Targeting FGFR4 Inhibits Hepatocellular Carcinoma in Preclinical Mouse Models

The fibroblast growth factor (FGF)-FGF receptor (FGFR) signaling system plays critical roles in a variety of normal developmental and physiological processes. It is also well documented that dysregulation of FGF-FGFR signaling may have important roles in tumor development and progression. The FGFR4–FGF19 signaling axis has been implicated in the development of hepatocellular carcinomas (HCCs) in mice, and potentially in humans. In this study, we demonstrate that FGFR4 is required for hepatocarcinogenesis; the progeny of FGF19 transgenic mice, which have previously been shown to develop HCCs, bred with FGFR4 knockout mice fail to develop liver tumors. To further test the importance of FGFR4 in HCC, we developed a blocking anti-FGFR4 monoclonal antibody (LD1). LD1 inhibited: 1) FGF1 and FGF19 binding to FGFR4, 2) FGFR4–mediated signaling, colony formation, and proliferation in vitro, and 3) tumor growth in a preclinical model of liver cancer in vivo. Finally, we show that FGFR4 expression is elevated in several types of cancer, including liver cancer, as compared to normal tissues. These findings suggest a modulatory role for FGFR4 in the development and progression of hepatocellular carcinoma and that FGFR4 may be an important and novel therapeutic target in treating this disease

In vivo efficacy of LD1.

<p><i>A</i>, LD1 inhibits FGF19-regulated <i>FOS</i> expression in mouse liver. The results are represented as fold expression relative to <i>FOS</i> levels in the livers of non-treated mice. <i>B</i>, LD1 (30 mg/kg; once weekly) inhibits HUH7 xenograft tumor growth in vivo. <i>C</i>, Effects of LD1 on the mRNA expression of <i>FGFR4</i>, <i>CYP7A1</i>, <i>FOS</i>, and <i>EGR1</i> in HUH7 xenograft tumors from Fig. 5B. <i>D</i>, Multiple, large, raised tumors (arrows) protruding from the hepatic surface of a DEN-accelerated FGF19-TG:FGFR4-WT mouse treated with a control antibody (upper panel). Liver of DEN-accelerated FGF19-TG:FGFR4-WT mouse treated with LD1 (lower panel). <i>E</i>, Liver weights of DEN–accelerated FGF19-TG:FGFR4-WT mice treated with control antibody, LD1, or 1A6 (anti-FGF19 antibody).</p

LD1 binds to FGFR4.

<p><i>A</i>, LD1 binds to human (h), mouse (m), and cynomolgus monkey (c) FGFR4, but does not bind to hFGFR1, hFGFR2, or hFGFR3. The binding of LD1 to immobilized FGFR-Fc chimeric proteins was determined by solid phase binding assay. <i>B</i>, Affinity of LD1 binding to mouse, cynomolgus monkey, and human FGFR4 as determined by surface plasmon resonance. <i>C</i>, Binding of LD1 to hFGFR4 expressed at the cell surface of stably transfected HEK293 cells as measured by FACS (RFU  =  Relative Fluorescence Unit). <i>D</i>, The binding of LD1 to immobilized hFGFR4-Flag chimeric proteins bearing point mutations as measured by a solid phase binding assay. <i>E</i>, The binding of LD1 to hFGFR4-Flag chimeric proteins bearing point mutations as evaluated by Western blot. Mutated proteins were electrophoresed and sequentially immunoblotted using LD1, an anti-FGFR4 (8G11), and an anti-Flag antibody. <i>F</i>, Dimer model illustrating the position of G165 (blue) on FGFR4 (red and yellow) bound to FGF19 (green).</p

LD1 inhibits FGFR4 activities.

<p><i>A</i>, LD1 inhibits FGFR4 binding to FGF1 and FGF19 as determined by solid phase binding assay. <i>B</i>, LD1 inhibits FGF1-stimulated proliferation of BaF3 cells stably expressing FGFR4/R1. <i>C</i>, LD1 inhibits FGFR4 signaling in L6 cells stably expressing FGFR4. <i>D</i>, Cell surface expression of FGFR4 protein in a subset of liver tumor cell lines as determined by FACS analysis using LD1.</p

FGFR4 is required for FGF19-mediated liver tumorigenesis.

<p><i>A</i>, Multiple, large, raised tumors (arrows) protruding from the hepatic surface of a 10-month-old FGF19-TG:FGFR4-WT mouse (left panel). Liver from a 10-month-old FGF19-TG:FGFR4-KO mouse (right panel). <i>B</i>, BrdU incorporation in female (left panel) and male (right panel) FGF19-TG or wild type mice bred with FGFR4-KO or FGFR4-WT mice. <i>C</i>, Prevalence of liver tumors in male and female FGF19-TG mice treated with DEN as determined by gross and histological examinations. <i>D</i>, Multiple, large, raised tumors (arrows) on the surface of the liver of a 4-month-old FGF19-TG:FGFR4-WT mouse treated with DEN. <i>E</i>, Liver weights from FGF19-TG or wild type female (left panel) and male (right panel) mice treated with DEN. The asterisk (*) indicates that the weight of the liver could not be measured from the 7-month time point for male FGF19-TG mice treated with DEN because none survived past 6 months of age. <i>F</i>, Liver weights of FGF19-TG or wild type female (left panel) and male (right panel) FGFR4-KO mice treated with DEN.</p

LD1 inhibits FGFR4 biological activities in liver cancer cell lines.

<p><i>A</i>, LD1 inhibits FGFR4 signaling in HEP3B cells as evaluated by Western blot. <i>B</i>, LD1 inhibits the FGFR4-regulated <i>CYP7A1</i> repression in HEP3B cells. <i>CYP7A1</i> levels are represented as fold expression relative to the level in untreated cells. <i>C</i>, LD1 inhibits FGFR4-regulated <i>FOS</i> expression in a panel of liver cancer cell lines. The results are represented as fold expression relative to the <i>FOS</i> level in untreated cells. <i>D</i>, Inhibition of colony formation by repression of FGFR4 expression in JHH5 cells stably transfected with an FGFR4 shRNA doxycycline-inducible vector. <i>E</i>, Enumeration of LD1-inhibited liver cancer cell line colony formation. The values are represented as percent of the number of colonies enumerated in the absence of added LD1. <i>F</i>, LD1 inhibits HCC cell line colony formation.</p

Approches et pratiques en évaluation de programmes

Tous les chapitres de cette nouvelle édition ont été écrits par des pédagogues, des enseignants universitaires et des formateurs rompus depuis de longues années à l'exercice du partage de connaissances en évaluation de programmes, tout en mettant l'accent sur la pratique plutôt que sur la théorie. Nous avons ajouté quatre nouveaux chapitres, car les connaissances en évaluation évoluent constamment, sur la stratégie de l'étude de cas, l'évaluation économique, les approches participatives ou encore l'approche dite réaliste. Il manquait dans la première édition des exemples relatifs à l'usage des méthodes mixtes, décrites dans la première partie. Deux nouveaux chapitres viennent donc combler cette lacune. Un défi essentiel auquel fait face tout enseignant en évaluation est lié à la maîtrise de la grande diversité des approches évaluatives et des types d'évaluation. La seconde partie de l'ouvrage présente quelques études de cas choisies pour montrer clairement comment les concepts exposés sont employés dans la pratique. Ces chapitres recouvrent plusieurs domaines disciplinaires et proposent divers exemples de pratiques évaluatives