In chronic kidney disease (CKD), serum concentrations of fibroblast growth factor 23 (FGF23) increase progressively as glomerular filtration rate declines, while renal expression of the FGF23 coreceptor Klotho decreases. Elevated circulating FGF23 levels are strongly associated with mortality and with left ventricular hypertrophy (LVH), which is a major cause of cardiovascular death in CKD patients. The cardiac FGF23/FGF receptor (FGFR) system and its role in the development of LVH in humans have not been addressed previously.

We conducted a retrospective case-control study in 24 deceased patients with childhood-onset end-stage renal disease (dialysis: n = 17; transplanted: n = 7), and 24 age- and sex-matched control subjects. Myocardial autopsy samples of the left ventricle were evaluated for expression of endogenous FGF23, FGFR isoforms, Klotho, calcineurin and nuclear factor of activated T-cells (NFAT) by immunohistochemistry, immunofluorescence microscopy, qRT-PCR and western blotting.

The majority of patients presented with LVH (67%). Human cardiomyocytes express full-length FGF23, and cardiac FGF23 is excessively high in patients with CKD. Enhanced myocardial expression of FGF23 in concert with Klotho deficiency strongly correlates with the presence of LVH. Cardiac FGF23 levels associate with time-averaged serum phosphate levels, up-regulation of FGFR4 and activation of the calcineurin-NFAT signaling pathway, an established mediator of cardiac remodelling and LVH. These changes are detected in patients on dialysis but not in those with a functioning kidney transplant.

Our results indicate a strong association between LVH and enhanced expression levels of FGF23, FGFR4 and calcineurin, activation of NFAT and reduced levels of soluble Klotho in the myocardium of patients with CKD. These alterations are not observed in kidney transplant patients.

This phase 2 study aimed to determine the efficacy and safety of the combination of bortezomib, melphalan, dexamethasone and intermittent thalidomide (VMDT) and its effect on bone remodeling and angiogenesis in relapsed/refractory myeloma. Bortezomib (1.0 mg/m(2)) was given on days 1, 4, 8, 11, oral melphalan (0.15 mg/kg) on days 1-4, whereas thalidomide (100 mg per day) and dexamethasone (12 mg/m(2)) were administered on days 1-4 and <em>17</em>-20 of a 28-day cycle, for four cycles. Patients without disease progression continued for up to eight cycles. VMDT effect on bone remodeling was evaluated by measuring osteoclast regulators (soluble receptor activator of nuclear <em>factor</em>-kappa B ligand/osteoprotegerin ratio, osteopontin, macrophage inflammatory protein-1alpha), dickkopf-1 protein, bone resorption and formation markers, whereas its effect on angiogenesis was assessed by measuring serum vascular endothelial <em>growth</em> <em>factor</em>, angiogenin, angiopoietin-2 and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, after four cycles and at the study end. A total of 62 patients were enrolled. The overall response rate was 66%: CR 13%, vgPR 27% and PR 26%. Median time to response was 35 days and median time to progression was 9.3 months. Common adverse events included cytopenias, peripheral neuropathy and infections. No patient experienced deep-vein thrombosis. VMDT reduced angiogenic cytokines, osteoclast regulators, dickkopf-1 and bone resorption. We conclude that VMDT with intermittent thalidomide is an active and well-tolerated regimen for relapsed/refractory myeloma, affecting abnormal bone remodeling and angiogenesis.

We have purified to near homogeneity a novel <em>17</em> kD <em>growth</em> <em>factor</em> from bovine uterus which we designated heparin-binding <em>growth</em> <em>factor</em>-8 (HBGF-8). The <em>growth</em> <em>factor</em> binds tightly to cation exchange resins and to Heparin-Sepharose and is stable to acetone precipitation and labile in acid. Based upon total activity in acetone extracts of bovine uterus stimulating 3H-thymidine incorporation into DNA of serum-starved NIH 313 cells, a 6940 fold purification was achieved with an overall yield of HBGF-8 activity of 0.4%, using extraction of acetone powders and chromatographic separations at neutral pH. Approximately 18 micrograms protein was obtained from 1.2 kg wet weight of tissue. HBGF-8 was clearly separated from <em>17</em>.5 kD bovine uterus basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) by purification and its N-terminal amino acid sequence analyzed. A polypeptide with a unique 25 N-terminal amino acid sequence was found. HBGF-8 was as active as acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) and slightly less active than bFGF in the mouse NIH 3T3 <em>fibroblast</em> mitogenic assay system with an intrinsic specific activity of 5000 dpm/ng under standard assay conditions.

BACKGROUND

Surgical excision of liver tumors represents the only curative treatment for primary and metastatic liver malignancies. It has been suspected that hepatectomy may stimulate growth of microscopic tumors. To determine whether local or systemic factors after hepatectomy are responsible for enhancement of tumor growth, the effects of hepatectomy on the experimental growth of liver or pulmonary tumors were examined.

METHODS

One hour after injection of 10(6) Morris hepatoma cells into either the portal or femoral vein, which produces isolated liver and lung tumors, respectively, animals were randomized to undergo 0%, 30%, or 70% partial hepatectomy (PH).

