Targeting FGF19 inhibits tumor growth in colon ... - Semantic Scholar

Oncogene (2008) 27, 85–97

& 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc

ORIGINAL ARTICLE

Targeting FGF19 inhibits tumor growth in colon cancer xenograft and FGF19 transgenic hepatocellular carcinoma models LR Desnoyers1, R Pai2, RE Ferrando2, K Ho¨tzel2, T Le3, J Ross3, R Carano3, A D’Souza1, J Qing1, I Mohtashemi4, A Ashkenazi1 and DM French2 1 Department of Molecular Oncology, Genentech Inc., South San Francisco, CA, USA; 2Department of Pathology, Genentech Inc., South San Francisco, CA, USA; 3Department of Biomedical Imaging, Genentech Inc., South San Francisco, CA, USA and 4 Department of Protein Chemistry, Genentech Inc., South San Francisco, CA, USA

Although fibroblast growth factor 19 (FGF19) can promote liver carcinogenesis in mice its involvement in human cancer is not well characterized. Here we report that FGF19 and its cognate receptor FGF receptor 4 (FGFR4) are coexpressed in primary human liver, lung and colon tumors and in a subset of human colon cancer cell lines. To test the importance of FGF19 for tumor growth, we developed an anti-FGF19 monoclonal antibody that selectively blocks the interaction of FGF19 with FGFR4. This antibody abolished FGF19-mediated activity in vitro and inhibited growth of colon tumor xenografts in vivo and effectively prevented hepatocellular carcinomas in FGF19 transgenic mice. The efficacy of the antibody in these models was linked to inhibition of FGF19-dependent activation of FGFR4, FRS2, ERK and b-catenin. These findings suggest that the inactivation of FGF19 could be beneficial for the treatment of colon cancer, liver cancer and other malignancies involving interaction of FGF19 and FGFR4. Oncogene (2008) 27, 85–97; doi:10.1038/sj.onc.1210623; published online 25 June 2007 Keywords: fibroblast growth factor; hepatocellular carcinoma; FGF19; colon adenocarcinoma; FGFR4; b-catenin

Introduction The fibroblast growth factor (FGF) family is composed of 22 structurally related polypeptides that bind to four receptor tyrosine kinases (FGFR1–4) and one kinasedeficient receptor (FGFR5) (Ornitz and Itoh, 2001; Sleeman et al., 2001; Eswarakumar et al., 2005). FGF interactions with FGFR1–4 result in receptor homodimerization and autophosphorylation, recruitment of cytosolic adaptors such as fibroblast growth factor receptor substrate 2 (FRS2) and initiation of multiple Correspondence: Dr DM French, Genentech Inc., MS 72B, 1 DNA Way, South San Francisco, CA 94080, USA. E-mail: [email protected] Received 10 January 2007; revised 20 April 2007; accepted 15 May 2007; published online 25 June 2007

signaling pathways (Powers et al., 2000). FGFs and FGFRs play important roles in development and tissue repair by regulating cell proliferation, migration, chemotaxis, differentiation, morphogenesis and angiogenesis. Amplification or overexpression of fgf2–4 and 8 is associated with the pathogenesis of leukemias and sarcomas as well as prostate, stomach, pancreas, bladder and colon cancers (Liscia et al., 1989; Huang et al., 1993; Dorkin et al., 1999; Marsh et al., 1999; Kiuru-Kuhlefelt et al., 2000; Krejci et al., 2001; Mattila et al., 2001; Ruohola et al., 2001; Shimokawa et al., 2003; Zaharieva et al., 2003; Holzmann et al., 2004). In addition, fgfr1–4 overexpression, translocation, polymorphisms and truncations are associated with neoplastic transformation in myeloma and breast, stomach, colon, bladder and cervical cancer (Jaakkola et al., 1993; Chesi et al., 1997, 2001; Richelda et al., 1997; Xiao et al., 1998; Cappellen et al., 1999; Popovici et al., 1999; Jang et al., 2000, 2001; Bange et al., 2002; Jeffers et al., 2002; Gowardhan et al., 2005). We previously showed that FGF19 ectopic expression in mice promotes hepatocyte proliferation, hepatocellular dysplasia and neoplasia (Nicholes et al., 2002). To investigate FGF19 association with human cancer, we evaluated its expression in primary tumors and tested the consequence of therapeutic FGF19 neutralization in relevant tumor models. We show here that FGF19 and its cognate receptor FGFR4 are coexpressed in primary human liver, lung and colon tumors. We evaluated FGF19 binding to FGFR5 and developed an antibody to neutralize this interaction. Administration of this antibody inhibited tumor growth in two human colon cancer models and FGF19 transgenic hepatocellular carcinomas (HCCs). These findings suggest that the therapeutic inactivation of FGF19 could be beneficial for the treatment of colon and liver cancer.

Results FGF19 and FGFR4 are expressed in human tumors Fgf19 was overexpressed in 6 out of 10 colon adenocarcinomas (Figure 1a) and in 7 out of 10 lung squamous cell carcinomas (SCC) relative to normal tissues (Figure 1b).

FGF19 and carcinomas in humans LR Desnoyers et al

86

Compared to normal tissues, fgfr4 expression was not significantly altered in colon tumors but appeared downregulated in SCC tumors (Figures 1a and b).

a

Colon Adenocarcinoma

Relative Expression

10,000

FGF19

To localize fgf19 and fgfr4 expression in tumor tissues, we performed in situ hybridization. Messenger RNA for both genes was prominent in neoplastic

d Lung Squamous Cell Carcinoma FGF19 ISH

FGFR4 Tumor Normal

1,000

FGFR4 ISH

100

10

FGF19 IHC 0

Tissue Samples (tumor and matched normal)

b

Lung Squamous Cell Carcinoma 10,000

FGF19

e

Hepatocellular Carcinoma

FGFR4

Relative Expression

FGF19 ISH Tumor Normal

1,000

100

FGFR4 ISH

10

FGF19 IHC 0

Tissue Samples (tumor and normal)

c

Colon Adenocarcinoma FGF19 ISH

f FGF19 ISH

FGFR4 ISH FGF19 IHC

FGF19 IHC

Oncogene

Cirrhotic Liver


87

a 80000

Relative Expression

FGF19

60000

FGFR4

50000 40000 30000 20000

MCF7

Breast

HT29

Colo205

SNU387

SNU453

SNU47S

SNU398

SW480 Colo201

Liver SW620

b

SW480

Colon

SNU185

Colo320

HT29

HCT116

SW620

Colo201

0

Colo205

10000

25 kDa 20

FGF19

15 FGFR4

c FGF19 and FGFR4 are expressed in human tumor cell lines and xenograft tissues To assess further FGF19 and FGFR4 association with human cancer and to identify potential preclinical models, we evaluated mRNA and protein expression in a panel of colon, breast and liver tumor cell lines. We found fgf19 expression in a subset of colon cancer cell lines, including Colo205, Colo201, SW620, HT29, HCT116 and SW480 (Figure 2a). SNU185, SNU398, MCF7 and all colon cancer cell lines tested expressed fgfr4. With the exception of HT29, which did not express FGF19 protein, the FGF19 and FGFR4 protein levels agreed with their mRNA expression in these cell lines (Figure 2b). The electrophoretic mobility of the secreted FGF19 was consistent with its expected molecular mass. However, we also detected several additional lower molecular mass bands possibly representing potential truncated protein. To verify that FGF19 expression is maintained in vivo, we immunostained colon cancer cell line-derived tumor xenografts (Figure 2c). Colo205 and Colo201 xenografts showed strong FGF19 staining in most

