1, Hui Huang 2, Gang Xiao , Zhi-you Zhou , Yang Chen€¦ · the heparan-like glycosaminoglycans (HLGAGs) of the extracellular matrix (ECM)(1). From this reservoir, FGFs may act directly

Anti-tumor Effect of Mutant Human FGFR2 ectodomain

1

Antitumor activity of a Recombinant soluble Ectodomain of Mutant Human Fibroblast

Gowth Factor Recptor-2 IIIc

Ju Wang 1, Xue-ting Liu 1, Hui Huang 2, Gang Xiao 1, Zhi-you Zhou 1, Yang Chen 1,

Zhi-hong Yu 1, Shui-lian He 1, An-an Chen 1, Ding-ding Wang 3 , Ying He 1, Zhi-cheng

Zhang 1 and An Hong 1

1Institute of Bioengineering, College of Life Science and Technology, Jinan University, Guangzhou , Guangdong 510632, China; 2Department of Cardiology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou, 510120 China and 3Department of Biotechnology, Institute of Life Science and Biological Pharmacy, Guangdong Pharmaceutical university, 280 Outer Ring Road East, Guangzhou Higher Education Mega Center, Guangzhou, 510006, China

1

1Financial support the Ministry of Science and Technology of China (2009ZX09103-632) and the National Natural Science Foundation of China (91029742). Requests for reprints: Ju Wang, Institute of Bioengineering, College of Life Science and Technology, Jinan University,

Guangzhou , Guangdong 510632, China; Tel: +86 20 85221983; Fax: +86 20 85221983;Email: [email protected]; or

An Hong, Institute of Bioengineering, College of Life Science and Technology, Jinan University, Guangzhou , Guangdong

510632, China;Tel: +86 20 85221983; Fax: +86 20 85221983; Email: [email protected]

Note Ju Wang and Xue-ting Liu contributed equally to this work.

on May 18, 2020. © 2011 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 12, 2011; DOI: 10.1158/1535-7163.MCT-11-0163

http://mct.aacrjournals.org/


2

Abstract

The fibroblast growth factor (FGF) signalling pathway is a recognised target of cancer

therapy. We have developed a strong inhibitor (S252W mutant soluble ectodomain of

fibroblast growth factor recptor-2 IIIc, msFGFR2) that binds FGFs and blocks the activation

of FGFRs. Thermodynamic binding studies indicated that msFGFR2 bound FGF-2 16.9 times

as strongly as wild-type soluble FGFR2IIIc ectodomain (wsFGFR2). It successfully

suppressed the growth, angiogenesis and metastasis of two tumour cell lines in vitro and in

vivo, and it potently inhibited cancer cell proliferation but not normal cell proliferation.

Therefore, msFGFR2 is a useful probe for FGF-dependent signalling pathways and a

potential broad-spectrum antitumour agent.

Key Words: sFGFR2; S252W; FGF-2; tumour inhibition; angiogenesis; metastasis

Abbreviations list: FGFs, Fibroblast growth factors; FGFR, Fibroblast growth factor

receptor; msFGFR2, S252W mutant soluble ectodomain of FGFR2IIIc; wsFGFR2, wild-type

soluble ectodomain of FGFR2IIIc; ITC, Isothermal Titration Calorimetry; CAM, Chicken

Chorioallantoic Membrane





3

Introduction

Fibroblast growth factors (FGFs) are small polypeptide growth factors. Many FGFs bind to

the heparan-like glycosaminoglycans (HLGAGs) of the extracellular matrix (ECM)(1). From

this reservoir, FGFs may act directly on target cells by binding to specific receptor tyrosine

kinases, and this binding induces receptor dimerization and activation, ultimately resulting in

the activation of various signal transduction cascades(2, 3). Some FGFs are potent angiogenic

factors and most of them play important roles in embryonic development and wound

healing(4, 5).