RESULTS

Animals that underwent portal injection of tumor had significantly increased liver tumor burden after PH (sham, 25 +/- 7 vs PH, 94 +/- 17; p < 0.01), whereas animals that underwent femoral injection had no change in lung tumor burden after PH. PH was associated with significantly increased levels of transforming growth factor-alpha, transforming growth factor-beta, and basic fibroblast growth factor in the liver but not in the lung.

CONCLUSIONS

Changes in liver cytokine-growth factor activation may contribute to enhanced tumor growth in the liver after hepatectomy.

Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive production of extracellular matrix (ECM) and understood to develop under the influence of certain <em>growth</em> <em>factors</em>. Connective tissue <em>growth</em> <em>factor</em> (CTGF) is a cysteine-rich mitogenic peptide that is implicated in various fibrotic disorders and induced in <em>fibroblasts</em> after activation with transforming <em>growth</em> <em>factor</em>-beta (TGF-beta). To better understand the mechanisms of persistent fibrosis seen in SSc, we previously established an animal model of skin fibrosis induced by exogenous application of <em>growth</em> <em>factors</em>. In this model, TGF-beta transiently induced subcutaneous fibrosis and serial injections of CTGF after TGF-beta caused persistent fibrosis. To further define the mechanisms of skin fibrosis induced by TGF-beta and CTGF in vivo, we investigated in this study, the effects of <em>growth</em> <em>factors</em> on the promoter activity of the proalpha2 (I) collagen (COL1A2) gene in skin fibrosis. For this purpose, we utilized transgenic reporter mice harboring the -<em>17</em> kb promoter sequence of the mouse COL1A2 linked to either a firefly luciferase gene or a bacterial beta-galactosidase gene. Serial injections of CTGF after TGF-beta resulted in a sustained elevation of COL1A2 mRNA expression and promoter activity compared with consecutive injection of TGF-beta alone on day 8. We also demonstrated that the number of <em>fibroblasts</em> with activated COL1A2 transcription was increased by serial injections of CTGF after TGF-beta in comparison with the injection of TGF-beta alone. Furthermore, the serial injections recruited mast cells and macrophages. The number of mast cells reached a maximum on day 4 and remained relatively high up to day 8. In contrast to the kinetics of mast cells, the number of macrophages was increased on day 4 and continued to rise during the subsequent consecutive CTGF injections until day 8. These results suggested that CTGF maintains TGF-beta-induced skin fibrosis by sustaining COL1A2 promoter activation and increasing the number of activated <em>fibroblasts</em>. The infiltrated mast cells and macrophages may also contribute to the maintenance of fibrosis.

Antiestrogens, such as tamoxifen, are widely used for endocrine treatment of estrogen receptor-positive breast cancer. However, as breast cancer progresses, development of tamoxifen resistance is inevitable. The mechanisms underlying this resistance are not well understood. To identify genes involved in tamoxifen resistance, we have developed a rapid screening method. To alter the tamoxifen-sensitive phenotype of human ZR-75-1 breast cancer cells into a tamoxifen-resistant phenotype, the cells were infected with retroviral cDNA libraries derived from human placenta, human brain, and mouse embryo. Subsequently, the cells were selected for proliferation in the presence of 4-hydroxy-tamoxifen (OH-TAM) and integrated cDNAs were identified by sequence similarity searches. From 155 OH-TAM-resistant cell colonies, a total of 25 candidate genes were isolated. Seven of these genes were identified in multiple cell colonies and thus cause antiestrogen resistance. The epidermal <em>growth</em> <em>factor</em> receptor, platelet-derived <em>growth</em> <em>factor</em> receptor-alpha, platelet-derived <em>growth</em> <em>factor</em> receptor-beta, colony-stimulating <em>factor</em> 1 receptor, neuregulin1, and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>17</em> that we have identified have been described as key regulators in the mitogen-activated protein kinase pathway. Therefore, this pathway could be a valuable target in the treatment of patients with breast cancer resistant to endocrine treatment. In addition, the putative gene LOC400500, predicted by in silico analysis, was identified. We showed that ectopic expression of this gene, designated as breast cancer antiestrogen resistance 4 (BCAR4), caused OH-TAM resistance and anchorage-independent cell <em>growth</em> in ZR-75-1 cells and that the intact open reading frame was required for its function. We conclude that retroviral transfer of cDNA libraries into human breast cancer cells is an efficient method for identifying genes involved in tamoxifen resistance.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> 21 is a member of endocrine FGFs subfamily, along with FGF19 and FGF23. It is emerging as a novel regulator with beneficial effects on a variety of metabolic parameters, including glucose and lipid control. FGF21 activity depends on membrane protein betaKlotho that physically complexes with various FGF receptors, thus conferring them the ability to bind FGF21 and activate downstream signaling pathways. FGF21, like other FGFs, folds to a beta-trefoil-like core region, with disordered N- and C-termini. In order to investigate their role in the activity of FGF21, we have constructed a series of deletion mutants and tested them for their ability to (1) bind betaKlotho, analyzed by surface plasmon resonance spectroscopy (2) signal through MAPK phosphorylation and inhibit apoptosis in 3T3-L1/betaKlotho <em>fibroblasts</em> (3) stimulate GLUT1 mRNA upregulation and glucose uptake in 3T3-L1 adipocytes. Binding studies with betaKlotho revealed that the interaction with the co-receptor involves the C-terminus, as progressive removal of amino acids from the carboxy end decreased affinity for betaKlotho. By contrast, removal of up to <em>17</em> amino acids from the N-terminus had no effect on the interaction with betaKlotho. Terminal deletions had greater effect on function, as deletions of six amino acids from the amino-terminus and only four from the carboxy-terminus each significantly impacted activity (10-fold). Of the extreme terminal truncations, with no detectable activity, DeltaN<em>17</em> acted as competitive antagonist while DeltaC20 did not. Our structure/function studies show that the C-terminus is important for betaKlotho interaction whereas the N-terminus likely interacts directly with FGF receptors.

