BACKGROUND

Lenvatinib is an oral inhibitor of multiple receptor tyrosine kinases (RTKs) targeting vascular endothelial growth factor receptor (VEGFR1-3), fibroblast growth factor receptor (FGFR1-4), platelet growth factor receptor α (PDGFR α), RET and KIT. Antiangiogenesis activity of lenvatinib in VEGF- and FGF-driven angiogenesis models in both in vitro and in vivo was determined. Roles of tumor vasculature (microvessel density (MVD) and pericyte coverage) as biomarkers for lenvatinib were also examined in this study.

METHODS

We evaluated antiangiogenesis activity of lenvatinib against VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. Effects of lenvatinib on in vivo angiogenesis, which was enhanced by overexpressed VEGF or FGF in human pancreatic cancer KP-1 cells, were examined in the mouse dorsal air sac assay. We determined antitumor activity of lenvatinib in a broad panel of human tumor xenograft models to test if vascular score, which consisted of high MVD and low pericyte coverage, was associated with sensitivity to lenvatinib treatment. Vascular score was also analyzed using human tumor specimens with 18 different types of human primary tumors.

RESULTS

Lenvatinib inhibited VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. In vivo angiogenesis induced by overexpressed VEGF (KP-1/VEGF transfectants) or FGF (KP-1/FGF transfectants) was significantly suppressed with oral treatments of lenvatinib. Lenvatinib showed significant antitumor activity in KP-1/VEGF and five 5 of 7 different types of human tumor xenograft models at between 1 to 100 mg/kg. We divided 19 human tumor xenograft models into lenvatinib-sensitive (tumor-shrinkage) and relatively resistant (slow-growth) subgroups based on sensitivity to lenvatinib treatments at 100 mg/kg. IHC analysis showed that vascular score was significantly higher in sensitive subgroup than relatively resistant subgroup (p < 0.0004). Among 18 types of human primary tumors, kidney cancer had the highest MVD, while liver cancer had the lowest pericyte coverage, and cancers in Kidney and Stomach had highest vascular score.

CONCLUSIONS

These results indicated that Lenvatinib inhibited VEGF- and FGF-driven angiogenesis and showed a broad spectrum of antitumor activity with a wide therapeutic window. MVD and pericyte-coverage of tumor vasculature might be biomarkers and suggest cases that would respond for lenvatinib therapy.

BACKGROUND

Although <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) can promote liver carcinogenesis in mice, its involvement in human hepatocellular carcinoma (HCC) has not been well investigated. FGF<em>19</em>, a member of the FGF family, has unique specificity for its receptor FGFR4. This study aimed to clarify the involvement of FGF<em>19</em> in the development of HCC.

METHODS

We investigated human FGF<em>19</em> and FGFR4 expression in 40 hepatocellular carcinoma specimens using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) analysis and immunohistochemistry. Moreover, we examined the expression and the distribution of FGF<em>19</em> and FGFR4 in 5 hepatocellular carcinoma cell lines (HepG2, HuH7, HLE, HLF, and JHH7) using RT-PCR and immunohistochemistry. To test the role of the FGF<em>19</em>/FGFR4 system in tumor progression, we used recombinant FGF<em>19</em> protein and small interfering RNA (siRNA) of FGF<em>19</em> and FGFR4 to regulate their concentrations.

RESULTS

We found that FGF<em>19</em> was significantly overexpressed in HCCs as compared with corresponding noncancerous liver tissue (P < 0.05). Univariate and multivariate analyses revealed that the tumor FGF<em>19</em> mRNA expression was an independent prognostic <em>factor</em> for overall and disease-free survival. Moreover, we found that the FGF<em>19</em> recombinant protein could increase the proliferation (P < 0.01, n = 12) and invasion (P < 0.01, n = 6) capabilities of human hepatocellular carcinoma cell lines and inhibited their apoptosis (P < 0.01, n = 12). Inversely, decreasing FGF<em>19</em> and FGFR4 expression by siRNA significantly inhibited proliferation and increased apoptosis in JHH7 cells (P < 0.01, n = 12). The postoperative serum FGF<em>19</em> levels in HCC patients was significantly lower than the preoperative levels (P < 0.01, n = 29).

CONCLUSIONS

FGF<em>19</em> is critically involved in the development of HCCs. Targeting FGF<em>19</em> inhibition is an attractive potential therapeutic strategy for HCC.

Small Heterodimer Partner (SHP) inhibits activities of numerous transcription <em>factors</em> involved in diverse biological pathways. As an important metabolic regulator, SHP plays a key role in maintaining cholesterol and bile acid homeostasis by inhibiting cholesterol conversion to bile acids. While SHP gene induction by increased bile acids is well established, whether SHP activity is also modulated remains unknown. Here, we report surprising findings that SHP is a rapidly degraded protein via the ubiquitin-proteasomal pathway and that bile acids or bile acid-induced intestinal <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) increases stability of hepatic SHP by inhibiting proteasomal degradation in an extracellular signal-regulated kinase (ERK)-dependent manner. SHP was ubiquitinated at Lys122 and Lys123, and mutation of these sites altered its stability and repression activity. Tandem mass spectrometry revealed that upon bile acid treatment, SHP was phosphorylated at Ser26, within an ERK motif in SHP, and mutation of this site dramatically abolished SHP stability. Surprisingly, SHP stability was abnormally elevated in ob/ob mice and diet-induced obese mice. These results demonstrate an important role for regulation of SHP stability in bile acid signaling in normal conditions, and that abnormal stabilization of SHP may be associated with metabolic disorders, including obesity and diabetes.