FGF signalling appears to play a role in tumour growth and angiogenesis, and autocrine

FGF signalling may be particularly important in the progression of steroid

hormone-dependent cancers to a hormone-independent state(6). FGFs secreted by tumours

act, on the one hand, in an autocrine manner promoting tumour growth, and, on the other

hand, in a paracrine manner on endothelial cells, thereby promoting tumour angiogenesis(7).

Both FGF2 and VEGF are known to play roles in both healthy and pathological

angiogenesis(8-10). In prostate cancer, FGF8 expression correlates with advanced tumour

stage and is associated with reduced survival(11, 12). In pancreatic cancer, high levels of

several FGFs (1, 2, 5 and 7) and most of the FGFRs have been observed, suggesting the

presence of autocrine activation of receptors and of downstream signalling pathways(13).

Elevated FGF2 expression in breast tumours is associated with more aggressive forms of the

disease(14, 15). Since FGF-2 is one of the most famous members of FGF family for its

proliferating and angiogenic effects, a lot of studies have evidenced that it is closely related to

tumors(16-18), we selected FGF-2 as a representative of FGF family to study the binding of

sFGFR2 to FGF ligands and the mechanisms of the inhibition of sFGFR2 on tumors.

FGFR overexpression is involved in the tumourigenesis and progression(19-21) of





4

multiple types of human cancers, such as breast and prostate cancers. Somatic gain of

function FGFR mutations are also present in multiple types of cancer, including bladder

cancer, endometrial carcinoma, lung squamous cell carcinoma, and cervical

carcinoma(22-24). Therefore, FGF signalling could provide interesting targets for anti-cancer

therapy.

The extracellular portion of FGFRs consists of three immunoglobulin (Ig)-like domains

(D1-3) and an acid box containing 4–8 amino acids between the D1 and D2 domains(25), and

it was named soluble FGFR (sFGFR). The acid box hinders the effective binding of ligands

by interacting with the D2-D3 domains to play a role in self-inhibition(26). sFGFR2

competitively binds to FGF ligands and inhibits the FGFR signalling pathway.

Two FGFR2 mutations located in the highly conserved linker region between D2 and D3,

S252W and P253R, are known to be associated with congenital skeletal disorders, such as

Apert syndrome (AS)(22). Crystal structure analysis of a complex consisting of FGF-2 and

mutant FGFR2 showed that both mutations increase the binding affinity between FGF2 and

FGFR2, resulting in sustained activation of the signalling pathway and symptoms associated

with the disorder(27, 28).

In this study, a vector construct was designed to express the extracellular domain of

FGFR2IIIc (sFGFR2) lacking both D1 and the acid box region and containing the S252W

mutation. This mutant sFGFR2 (msFGFR2) was bacterially expressed and subsequently





5

renatured. The binding affinity of msFGFR2 for FGF-2 was found to be 16.9 times as high as

that of wild-type sFGFR2 (wsFGFR2) using an ITC assay. In vivo studies showed that

treatment with msFGFR2 resulted in significant inhibition of tumour growth and improved

the overall survival of tumour-bearing nude mice.

Hereafter, sFGFR2 is used to refer to both wsFGFR2 and msFGFR2.

Materials and Methods

Reagents

Anti-� actin and anti-phospho-FGFR (Y653/Y654) were from Cell Signaling Technology.

Anti-Erk1/2, anti-phospho-Erk1/2, anti-FGFR2, HRP-goat anti-rabbit conjugate and

HRP-goat anti-mouse conjugate were from Santa Cruz. Human Fibroblast Growth

Factor-basic ELISA construction kits were from R&D Systems.

Cell lines and animals

Human breast cancer cell line MCF-7 and human prostate cancer cell line DU145 were from

the American Type Culture Collection (ATCC). Mouse mammary carcinoma cell line 4T1

and mouse embryonic fibroblast cell line NIH/3T3 were provided by Dr. Sun Fen-yong in our

lab, also from ATCC. All cells were passaged fewer than 6 months after resuscitation and

cultured using the protocol provided by ATCC. Sera and media were purchased from

Invitrogen and American Type Culture.