Activation of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) receptor 3 (FGFR3) has been linked to the development of human cancers by mechanisms that are not well understood. The MUC1 oncoprotein is aberrantly overexpressed by certain hematologic malignancies and most human carcinomas. The present studies show that MUC1 associates with FGFR3. Stimulation of cells with FGF1 increased the interaction between MUC1 and FGFR3. FGF1 stimulation also induced c-Src-dependent tyrosine phosphorylation of the MUC1 cytoplasmic domain on a YEKV motif. FGF1-induced tyrosine phosphorylation of MUC1 was associated with increased binding of MUC1 to beta-catenin and targeting of MUC1 and beta-catenin to the nucleus. FGF1 also induced binding of MUC1 to the heat shock protein 90 (HSP90) chaperone by a mechanism dependent on phosphorylation of the YEKV motif. Notably, beta-catenin and HSP90 compete for binding to the MUC1 cytoplasmic domain, indicating that MUC1 forms mutually exclusive complexes with these proteins. The results also show that inhibition of HSP90 with geldanamycin or <em>17</em>-(allylamino)-<em>17</em>-demethoxygeldanamycin attenuates FGF1-induced binding of MUC1 to HSP90 and targeting of MUC1 to the mitochondrial outer membrane. These findings indicate that FGF1 induces phosphorylation of MUC1 on YEKV and thereby activates two distinct pathways: (a) nuclear localization of MUC1 and beta-catenin and (b) delivery of MUC1 to mitochondria by HSP90.

OBJECTIVE

To determine the vitreous levels of 27 types of cytokines in eyes with retinopathy of prematurity (ROP).

METHODS

Retrospective case-control study.

METHODS

Twenty-seven eyes of 19 infants with stage 4 ROP were studied. Six eyes of 5 patients with congenital cataract who underwent lensectomy were used as controls.

METHODS

The ROP eyes were divided into 2 groups according to vascular activity: 12 eyes with vascularly active ROP and 15 eyes with vascularly inactive ROP. Undiluted vitreous samples were collected, and the vitreous concentrations of 27 types of cytokines were determined by a multiplex bead analysis system: interleukin (IL)-1b, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-<em>17</em>, Eotaxin, <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) basic, granulocyte-colony stimulating <em>factor</em> (G-CSF), granulocyte macrophage colony-stimulating <em>factor</em> (GM-CSF), interferon-r, interferon-gamma-inducible protein (IP)-10, monocyte chemoattractant protein 1, macrophage inflammatory protein (MIP)-1a, MIP-1b, platelet-derived <em>growth</em> <em>factor</em> bb, regulated on activation, normal T cell expressed and secreted (RANTES), tumor necrosis <em>factor</em> alpha, and vascular endothelial <em>growth</em> <em>factor</em> (VEGF).

METHODS

The vitreous levels of the 27 types of cytokines and a comparison of the levels in the 3 groups.

RESULTS

The postmenstrual age at vitrectomy was significantly younger in the vascularly active ROP eyes than in vascularly inactive ROP eyes. The cytokines that had significantly different vitreous levels among the 3 groups were: IL-6, IL-7, IL-10, IL-15, Eotaxin, FGF basic, G-CSF, GM-CSF, IP-10, RANTES, and VEGF (P<0.05). The vitreous levels of IL-6, IL-7, IL-15, Eotaxin, G-CSF, IP-10, and RANTES were significantly higher (P<0.05) in both vascularly active and inactive ROP eyes than in control eyes, whereas the vitreous level of VEGF was significantly higher (P<0.05) only in vascularly active ROP eyes than in control eyes. There was a significantly negative correlation (r = -0.382; P = 0.0495) between the VEGF level and the postmenstrual age at vitrectomy.

CONCLUSIONS

These results indicate that, although cytokines other than VEGF may be involved in the pathologic changes in eyes with ROP, VEGF is likely to have the strongest correlation with the vascular activity in ROP eyes among these cytokines.

