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. 2012;7(5):e36713.
doi: 10.1371/journal.pone.0036713. Epub 2012 May 15.

Targeting FGFR4 inhibits hepatocellular carcinoma in preclinical mouse models

Dorothy M French  1 , Benjamin C Lin, Manping Wang, Camellia Adams, Theresa Shek, Kathy Hötzel, Brad Bolon, Ronald Ferrando, Craig Blackmore, Kurt Schroeder, Luis A Rodriguez, Maria Hristopoulos, Rayna Venook, Avi Ashkenazi, Luc R Desnoyers
Affiliations

Targeting FGFR4 inhibits hepatocellular carcinoma in preclinical mouse models

Dorothy M French et al. PLoS One. 2012.

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have the following competing interest: Dorothy M. French, Benjamin C. Lin, Manping Wang, Camellia Adams, Theresa Shek, Kathy Hötzel, Ronald Ferrando, Craig Blackmore, Kurt Schroeder, Luis A. Rodriguez, Maria Hristopoulos, Rayna Venook, Avi Ashkenazi, and Luc R. Desnoyers were full-time employees of Genentech, Inc. at the time this work was completed. Brad Bolon is a full-time employee of GEMpath, Inc. The affiliations with Genentech, Inc. and GEMpath, Inc. do not alter the authors’ adherence to all PLoS ONE policies on sharing data and materials. A patent is pending for the results described herein.

Figures

Figure 1
Figure 1. FGFR4 is required for FGF19-mediated liver tumorigenesis.
A, 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). B, BrdU incorporation in female (left panel) and male (right panel) FGF19-TG or wild type mice bred with FGFR4-KO or FGFR4-WT mice. C, Prevalence of liver tumors in male and female FGF19-TG mice treated with DEN as determined by gross and histological examinations. D, Multiple, large, raised tumors (arrows) on the surface of the liver of a 4-month-old FGF19-TG:FGFR4-WT mouse treated with DEN. E, 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. F, Liver weights of FGF19-TG or wild type female (left panel) and male (right panel) FGFR4-KO mice treated with DEN.
Figure 2
Figure 2. LD1 binds to FGFR4.
A, 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. B, Affinity of LD1 binding to mouse, cynomolgus monkey, and human FGFR4 as determined by surface plasmon resonance. C, Binding of LD1 to hFGFR4 expressed at the cell surface of stably transfected HEK293 cells as measured by FACS (RFU  =  Relative Fluorescence Unit). D, The binding of LD1 to immobilized hFGFR4-Flag chimeric proteins bearing point mutations as measured by a solid phase binding assay. E, 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. F, Dimer model illustrating the position of G165 (blue) on FGFR4 (red and yellow) bound to FGF19 (green).
Figure 3
Figure 3. LD1 inhibits FGFR4 activities.
A, LD1 inhibits FGFR4 binding to FGF1 and FGF19 as determined by solid phase binding assay. B, LD1 inhibits FGF1-stimulated proliferation of BaF3 cells stably expressing FGFR4/R1. C, LD1 inhibits FGFR4 signaling in L6 cells stably expressing FGFR4. D, Cell surface expression of FGFR4 protein in a subset of liver tumor cell lines as determined by FACS analysis using LD1.
Figure 4
Figure 4. LD1 inhibits FGFR4 biological activities in liver cancer cell lines.
A, LD1 inhibits FGFR4 signaling in HEP3B cells as evaluated by Western blot. B, LD1 inhibits the FGFR4-regulated CYP7A1 repression in HEP3B cells. CYP7A1 levels are represented as fold expression relative to the level in untreated cells. C, LD1 inhibits FGFR4-regulated FOS expression in a panel of liver cancer cell lines. The results are represented as fold expression relative to the FOS level in untreated cells. D, Inhibition of colony formation by repression of FGFR4 expression in JHH5 cells stably transfected with an FGFR4 shRNA doxycycline-inducible vector. E, 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. F, LD1 inhibits HCC cell line colony formation.
Figure 5
Figure 5. In vivo efficacy of LD1.
A, LD1 inhibits FGF19-regulated FOS expression in mouse liver. The results are represented as fold expression relative to FOS levels in the livers of non-treated mice. B, LD1 (30 mg/kg; once weekly) inhibits HUH7 xenograft tumor growth in vivo. C, Effects of LD1 on the mRNA expression of FGFR4, CYP7A1, FOS, and EGR1 in HUH7 xenograft tumors from Fig. 5B. D, 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). E, Liver weights of DEN–accelerated FGF19-TG:FGFR4-WT mice treated with control antibody, LD1, or 1A6 (anti-FGF19 antibody).
Figure 6
Figure 6. FGFR4 expression is deregulated in cancer.
A, Whisker-box plots show FGFR4 expression in human tumors and normal tissues as determined by mRNA analysis of the BioExpress database. The center line indicates the median; the box represents the interquartile range between the first and third quartiles. “Whiskers” extend from the interquartile to the positions of extreme values. B, FGFR4 immunostaining in samples of breast (×100 magnification) and pancreatic (×100 magnification) adenocarcinomas, and hepatocellular carcinoma (×200 magnification and ×400 magnification). C, FGFR4 mRNA expression in a panel of human normal liver and liver tumors as determined by qRT-PCR. The value for each sample is represented as fold expression relative to the level observed in sample N1.
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