Balb/c nude mice and Balb/c mice (4–5 weeks) were obtained from the Experimental Animal





6

Research Centre of Zhongshan University and were maintained in its SPF lab.

sFGFR2 expression, renaturation, and purification

cDNA was synthesised from human placenta mRNA by reverse transcription. The DNA

region from D2 to D3 of the FGFR2IIIc isoform (amino acids 147-366 of hBEK) was

amplified from cDNA, and S252W mutation was introduced by splice-overlap PCR method.

These two gene fragments with or without mutation were cloned into the pET3c vector and

expressed in E. coli. Bacteria were collected and lysed, and inclusion bodies were isolated.

After washing the inclusion bodies twice, protein refolding was performed on a gel filtration

column(GE Healthcare, Piscataway, NJ, USA). sFGFR2 showing more than 95% purity was

harvested through heparin affinity chromatography(GE).

Isothermal titration calorimetry measurements

Titration calorimetry (ITC) measurements were performed with an Auto-iTC200 (GE). A

typical titration consisted of injecting 2 �L aliquots of 93 �M ligand (FGF-2) solution into 35

�M msFGFR2 or wsFGFR2 solution after every 2.5 min to ensure that the titration peak

returned to the baseline prior to the next injection. Aliquots of more concentrated ligand

solutions were injected into only the reaction buffer (25 mM HEPES containing 600 mM

NaCl and 5% glycerol, pH 7.5) in separate ITC runs to measure the heats of dilution of the

ligands.

Western blotting

To determine the effect of msFGFR2 on FGFRs and ERK activation, DU145 and MCF-7

cells were starved in serum-free medium overnight to knockdown the endogenous level of





7

phosphorylated kinases. Cells pretreated with 20 ng/ml FGF-2 for 15 min were then treated

with 5 �g/ml msFGFR2 for 15 min. Then they were solubilized by incubation at 4°C for 15

min in cell lysis solution(UpState) containing protease inhibitor cocktail(Roche). The protein

concentration of the soluble extracts was determined by BCA protein assay kit(Thermo Fisher

Scientific). Separation of 50 �g of total protein was done on 12% SDS-polyacrylamide gels

and transferred to a PVDF membrane before immunoblotting with primary antibodies as

indicated. The equal loading of protein samples was verified with antibodies against total

FGFR2 and ERK. The specific proteins were detected by the enhanced chemiluminescence

detection system (Thermo Fisher Scientific).

FGF2 detection by ELISA

MCF-7 and DU145 cells were seeded into 100-mm dishes at a density of 2 106 per dish and

incubated in regular medium overnight then placed in serum-free medium for 24 h. The

medium was then removed and stored at -70°C. The FGF2 level in the medium could be

detected using an FGF-2 ELISA assay (Quantikine™, R&D, Minneapolis, MN) according to

the manufacturer’s instructions.

Detection of cell proliferation

Firstly, since sFGFR2 could specifically inhibit the proliferation of NIH3T3 stimulated by

FGF-2 and NIH3T3 cell line was used to detect FGF-2 activity standardly(29, 30), the

investigation of the activity of sFGFR2 was done in NIH3T3 cells. Then, we used MCF-7

and DU145 cell line to detect the anti-tumor effects of sFGFR2. Cells were cultured in

96-well plates (approximately 5000 cells/well) for 24 h. Cells were then starved with phenol

red-free DMEM plus 1% dialysed foetal calf serum (A15-107) (PAA laboratories) for 24 h.

The experiment included a control group, a FGF-2 group (20 ng/ml), a sFGFR2 group (10, 20,

40, 80, 160, 320, 640, and 5000 ng/ml), and a FGF-2 plus sFGFR2 group. After induction





8

48h by sFGFR2, cell growth was measured by a

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay. Absorbance was

recorded at 570 nm with a reference at 630 nm serving as the blank. The effect of sFGFR2 on

cell viability was assessed as percent cell viability compared with control cells, which were

arbitrarily assigned 100% viability. The mean value of five wells was calculated, and each

experiment was repeated three times.