During early vertebrate embryogenesis, mesoderm is specified by a signal emanating from prospective endoderm. This signal can respecify Xenopus prospective ectoderm as mesoderm, and can be mimicked by members of the <em>fibroblast</em> <em>growth</em> <em>factor</em> and transforming <em>growth</em> <em>factor</em>-beta families. In other systems, the p21c-ras proto-oncogene product has been implicated in signal transduction for various polypeptide <em>growth</em> <em>factors</em>. We report here that a dominant inhibitory ras mutant blocks the mesoderm-inducing activity of <em>fibroblast</em> <em>growth</em> <em>factor</em> and activin, as well as the endogenous inducing activity of prospective endoderm. A constitutively active ras mutant partially mimics these activities. These results indicate that p21ras may have a central role in the transduction of the mesoderm inductive signal. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and activin have emerged as candidates for endogenous mesoderm-inducing molecules. The character of the mesoderm induced by these two <em>factors</em> is overlapping but distinct when assessed both by histological and molecular criteria. The signal transduction pathways used during induction by these <em>factors</em> are unknown. We used messenger RNA microinjection of Xenopus eggs to express a dominant inhibitory mutant ras, p21(Asn <em>17</em>)Ha-ras, in cells competent to respond to inducing <em>factors</em> to examine the role of p21ras in this response. This mutant, which has a reduced affinity for GTP relative to GDP, blocks a variety of mitogenic signals in 3T3 <em>fibroblasts</em> as well as the differentiation of pheochromocytoma cells in response to nerve <em>growth</em> <em>factor</em>.

Expression of p21rasAsn-<em>17</em>, a dominant negative mutant of p21ras that blocks p21ras activation by <em>growth</em> <em>factors</em>, inhibits activation of extracellular signal-regulated kinase 2 (ERK2) by insulin and platelet-derived <em>growth</em> <em>factor</em> in rat-1 cells [A. M. M. de Vries-Smits, B. M. T. Burgering, S. J. Leevers, C. J. Marshall, and J. L. Bos, Nature (London) 357:602-604, 1992]. Here we report that expression of p21rasAsn-<em>17</em> does not abolish epidermal <em>growth</em> <em>factor</em> (EGF)-induced phosphorylation of ERK2 in <em>fibroblasts</em>. Since EGF activates p21ras in these cells, this indicates that EGF induces a p21ras-independent pathway for the phosphorylation of ERK2 as well. We investigated whether activation of protein kinase C (PKC) or increase in intracellular calcium could be involved in p21ras-independent signaling. In rat-1 cells, inhibition of either PKC, by prolonged 12-O-tetradecanoylphorbol-13-acetate (TPA) pretreatment, or calcium influx, by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) pretreatment, did not abolish EGF-induced ERK2 phosphorylation. However, a combined inhibition of both p21ras and calcium influx, but not PKC, resulted in a complete inhibition of EGF-induced ERK2 phosphorylation. In contrast, in Swiss 3T3 cells, inhibition of both p21ras activation and TPA-sensitive PKC, but not calcium influx, inhibited EGF-induced ERK2 phosphorylation. These results demonstrate that in <em>fibroblasts</em>, EGF induces alternative pathways of ERK2 phosphorylation in a cell-type-specific manner.

Fibroblast growth factors are essential molecules for development. Here we characterize Fgfl7, a new member of the fibroblast growth factor (FGF) family. The Fgfl7 gene maps to mouse chromosome 14 and is highly conserved between mouse and human (93% identity). It exhibits 60% amino acid identity with Fgf8 and 50% identity with Fgf8. Both Fgf8 and Fgf17 have a similar structure and a similar pattern of alternative splicing in the 5' coding region. When expressed in 3T3 fibroblasts, mouse FGF17 is transforming, indicating that it can activate the 'c' splice form of either FGF receptor (FGFR) one or two. During midgestation embryogenesis, in situ hybridization analysis localized Fgf17 expression to specific sites in the midline structures of the forebrain, the midbrain-hindbrain junction, the developing skeleton and in developing arteries. Comparison to Fgf8 revealed a striking similarity in expression patterns, especially in the central nervous system (CNS), suggesting that both genes may be important for CNS development, although Fgf17 is expressed somewhat later than Fgf8. In the developing skeleton, both genes are expressed in costal cartilage while Fgf8 is preferentially expressed in long bones. In the developing great vessels Fgfl7 is preferentially expressed, suggesting that it may have a more prominent role in vascular growth.

We have previously shown that bone organ cultures produce large amounts of latent transforming <em>growth</em> <em>factor</em> beta (TGF beta), which lacks latent TGF beta-binding protein (LTBP). In this study we used the known osteoblast-like cell lines UMR-106, ROS <em>17</em>/2.8, and MG63 as models to further examine latent TGF beta expression in bone. We found that the osteosarcoma cell line UMR-106 secreted latent TGF beta almost exclusively as a 100-kDa complex lacking LTBP. ROS <em>17</em>/2.8 cells produced both the 100-kDa complex and also a 290-kDa complex containing the <em>fibroblast</em>ic (190 kDa) form of LTBP. MG63 cells (like human foreskin <em>fibroblasts</em>) expressed almost exclusively the 290-kDa complex. To investigate the regulation of latent TGF beta complexes in bone cells we assessed the effects of TGF beta 1 treatment on expression of active and latent TGF beta. TGF beta 1 induced secretion of latent but not active TGF beta in all cell types examined. In human foreskin <em>fibroblast</em> cells, TGF beta 1 and LTBP mRNA were expressed concomitantly. In contrast, in osteosarcoma cell lines autoinduction of TGF beta 1 mRNA was associated with either a delayed increase or no change in LTBP mRNA. In UMR-106 cells LTBP message was virtually undetectable. We postulate that the expression of different latent TGF beta forms by osteoblast-like cells may reflect their maturation states and that different latent TGF beta complexes may have different functions, for example as secretory forms or as matrix storage forms.