The purpose of this study was to test the hypothesis that specific cytokines are involved in the initiation and evolution of the fibrotic process in adhesive capsulitis of the shoulder. After approval from the Institutional Review Board, biopsies of shoulder capsule and synovium were collected during shoulder arthroscopy from <em>19</em> patients with adhesive capsulitis, 14 patients with nonspecific synovitis and no fibrosis or clinical evidence of adhesive capsulitis, and seven patients undergoing surgery for another pathology who had a normal capsule and synovium. Immunohistochemical localization with monoclonal antibodies to transforming <em>growth</em> <em>factor</em>-beta and its receptor, platelet-derived <em>growth</em> <em>factor</em> and its receptor, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, interleukin-1 beta, tumor necrosis <em>factor</em>-alpha, and hepatocyte <em>growth</em> <em>factor</em> was performed using standard immunoperoxidase techniques. The frequency of cytokine staining was correlated with the clinical diagnosis. Synovial cells, <em>fibroblasts</em>, T-cells, and B-cells were identified with specific antibodies, and newly synthesized matrix was examined for type-I and type-III collagen by immunohistochemical staining. The predominant cell types present were synovial cells and <em>fibroblasts</em>. Staining for type-III collagen in adhesive capsulitis tissues indicated new deposition of collagen in the capsule. There was staining for transforming <em>growth</em> <em>factor</em>-beta and its receptor, platelet-derived <em>growth</em> <em>factor</em> and its receptor, interleukin-1 beta, and tumor necrosis <em>factor</em>-alpha in adhesive capsulitis and nonspecific synovitis tissues, compared with minimal staining in normal capsule. Staining was more frequent in synovial cells than in capsular cells. The frequency of cell and matrix staining for transforming <em>growth</em> <em>factor</em>-beta, platelet-derived <em>growth</em> <em>factor</em>, and hepatocyte <em>growth</em> <em>factor</em> was greater in adhesive capsulitis tissues than in those from patients with nonspecific synovitis. No difference in the frequency of staining between primary (idiopathic) and secondary adhesive capsulitis was found. The results of this study indicate that adhesive capsulitis involves both synovial hyperplasia and capsular fibrosis. Cytokines such as transforming <em>growth</em> <em>factor</em>-beta and platelet-derived <em>growth</em> <em>factor</em> may be involved in the inflammatory and fibrotic processes in adhesive capsulitis. Matrix-bound transforming <em>growth</em> <em>factor</em>-beta may act as a persistent stimulus, resulting in capsular fibrosis. Understanding the basic pathophysiology of adhesive capsulitis is an important step in the development of clinically useful antifibrotic agents that may serve as novel treatments for patients with this conditions.

OBJECTIVE

Roux-en-Y gastric bypass (RYGB) in humans can remit type 2 diabetes, but the operative mechanism is not completely understood. In mice, fibroblast growth factor (FGF) 15 (FGF19 in humans) regulates hepatic bile acid (BA) production and can also resolve diabetes. In this study, we tested the hypothesis that the FGF19-BA pathway plays a role in the remission of human diabetes after RYGB surgery.

METHODS

Cohorts of diabetic and nondiabetic individuals of various body weights were used. In addition, RYGB patients without diabetes (No-Diabetes), RYGB patients with diabetes who experienced remission for at least 12 months after surgery (Diabetes-R), and RYGB patients with diabetes who did not go into remission after surgery (Diabetes-NoR) were studied. Circulating FGF19 and BA levels, hepatic glycogen content, and expression levels of genes regulating the FGF19-BA pathway were compared among these groups of patients using pre- and postoperative serum samples and intraoperative liver biopsies.

RESULTS

Preoperatively, patients with diabetes had lower FGF19 and higher BA levels than nondiabetic patients, irrespective of body weight. In diabetic patients undergoing RYGB, lower FGF19 levels were significantly correlated with increased hepatic expression of the cholesterol 7alpha-hydroxylase 1 (CYP7A1) gene, which modulates BA production. Following RYGB surgery, however, FGF19 and BA levels (particularly cholic and deoxycholic acids) exhibited larger increases in Diabetic-R patients compared with nondiabetic and Diabetic-NoR patients.

CONCLUSIONS

Taken together, the baseline and postoperative data implicate the FGF19-CYP7A1-BA pathway in the etiology and remission of type 2 diabetes following RYGB surgery.