Cell Cycle Analysis

DU145 or MCF-7 cells in the logarithmic phase of growth were incubated with 5 �g/ml

sFGFR2 for 24 h. For cell cycle analysis, samples (1×106 cells) were fixed and permeabilized

by addition of 1 ml of ice-cold 70% ethanol for 15 min on ice. Then after washing, the cells

were resuspended in 125 �l of 1.12% (w/v) sodium citrate containing 0.2 mg/ml RNase

(Roche) and incubated at 37°C for 15 min. 50 �g/ml propidium iodide (Sigma-Aldrich) was

added to the cells for 30 min at room temperature in the dark. The cells were stored at 4°C

until assay by flow cytometry (FACSCalibur, BD Biosciences). Cell cycle analysis was done

using ModFit LT software (Verity).

Quantification of Apoptotic Cells

msFGFR2-induced apoptosis in DU145, MCF-7 and NIH3T3 cells was determined by flow

cytometry using the Annexin V–conjugated Alexa Fluor 488 (Alexa488) Apoptosis Detection

Kit (Millipore) following the instructions of the manufacturer. Briefly, after overnight serum

starvation, cells were treated with msFGFR2 (5 �g/mL) for 48 hours. The cells were then

harvested, washed in PBS, and incubated with Alexa488 and propidium iodide for cellular

staining at room temperature for 10 minutes in the dark. The stained cells were analyzed by

FACS using a FACSCalibur instrument (BD Biosciences) equipped with CellQuest 3.3

software.





9

Inhibitory effects of msFGFR2 on breast cancer and prostate cancer xenografts in nude

mice

A total of 2.5 × 107 MCF-7 or 5 × 106 DU145 cells were subcutaneously injected into

BALB/c athymic nude mice. MCF-7 cells were injected into female mice. The presence of

tumours was apparent on day 7 after the injection of MCF-7 cells or on day 21 after injection

of DU145 cells. From then on, all mice were given PBS (vehicle) or 0.1 mg msFGFR2 daily

by intravenous injection for 28 days. The length, width, and height of the subcutaneous

tumours were measured everyday with callipers. The tumour volume (mm3) was calculated

with the following formula: (a: the short diameter [mm]; b: the long diameter [mm]).

After the mice were sacrificed, the tumours were weighed and then incubated in formalin.

The mortality rate of the mice was also calculated.

Effect of sFGFR2 on tumour cell invasion

Quantitative analysis of 4T1 tumour cell invasion was performed using a 96-well chamber

system (QCM 96-Well Cell Invasion assay; Chemicon International/Millipore, Darmstadt,

Germany) according to the instructions of the manufacturer. Briefly, 20,000 cells/well were

resuspended in serum-free medium and allowed to attach to inserts coated with a layer of

extracellular matrix (ECM; ECMatrix solution is a solid gel of basement membrane proteins

prepared from the Engelbreth Holm-Swarm mouse tumor). The inserts were placed into a

feeder tray containing cell culture medium in the presence of chemoattractant (5 �g/ml

sFGFR2 in 10% foetal bovine serum). After 24 h, invasive cells which migrated through the

ECM-coated membrane (8 �m pore size) were washed in PBS, detached from the bottom of





10

the inserts, lysed and stained with a fluorescent dye for fluorimetric quantification. Six wells

were analyzed for each cell clone.

In vivo inhibition of tumour metastasis by msFGFR2

BALB/c female mice 4 to 6 weeks old weighing 18 to 22 g, were intravenously injected with

3 × 104 4T1 mouse breast cancer cells and divided into two groups randomly. Then each

group contained 8 mice treated with PBS(vehicle) or 0.1 mg msFGFR2 via the tail vein daily

for 14 d.. After the animals were sacrificed, lung tumour nodules were counted, and lung

tissues were incubated in formalin.

Hematoxylin and eosin (H&E) staining and immunohistochemistry

After formalin-fixed tumour sections were prepared, the tissues were stained with H&E to

observe the cell distribution in the tumours. Rat monoclonal antibodies against CD34,

p-FGFRs, Ki67, E-Cadherin, Vimentin, and �-SMA were used for immunohistochemical

analysis.