We have previously reported that serotonin (5-hydroxytryptamine [5HT]) alters cultured bovine pulmonary artery smooth muscle cell (SMC) configuration through two different regulatory mechanisms. We now report that 5HT also regulates SMC <em>growth</em> through these same two mechanisms--a stimulatory event initiated intracellularly and inhibition of <em>growth</em> resulting from a cell surface action. 5HT (1 microM) plus 0.1 mM iproniazid (a 5HT metabolic inhibitor) produced a severalfold stimulation of DNA synthesis (as measured by [3H]thymidine incorporation) of SMCs after a <em>17</em>-24-hour incubation with only a slight elevation of cellular cAMP. This stimulatory effect responded synergistically with other <em>growth</em> <em>factors</em> including platelet-derived <em>growth</em> <em>factor</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em>, and epidermal <em>growth</em> <em>factor</em> and was effectively reversed by 5HT uptake inhibition. It was not produced by 5-hydroxyindoleacetic acid, a metabolite of 5HT. In the presence of 1 microM 5HT plus 0.1 mM isobutylmethylxanthine (IBMX), cAMP was elevated eightfold, dendritic formation occurred, and [3H]thymidine labeling of SMCs was inhibited. Inhibition of labeling by [3H]thymidine was mimicked by other agents that elevated cellular cAMP (10 microM histamine, 1 microM isoproterenol plus 0.1 mM IBMX, and 10 microM forskolin) and by 1 mM dibutyryl cAMP. This inhibitory effect was not blocked by either inhibition of 5HT uptake or 5HT-receptor antagonists ketanserin (5HT2); methiothepin, spiperone, and mianserin (5HT1/5HT2); and 3-tropanyl-indole-3-carboxylate and 3-tropanyl-3,5-dichlorobenzoate (5HT3). However, similar to 5HT, the 5HT1A agonist, (+/-)-8-hydroxy-(+/-)-2-dipropylamino-8-hydroxy-1,2,3, 4-tetrahydronaphthalenehydrobromide, in association with IBMX, produced an elevation in cAMP and inhibition of labeling by [3H]thymidine. 5HT, in the presence of either iproniazid or IBMX, did not alter [Ca2+]i, indicating that [Ca2+]i was not a signal for either of these actions.(ABSTRACT TRUNCATED AT 250 WORDS)

The proper guidance of the Caenorhabditis elegans hermaphrodite sex myoblasts (SMs) requires the genes egl-15 and egl-<em>17</em>. egl-15 has been shown to encode the C. elegans orthologue of the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR). Here we clone egl-<em>17</em> and show it to be a member of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family, one of the first functional invertebrate FGFs known. egl-<em>17</em> shares homology with other FGF members, conserving the key residues required to form the distinctive tertiary structure common to FGFs. Genetic and molecular evidence demonstrates that the SM migration defect seen in egl-<em>17</em> mutant animals represents complete loss of egl-<em>17</em> function. While mutations in egl-<em>17</em> affect only SM migration, mutations in egl-15 can result in larval arrest, scrawny body morphology, and the ability to suppress mutations in clr-1. We propose that EGL-<em>17</em> (FGF) acts as a ligand for EGL-15 (FGFR) specifically during SM migration and that another ligand(s) activates EGL-15 for its other functions.

BACKGROUND

Biliary tract cancers (BTCs) typically present at an advanced stage, and systemic chemotherapy is often of limited benefit.

METHODS

Hybrid capture-based comprehensive genomic profiling (CGP) was performed for 412 intrahepatic cholangiocarcinomas (IHCCAs), 57 extrahepatic cholangiocarcinomas (EHCCAs), and 85 gallbladder carcinomas (GBCAs). The mutational profile was correlated with the clinical outcome of standard and experimental therapies for 321 patients. Clinical variables, detected mutations, and administered therapies were correlated with overall survival (OS) in a Cox regression model.

RESULTS

The most frequent genetic aberrations (GAs) observed were tumor protein 53 (TP53; 27%), cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B; 27%), KRAS (22%), AT-rich interactive domain-containing protein 1A (ARID1A; 18%), and isocitrate dehydrogenase 1 (IDH1; 16%) in IHCCA; KRAS (42%), TP53 (40%), CDKN2A/B (<em>17</em>%), and SMAD4 (21%) in EHCCA; and TP53 (59%), CDKN2A/B (19%), ARID1A (13%), and ERBB2 (16%) in GBCA. <em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR; 11%) and IDH mutations (20%) were mostly limited to IHCCA but appeared to be mutually exclusive. In the IHCCA group, TP53 and KRAS mutations were associated significantly with poor OS, whereas FGFR2 mutations were associated with improved OS (P = .001), a younger age at onset, and female sex. IDH1/2 mutations were not prognostic. In a multivariate model, the effects of TP53 and FGFR GAs remained significant (P < .05). Patients with FGFR GAs had superior OS with FGFR-targeted therapy versus standard regimens (P = .006). Targeted therapy in IHCCA was associated with a numerical OS improvement (P = .07).