70000

HCT116

epithelial cells in colon adenocarcinomas and lung SCC (Figures 1c and d). In a tissue microarray comprising 35 colon adenocarcinoma cases, 26 (74%) had positive signal for fgf19 messenger RNA (mRNA) and 27 (77%) had positive signal for fgfr4 mRNA. Of 14 lung SCC cases, 14 (100%) had weak to moderate positive signal for fgf19 mRNA and 13 (93%) had weak positive signal for fgfr4 mRNA. In addition, neoplastic epithelial cells showed strong FGF19 staining by immunohistochemistry in both colon adenocarcinomas (Figure 1c) and lung SCC (Figure 1d). Because systemic FGF19 expression in transgenic mice promotes HCC (Nicholes et al., 2002), we also evaluated fgf19 and fgfr4 mRNA expression in liver samples. Of 50 cases of HCC, 23 (46%) demonstrated positive signal for fgf19 and 30 (60%) for fgfr4. Both genes were expressed in the neoplastic hepatocytes (Figure 1e). The neoplastic hepatocytes also showed strong FGF19 staining by immunohistochemistry (Figure 1e). These results demonstrate that FGF19 and FGFR4 are expressed in several types of human cancers. Because cirrhosis often precedes HCC, we evaluated FGF19 expression in cirrhotic liver. These samples showed strong FGF19 mRNA and protein signals in regenerative nodule hepatocytes (Figure 1f), suggesting that FGF19 expression occurs early during liver neoplastic progression.

100 kDa

Colo205

Colo201

T T S

S SW620

HCT116

T

T

Figure 2 FGF19 and FGFR4 expression in cell lines and xenograft tissue. (a) Fgf19 and fgfr4 mRNA expression in a panel of cancer cell lines evaluated by semi quantitative reverse transcription (RT)–PCR. (b) Western blot of FGF19 and FGFR4 protein expressed in a panel of colon cancer cell lines. (c) FGF19 immunostaining of a subset of colon cancer cell lines xenografted subcutaneously into nude mice and excised after 3 weeks (T ¼ tumor; S ¼ stroma). Images of representative fields at 400 with inset at 600 are shown (bar ¼ 25 mm). FGF19, fibroblast growth factor 19; FGFR4, FGF receptor 4.

Figure 1 FGF19 and FGFR4 are expressed in cancer. Fgf19 and fgfr4 mRNA expression in normal and tumor samples from colon (a) and lung (b) evaluated by semi quantitative reverse transcription (RT)–PCR (normal tissues are indicated with an ‘N’ and tumor tissues are indicated with a ‘T’). Samples evaluated for fgf19 expression are on the left and the same samples evaluated for fgfr4 expression are on the right. For (a) the normal and tumor tissue are from the same patient; for (b) the normal and tumor tissue are from different patients. Bright-field (top left panels) and dark-field (top right panels) illumination of in situ hybridization with fgf19 or fgfr4 riboprobes ( 100 magnification for colon and cirrhotic liver; 200 magnification for Lung and HCC) and FGF19 IHC (lower left panel 100 magnification and lower right panel 600 magnification) in colon adenocarcinoma (c), lung squamous cell carcinoma (d), hepatocellular carcinoma (e), and cirrhotic liver nodules (f). Arrows in (d) delineate positive neoplastic cells. FGF19, fibroblast growth factor 19; FGFR4, FGF receptor 4; HCC, hepatocellular carcinoma. Oncogene


88

neoplastic epithelial cells but not in the associated mouse stroma or adjacent normal tissue. SW620 and HCT116 tumors showed FGF19 immunoreactivity in scattered neoplastic cells. These results confirm that colon cancer cell lines express FGF19 upon growing in vitro in culture dishes as well as in vivo in a subcutaneous xenograft setting.

antibodies (1A1, 1A6 and 1D1) were characterized further. In enzyme-linked immunosorbent assays, 1A1 and 1A6 bound to FGF19 with a comparable EC50 of 40 pM, whereas 1D1 bound with an EC50 of 400 pM (Figure 4a). In a solid-phase receptor binding assay, 1A6 blocked FGF19 binding to FGFR4 with an IC50 of 3 nM (Figure 4b). 1A1, 1D1 and an irrelevant control antibody did not inhibit this interaction. We then analysed antibody neutralization of FGF19induced FGF pathway activation in HCC Hep3B cells (Schlessinger, 2004). 1A6 inhibited FGF19-induced FRS2 and mitogen-activated protein kinase (MAPK) phosphorylation at all doses tested, whereas 1D1 and the control antibody did not show significant inhibitory activity (Figure 4c). FGF19 plays a role in cholesterol homeostatis by repressing hepatic expression of cholesterol-7-a-hydroxylase 1 (Cyp7a1), the rate-limiting enzyme for cholesterol and bile acid synthesis (Holt et al., 2003; Yu et al., 2005). To evaluate the FGF19 antibodies further, we examined their effect on FGF19-induced Cyp7a1 downregulation in the hepatoblastoma HEPG2 cell line. In the absence of antibody, FGF19 reduced Cyp7a1 expression by 75% (Figure 4d). The addition of 1.1 mg/ ml 1A6 abolished FGF19 activity completely, whereas 1A1 reduced this activity only by 50% at 10 mg/ml but not at lower antibody concentrations. Treatment with a control antibody did not affect FGF19 activity.