Chick chorioallantoic membrane (CAM) assay

Fertilized chicken eggs were maintained under constant humidity at 37°C. On day 4 of

incubation, a square window was opened into the shell. On day 9 of incubation, a sterile

gelatin sponge (1mm3) was placed onto the CAM immediately followed by topical

administration of msFGFR2 or wsFGFR2 (both at 3�g /egg). After 3 days, quantification of

CAM vascular network was assessed by counting the number of vessels converging toward

the implant under a stereomicroscope and the experiment was repeated three times.

Statistical analysis.

Statistical analysis was performed using the Statistical Package for Social Sciences 13.0





11

(SPSS). Data are presented as mean ± s.e.m. Student’s t test (two-tailed) was used to compare

two groups for independent samples assuming equal variances among all experimental data

sets. Statistical significance was assumed if P <0.05.

Results

sFGFR2 protein expression, purification, and identification

The FGFR2 extracellular domain was expressed in E. coli and the recombinant protein

accumulated in bacteria as inclusion bodies. The protein was examined by SDS-PAGE and

western blot analysis, revealing the presence of a single band following renaturation and

purification (Fig. 1B). The purity of sFGFR2 was more than 95% (Fig. 1C).

MTT assays showed that sFGFR2 (10–5000 ng/ml) significantly inhibited the

stimulatory effect of FGF-2 (20 ng/ml) on NIH3T3 cell proliferation (Fig. 1D). The inhibition

rates of 20 ng/ml msFGFR2 and wsFGFR2 were 59% and 36.5%, respectively. Thus,

recombinant sFGFR2 successfully refolded and inhibited mitogenic activity by competing

with membrane-bound FGFR for binding to FGF-2. The inhibitory effect of sFGFR2 was

dose-dependent. However, in the absence of FGF-2, sFGFR2 did not show any significant

inhibitory effect (Fig. 1D).

sFGFR2 inhibits FGFR signalling by binding to its ligands

The binding of sFGFR2 to FGF-2 was measured by isothermal titration calorimetry (ITC)

(Fig. 2A). The dissociation constant (Kd) of wsFGFR2 for FGF-2 was 4.93 × 10-6 mol/L,





12

whereas that of msFGFR2 was 2.92 × 10-7mol/L. Thus, the binding capacity of msFGFR2 for

FGF-2 was 16.9 times higher.

In Du145 cells, western blotting revealed that the phosphorylation levels of membrane

FGFRs and ERK1/2 activated by FGF-2 significantly declined in the presence of sFGFR2

compared with control conditions (Fig. 2B), and similar results were obtained in MCF-7

cells(Fig. 2C).

This result suggested that sFGFR2 inhibited FGF signalling by binding to its ligands.

The binding of msFGFR2 was found to be stronger than that of wsFGFR2. The MTT assay

described above showed that msFGFR2 inhibited cell proliferation more profoundly than

does wsFGFR2. Therefore, msFGFR2 was tested in the following in vitro and in vivo

experiments.

Effects of sFGFR2 on the proliferation and apoptosis of DU145, MCF-7 and NIH3T3 cells

sFGFR2 significantly inhibited the growth of DU145 and MCF-7 cells. In the absence of

FGF-2, sFGFR2 was effective when its dose was 20-160ng/ml, but the most effective dose

was 5�g/ml (Fig 3A). After 20 ng/ml FGF-2 was added, 40 ng/ml msFGFR2 was the most

effective dose, decreased the proliferation of DU145 cells from 140% to 65%, MCF-7 cells

from 120% to about 95%.

ELISA assays revealed that after 24 h of culture, the FGF-2 concentration reached 26

pg/ml in the DU145 cell culture medium, whereas it was 1 pg/ml in the MCF-7 cell culture





13

medium (Fig. 3B). The MTT results were consistent with the ELISA results, which suggested

that sFGFR2 antagonised the positive influence of FGF-2 on the proliferation of DU145 and

MCF-7 cells.

Flow cytometric analysis showed that msFGFR2 increased the percentage of DU145

cells in G1 phase from 59.2% to 75.5% and that of MCF-7 cells from 69.1% to 85.7% (Fig.

3C).

Annexin V/PI double staining showed that msFGFR2 increased the percentage of DU145

cells undergoing early apoptosis from 7.91% to 17.78% and the percentage of MCF-7 cells

undergoing late apoptosis from 3.13% to 22.01% (Fig. 3D).

sFGFR2 did not have a significant effect on the G1 arrest and apoptosis of NIH3T3 cells,

for reasons that remain unclear (Fig. 3C and 3D).

These results indicate that sFGFR2 significantly inhibited the proliferation of tumour cells

only when it was FGF-2-dependent. In addition, sFGFR2 increased the G1-phase arrest and

apoptosis of tumour cells, both of which could contribute to the inhibitory effect of sFGFR2

on tumour cell proliferation.

The inhibition of CAM angiogenesis by msFGFR2

CAM angiogenesis was significantly inhibited by treatment with msFGFR2 (3 �g/egg) for 72

h (Fig. 4). There were fewer and thinner veins in the regions covered with the msFGFR2

cellulose membrane. Quantitative analysis of vascular density showed that the inhibitory rate





14

of msFGFR2 on CAM angiogenesis was 90%.

Inhibition of tumour growth in vivo by msFGFR2

In nude mice bearing DU145 xenografts, the average tumour weight in the msFGFR2

group (n = 8) and control group (n = 5) was 0.173 g and 0.353 g, respectively (Fig. 5B). Thus,

the inhibition of the average tumour weight was 51.0%. On Day 28 after the treatment with

sFGFR2, the survival rate was 62.5% in the control group and 100% in the msFGFR2 group

(Fig. 5C). Similarly, in nude mice bearing MCF-7 xenografts, msFGFR2 inhibited the

average tumour weight by 36.1% compared to control animals.

Immunohistochemical analysis showed that msFGFR2 significantly inhibited the

phosphorylation of FGFRs and the expression of both Ki67 (a tumour proliferation marker)

and CD34 (an angiogenesis marker) in tumour tissues (Fig. 5D).

These results indicated that msFGFR2 significantly inhibited tumour growth. The

suppressive effect was stronger on DU145 cells than on MCF-7 cells, which suggested that

the high level of FGF-2 secreted by DU145 might be responsible for this effect.

The inhibition of tumour metastasis in vivo by msFGFR2

sFGFR2 significantly inhibited the migration of 4T1 mouse breast cancer cells in the

Chemicon QCM 96-well Invasion Assay (Fig. 6A). Next, we injected 4T1 cells into Balb/C

mice via the tail vein, and the average number of lung nodules in the msFGFR2 and control

group was 26.9 and 60.5, respectively (Fig. 6B); thus, msFGFR2 showed a significant





15

inhibitory rate of 55.5%. H&E staining of lung tissues yielded similar results (Fig. 6C).

Immunohistochemical staining revealed that msFGFR2 significantly inhibited the

phosphorylation of FGFRs and the expression of CD34 (a new vessel marker), vimentin and

�-SMA (mesenchymal cell markers), but E-Cadherin expression (an epithelial cell marker)

was unchanged in the tumour tissues (Fig. 6C). These results suggest that msFGFR2 may

inhibit angiogenesis, vimentin and �-SMA-mediated migration. The molecular mechanism

should be explored further.

Discussion

Soluble forms of the extracellular domain of FGFRs (sFGFRs) are known for their roles in

suppressing FGF signalling(31) and inhibiting tumour proliferation(32-34). The msFGFR2

used in this study does not contain the D1 domain and the acid box and includes the S252W

mutation. Its Kd value, which shows the binding affinity of msFGFR2 for FGF-2, is 16.9

times as high as that of wsFGFR2. In vivo experiments showed that the inhibition of DU145

tumour growth following msFGFR2 administration was as high as 55.4%.

In a study by Omar et al(27), mutation of S252W in FGFR2IIIc caused an 8.6- to 36-fold

increase in the binding affinity of FGFR2IIIc for FGF9/16/20, a 4.7- to 7.2-fold increase in

affinity for FGF1/2, a 6.3- to 8.2-fold increase of affinity for FGF3/5, a 4.9- to 6-fold increase

of affinity for FGF4/6, and a 4.2-fold increase of affinity for FGF23; these results indicated

that msFGFR2 has increased binding affinity for most FGFR2IIIc ligands. Based on these





16

data, we speculate that considering the numerous members of the FGF family, the mechanism

the inhibitory effects of msFGFR2 on tumours involves blocking almost every branch of the

FGF signalling pathway, which makes msFGFR2 superior as an antitumor agent compared

with FGF antibodies(35).

msFGFR2 significantly inhibited NIH/3T3 and tumour cell proliferation only when

exogenous FGF-2 was administered. Similarly, msFGFR2 markedly induced apoptosis in

DU145 and MCF-7 cells, but not in NIH/3T3 cells. Such effects seem to be related to the

expression levels of FGF2 and even of FGFRs, which are often much higher in tumour

tissues than in normal tissues. Therefore, inhibition of the FGF pathway in cancer might be

an effective and selective means of inhibiting cancer cell growth with low toxicity to

noncancerous cells.

Our animal experiment results showed that msFGFR2 is able to inhibit tumour metastasis.

The relationship between FGFRs and tumour metastasis has been extensively studied(36).

Some studies have shown that FGFR1 promotes EMT and causes tumour cell invasion(37,

38). Another study showed that FGFR2IIIc may be related to MET and may favour secondary

tumour growth(39). Our study showed that msFGFR2 significantly inhibits the seeding of

mouse breast cancer cells 4T1 in lungs in vivo, which suggests that FGF signalling could be

related to the motility of tumour cells, and the underlying mechanism should be addressed

further.





17

As an inhibitor of FGFs, msFGFR2 can suppress the growth of multiple types of tumours.

In our study, msFGFR2 was a stronger inhibitor of DU145 cells and xenografts in mice than

of MCF-7 cells and xenografts. We also found that msFGFR2 binds to membrane FGFRs of

tumour cells in the presence of FGF-2 (data not shown), which might suggest that the effects

of msFGFR2 are related to FGF ligands and FGFR expression levels.

Overall, msFGFR2 has strong inhibitory effects on tumour growth and metastasis,

especially in regard to angiogenesis in vitro and in vivo. Because excessive FGFR signalling

is present in most tumours, msFGFR2 demonstrates significant potential for use in clinical

applications.

Acknowledgement

This work was funded by the Ministry of Science and Technology of China

(2009ZX09103-632) and by the National Natural Science Foundation of China (91029742).

We thank Dr. Sun Fenyong, Dr. Xie Qiu-ling, Dr. Chen Xiao-jia, and members of

Guangdong Provincial Key Laboratory of Bio-engineering Medicine (National Engineering

Research Centre of Genetic Medicine) for technical help, invaluable discussions, and

reagents.





18

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Figure 1. Design, production, and identification of msFGFR2

A. The structure of the wild-type and mutant extracellular domain of FGFR2IIIc (sFGFR2)

and full-length FGFR2IIIc receptor. The heparin-binding domain, the alternative-splicing

domain (in the D3 domain), and the location of the AS mutation (S252W) (in the D2 domain)

are labelled with thick black lines, thick gray lines and an arrow, respectively.

B. SDS-PAGE and western blot analysis showing that sFGFR2 was highly expressed in E.

coli and its purity was more than 95% after renaturation and purification. Lane 1, protein

molecular mass markers (kDa); lanes 2-3, cell lysates of uninduced (lane 2) and

IPTG-induced (lane 3) BL21 (+) cells containing the wild-type recombinant DNA expression

vector wsFGFR2; lanes 4-5, the same as lanes 2-3 except BL21 (+) cells containing the

mutant recombinant DNA expression vector msFGFR2; lanes 6-7, wsFGFR2 (lane 6) and

msFGFR2 (lane 7) proteins following purification by heparin affinity chromatography; lane

8-9, western blot analysis of wsFGFR2 and msFGFR2 proteins, respectively.

C. Mass spectrometric analysis of wsFGFR2 and msFGFR2.

D. Inhibitory effect of renatured sFGFR2 on the proliferation of NIH3T3 cells (murine

fibroblasts) based on MTT assays.





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Figure 2. Inhibition of FGFR signalling by sFGFR2 binding to its ligands

A. Thermodynamic analysis of FGF-2 binding to msFGFR2 and wsFGFR2.

Top graph: raw data (dq/dt) for injections of FGF-2 into a solution of sFGFR2 in 25 mM

HEPES containing 600 mM NaCl and 5% glycerol (pH 7.5). Bottom graph: data after peak

integration and concentration normalisation. The sample cell contained 35 �M msFGFR2 or

wsFGFR2, and FGF-2 was titrated from a 93 �M stock solution.

B. The phosphorylation of both FGFRs and ERK1/2 was inhibited by wsFGFR2 and

msFGFR2 (5 �g/ml) in DU145 cells and MCF-7 cells.

Figure 3. Effects of sFGFR2 on the proliferation and apoptosis of DU145 and MCF-7

cells

A. Inhibition of the proliferation of MCF-7 and DU145 cells by sFGFR2 was measured using

an MTT assay. When 20 ng/ml FGF-2 was added, 40 ng/ml sFGFR2 showed the highest

inhibition of cell proliferation. The average value of five wells was calculated, and the

experiment was repeated three times.

B. An ELISA assay was utilised to determine the FGF-2 concentration in the culture media of

DU145 and MCF-7 cells.

C. Effects of sFGFR2 (5 �g/ml) on the cell cycle were examined by fluorescence activated

cell sorting. Treatment with either msFGFR2 or wsFGFR2 resulted in G1 arrest and the

inhibition of cell proliferation in DU145, MCF-7 and NIH3T3 cells.

D. Effects of sFGFR2 (5 �g/ml) on the apoptosis of DU145, MCF-7 and NIH3T3 cells were





23

measured by annexin V/PI double staining. MsFGFR2 was found to significantly induce

apoptosis.

Figure 4. Inhibition of angiogenesis by msFGFR2 as detected using a chick

chorioallantoic membrane assay

msFGFR2 was applied to a cellulose acetate membrane, and 72 h after fertilisation, eggs were

covered with the membrane for 3 days. Strong inhibition of angiogenesis by msFGFR2 was

observed.

Figure 5. Inhibition of MCF-7 and DU145 xenografts in nude mice by msFGFR2.

A Tumour growth curves of the msFGFR2 group and control group.

B Tumour weights of the msFGFR2 group and control group.

C Survival analysis of the msFGFR2 group and control group.

D Photos of mice, photos of tumours, H&E staining and immunohistochemistry of tumours in

the msFGFR2 group and control group. Tumour tissues and the expression of pFGFRs, Ki-67

and CD34 were examined.

Figure 6. Inhibition of tumour metastasis in vitro and in vivo by msFGFR2

A. Inhibition of 4T1 cell invasion by sFGFR2.





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B. The influence of msFGFR2 on tumour metastasis was examined in an experimental lung

metastasis model with 4T1 breast cancer cells. One-hundred micrograms of msFGFR2 was

given to each mouse each day for 14 days. The number of lung metastases in the msFGFR2

group was less than that in the control group. Statistical analysis showed that the number of

lung metastases in mice receiving msFGFR2 was significantly less than that in the control

group. Data are expressed as mean ± SD from eight mice in each group.

C. H&E staining of lung biopsies and immunohistochemistry of tumours in the msFGFR2 group

and control group. The expression of pFGFRs, -SMA, Vimentin, E-Cadherin and CD34 were

examined.






















Published OnlineFirst July 12, 2011.Mol Cancer Ther Ju Wang, Xue-ting Liu, Hui Huang, et al. Mutant Human Fibroblast Growth Factor Receptor-2 IIIcAntitumor activity of a Recombinant soluble Ectodomain of

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