in human malignant pleural mesothelioma tumor-initiating cells.

BACKGROUND: Malignant pleural mesothelioma is an aggressive cancer, characterized by rapid progression and high mortality. Persistence of tumor-initiating cells (TICs, or cancer stem cells) after cytotoxic drug treatment is responsible for tumor relapse, and represents one of the main reasons for the poor prognosis of mesothelioma. In fact, identification of the molecules affecting TIC viability is still a significant challenge. METHODS: TIC-enriched cultures were obtained from 10 human malignant pleural mesotheliomas and cultured in vitro. Three fully characterized tumorigenic cultures, named MM1, MM3, and MM4, were selected and used to assess antiproliferative effects of the multi-kinase inhibitor sorafenib. Cell viability was investigated by MTT assay, and cell cycle analysis as well as induction of apoptosis were determined by flow cytometry. Western blotting was performed to reveal the modulation of protein expression and the phosphorylation status of pathways associated with sorafenib treatment. RESULTS: We analyzed the molecular mechanisms of the antiproliferative effects of sorafenib in mesothelioma TIC cultures. Sorafenib inhibited cell cycle progression in all cultures, but only in MM3 and MM4 cells was this effect associated with Mcl-1-dependent apoptosis. To investigate the mechanisms of sorafenib-mediated antiproliferative activity, TICs were treated with epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) causing, in MM3 and MM4 cells, MEK, ERK1/2, Akt, and STAT3 phosphorylation. These effects were abolished by sorafenib only in bFGF-treated cells, while a modest inhibition occurred after EGF stimulation, suggesting that sorafenib effects are mainly due to FGF receptor (FGFR) inhibition. Indeed, FGFR1 phosphorylation was inhibited by sorafenib. Moreover, in MM1 cells, which release high levels of bFGF and showed autocrine activation of FGFR1 and constitutive phosphorylation/activation of MEK-ERK1/2, sorafenib induced a more effective antiproliferative response, confirming that the main target of the drug is the inhibition of FGFR1 activity. CONCLUSIONS: These results suggest that, in malignant pleural mesothelioma TICs, bFGF signaling is the main target of the antiproliferative response of sorafenib, acting directly on the FGFR1 activation. Patients with constitutive FGFR1 activation via an autocrine loop may be more sensitive to sorafenib treatment and the analysis of this possibility warrants further clinical investigation.

Tyrosine Kinase Receptor Expression in Canine Liposarcoma.

The expression of tyrosine kinase receptors is attracting major interest in human and veterinary oncological pathology because of their role as targets for adjuvant therapies. Little is known about tyrosine kinase receptor (TKR) expression in canine liposarcoma (LP), a soft tissue sarcoma. The aim of this study was to evaluate the immunohistochemical expression of the TKRs fibroblast growth factor receptor 1 (FGFR1) and platelet-derived growth factor receptor-ß (PDGFRß); their ligands, fibroblast growth factor 2 (FGF2) and platelet-derived growth factor B (PDGFB); and c-kit in canine LP. Immunohistochemical labeling was categorized as high or low expression and compared with the mitotic count and MIB-1-based proliferation index. Fifty canine LPs were examined, classified, and graded. Fourteen cases were classified as well differentiated, 7 as myxoid, 25 as pleomorphic, and 4 as dedifferentiated. Seventeen cases were grade 1, 26 were grade 2, and 7 were grade 3. A high expression of FGF2, FGFR1, PDGFB, and PDGFRß was identified in 62% (31/50), 68% (34/50), 81.6% (40/49), and 70.8% (34/48) of the cases, respectively. c-kit was expressed in 12.5% (6/48) of the cases. Mitotic count negatively correlated with FGF2 ( R = -0.41; P < .01), being lower in cases with high FGF2 expression, and positively correlated with PDGFRß ( R = 0.33; P < .01), being higher in cases with high PDGFRß expression. No other statistically significant correlations were identified. These results suggest that the PDGFRß-mediated pathway may have a role in the progression of canine LP and may thus represent a promising target for adjuvant cancer therapies.

Tumour initiating cells and IGF/FGF signalling contribute to sorafenib resistance in hepatocellular carcinoma.