FGF19 receptor binding To examine FGF19’s binding specificity more completely, we assessed its interaction with all known human FGFRs in their different alternatively spliced forms including the recently identified FGFR5 (FGFR1L) (Sleeman et al., 2001). In a solid-phase assay, FGF19 dose-dependent binding was restricted to FGFR4 (Figure 3a). In a receptor pull-down assay, FGF1 bound to all FGFRs, whereas FGF19 interaction was limited to FGFR4 (Figure 3b). The dissociation constant of FGF19 binding to FGFR4 determined using Scatchard analysis indicated a high-affinity interaction, with Kd 0.25 nM (Figure 3c). These results are consistent with previous findings (Ornitz et al., 1996; Xie et al., 1999) and further emphasize the unique binding specificity of FGF19 for FGFR4. Generation of an FGF19-blocking antibody To develop an FGF19-neutralizing agent we generated a panel of monoclonal antibodies. From this panel, three

4

3IIIc

3IIIb

2βIIIc

2βIIIb

– 2αIIIc

2αIIIb

1βIIIc

1βIIIb

FGFRs

+

4

Heparin 1αIIIc

3

b

FGFR4 FGFR1-FGFR3 FGFR5

1αIIIb

Binding (O.D. 450nm)

a 4

FGF19

2

FGF1 1

0 0.001

0.01

0.1

1

[FGF19] (µg/ml) Bound (cpm × 10-3)

Bound/Free

c 0.12

0.08

12

total specific

8 4 0 0.0

0.5

1.0

1.5

125

[

I-FGF19] (pM)

0.04

0.00 0

5

10 15 Bound (µM)

20

25

Figure 3 FGF19 binding to FGFR5. (a) FGF19 binds specifically to FGFR4 in a solid-phase assay where FGFR1–4 Fc chimeric proteins were immobilized and binding was detected using a biotinylated FGF19-specific polyclonal antibody. (b) A receptor pulldown assay demonstrated FGF1 bound to all FGFRs whereas FGF19 interaction was limited to FGFR4. (c) Scatchard analysis of 125IFGF19 binding to solid phase captured FGFR4-IgG. FGF, fibroblast growth factor; FGFR, FGF receptor. Oncogene


4

b

1A1 1A6 1D1 Ctl Ab

Binding (% Control)

5

Binding (O.D. 450 nm)

89

a

3 2 1 0 0.0001

150

100 1A1 1D1 Ctl Ab 1A6

50

0 0.01

1

0.1

1

[MAbs] (µg/ml)

c

d –

FGF19 -

Control +

-

+

1A6 +

+

1D1 +

+

+

100

Relative Cyp7α1 Expression (%)

Antibodies

10

[MAbs] (µg/ml)

+ p-FRS2

p-MAPK

MAPK

1A6 1A1 Ctl Ab

75

50

25

0 [Abs] (µg/ml)

0

0 0.04 0.12 0.3 1.1 3.3 10 FGF19

e

p = 0.012

p = 0.0055

Migration (number of cells)

250 p = 0.0007 200 150 100 50 0 FGF19 0.1 1

5

1A6 (µg/ml)

Figure 4 1A6 inhibits FGF19 biological activities. (a) Binding of anti-FGF19 monoclonal antibodies to FGF19. (b) Inhibition of FGF19 binding to solid phase captured FGFR4-Fc by anti-FGF19 monoclonal antibody 1A6. (c) Western blot analysis of FGF19induced FRS2 and MAPK phosphorylation in HEP3B cells in the presence of anti-FGF19 monoclonal antibodies. (d) Inhibition of FGF19-induced repression of CYP7a1 expression in HEPG2 cells by monoclonal antibody 1A6. CYP7a1 expression was evaluated by semi-quantitative reverse transcription (RT) –PCR using gene-specific primers and probes and normalized to GAPDH expression. (e) Inhibition of FGF19-induced cell migration by anti-FGF19 monoclonal antibody 1A6 using a Boyden chamber transwell migration assay. FGF19, fibroblast growth factor 19; FGFR4, FGF receptor 4; CYP7a1, cholesterol-7-a-hydroxylase 1.

Because FGFR4 plays a role in cell migration, we evaluated the chemotactic activity of FGF19 (Wang et al., 2004; Spinola et al., 2005). In a modified Boyden chamber assay, FGF19 promoted migration of HCT116 cells in a dose-dependent fashion, reaching a maximum at 16 ng/ml (Supplementary Figure S1). 1A6 inhibited FGF19-induced cell migration (Figure 4e). At higher concentrations, 1A6 reduced cell migration to 20% below the basal level, likely by inhibiting both exogenously added and endogenously produced FGF19. Together, these results demonstrate that 1A6 is a potent inhibitor of FGF19 activity in vitro.

1A6 epitope is localized in the FGF19-binding interface with FGFR4 To characterize further the FGF19 antibodies we localized their epitopes and evaluated whether FGF19 conformational components contribute to their binding. We first attempted to isolate an epitope-containing peptide from an FGF19 tryptic digest using an agarosecoupled 1A6 affinity matrix. This approach was unsuccessful possibly because the FGF19 fragmentation compromised the conformational integrity of 1A6 epitope. We therefore modified the procedure and bound FGF19 to the agarose-coupled antibody before Oncogene


90

trypsin digestion. Analysis of the total digest demonstrated a complete fragmentation of the adsorbed FGF19 without 1A6 masking of trypsin cleavage sites (Figure 5a). Bound peptides were eluted, identified by mass spectrometry and sequenced. We identified the non-specifically adsorbed peptides using an irrelevant control antibody coupled to agarose in a parallel experiment. The agarose-coupled 1A6 specifically isolated the FGF19 peptide G133-R155 (Figure 5b and Supplementary Figure S2a). Using agarose-coupled 1A1, we specifically isolated the FGF19 peptide G156-R180 (Figure 5c and Supplementary Figure S2b). Because agarose conjugation of 1D1 abolished its binding to FGF19, we used a peptide competition assay to identify its epitope. Only a peptide including the nine most C-terminal FGF19 amino acids competed 1D1 binding to the solid-phase adsorbed antigen (Figure 5d). This peptide did not compete for 1A1 binding to FGF19

(data not shown). Because overlapping peptides were used in this competition assay, we surmise that the 1D1 epitope is located in the last four FGF19 amino acids (SFEK). We mapped the antibody epitopes on the previously described structural model of FGF19 bound to FGFR4 (Harmer et al., 2004). The epitopes of 1A1 and 1D1 are located in a distal portion of FGF19, which is not represented on this model due to its lack of ordered structure. However, the 1A6 epitope is localized in the FGF19-binding interface with FGFR4 (Figure 5e). These results suggest that 1A6 directly occludes the receptor-binding site of FGF19. 1A6 inhibits the growth of human colon cancer xenografts To determine whether FGF19 neutralization could inhibit tumor growth in vivo, we selected the colon cancer cell lines HCT116 and Colo201, both of which

a

Binding (O.D.450 nm)

[T156-180]

[T156-180]