Given the high prevalence and rising incidence of non-alcoholic fatty liver disease (NAFLD), the absence of approved therapies is striking. Although the mainstay of treatment of NAFLD is weight loss, it is hard to maintain, prompting the need for pharmacotherapy as well. A greater understanding of disease pathogenesis in recent years was followed by development of new classes of medications, as well as potential repurposing of currently available agents. NAFLD therapies target four main pathways. The dominant approach is targeting hepatic fat accumulation and the resultant metabolic stress. Medications in this group include peroxisome proliferator-activator receptor agonists (eg, pioglitazone, elafibranor, saroglitazar), medications targeting the bile acid-farnesoid X receptor axis (obeticholic acid), inhibitors of de novo lipogenesis (aramchol, NDI-010976), incretins (liraglutide) and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-21 or FGF-<em>19</em> analogues. A second approach is targeting the oxidative stress, inflammation and injury that follow the metabolic stress. Medications from this group include antioxidants (vitamin E), medications with a target in the tumour necrosis <em>factor</em> α pathway (emricasan, pentoxifylline) and immune modulators (amlexanox, cenicriviroc). A third group has a target in the gut, including antiobesity agents such as orlistat or gut microbiome modulators (IMM-124e, faecal microbial transplant, solithromycin). Finally, as the ongoing injury leads to fibrosis, the harbinger of liver-related morbidity and mortality, antifibrotics (simtuzumab and GR-MD-02) will be an important element of therapy. It is very likely that in the next few years several medications will be available to clinicians treating patients with NAFLD across the entire spectrum of disease.

Defects in heart development are the most common congenital abnormalities in humans, providing a strong incentive to learn more about the underlying causes. Previous studies have implicated the metalloprotease-disintegrins ADAMs (a disintegrin and metalloprotease) 17 and <em>19</em> as well as heparin binding EGF-like <em>growth</em> <em>factor</em> (HB-EGF) and neuregulins in heart development in mice. Here, we show that mice lacking both ADAMs 17 and <em>19</em> have exacerbated defects in heart development compared to mice lacking either ADAM, providing the first evidence for redundant or compensatory functions of ADAMs in development. Moreover, we identified additional compensatory or redundant roles of ADAMs 9 and <em>19</em> in morphogenesis of the mitral valve and cardiac outflow tract. Cell biological studies designed to address the functions of these ADAMs in shedding of HB-EGF uncovered a contribution of ADAM<em>19</em> to this process, but this was only evident in the absence of the major HB-EGF sheddase, ADAM17. In addition, ADAM17 emerged as the major sheddase for neuregulins beta1 and beta2 in mouse embryonic <em>fibroblasts</em>. These results raise the possibility that ADAMs 9, 17, and <em>19</em> contribute to heart development in humans and have implications for understanding the mechanisms underlying congenital heart disease.

BACKGROUND

Klotho and fibroblast growth factor 23 (FGF23) are key regulators of mineral metabolism in renal insufficiency. FGF23 levels have been shown to increase early in chronic kidney disease (CKD); however, the corresponding soluble Klotho levels at the different CKD stages are not known.

METHODS

Soluble Klotho, FGF23, parathyroid hormone (PTH), 1,25-dihydroxy vitamin D(3) (1,25D) and other parameters of mineral metabolism were measured in an observational cross-sectional study in 87 patients. Locally weighted scatter plot smoothing function of these parameters were plotted versus estimated glomerular filtration rate (eGFR) to illustrate the pattern of the relationship. Linear and non-linear regression analyses were performed to estimate changes in mineral metabolism parameters per 1mL/min/1.73 m(2) decline.

RESULTS

In CKD 1-5, Klotho and 1,25D linearly decreased, whereas both FGF23 and PTH showed a baseline at early CKD stages and then a curvilinear increase. Crude mean Klotho level declined by 4.8 pg/mL (95% CI 3.5-6.2 pg/mL, P < 0.0001) and 1,25D levels by 0.30 ng/L (95% CI 0.18-0.41 ng/L, P < 0.0001) as GFR declined by 1 mL/min/1.73 m(2). After adjustment for age, gender, serum 25-hydroxyvitamin D levels and concomitant medications (calcium, supplemental vitamin D and calcitriol), we estimated that the mean Klotho change was 3.2 pg/mL (95% CI 1.2-5.2 pg/mL, P = 0.0019) for each 1 mL/min/1.73 m(2) GFR change. FGF23 departed from the baseline at an eGFR of 47 mL/min/1.73 m(2) (95% CI 39-56 mL/min/1.73 m(2)), whereas PTH departed at an eGFR of 34 mL/min/1.73 m(2) (95% CI 19-50 mL/min/1.73 m(2)).

CONCLUSIONS

Soluble Klotho and 1,25D levels decrease and FGF23 levels increase at early CKD stages, whereas PTH levels increase at more advanced CKD stages.

The early <em>growth</em> response 1 (Egr1) gene is a transcription <em>factor</em> that acts as both a tumor suppressor and a tumor promoter. Egr1-null mouse embryo <em>fibroblasts</em> bypass replicative senescence and exhibit a loss of DNA damage response and an apparent immortal <em>growth</em>, suggesting loss of p53 functions. Stringent expression analysis revealed 266 transcripts with >2-fold differential expression in Egr1-null mouse embryo <em>fibroblasts</em>, including 143 known genes. Of the 143 genes, program-assisted searching revealed 66 informative genes linked to Egr1. All 66 genes could be placed on a single regulatory network consisting of three branch points of known Egr1 target genes: TGFbeta1, IL6, and IGFI. Moreover, <em>19</em> additional genes that are known targets of p53 were identified, indicating that p53 is a fourth branch point. Electrophoretic mobility shift assay as well as chromatin immunoprecipitation confirmed that p53 is a direct target of Egr1. Because deficient p53 expression causes tumors in mice, we tested the role of Egr1 in a two-step skin carcinogenesis study (144 mice) that revealed a uniformly accelerated development of skin tumors in Egr1-null mice (P < 0.005). These studies reveal a new role for Egr1 as an in vivo tumor suppressor.