OBJECTIVE: Sorafenib is effective in hepatocellular carcinoma (HCC), but patients ultimately present disease progression. Molecular mechanisms underlying acquired resistance are still unknown. Herein, we characterise the role of tumour-initiating cells (T-ICs) and signalling pathways involved in sorafenib resistance. DESIGN: HCC xenograft mice treated with sorafenib (n=22) were explored for responsiveness (n=5) and acquired resistance (n=17). Mechanism of acquired resistance were assessed by: (1) role of T-ICs by in vitro sphere formation and in vivo tumourigenesis assays using NOD/SCID mice, (2) activation of alternative signalling pathways and (3) efficacy of anti-FGF and anti-IGF drugs in experimental models. Gene expression (microarray, quantitative real-time PCR (qRT-PCR)) and protein analyses (immunohistochemistry, western blot) were conducted. A novel gene signature of sorafenib resistance was generated and tested in two independent cohorts. RESULTS: Sorafenib-acquired resistant tumours showed significant enrichment of T-ICs (164 cells needed to create a tumour) versus sorafenib-sensitive tumours (13 400 cells) and non-treated tumours (1292 cells), p<0.001. Tumours with sorafenib-acquired resistance were enriched with insulin-like growth factor (IGF) and fibroblast growth factor (FGF) signalling cascades (false discovery rate (FDR)<0.05). In vitro, cells derived from sorafenib-acquired resistant tumours and two sorafenib-resistant HCC cell lines were responsive to IGF or FGF inhibition. In vivo, FGF blockade delayed tumour growth and improved survival in sorafenib-resistant tumours. A sorafenib-resistance 175 gene signature was characterised by enrichment of progenitor cell features, aggressive tumorous traits and predicted poor survival in two cohorts (n=442 patients with HCC). CONCLUSIONS: Acquired resistance to sorafenib is driven by T-ICs with enrichment of progenitor markers and activation of IGF and FGF signalling. Inhibition of these pathways would benefit a subset of patients after sorafenib progression.

A place for precision medicine in bladder cancer: targeting the FGFRs.

Bladder tumors show diverse molecular features and clinical outcome. Muscle-invasive bladder cancer has poor prognosis and novel approaches to systemic therapy are urgently required. Non-muscle-invasive bladder cancer has good prognosis, but high recurrence rate and the requirement for life-long disease monitoring places a major burden on patients and healthcare providers. Studies of tumor tissues from both disease groups have identified frequent alterations of FGFRs, including mutations of FGFR3 and dysregulated expression of FGFR1 and FGFR3 that suggest that these may be valid therapeutic targets. We summarize current understanding of the molecular alterations affecting these receptors in bladder tumors, preclinical studies validating them as therapeutic targets, available FGFR-targeted agents and results from early clinical trials in bladder cancer patients.

Antibody-Mediated Inhibition of the FGFR1c Isoform Induces a Catabolic Lean State in Siberian Hamsters.

Hypothalamic tanycytes are considered to function as sensors of peripheral metabolism. To facilitate this role, they express a wide range of receptors, including fibroblast growth factor receptor 1 (FGFR1). Using a monoclonal antibody (IMC-H7) that selectively antagonizes the FGFR1c isoform, we investigated possible actions of FGFR1c in a natural animal model of adiposity, the Siberian hamster. Infusion of IMC-H7 into the third ventricle suppressed appetite and increased energy expenditure. Likewise, peripheral treatment with IMC-H7 decreased appetite and body weight and increased energy expenditure and fat oxidation. A greater reduction in body weight and caloric intake was observed in response to IMC-H7 during the long-day fat state as compared to the short-day lean state. This enhanced response to IMC-H7 was also observed in calorically restricted hamsters maintained in long days, suggesting that it is the central photoperiodic state rather than the peripheral adiposity that determines the response to FGFR1c antagonism. Hypothalamic thyroid hormone availability is controlled by deiodinase enzymes (DIO2 and DIO3) expressed in tanycytes and is the key regulator of seasonal cycles of energy balance. Therefore, we determined the effect of IMC-H7 on hypothalamic expression of these deiodinase enzymes. The reductions in food intake and body weight were always associated with decreased expression of DIO2 in the hypothalamic ependymal cell layer containing tanycytes. These data provide further support for the notion the tanycytes are an important component of the mechanism by which the hypothalamus integrates central and peripheral signals to regulate energy intake and expenditure.