% Intensity

[T133-155] [T133-155]

d 3.0

1D1

2.5 2.0 1.5 1.0 0.5

A183-G192 D173-E182 V178-P187

0.0 0.01 0.1 1 10 100 1000 [Peptides] (µg/ml)

1A6

Non-specific

e

[T133-155]

% Intensity

b

2665.9

1A1

Non-specific

[T156-180]

% Intensity

c

3240.3

Mass (m/z) Figure 5 Identification of epitopes recognized by FGF19 antibodies. (a) Mass spectroscopic analysis of soluble fraction of trypsindigested FGF19 bound to agarose-coupled 1A6. (b) Mass spectroscopic analysis of FGF19 tryptic peptide eluted from agarose-coupled 1A6. (c) Mass spectroscopic analysis of FGF19 tryptic peptide eluted from agarose-coupled 1A1. (d) Peptide competition assay to identify the 1D1 epitope. (e) Mapping of 1A6 epitope (orange) onto the FGF19–FGFR4 interaction model. FGFR4 surface is shown in blue and pink whereas FGF19 ribbon representations are in green and yellow. FGF19, fibroblast growth factor 19; FGFR4, FGF receptor 4. Oncogene


91

express FGF19 and FGFR4 (Figure 2a) and form tumors in vivo. We treated mice with established HCT116 xenograft tumors twice weekly with 5 mg/kg of either 1A6 or a control antibody. At day 35, 1A6 suppressed tumor growth by 57% (P ¼ 0.07, n ¼ 5) compared to the control group (Figure 6a). We repeated this study with a higher dose of 1A6 (15 mg/kg; 2 / week) and a statistically significant suppression of tumor growth was observed (60% growth inhibition, P ¼ 0.01, n ¼ 5). To verify that 1A6 inhibited tumor growth by blocking FGF19 activity, we examined tumors at the end of the study for markers of FGFR4 signaling. Activation of FGFR4, FRS2, extracellular signalregulated kinase (ERK) and b-catenin was significantly decreased in xenograft tumors from animals treated with 1A6 compared to the control antibody (Figure 6b). In one case, the control-treated xenograft tumor showed low pFRS2. The relative lack of FRS2 phosphorylation correlates with the small tumor size and is consistent with our hypothesis that this pathway is important for tumor growth. Next, we used xenografts of Colo201, a colon cancer cell line expressing higher FGF19 levels than HCT116 (Figure 2a). 1A6 treatment (30 mg/kg; 2 /week) significantly suppressed the growth of established tumors (at day 27, 64% growth inhibition, P ¼ 0.03, n ¼ 5) compared to the control antibody (Figure 6c). Analysis of the excised tumors showed that 1A6 significantly decreased FGFR4, FRS2, ERK and b-catenin activation compared to the control antibody (Figure 6d). Together, these results demonstrate 1A6 anti-tumor efficacy in colon cancer models and associate its activity with inhibition of FGF19-dependent FGFR4, FRS2, ERK and b-catenin activation. 1A6 prevents hepatocellular carcinomas in FGF19 transgenic mice We demonstrated previously that overexpression of FGF19 in the skeletal muscle results in development of HCCs by 10–12 months of age (Nicholes et al., 2002). To confirm that FGF19 is acting as a tumor promoter in this model, we treated the FGF19 transgenic mice with a tumor initiator, diethylnitrosamine (DEN), which accelerated tumor formation by 50% (unpublished data). To determine whether 1A6 could prevent HCCs, DEN-accelerated FGF19 transgenic mice were treated with either 1A6 or control antibody (gp120) for 6 months. At the end of the treatment, all of the control-treated mice had grossly evident multifocal, large HCCs throughout the liver lobes, whereas 1A6treated animals had either no liver tumors or, in one case (#1862), a single small tumor present on the diaphragmatic surface of the median lobe (Figure 7a). Liver weights from 1A6-treated mice (mean ¼ 1.7170.05 g) were significantly lower than liver weights from controltreated mice (mean ¼ 3.1570.58 g; P ¼ 0.014), but were not significantly different from those of normal FVB wildtype mice (mean ¼ 1.5670.08 g; P ¼ 0.82). In addition, tumor volume was determined by micro-computed tomography (micro-CT) image analysis, corrected for tumor volume with normal FVB liver, and graphed as

a percent of total liver volume (Figure 7b). Percent tumor volume of 1A6-treated mice (mean ¼ 7.573.2%) was significantly lower than control gp120-treated mice (mean ¼ 23.876.8%; P ¼ 0.05). Furthermore, tumor weights strongly correlated with percent tumor volume (r2 ¼ 0.993702). These data clearly demonstrate that 1A6 effectively neutralizes circulating FGF19 to prevent tumor formation in FGF19 transgenic mice. Discussion Systemic FGF19 expression in transgenic mice promotes hepatocellular carcinogenesis (Nicholes et al., 2002). However, no report as yet has confirmed FGF19 in human cancer. Our results show that FGF19 is expressed in several human tumors. Neoplastic cells of colon adenocarcinoma, lung squamous cell carcinoma and HCC highly expressed FGF19. FGF19 was also detected in cirrhosis, a preneoplastic condition that often leads to liver carcinoma (Anthony, 1979; Kobayashi et al., 2006). These data raise the possibility that FGF19 may be involved in tumor pathogenesis and, particularly for the liver, in early neoplastic progression. Previous work suggested that FGF19 binds selectively to FGFR4 (Xie et al., 1999). FGFR4 deregulation has been linked to cancer pathogenesis. FGFR4 truncation and cytoplasmic localization correlates with malignancy in pituitary adenomas (Qian et al., 2004). FGFR4 expression predicts short survival in astrocytoma (Yamada et al., 2002). A single nucleotide polymorphism at codon 388 of FGFR4 strongly correlates with the prognosis of prostate and lung adenocarcinoma, head and neck squamous cell carcinoma and soft tissue sarcoma (Morimoto et al., 2003; Streit et al., 2004; Wang et al., 2004). Our data demonstrate that neoplastic cells of colon adenocarcinoma, lung squamous cell carcinoma and HCC often express FGFR4 in conjunction with FGF19, suggesting that their interaction may contribute to tumor progression. The importance of FGF19 in tumor growth was further examined by generating anti-FGF19 monoclonal antibodies. Among these, 1A6 blocked the interaction of FGF19 with FGFR4. In HCC HEP3B cells, 1A6 inhibited FGF19 stimulation of FGFR4-mediated events including FGFR4, FRS2 and MAPK phosphorylation and CYP7a1 gene repression. 1A6 also effectively inhibited FGF19 chemotactic activity toward HCT116 cells. To define how 1A6 inhibits FGF19 at the molecular level, we identified and compared the epitopes of three FGF19 antibodies. 1A1 recognized an epitope located between the amino acids 156–180, whereas 1D1 bound to the most C-terminal five residues. The regions represented by these epitopes are absent from the recently reported crystal structure of FGF19 (Harmer et al., 2004). The 1A6-binding epitope mapped to the b12 strand of FGF19. Based on our structural model of the FGF19–FGFR4 complex, the b12 strand is located on the surface of the ligand’s interface with the receptor Oncogene