CONCLUSIONS

This is the largest clinically annotated data set of BTC cases with CGP and indicates the potential of CGP for improving outcomes. CGP should be strongly considered in the management of BTC patients. Cancer 2016;122:3838-3847. © 2016 American Cancer Society.

Disintegrin metalloproteases of the ADAM family form a large (at present>> 40 members in mammals) family of multidomain membrane proteins that in their ectodomain combine a cystein-rich, disintegrin and a zinc metalloprotease domain. Via their metalloprotease domain, ADAMs are often implicated in ectodomain shedding, either to release e.g. <em>growth</em> <em>factors</em> or to initiate further intracellular signalling via regulated intramembrane proteolysis. Mainly based upon overexpression studies in vehicle cells, three of them, ADAMs 9, 10 and <em>17</em>, have been proposed to act as alpha-secretases for amyloid precursor protein (APP). It is striking thereby that this role has since then remained somewhat ill-defined, as APP processing in ADAM9 deficient neurons is unaltered, and also ADAM10 deficient murine embryonic <em>fibroblasts</em> exhibit at best a highly variable reduction in alpha-secretase activity. However, during the past years, numerous other substrates have been linked to all three sheddases, the cleavage of which in some cases appears to be strikingly more important for the organism than APP processing. Most notably, the perinatally lethal phenotype of ADAM<em>17</em> knockout mice is dominated by a loss of <em>growth</em> <em>factor</em> shedding, while the even earlier fatal effects of ADAM10 deficiency exhibit key features of disabled Notch signalling and possibly also cadherin processing defects. In this review, we will summarize the published data on the "non-APP" functions of all three ADAMs, the further evaluation of which may be crucial when attempting to treat Alzheimer s Disease by increasing their expression and/or activity. As the knockouts of ADAM10 and ADAM<em>17</em> are only informative for their roles in (early) development, while a number of recently assigned new substrates play crucial roles in the normal and/or diseased adult organism as well, work on conditional knockout models will be crucial to fully characterize both the full functional portfolio of (candidate) alpha-secretases as well as their clinical relevance, which may go way beyond Alzheimer s Disease.

The expression of mRNAs for vascular endothelial <em>growth</em> <em>factor</em> (VEGF) was examined in 42 cases of primary lung cancer tissues (18 adenocarcinomas, 18 squamous cell carcinomas, 2 large cell carcinomas, 3 small cell carcinomas, and 1 adenoid cystic carcinoma) and 4 human lung cancer cell lines. As seen by reverse transcription-PCR analysis, VEGF mRNAs were expressed predominantly as transcripts for the secretory forms of VEGF (VEGF121 and VEGF165), both in resected lung cancer tissues and in human lung cancer cell lines. The positive ratios of VEGF mRNA according to pathological type were 66.7% (12 of 18) in adenocarcinoma, 72.2% (13 of 18) in squamous cell carcinoma, 100% (2 of 2) in large cell carcinoma, and 67% (2 of 3) in small cell carcinoma. The relative antigen levels of VEGF detected by immunohistochemical examination almost coincided with the relative VEGF mRNA expression levels. Also, we examined the expression of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> mRNA in the same tumor specimens. However, no significant correlation was found between the VEGF and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> mRNA expression levels. We assessed the relationship between the VEGF121 mRNA expression level and the survival period in patients (n = <em>17</em>) who underwent a curative operation at stage I of the disease. The median survival of the VEGF high-expression group was 8 months, and that of the VEGF low-expression group was 151 months. The 3- and 5-year survival rates of the high-expression group (n = 6) were 50.0% and 16.7%, respectively. On the other hand, those of the low expression group (n = 11) were 90.9% and 77.9%, respectively. The difference in survival between the two groups was significant (P < 0.05). Among eight cases of long-term survival beyond 5 years, seven cases had low or no VEGF121 mRNA expression. In contrast, among 18 cases with VEGF121 mRNA overexpression, <em>17</em> cases died due to recurrence. As a marker of tumor angiogenesis, the VEGF121 mRNA expression level may be a significant prognostic indicator of lung cancers in early stages.