OBJECTIVE

Cholestasis is a liver disorder characterized by impaired bile flow, reduction of bile acids (BAs) in the intestine, and retention of BAs in the liver. The farnesoid X receptor (FXR) is the transcriptional regulator of BA homeostasis. Activation of FXR by BAs reduces circulating BA levels in a feedback mechanism, repressing hepatic cholesterol 7α-hydroxylase (Cyp7a1), the rate-limiting enzyme for the conversion of cholesterol to BAs. This mechanism involves the hepatic nuclear receptor small heterodimer partner and the intestinal <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>19</em> and 15. We investigated the role of activation of intestine-specific FXR in reducing hepatic levels of BAs and protecting the liver from cholestasis in mice.

METHODS

We generated transgenic mice that express a constitutively active FXR in the intestine. Using FXR gain- and loss-of-function models, we studied the roles of intestinal FXR in mice with intrahepatic and extrahepatic cholestasis.

RESULTS

Selective activation of intestinal FXR induced FGF15 and repressed hepatic Cyp7a1, reducing the pool size of BAs and changing the BA pool composition. Activation of intestinal FXR protected mice from obstructive extrahepatic cholestasis after bile duct ligation or administration of α-naphthylisothiocyanate. In Mdr2(-/-) mice, transgenic expression of activated FXR in the intestine protected against liver damage, whereas absence of FXR promoted progression of liver disease.

CONCLUSIONS

Activation of FXR transcription in the intestine protects the liver from cholestasis in mice by inducing FGF15 expression and reducing the hepatic pool of BA; this approach might be developed to reverse cholestasis in patients.

The transcription <em>factor</em> nuclear <em>factor</em>-kappaB (NF-kappaB) regulates genes important for tumor invasion, metastasis and chemoresistance. Normally, NF-kappaB remains sequestered in an inactive state by cytoplasmic inhibitor-of-kappaB (IkappaB) proteins. NF-kappaB translocates to nucleus and activates gene expression upon exposure of cells to <em>growth</em> <em>factors</em> and cytokines. We and others have shown previously that NF-kappaB is constitutively active in a subset of breast cancers. In this study, we show that constitutive activation of NF-kappaB leads to overexpression of the anti-apoptotic genes c-inhibitor of apoptosis 2 (c-IAP2) and manganese superoxide dismutase (Mn-SOD) in breast cancer cells. Furthermore, expression of the anti-apoptotic tumor necrosis <em>factor</em> receptor associated <em>factor</em> 1 (TRAF1) and defender-against cell death (DAD-1) is regulated by NF-kappaB in certain breast cancer cells. We also demonstrate that NF-kappaB-inducible genes protect cancer cells against paclitaxel as MDA-MB-231 breast cancer cells modified to overexpress IkappaBalpha required lower concentrations of paclitaxel to arrest at the G2/M phase of the cell cycle and undergo apoptosis when compared to parental cells. The effect of NF-kappaB on paclitaxel-sensitivity appears to be specific to cancer cells because normal <em>fibroblasts</em> derived from embryos lacking p65 subunit of NF-kappaB and wild type littermate embryos were insensitive to paclitaxel-induced G2/M cell cycle arrest. Parthenolide, an active ingredient of herbal remedies such as feverfew (tanacetum parthenium), mimicked the effects of IkappaBalpha by inhibiting NF-kappaB DNA binding activity and Mn-SOD expression, and increasing paclitaxel-induced apoptosis of breast cancer cells. These results suggest that active ingredients of herbs with anti-inflammatory properties may be useful in increasing the sensitivity of cancers with constitutively active NF-kappaB to chemotherapeutic drugs. Oncogene (2000) <em>19</em>, 4159 - 4169

We have identified a novel <em>fibroblast</em> <em>growth</em> <em>factor</em>, FGF-<em>19</em>, the most distant member of the FGF family described to date. FGF-<em>19</em> is a high affinity, heparin dependent ligand for FGFR4 and is the first member of the FGF family to show exclusive binding to FGFR4. Human FGF-<em>19</em> maps to chromosome 11 q13.1, a region associated with an osteoporosis-pseudoglioma syndrome of skeletal and retinal defects. FGF-<em>19</em> message is expressed in several tissues including fetal cartilage, skin, and retina, as well as adult gall bladder and is overexpressed in a colon adenocarcinoma cell line.