92

b

HCT116 Xenograft gp120

1A6

pFGFR4

a

HCT116 Xenograft

FGFR4

1200 Control Ab Tumor Volume (mm3)

1000

pFRS2

1A6

800 FRS2 600 pERK 400 ERK 200

non-phosph. β-catenin

0 25 30 Treatment Days

35

c

223

89

737

Tumor Volume

785

β-catenin 181

20

1398

15

Colo201 Xenograft 800

Colo201 Xenograft

d Control Ab

gp120

1A6

Tumor Volume (mm3)

1A6 600

pFGFR4

FGFR4

400

pFRS2 200 FRS2 0 0

5

10

15

20

25

30

pERK

Treatment Days ERK non-phosph. β-catenin

197

925

253

398

389

Tumor Volume

1308

β-catenin

Figure 6 Effect of 1A6 on colon tumor xenografts. Athymic mice were subcutaneously inoculated with 5 106 HCT116 (a) or Colo201 (c) cells. Mice bearing established tumors of equivalent volumes (B100 mm3) were randomized into groups and treated intraperitoneally twice weekly with 1A6 or a control antibody. Results are given as mean tumor volume 7s.e.m. At the end of the studies HCT116 xenograft tumors (b) and Colo201 xenograft tumors (d) from 1A6 or control antibody-treated mice were excised, homogenized and analysed for FGFR4, FRS2, ERK and b-catenin activation by western blot. ERK, extracellular signal-regulated kinase; FGFR4, FGF receptor 4; FRS2, fibroblast growth factor receptor substrate 2. Oncogene


93

a

b

Control Ab treated

1A6 treated

Control Ab treated

Total Tumor Volume to Total Liver Volume 70

1A6 treated

Percentage Tumor Volume to Total Liver Volume (%)

60 50 40 30 20 10 0 -10 1A6

control Ab

Treatment Figure 7 Effect of 1A6 on FGF19 transgenic hepatocellular carcinomas. (a) FGF19 transgenic mice were treated with either a control antibody or 1A6 and liver was collected for gross evaluation. (b) Micro-CT analysis using an iodinated triglyceride for enhancement of normal hepatocytes demonstrates increased unenhanced tumor volume in control-treated versus 1A6-treated liver. FGF19, fibroblast growth factor 19; micro-CT, micro-computed tomography.

(Figure 6e). Thus, 1A6 directly competes with FGFR4 for FGF19 interaction. We then tested the anti-tumor efficacy of 1A6 in vivo using colon cancer xenograft and FGF19 transgenic HCC models. 1A6 treatment significantly reduced growth of HCT116 and Colo201 xenografts compared to a control antibody. In conjunction, 1A6 substantially reduced FGFR4, FRS2, ERK and b-catenin activation, suggesting that FGF19 plays a role in the growth of these tumors. Since FGF19 does not have a direct murine ortholog that is bound by 1A6, the anti-tumor activity of 1A6 is likely mediated by neutralization of tumor-produced FGF19. In addition, the ability of 1A6 to prevent HCCs in FGF19 transgenic mice where FGF19 is ectopically expressed in the skeletal muscle further confirms that 1A6 effectively neutralizes the oncogenic activity of FGF19. The FGF19 transgenic hepatocellular tumors have a high frequency of b-catenin nuclear localization and activating mutations (Nicholes et al., 2002). b-Catenin pathway deregulation promotes tumor development in

several types of cancer, including colorectal adenocarcinoma and HCC. Activation of this pathway impacts neoplastic cell proliferation, epithelial mesenchymal transition, invasion and metastasis. Repression of b-catenin activation by 1A6 in colon tumor xenografts provides further evidence for a link between FGF19 and wnt pathway activity in the context of cancer. FGF19 is potentially an attractive target for cancer therapy for several reasons. FGF19 expression in the adult is restricted to the gall bladder (Xie et al., 1999) and is specific in cancer tissues to the neoplastic cells. FGF19 binds selectively to FGFR4, which has been associated with cancer progression. Knockout mice deficient in FGFR4 or the FGF19 murine ortholog, FGF15, develop normally and do not show gross abnormalities, suggesting that inhibition of FGF19 may not have significant safety limitations . Finally, the activity of the FGF19-FGFR4 axis in colorectal and perhaps other tumors may provide useful markers for identifying patients who could benefit most from antiFGF19 therapy. Oncogene


94 Materials and methods Gene expression Total RNA from frozen tissue samples was extracted using RNA STAT-60 according to the manufacturer’s protocol (Tel-test ‘B’ Inc., Friendswood, TX, USA). Total RNA from cultured cells was isolated with RNeasy kit using the manufacturer’s protocol (Qiagen, Valencia, CA, USA), treated with DNAse-I (Ambion, Austin, TX, USA) and used for realtime PCR. Specific primers and fluorogenic probes for human FGF19, FGFR4 and RPL19 (Supplemental Data Table 1) were designed using Primer Express 1.1 (PE, Applied Biosystems, Foster City, CA, USA). Gene-specific signals, evaluated in triplicate, were normalized to the RPL19. In situ hybridization 33 P-UTP-labeled sense or antisense probes corresponding to human FGFR4 (nucleotides 435–1183 of NM_022963) or FGF19 (nucleotides 495–1132 of NM_005117) were generated by PCR (Mauad et al., 1994). Sections were processed for in situ hybridization as described previously (Jubb et al., 2006). Tissue microarrays were obtained from the following sources: multi-tumor TMA was made at Genentech Inc., HCC TMAs were from Pentagen (Seoul, Korea) (catalog nos. A217 and A204) or Cybrdi (Frederick, MD, USA) (catalog nos. CC03-01-003 and CC03-02-01-001). Immunohistochemistry Formalin-fixed paraffin-embedded tissue sections were treated for antigen retrieval using Trilogy (Cell Marque, Rocklin, CA, USA) and then incubated with 10 ug/ml FGF19 antibody (1D1; Genentech Inc.). The immunostaining was accomplished using a biotinylated secondary antibody, an ABC-HRP reagent (Vector Labs, Burlingame, CA, USA) and a metalenhanced DAB colorimetric peroxydase substrate (Pierce Laboratories, Rockford, IL, USA). Immunoprecipitation and immunoblotting Total proteins from cultured cells were extracted on ice for 30 min in extraction buffer (20 mM Tris, pH 8, 137 mM NaCl, 1 mM ethylene glycol tetraocetic acid (EGTA), 1% Triton X100, 10% glycerol, 1.5 mM MgCl2, complete protease inhibitor cocktail (Roche Applied Sciences, Indianapolis, IN, USA)). Lysates (100 mg protein) were incubated in 1 ml PBS/0.1% Triton with agarose-coupled antibodies for 1 h at 41C, then washed with buffer and eluted with 10 ml Elution buffer (Pierce Biotechnology, Rockford, IL, USA) and analysed by immunoblot using biotinylated FGF19 antibody (BAF969; R&D Systems, Minneapolis, MN, USA), FGFR4 antibody (1G7; Genentech Inc.) and IRDye 800-conjugated secondary reagents and visualized using the Odyssey scanner (Li-Cor Biotechnology, Lincoln, NE, USA). Solid-phase receptor binding assay Maxisorb 96-well plates were coated overnight at 41C with 50 ml of 2 mg/ml anti-human immunoglobulin Fcg fragment specific (Jackson Immunoresearch, West Grove, PA, USA) and used to capture 1 mg/ml FGFR-Fc chimeric proteins (R&D Systems). After blocking with phosphate-buffered saline (PBS)/3% bovine serum albumin (BSA), FGF19 and heparin were added and incubated 2 h (Seikagaku Corporation, Tokyo, Japan). A biotinylated FGF19-specific polyclonal antibody (BAF969; R&D Systems) followed by streptavidinHRP and TMB was used for detection. Oncogene