Treatment of L929 cells with tumor necrosis <em>factor</em> alpha (TNFalpha) activates a programmed cell death pathway resulting in apoptosis. We investigated the intracellular signaling pathways activated in L929 cells by TNFalpha. TNFalpha robustly activates Jun kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family. In addition, p42(MAPK) is activated, but a 10-fold greater concentration of TNFalpha was required for substantial MAPK activation than was needed for maximal JNK stimulation. Simultaneous treatment of L929 cells with <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) significantly reduced the apoptotic response to TNFalpha. FGF-2 substantially activated the Raf/MEK/MAPK (where MEK is mitogen-activated protein kinase kinase) pathway but did not affect TNFalpha activation of JNK. These results indicate that although JNK may play an important role in transmitting the TNFalpha signal from the cell surface to the nucleus, activation of the JNK pathway is not sufficient to induce apoptosis. Expression of dominant-negative Asn-<em>17</em> Ras in L929 cells diminished the FGF-2 stimulation of p42(MAPK) and eliminated the protective effect of FGF-2. Asn-<em>17</em> Ras expression did not affect JNK activity and had no effect on TNFalpha activation of JNK. Pharmacological inhibition of MEK-1 activity by incubation of cells with the compound PD 098059 blocked p42(MAPK) activation and FGF-2 protection against apoptosis. Interestingly, activated Val-12 Ras expression substantially enhanced TNFalpha-mediated apoptosis in L929 cells, but Val-12 Ras did not constitutively activate MAPK in L929 cells and FGF-2 partially protected Val-12 Ras-expressing cells from TNFalpha-mediated apoptosis. Our data indicate that activation of the MAPK pathway mediates an FGF-2 protective effect against apoptosis and highlights the important role that integration of multiple intracellular signaling pathways plays in the regulation of cell <em>growth</em> and death.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 (FGFR2) mutations have been associated with the craniosynostotic conditions Crouzon, Jackson-Weiss, and Pfeiffer syndromes. Previously, mutations were described in the exons IIIa and IIIc, which form the extracellular, third immunoglobulin-like domain (IgIII) and adjacent linker regions, both of which are normally involved in ligand binding. For all three conditions, mutations were found in exon IIIc. Only in Crouzon syndrome were mutations identified in exon IIIa. In this study, 39 cases with one of these three conditions were screened for exon IIIa or IIIc mutations. Eleven mutations are reported in <em>17</em> unrelated cases. Mutations in exon IIIa are identified for not only Crouzon but also Jackson-Weiss and Pfeiffer syndromes. Four mutations in either exon IIIa or exon IIIc reported only in Crouzon syndrome are present also in one of the other two syndromes. Two insertions, one in exon IIIa in a Crouzon syndrome patient and the other in exon IIIc in a Pfeiffer syndrome patient, were observed. The latter mutation has the same alternative RNA splicing effect as a reported synonymous mutation for Crouzon syndrome. A missense mutation was detected in one Pfeiffer syndrome family in which two members had craniosynostosis without limb anomalies. The inter- and intrafamilial variability in expression of FGFR2 mutations suggests that these three syndromes, presumed to be clinically distinct, are instead representative of a spectrum of related craniosynostotic and digital disorders.

BACKGROUND

Lenvatinib is an oral, multitargeted tyrosine kinase inhibitor of the vascular endothelial growth factor receptors 1 through 3 (VEGFR1-VEGFR3), fibroblast growth factor receptors 1 through 4 (FGFR1-FGFR4), platelet-derived growth factor receptor α (PDGFRα), ret proto-oncogene (RET), and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) signaling networks implicated in tumor angiogenesis. Positive phase 1 results in solid tumors prompted a phase 2 trial in patients with advanced, radioiodine-refractory, differentiated thyroid cancer (RR-DTC).

METHODS

Fifty-eight patients with RR-DTC who had disease progression during the previous 12 months received lenvatinib 24 mg once daily in 28-day cycles until disease progression, unmanageable toxicity, withdrawal, or death. Previous VEGFR-targeted therapy was permitted. The primary endpoint was the objective response rate (ORR) based on independent imaging review. Secondary endpoints included progression-free survival (PFS) and safety. Serum levels of 51 circulating cytokines and angiogenic factors also were assessed.

RESULTS

After ≥14 months of follow-up, patients had an ORR of 50% (95% confidence interval [CI], 37%-63%) with only partial responses reported. The median time to response was 3.6 months, the median response duration was 12.7 months, and the median PFS was 12.6 months (95% CI, 9.9-16.1 months). The ORR for patients who had received previous VEGF therapy (n = 17) was 59% (95% CI, 33%-82%). Lower baseline levels of angiopoietin-2 were suggestive of tumor response and longer PFS. Grade 3 and 4 treatment-emergent adverse events, regardless of their relation to treatment, occurred in 72% of patients and most frequently included weight loss (12%), hypertension (10%), proteinuria (10%), and diarrhea (10%).

CONCLUSIONS

In patients with and without prior exposure to VEGF therapy, the encouraging response rates, median time to response, and PFS for lenvatinib have prompted further investigation in a phase 3 trial. Cancer 2015;121:2749-2756. © 2015 American Cancer Society.

BACKGROUND

Inflammation is a well-known etiological <em>factor</em> for colorectal cancer, but mechanisms underlying the linkage between inflammation and cancer are incompletely understood. We hypothesized that two pro-inflammatory cytokines, TNFα and IL-<em>17</em>, might play a role in promoting colorectal carcinogenesis. Aerobic glycolysis is a metabolic adaptation that promotes the survival/proliferation of cancer cells. Paracrine signaling between tumor cells and cancer-associated <em>fibroblasts</em> also plays a role in carcinogenesis.