Aberrant signaling through the <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>)/<em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 4 (FGFR 4) signaling complex has been shown to cause hepatocellular carcinoma (HCC) in mice and has been implicated to play a similar role in humans. We have developed BLU9931, a potent and irreversible small-molecule inhibitor of FGFR4, as a targeted therapy to treat patients with HCC whose tumors have an activated FGFR4 signaling pathway. BLU9931 is exquisitely selective for FGFR4 versus other FGFR family members and all other kinases. BLU9931 shows remarkable antitumor activity in mice bearing an HCC tumor xenograft that overexpresses FGF<em>19</em> due to amplification as well as a liver tumor xenograft that overexpresses FGF<em>19</em> mRNA but lacks FGF<em>19</em> amplification. Approximately one third of patients with HCC whose tumors express FGF<em>19</em> together with FGFR4 and its coreceptor klotho β (KLB) could potentially respond to treatment with an FGFR4 inhibitor. These findings are the first demonstration of a therapeutic strategy that targets a subset of patients with HCC.

CONCLUSIONS

This article documents the discovery of BLU9931, a novel irreversible kinase inhibitor that specifically targets FGFR4 while sparing all other FGFR paralogs and demonstrates exquisite kinome selectivity. BLU9931 is efficacious in tumors with an intact FGFR4 signaling pathway that includes FGF<em>19</em>, FGFR4, and KLB. BLU9931 is the first FGFR4-selective molecule for the treatment of patients with HCC with aberrant FGFR4 signaling.

OBJECTIVE

To compare cell cycle kinetics in corneal endothelial cells from young and old donors.

METHODS

Human corneas were obtained from the eye bank and separated into two groups: young (<em>19</em> corneas, <30 years of age) and old (40 corneas, >50 years of age). Corneas were cut in quarters, and the endothelium was released from contact inhibition by producing a 2-mm scrape wound. Unwounded endothelium acted as a negative control. Corneal pieces were exposed for 24, 36, 48, 60, 72, and 84 hours to medium containing 10% fetal bovine serum, 20 ng/ml <em>fibroblast</em> <em>growth</em> <em>factor</em>, and 50 mg/ml gentamicin or the same medium supplemented with 10 ng/ml epidermal <em>growth</em> <em>factor</em> (EGF). Tissue was fixed, immunostained for Ki67 (a marker for the late G1-through M-phase) or for 5-bromo-2'-deoxyuridine (BrdU; a marker for the S-phase), and mounted in medium containing propidium iodide (PI) to visualize all nuclei. Confocal images were evaluated using an image analysis program to count Ki67-positive and PI-stained cells and to evaluate cell cycle position. Cells were counted in 15x100 microm2 areas randomly selected from each wound, and the mean was used for subsequent calculations.

RESULTS

Human corneal endothelial cells could be reliably scored for their position within the cell cycle using Ki67 staining patterns. In both age groups, cells repopulating the wound area stained positively for Ki67, whereas no Ki67 staining was observed in unwounded areas under any condition tested. Cells from old donors treated with fetal bovine serum and FGF stained positively for Ki67, indicating that these cells were actively cycling. Compared with cells from young donors, old cells entered the cell cycle more slowly (48 versus 36 hours), the peak of Ki67 staining occurred later (72 versus 60 hours), and fewer cells proliferated (23% versus 47%) or exhibited mitotic figures (4% versus 7%). Addition of EGF to the culture medium increased Ki67 staining in both groups, but the effect on old cells was more dramatic. More cells from old donors entered the cell cycle by 36 hours after wounding, the number of proliferating cells increased 1.6-fold, and the relative number of mitotic figures increased 2.5-fold over cells treated in the absence of EGF.

CONCLUSIONS

Regardless of donor age, corneal endothelial cells can enter and complete the cell cycle. In the presence of fetal bovine serum and FGF, cells from old donors can proliferate but respond more slowly and to a lesser extent than cells from young donors. EGF added to the medium stimulates cells from old donors to enter the cell cycle faster, increases the relative number of actively cycling cells, and increases the number of cells exhibiting mitotic figures. The resultant hypothesis is that it is possible to stimulate a significant proliferative response in corneal endothelial cells from old individuals. Administration of an optimal combination of stimulatory growth factors is required under conditions in which cells have been transiently released from contact inhibition.

Bile acids are regulators of lipid and glucose metabolism, and modulate inflammation in the liver and other tissues. Primary bile acids such as cholic acid and chenodeoxycholic acid (CDCA) are produced in the liver, and converted into secondary bile acids such as deoxycholic acid (DCA) and lithocholic acid by gut microbiota. Here we investigated the possible roles of bile acids in non-alcoholic fatty liver disease (NAFLD) pathogenesis and the impact of the gut microbiome on bile acid signalling in NAFLD.

Serum bile acid levels and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>), liver gene expression profiles and gut microbiome compositions were determined in patients with NAFLD, high-fat diet-fed rats and their controls.

Serum concentrations of primary and secondary bile acids were increased in patients with NAFLD. In per cent, the farnesoid X receptor (FXR) antagonistic DCA was increased, while the agonistic CDCA was decreased in NAFLD. Increased mRNA expression for cytochrome P450 7A1, Na+-taurocholate cotransporting polypeptide and paraoxonase 1, no change in mRNA expression for small heterodimer partner and bile salt export pump, and reduced serum FGF<em>19</em> were evidence of impaired FXR and <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 4 (FGFR4)-mediated signalling in NAFLD. Taurine and glycine metabolising bacteria were increased in the gut of patients with NAFLD, reflecting increased secondary bile acid production. Similar changes in liver gene expression and the gut microbiome were observed in high-fat diet-fed rats.

The serum bile acid profile, the hepatic gene expression pattern and the gut microbiome composition consistently support an elevated bile acid production in NAFLD. The increased proportion of FXR antagonistic bile acid explains, at least in part, the suppression of hepatic FXR-mediated and FGFR4-mediated signalling. Our study suggests that future NAFLD intervention may target the components of FXR signalling, including the bile acid converting gut microbiome.

OBJECTIVE

BIBF 1120 is an oral, potent angiokinase inhibitor targeting receptors of the vascular endothelial growth factors, platelet-derived growth factors, and fibroblast growth factors. This phase I, accelerated titration study assessed the maximum tolerated dose, safety, pharmacokinetics, and pharmacodynamic effects of BIBF 1120.

METHODS

Sixty-one patients with advanced cancers received BIBF 1120 in successive cohorts. Twenty-five received 50 to 450 mg once daily and 36 received 150 to 300 mg twice daily in 4-week treatment courses interspersed by 1 week of washout. Dynamic contrast-enhanced magnetic resonance imaging assessed antiangiogenic effect in 42 patients.

RESULTS

Most frequent BIBF 1120-related adverse events were mostly mild to moderate (Common Toxicity Criteria grade 1-2) nausea (68.9%), vomiting (45.9%), and diarrhea (44.3%). The majority of dose-limiting adverse events of Common Toxicity Criteria grade 3 or 4 were reversible liver enzyme elevations. The maximum tolerated dose was 250 mg of BIBF 1120 for once and twice daily dosing. BIBF 1120 was absorbed moderately fast (t(max) = 1-3 hours at steady state), with no deviation from dose linearity and no decrease of exposure over time. The gMean terminal half-life was from 13 to 19 hours. One complete and two partial responses occurred in patients with renal cell cancer (n = 2) and colorectal cancer (n = 1). Dynamic contrast-enhanced magnetic resonance imaging showed a significant reduction in tumor blood flow in 55% of evaluable patients.

CONCLUSIONS

BIBF 1120 dosed continuously displayed a favorable safety and pharmacokinetics profile, and first efficacy signals were observed. Twice daily dosing permitted increased drug exposure without additional toxicity. Two hundred milligrams BIBF 1120 twice daily is the recommended dose for phase II monotherapy studies.

The C57BL/6J strain is one of the most widely used animal models for biomedical research, and individual mice within the strain are often assumed to be genetically identical after more than 70 yr of inbreeding. Using a single nucleotide polymorphism (SNP) genotyping panel, we assessed if copy number variations (CNVs) could be detected within the C57BL/6J strain by comparing relative allele frequencies in first generation (F(1)) progeny of C57BL/6J mice. Sequencing, quantitative PCR, breeding, and array comparative genomic hybridization (CGH) together confirmed the presence of two CNVs. Both CNVs span genes encoded on chromosome <em>19</em>, and quantitative RT-PCR demonstrated that they result in altered expression of the insulin-degrading enzyme (Ide) and <em>fibroblast</em> <em>growth</em> <em>factor</em> binding protein 3 (Fgfbp3) genes. Analysis of 39 different C57BL/6J breeders revealed that 64% of mice from the Jackson Laboratory colony were heterozygous for the CNV spanning Ide. Homozygotes with and without the duplication were present in concordance with Hardy-Weinberg equilibrium (13% and 23%, respectively), and analysis of archived samples from the C57BL/6J colony suggests that the duplication has rapidly reached a high frequency in the colony since <em>19</em>94. The identification of two CNVs in the small portion of the genome screened demonstrates that individual mice of highly inbred strains are not isogenic and suggests other CNVs may be segregating within C57BL/6J as well as other carefully maintained inbred strains. These differences can influence interpretations of physiological, biomedical, and behavioral experiments and can be exploited to model CNVs apparent in the human genome.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family signaling largely controls cellular homeostasis through short-range intercell paracrine communication. Recently FGF15/<em>19</em>, 21, and 23 have been implicated in endocrine control of metabolic homeostasis. The identity and location of the FGF receptor isotypes that mediate these effects are unclear. The objective was to determine the role of FGFR4, an isotype that has been proposed to mediate an ileal FGF15/<em>19</em> to hepatocyte FGFR4 axis in cholesterol homeostasis, in metabolic homeostasis in vivo.

METHODS

FGFR4(-/-) mice-mice overexpressing constitutively active hepatic FGFR4--and FGFR4(-/-) with constitutively active hepatic FGFR4 restored in the liver were subjected to a normal and a chronic high-fat diet sufficient to result in obesity. Systemic and liver-specific metabolic phenotypes were then characterized.

RESULTS

FGFR4-deficient mice on a normal diet exhibited features of metabolic syndrome that include increased mass of white adipose tissue, hyperlipidemia, glucose intolerance, and insulin resistance, in addition to hypercholesterolemia. Surprisingly, the FGFR4 deficiency alleviated high-fat diet-induced fatty liver in obese mice, which is also a correlate of metabolic syndrome. Restoration of FGFR4, specifically in hepatocytes of FGFR4-deficient mice, decreased plasma lipid levels and restored the high-fat diet-induced fatty liver but failed to restore glucose tolerance and sensitivity to insulin.

CONCLUSIONS

FGFR4 plays essential roles in systemic lipid and glucose homeostasis. FGFR4 activity in hepatocytes that normally serves to prevent systemic hyperlipidemia paradoxically underlies the fatty liver disease associated with chronic high-fat intake and obesity.

Previous studies have shown that administration of <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>19</em> (FGF-<em>19</em>) reverses diabetes, hepatic steatosis, hyperlipidemia, and adipose accretion in animal models of obesity. To investigate the mechanism for this effect, we determined whether FGF-<em>19</em> modulated hepatic fatty acid synthesis, a key process controlling glucose tolerance and triacylglycerol accumulation in liver, blood, and adipose tissue. Incubating primary hepatocyte cultures with recombinant FGF-<em>19</em> suppressed the ability of insulin to stimulate fatty acid synthesis. This effect was associated with a reduction in the expression of lipogenic enzymes. FGF-<em>19</em> also suppressed the insulin-induced expression of sterol regulatory element-binding protein-1c (SREBP-1c), a key transcriptional activator of lipogenic genes. FGF-<em>19</em> inhibition of lipogenic enzyme expression was not mediated by alterations in the activity of the insulin signal transduction pathway or changes in the activity of ERK, p38 MAPK, and AMP-activated protein kinase (AMPK). In contrast, FGF-<em>19</em> increased the activity of STAT3, an inhibitor of SREBP-1c expression and decreased the expression of peroxisome proliferator-activated receptor-gamma coactivator-1beta (PGC-1beta), an activator of SREBP-1c activity. FGF-<em>19</em> also increased the expression of small heterodimer partner (SHP), a transcriptional repressor that inhibits lipogenic enzyme expression via a SREBP-1c-independent mechanism. Inhibition of SREBP-1c activity by changes in STAT3 and PGC-1beta activity and inhibition of gene transcription by an elevation in SHP expression can explain the inhibition of lipogenesis caused by FGF-<em>19</em>. In summary, the inhibitory effect of FGF-<em>19</em> on insulin activation of hepatic fatty acid synthesis constitutes a mechanism that would explain the beneficial effect of FGF-<em>19</em> on metabolic syndrome.

OBJECTIVE

Variations in genes that regulate bile acid (BA) synthesis are associated with colonic transit in patients with irritable bowel syndrome (IBS). We investigated features of BA synthesis and excretion and genetic features of patients with different types of IBS.

METHODS

In 26 healthy volunteers, 26 patients with IBS and constipation (IBS-C), and 26 with IBS and diarrhea (IBS-D), we measured serum levels of 7α-hydroxy-4-cholesten-3-one (C4; a surrogate for BA synthesis) and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>19</em> (an ileal hormone that downregulates BA synthesis). For stool samples, we measured concentration of BA, weight, and amount of fat when participants were given high-fat diets. Spearman correlations were used to explore relationships among <em>factors</em>. We analyzed 1 polymorphism in Klotho-β (KLB) and 3 in <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor-4 (FGFR4) for all members of each group using analysis of covariance.

RESULTS

The concentration of BA in stool was associated with group (for a comparison of 3 groups; P = .057); it was higher in patients with IBS-D than IBS-C (P = .017). The serum level of C4 was higher in patients with IBS-D than IBS-C (P = .02) or healthy volunteers (P = .01); 38% of patients with IBS-D had increased serum levels of C4, compared with healthy volunteers. Serum level of C4 correlated with stool concentration of BA (rs = 0.606; P < .001), serum FGF<em>19</em> (rs = -0.324; P = .007), and stool weight (rs = 0.366; P = .003). Stool concentration of BA correlated with weight (rs = 0.737; P < .001) and level of fat (rs = 0.528; P < .001). Body mass index correlated with serum level of C4 (rs = 0.423, P < .001) and stool concentration of BA (rs = 0.507, P < .001), and was higher in patients with IBS-D compared with other groups (overall P = .036). FGFR4 rs<em>19</em>66265 was associated with stool level of BA (P = .032).

CONCLUSIONS

Patients with IBS-D have greater body mass index and synthesize and excrete higher levels of BA than individuals with IBS-C or healthy volunteers. Serum levels of C4 might be used to identify patients with IBS-D who have BA malabsorption; studies are needed to determine if some patients have a genetic predisposition to this disorder.

OBJECTIVE

Brivanib, a selective dual inhibitor of fibroblast growth factor and VEGF signaling, has recently been shown to have activity as first-line treatment for patients with advanced hepatocellular carcinoma (HCC). This phase II open-label study assessed brivanib as second-line therapy in patients with advanced HCC who had failed prior antiangiogenic treatment.

METHODS

Brivanib was administered orally at a dose of 800 mg once daily. The primary objectives were tumor response rate, time to response, duration of response, progression-free survival, overall survival (OS), disease control rate, time to progression (TTP), and safety and tolerability.