Receptor pull-down assay FGFR-Fc chimeric proteins (400 ng) were incubated with FGF19 or FGF1 and heparin (200 ng) in 50:50 Dulbecco’s modified essential media Ham F12 containing 10 mM HEPES, pH 7.4, and 0.1% BSA for 1 h followed by Protein G-agarose (20 ml) for 30 min. The matrix was washed with PBS/0.1% Triton X-100, eluted with sodium dodecyl sulphate– polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer containing reducing agent and analysed by western blotting using biotinylated FGF19 antibody (BAF969) or biotinylated FGF1 antibody (BAF232; R&D Systems). Generation of FGF19 monoclonal antibodies Balb/c mice were immunized with FGF19-His. Spleens were harvested after 8 weeks and hybridomas were generated. Selected FGF19 antibody producing hybridomas were subcloned twice to insure monoclonality. The clones were inoculated for ascites production and antibodies were purified by Protein A-agarose affinity chromatography. Solid-phase antibody binding assay Non-specific binding sites of HisGrab nickel-coated plates (Pierce) were saturated with PBS/3% BSA, and then incubated with 1 mg/ml FGF19-His for 1 h. The plates were washed and incubated for 1 h with FGF19 antibodies in the presence or the absence of FGF19 peptides and detected using a HRPconjugated anti-mouse IgG (Jackson Immunoresearch) and the TMB peroxidase colorigenic substrate (KPL, Gaithersburg, MD, USA). CYP7a1 expression analysis HEPG2 cells were starved overnight in serum-free media and treated with 100 ng/ml FGF19 for 6 h in the presence or absence of antibodies. CYP7a1 expression was evaluated in triplicate by semi-quantitative reverse transcription (RT)–PCR (Taqman ABI PRISM 7700, Applied Biosystems) and normalized to GAPDH expression. FGFR4/MAPK phosphorylation HEP3B cells starved overnight in serum-free media were treated with 40 ng/ml FGF19 for 10 min in the presence or the absence of antibodies. Cells were lysed in R27A buffer (Upstate, Charlottesville, VA, USA) with 10 mM NaF, 1 mM sodium orthovanadate, and Complete protease inhibitor tablet (Roche). Lysates were prepared, electrophoresed and analysed by Immunoblot using phospho-FRS2 (Santa Cruz, Santa Cruz, CA, USA), phospho-MAPK and MAPK-specific antibodies (Cell Signaling, Danvers, CA, USA). Cell migration assay Cell motility was measured using a modified Boyden chamber assay (Eriksson et al., 1992). Briefly the surface of 8 mm porosity 24-well format PET membrane filters (BD Biosciences, Bedford, MA, USA) was coated overnight at 41C with 50 ml of type 1 collagen (50 mg/ml; Sigma, St Louis, MO, USA) in 0.02 M acetic acid. Cells (5 104) in serum-free minimal essential medium containing 0.1% BSA were added to the upper chamber. The lower chamber was filled with the same media and the plates were incubated at 371C. The next day the upper chamber was wiped with a cotton swab and the cells that migrated to the lower side of the insert were stained and counted under a microscope. Triplicate sets of data were averaged for each condition. Antibody epitope excision FGF19 (10 mg) was incubated for 2 h in 50 mM Tris, pH 7.4 with 50 ml of agarose-coupled antibody. The resin was washed


95 and digested with 0.1 mg trypsin (Promega, Madison, WI, USA) overnight at 371C in 100 mM ammonium bicarbonate (pH 8). The gel slurry was washed and the bound peptides were eluted with 10% trifluoroacetic acid and analysed by MALDITOF-MS (Voyager; Applied Biosystems). Candidate peptides were subjected to collision-induced dissociation (QSTAR) and manually sequenced to confirm the peptide mass mapping identification (Supplementary Figure S2). Xenograft experiments All animal protocols were approved by an Institutional Animal Care and Use Committee. Six- to eight-week-old athymic BALB/c female mice (Charles Rivers Inc., Wilmington, MA, USA) were inoculated subcutaneously with 5 106 cells (200 ml/mouse). After 7 days, mice bearing tumors of equivalent volumes (B100 mm3) were randomized into groups (n ¼ 10) and treated intraperitoneally twice weekly. Tumors were measured with an electronic caliper (Fowler Sylvac UltraCal Mark III) and average tumor volume was calculated using the formula (W2 L)/2, where W is the smaller diameter and L is the larger diameter. FGFR4, FRS2, ERK and b-catenin phosphorylation in xenograft tumors Tumors excised from treated animals were homogenized in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP40, 1 mM EDTA, 0.25% sodium deoxycholate, 1 mM NaF, 1 mM sodium orthovanadate and complete protease inhibitor (Roche)). Equal amounts of proteins were incubated with 1 mgspecific FGFR4 (1G7; Genentech Inc.) or FRS2 (UpState) antibody immobilized onto Protein A-Sepharose for 2 h at 41C then washed with lysis buffer and eluted with 2 Laemmli buffer, boiled, and microcentrifuged. Immunoblots were done with phosphotyrosine antibody (4G10, UpState), phosphoERK2 antibody (Santa Cruz Biotech) or N-terminally dephosphorylated b-catenin antibody (UpState). Membranes were stripped (Pierce) and reprobed with appropriate antibodies to determine total proteins.