METHODS

The effect of TNFα and IL-<em>17</em> on aerobic glycolysis and growth <em>factor</em> production in cultured human colorectal cancer cells was investigated. Glucose utilization and lactate production were quantified by measuring the disappearance of glucose and appearance of lactate in the culture medium. Glucose transporter and glycolytic enzyme expression levels were measured by immunoblotting.

RESULTS

TNFα and IL-<em>17</em> cooperatively stimulated glycolysis in HT-29, T84, Caco-2 and HCT116 colorectal cancer cells. Treatment of HT-29 cells with TNFα plus IL-<em>17</em> also increased the expression of HIF-1α and c-myc, two <em>factor</em>s know to induce the transcription of genes encoding components of the glycolytic pathway. To further investigate mechanisms for cytokine-stimulated glycolysis, the effects of TNFα and IL-<em>17</em> on expression of six members and one regulator of the glycolytic pathway were investigated. TNFα and IL-<em>17</em> cooperatively increased the expression of the glucose transporter SLC2A1 and hexokinase-2 but did not regulate expression of glucose transporter SLC2A3, enolase-1, pyruvate kinase M2, lactate dehydrogenase A, or 6-phoshofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3). Experiments with inhibitors indicated that HIF-1α played a role in induction of SLC2A1 and that the transcription <em>factor</em> NF-κB played a role in induction of hexokinase-2 by TNFα and IL-<em>17</em>. TNFα and IL-<em>17</em> also synergistically stimulated production by HT-29 cells of a growth <em>factor</em> that simulated proliferation/survival of NIL8 fibroblastic cells. The activity of this <em>factor</em> was not specifically inhibited by the EGFR inhibitor AG1478, indicating that it is not an EGFR ligand.

CONCLUSIONS

Chronic inflammation is known to promote colorectal tumorigenesis. The pro-inflammatory cytokines TNFα and IL-<em>17</em> may contribute to this effect by stimulating glycolysis and growth <em>factor</em> production in colorectal cancer cells.

Fibrin sealant products are used in hemostasis and tissue sealing, and potentially as a cell delivery vehicle. In this study, fibrin sealant was evaluated as a delivery vehicle for human dermal <em>fibroblasts</em>. <em>Fibroblast</em> proliferation and migration were assessed in various dilutions of fibrin sealant by changing the fibrinogen and thrombin concentration. <em>Fibroblasts</em> proliferated well within three-dimensional (3-D) fibrin clots consisting of fibrinogen (5-<em>17</em> mg/mL) and thrombin (1-167 U/mL). These <em>fibroblasts</em> also retained good morphology and <em>growth</em> characteristics after migrating out of the 3-D fibrin clots. Furthermore, using Western blot and fluorescence-activated cell-sorting analysis, we found that the expression of <em>growth</em> <em>factors</em> and interleukins in the entire <em>fibroblast</em>-fibrin construct was dependent on the fibrin sealant formulation. For example, in a formulation in which <em>fibroblasts</em> showed modest proliferation and migration, interleukin 8 was secreted to a lesser extent than in a formulation that supported robust proliferation and migration. To our knowledge, this is the first time that it has been shown that modifying the concentration of fibrinogen and thrombin affects <em>fibroblast</em> behavior within formed 3-D fibrin clots. In addition, some of these formulations present an ideal delivery vehicle for <em>fibroblasts</em> that could be used for the treatment of chronic wounds.

BACKGROUND

Kallmann syndrome is a clinically and genetically heterogeneous disorder. To date, loss-of-function mutations in the genes encoding anosmin-1 (KAL1) and fibroblast growth factor receptor 1 (FGFR1) have been described in the X-linked and autosomal dominant forms of this syndrome, respectively.

OBJECTIVE

The objective was to investigate genetic defects in the KAL1 and FGFR1 genes in patients with congenital isolated hypogonadotropic hypogonadism (IHH).

METHODS

Eighty patients (71 males and nine females) with IHH were studied, of which 30 were familial. Forty-six of them had olfactory abnormalities.

METHODS

The coding regions of both KAL1 and FGFR1 genes were amplified and automatically sequenced. The KAL1 mutations were investigated only in patients with olfactory abnormalities, whereas FGFR1 was studied in the entire group.

RESULTS

Two novel KAL1 mutations, an intragenic deletion of exons 3-6 and a splicing mutation IVS7 + 1G>A, were identified in two of 46 patients with Kallmann syndrome. Eight novel heterozygous FGFR1 mutations (G48S, L245P, R250W, A343V, P366L, K618fsX654, P722S, and V795I) were identified in nine of 80 patients with IHH. Eight of them had olfactory abnormalities. Interestingly, the G48S mutation was identified in a normosmic IHH patient. Two unrelated females, who carried FGFR1 mutations, had anosmia and normal reproductive function.

CONCLUSIONS

We identified novel mutations in KAL1 and FGFR1 genes in IHH patients. FGFR1 mutations were identified in 17% of the patients with olfactory abnormalities and in one of 34 normosmic IHH patients. In addition, isolated anosmia was identified in two unrelated females as a partial phenotypic manifestation of FGFR1 defects.