RESULTS

Forty-six patients were treated. Best responses to treatment with brivanib (N = 46 patients) using modified World Health Organization criteria were partial responses for two patients (4.3%), stable disease for 19 patients (41.3%), and progressive disease for 19 patients (41.3%). The tumor response rate was 4.3%; the disease control rate was 45.7%. Median OS was 9.79 months. Median TTP as assessed by study investigators following second-line treatment with brivanib was 2.7 months. The most common adverse events were fatigue, decreased appetite, nausea, diarrhea, and hypertension.

CONCLUSIONS

Brivanib had a manageable safety profile and is one of the first agents to show promising antitumor activity in advanced HCC patients treated with prior sorafenib.

Bile acids (BAs) are essential for fat absorption and appear to modulate glucose and energy metabolism. Colesevelam, a BA sequestrant, improves glycemic control in type 2 diabetes mellitus (T2DM). We aimed to characterize the alterations in BA metabolism associated with T2DM and colesevelam treatment and to establish whether metabolic consequences of T2DM and colesevelam are related to changes in BA metabolism. Male subjects with T2DM (n = 16) and controls (n = 12) were matched for age and body mass index. BA pool sizes and synthesis/input rates were determined before and after 2 and 8 weeks of colesevelam treatment. T2DM subjects had higher cholic acid (CA) synthesis rate, higher deoxycholic acid (DCA) input rate, and enlarged DCA pool size. Colesevelam resulted in a preferential increase in CA synthesis in both groups. CA pool size was increased whereas chenodeoxycholic acid and DCA pool sizes were decreased upon treatment. Fasting and postprandial <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) levels did not differ between controls and diabetics, but were decreased by treatment in both groups. Colesevelam treatment reduced hemoglobin A1C by 0.7% (P < 0.01) in diabetics. Yet, no relationships between BA kinetic parameters and changes in glucose metabolism were found in T2DM or with colesevelam treatment.

CONCLUSIONS

Our results reveal significant changes in BA metabolism in T2DM, particularly affecting CA and DCA. Colesevelam treatment reduced FGF<em>19</em> signaling associated with increased BA synthesis, particularly of CA, and resulted in a more hydrophilic BA pool without altering total BA pool size. However, these changes could not be related to the improved glycemic control in T2DM.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) is a hormone-like protein that regulates carbohydrate, lipid and bile acid metabolism. At supra-physiological doses, FGF<em>19</em> also increases hepatocyte proliferation and induces hepatocellular carcinogenesis in mice. Much of FGF<em>19</em> activity is attributed to the activation of the liver enriched FGF Receptor 4 (FGFR4), although FGF<em>19</em> can activate other FGFRs in vitro in the presence of the coreceptor βKlotho (KLB). In this report, we investigate the role of FGFR4 in mediating FGF<em>19</em> activity by using Fgfr4 deficient mice as well as a variant of FGF<em>19</em> protein (FGF<em>19</em>v) which is specifically impaired in activating FGFR4. Our results demonstrate that FGFR4 activation mediates the induction of hepatocyte proliferation and the suppression of bile acid biosynthesis by FGF<em>19</em>, but is not essential for FGF<em>19</em> to improve glucose and lipid metabolism in high fat diet fed mice as well as in leptin-deficient ob/ob mice. Thus, FGF<em>19</em> acts through multiple receptor pathways to elicit pleiotropic effects in regulating nutrient metabolism and cell proliferation.

Although there is experimental evidence supporting the involvement of hepatic stem cells in the pathogenesis of liver cancers, the detection and isolation of these cells remains elusive. A logical approach to detecting these cells would take advantage of their ability to differentiate (or to give rise to cells that differentiate) into hepatocytes. This approach requires an assay system that is conducive to hepatocytic differentiation. Here, we report the development of an in vitro system consisting of a three-dimensional collagen gel matrix and a <em>fibroblast</em> feeder layer that supports hepatocytic differentiation from precursor epithelial (oval) cell lines. The LE/2 and LE/6 oval cell lines used in this study are nontumorigenic cells that are derived from the livers of adult rats fed a choline-deficient diet containing 0.1% ethionine for 2 and 6 weeks, respectively. These lines consist of small cells that are phenotypically immature with few cytoplasmic organelles and a high nuclear-to-cytoplasmic ratio. After 4 weeks in the three-dimensional culture system, these cells acquired typical hepatocytic morphology. By electron microscopy, the cells formed canalicular structures that are typical of hepatocytes and were organelle rich, displaying peroxisomes, abundant mitochondria, and rough endoplasmic reticulum. The cells produced albumin and displayed a cytokeratin (CK) pattern typical of hepatocytes (CK 8 and CK 18-positive and CK <em>19</em>-negative). The presence of a mesenchymal cell feeder layer was essential for supporting hepatocytic differentiation. Without a feeder layer but in the presence of hepatocyte <em>growth</em> <em>factor</em> and/or keratinocyte <em>growth</em> <em>factor</em>, the precursor cells formed ductal structures, suggestive of differentiation along the bile duct lineage. The three-dimensional system described provides direct proof of the lineage generation capacity of oval cells. It offers a model to study <em>factors</em> that may be important for hepatocytic differentiation from precursor cells and a means to assay cell populations for their ability to give rise to normal and transformed hepatocytes.