Micro-CT imaging and analysis of hepatocellular carcinomas in FGF19 TG mice Liver tumors were identified by micro-CT imaging with Fenestra-LC (Lee et al., 1997). At 6 months of age, FGF19 transgenic mice were injected with Fenestra-LC (Advanced Research Technologies Inc., Saint-Laurent, Quebec, Canada), 20 ml/g intravenously, and a conscious 3-h hepatic uptake was allowed before mice were euthanized and livers analysed (mCT 40 system; Scanco Medical, Bassersdorf, Switzerland). Whole livers were submerged in soybean oil (Sigma-Aldrich, St Louis, MO, USA) in preparation for micro-CT imaging. Ninety-min scans were obtained at 30-mm isotropic voxel size, with 512 projections at an integration time of 300 ms, energy of 45 keV and tube current of 177 mA. Volumetric image files were analysed using image analysis software from AnalyzeDirect (Lenexa, KS, USA). An intensity threshold of 16 Houndsfield Units (HU) was used to segment the tissue mass from the background signal (soybean oil). A second threshold (26 HU) was used for functional hepatic tissue volumes that accumulated contrast. An average of the total low-intensity hepatic volumes from wild-type FVB mice was subtracted to obtain volumes associated only with tumors. Data are expressed as a percentage of tumor volume of total liver volume. Statistical analysis Statistical significance was analysed using the unpaired twotailed Student’s t-test. Values of Po0.05 were considered significant. Data are expressed as the mean7s.e.m.

Acknowledgements We thank Chris Wiesmann for structural modeling; Robin E Taylor and Julio Ramirez for paraffin embedding and sectioning; Sheila Bheddah for immunohistochemistry assistance; and Hartmut Koeppen, Paul Polakis, and Vishva Dixit for helpful discussions.

References Anthony PP. (1979). Precancerous changes in the human liver. J Toxicol Environ Health 5: 301–313. Bange J, Prechtl D, Cheburkin Y, Specht K, Harbeck N, Schmitt M et al. (2002). Cancer progression and tumor cell motility are associated with the fgfr4 arg(388) allele. Cancer Res 62: 840–847. Cappellen D, De Oliveira C, Ricol D, de Medina S, Bourdin J, Sastre-Garau X et al. (1999). Frequent activating mutations of fgfr3 in human bladder and cervix carcinomas. Nat Genet 23: 18–20. Chesi M, Brents LA, Ely SA, Bais C, Robbiani DF, Mesri EA et al. (2001). Activated fibroblast growth factor receptor 3 is an oncogene that contributes to tumor progression in multiple myeloma. Blood 97: 729–736. Chesi M, Nardini E, Brents LA, Schrock E, Ried T, Kuehl WM et al. (1997). Frequent translocation t(4;14) (p16.3;q32.3) in multiple myeloma is associated with increased expression and activating mutations of fibroblast growth factor receptor 3. Nat Genet 16: 260–264. Dorkin TJ, Robinson MC, Marsh C, Bjartell A, Neal DE, Leung HY. (1999). Fgf8 over-expression in prostate cancer is associated with decreased patient survival and persists in androgen independent disease. Oncogene 18: 2755–2761.

Eriksson A, Siegbahn A, Westermark B, Heldin CH, ClaessonWelsh L. (1992). Pdgf alpha- and beta-receptors activate unique and common signal transduction pathways. EMBO J 11: 543–550. Eswarakumar VP, Lax I, Schlessinger J. (2005). Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev 16: 139–149. Gowardhan B, Douglas DA, Mathers ME, McKie AB, McCracken SR, Robson CN et al. (2005). Evaluation of the fibroblast growth factor system as a potential target for therapy in human prostate cancer. Br J Cancer 92: 320–327. Harmer NJ, Pellegrini L, Chirgadze D, Fernandez-Recio J, Blundell TL. (2004). The crystal structure of fibroblast growth factor (fgf) 19 reveals novel features of the fgf family and offers a structural basis for its unusual receptor affinity. Biochemistry 43: 629–640. Holt JA, Luo G, Billin AN, Bisi J, McNeill YY, Kozarsky KF et al. (2003). Definition of a novel growth factor-dependent signal cascade for the suppression of bile acid biosynthesis. Genes Dev 17: 1581–1591. Holzmann K, Kohlhammer H, Schwaenen C, Wessendorf S, Kestler HA, Schwoerer A et al. (2004). Genomic DNA-chip hybridization reveals a higher incidence of genomic Oncogene


96 amplifications in pancreatic cancer than conventional comparative genomic hybridization and leads to the identification of novel candidate genes. Cancer Res 64: 4428–4433. Huang YQ, Li JJ, Nicolaides A, Zhang WG, Friedman-Kien AE. (1993). Increased expression of fibroblast growth factors (fgfs) and their receptor by protamine and suramin on kaposi’s sarcoma-derived cells. Anticancer Res 13: 887–890. Jaakkola S, Salmikangas P, Nylund S, Partanen J, Armstrong E, Pyrhonen S et al. (1993). Amplification of fgfr4 gene in human breast and gynecological cancers. Int J Cancer 54: 378–382. Jang JH, Shin KH, Park JG. (2001). Mutations in fibroblast growth factor receptor 2 and fibroblast growth factor receptor 3 genes associated with human gastric and colorectal cancers. Cancer Res 61: 3541–3543. Jang JH, Shin KH, Park YJ, Lee RJ, McKeehan WL, Park JG. (2000). Novel transcripts of fibroblast growth factor receptor 3 reveal aberrant splicing and activation of cryptic splice sequences in colorectal cancer. Cancer Res 60: 4049–4052. Jeffers M, LaRochelle WJ, Lichenstein HS. (2002). Fibroblast growth factors in cancer: therapeutic possibilities. Expert Opin Ther Targets 6: 469–482. Jubb AM, Pham TQ, Frantz GD, Peale Jr FV, Hillan KJ. (2006). Quantitative in situ hybridization of tissue microarrays. Methods Mol Biol 326: 255–264. Kiuru-Kuhlefelt S, Sarlomo-Rikala M, Larramendy ML, Soderlund M, Hedman K, Miettinen M et al. (2000). Fgf4 and int2 oncogenes are amplified and expressed in kaposi’s sarcoma. Mod Pathol 13: 433–437. Kobayashi M, Ikeda K, Hosaka T, Sezaki H, Someya T, Akuta N et al. (2006). Dysplastic nodules frequently develop into hepatocellular carcinoma in patients with chronic viral hepatitis and cirrhosis. Cancer 106: 636–647. Krejci P, Dvorakova D, Krahulcova E, Pachernik J, Mayer J, Hampl A et al. (2001). Fgf-2 abnormalities in b cell chronic lymphocytic and chronic myeloid leukemias. Leukemia 15: 228–237. Lee Jr FT, Chosy SG, Naidu SG, Goldfarb S, Weichert JP, Bakan DA et al. (1997). Ct depiction of experimental liver tumors: Contrast enhancement with hepatocyte-selective iodinated triglyceride versus conventional techniques. Radiology 203: 465–470. Liscia DS, Merlo GR, Garrett C, French D, MarianiCostantini R, Callahan R. (1989). Expression of int-2 mrna in human tumors amplified at the int-2 locus. Oncogene 4: 1219–1224. Marsh SK, Bansal GS, Zammit C, Barnard R, Coope R, Roberts-Clarke D et al. (1999). Increased expression of fibroblast growth factor 8 in human breast cancer. Oncogene 18: 1053–1060. Mattila MM, Ruohola JK, Valve EM, Tasanen MJ, Seppanen JA, Harkonen PL. (2001). Fgf-8b increases angiogenic capacity and tumor growth of androgen-regulated s115 breast cancer cells. Oncogene 20: 2791–2804. Mauad TH, van Nieuwkerk CM, Dingemans KP, Smit JJ, Schinkel AH, Notenboom RG et al. (1994). Mice with homozygous disruption of the mdr2 p-glycoprotein gene. A novel animal model for studies of nonsuppurative inflammatory cholangitis and hepatocarcinogenesis. Am J Pathol 145: 1237–1245. Morimoto Y, Ozaki T, Ouchida M, Umehara N, Ohata N, Yoshida A et al. (2003). Single nucleotide polymorphism in fibroblast growth factor receptor 4 at codon 388 is associated with prognosis in high-grade soft tissue sarcoma. Cancer 98: 2245–2250. Oncogene

Nicholes K, Guillet S, Tomlinson E, Hillan K, Wright B, Frantz GD et al. (2002). A mouse model of hepatocellular carcinoma : Ectopic expression of fibroblast growth factor 19 in skeletal muscle of transgenic mice. Am J Pathol 160: 2295–2307. Ornitz DM, Itoh N. (2001). Fibroblast growth factors. Genome Biol 2: REVIEWS3005. Ornitz DM, Xu J, Colvin JS, McEwen DG, MacArthur CA, Coulier F et al. (1996). Receptor specificity of the fibroblast growth factor family. J Biol Chem 271: 15292–15297. Popovici C, Zhang B, Gregoire MJ, Jonveaux P, LafagePochitaloff M, Birnbaum D et al. (1999). The t(6;8)(q27;p11) translocation in a stem cell myeloproliferative disorder fuses a novel gene, fop, to fibroblast growth factor receptor 1. Blood 93: 1381–1389. Powers CJ, McLeskey SW, Wellstein A. (2000). Fibroblast growth factors, their receptors and signaling. Endocr Relat Cancer 7: 165–197. Qian ZR, Sano T, Asa SL, Yamada S, Horiguchi H, Tashiro T et al. (2004). Cytoplasmic expression of fibroblast growth factor receptor-4 in human pituitary adenomas: Relation to tumor type, size, proliferation, and invasiveness. J Clin Endocrinol Metab 89: 1904–1911. Richelda R, Ronchetti D, Baldini L, Cro L, Viggiano L, Marzella R et al. (1997). A novel chromosomal translocation t(4; 14)(p16.3; q32) in multiple myeloma involves the fibroblast growth-factor receptor 3 gene. Blood 90: 4062–4070. Ruohola JK, Viitanen TP, Valve EM, Seppanen JA, Loponen NT, Keskitalo JJ et al. (2001). Enhanced invasion and tumor growth of fibroblast growth factor 8b-overexpressing mcf-7 human breast cancer cells. Cancer Res 61: 4229–4237. Schlessinger J. (2004). Common and distinct elements in cellular signaling via egf and fgf receptors. Science 306: 1506–1507. Shimokawa T, Furukawa Y, Sakai M, Li M, Miwa N, Lin YM et al. (2003). Involvement of the fgf18 gene in colorectal carcinogenesis, as a novel downstream target of the beta-catenin/t-cell factor complex. Cancer Res 63: 6116–6120. Sleeman M, Fraser J, McDonald M, Yuan S, White D, Grandison P et al. (2001). Identification of a new fibroblast growth factor receptor, fgfr5. Gene 271: 171–182. Spinola M, Leoni V, Pignatiello C, Conti B, Ravagnani F, Pastorino U et al. (2005). Functional fgfr4 gly388arg polymorphism predicts prognosis in lung adenocarcinoma patients. J Clin Oncol 23: 7307–7311. Streit S, Bange J, Fichtner A, Ihrler S, Issing W, Ullrich A. (2004). Involvement of the fgfr4 arg388 allele in head and neck squamous cell carcinoma. Int J Cancer 111: 213–217. Wang J, Stockton DW, Ittmann M. (2004). The fibroblast growth factor receptor-4 arg388 allele is associated with prostate cancer initiation and progression. Clin Cancer Res 10: 6169–6178. Xiao S, Nalabolu SR, Aster JC, Ma J, Abruzzo L, Jaffe ES et al. (1998). Fgfr1 is fused with a novel zinc-finger gene, znf198, in the t(8;13) leukaemia/lymphoma syndrome. Nat Genet 18: 84–87. Xie MH, Holcomb I, Deuel B, Dowd P, Huang A, Vagts A et al. (1999). Fgf-19, a novel fibroblast growth factor with unique specificity for fgfr4. Cytokine 11: 729–735. Yamada SM, Yamada S, Hayashi Y, Takahashi H, Teramoto A, Matsumoto K. (2002). Fibroblast growth factor receptor (fgfr) 4 correlated with the malignancy of human astrocytomas. Neurol Res 24: 244–248.


97 Yu C, Wang F, Jin C, Huang X, McKeehan WL. (2005). Independent repression of bile acid synthesis and activation of c-jun n-terminal kinase (jnk) by activated hepatocyte fibroblast growth factor receptor 4 (fgfr4) and bile acids. J Biol Chem 280: 17707–17714.

Zaharieva BM, Simon R, Diener PA, Ackermann D, Maurer R, Alund G; et al. (2003). High-throughput tissue microarray analysis of 11q13 gene amplification (ccnd1, fgf3, fgf4, ems1) in urinary bladder cancer. J Pathol 201: 603–608.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene