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Annals of Oncology 13: 15981604, 2002

Original article DOI: 10.1093/annonc/mdf248

2002 European Society for Medical Oncology

Clinical implications of expression of ETS-1 related to

angiogenesis in uterine cervical cancers

J. Fujimoto*, I. Aoki, H. Toyoki, S. Khatun & T. Tamaya

*Correspondence to: Dr J. Fujimoto, Department of Obstetrics andGynecology, Gifu University School of Medicine, 40 Tsukasa-machi,Gifu City 500-8705, Japan. Tel: +81-58-267-2631;

Fax: +81-58-265-9006; E-mail: jf@cc.gifu-u.ac.jp

Department of Obste trics and Gynecology, Gifu Univers ity School of Medicine, Gifu City, Japan

Received 6 November 2001; revised 18 February 2002; accepted 26 March 2002

Background: Angiogenesis is essential for development, growth and advancement of solid tumors.

ETS-1 has been recognized as a candidate for tumor angiogenic transcription factor. This prompted us

to study the clinical implications of ETS-1-related angiogenesis in uterine cervical cancers.

Patients and methods: Fifty patients underwent curative resection for uterine cervical cancers. The

patients prognoses were analyzed with a 24-month survival rate. In the tissue of 60 uterine cervical

cancers, the levels of ets-1 mRNA, vascular endothelial growth factor (VEGF), basic fibroblast growth

factor (bFGF), platelet-derived endothelial cell growth factor (PD-ECGF) and interleukin (IL)-8 were

determined by competitive reverse transcriptionpolymerase chain reaction using recombinant RNA

and enzyme immunoassay, and the localization and counts of microvessels were determined by

immunohistochemistry.

Results: There was a significant correlation between microvessel counts and ets-1 gene expression

levels in uterine cervical cancers. Immunohistochemical staining revealed that the localization of

ETS-1 was similar to that of vascular endothelial cells. The level of ets-1 mRNA correlated with the

levels of PD-ECGF and IL-8 among angiogenic factors. Furthermore, the prognosis of the 25 patients

with high ets-1 mRNA expression in uterine cervical cancers was extremely poor, while the 24-month

survival rate of the other 25 patients with low ets-1 mRNA expression was 92%.

Conclusions: ETS-1 might be a prognostic indicator as an angiogenic mediator in uterine cervical

cancers.

Key words: angiogenesis, ets-1, IL-8, PD-ECGF, uterine cervical cancer

Introduction

Angiogenesis is essential for development, growth and

advancement of solid tumors [1]. The angiogenic factors

vascular endothelial growth factor (VEGF), basic fibroblast

growth factor (bFGF), platelet-derived endothelial cell growth

factor (PD-ECGF), identified with thymidine phosphorylase

(TP), and interleukin (IL)-8 work on angiogenesis in uterine

cervical cancers [28]. VEGF, particularly its VEGF165 and

VEGF121 isomers, was dominantly expressed in cancer cells,

especially in adenocarcinomas, but its levels did not correlate

with patient prognosis [2]. Basic FGF was expressed in both

cancer and interstitial cells of uterine cervix, and its levels cor-

related with clinical stage, but not in an obvious manner with

patient prognosis [3]. PD-ECGF was dominantly expressed in

interstitial cells of uterine cervical cancers, and its levels cor-

related with patient prognosis in the primary tumor of uterine

cervical cancers, especially in the metastatic lymph nodes

[4, 5]. Most noteworthy, serum PD-ECGF can be used as a

tumor marker of both squamous cell carcinoma and adeno-

carcinoma of the uterine cervix [6]. IL-8 was supplied from

tumor-associated macrophages infiltrated near cancer cells,

and its levels correlated with patient prognosis [7]. Therefore,

if PD-ECGF and IL-8 can be suppressed as a tumor dormancy

therapy, patient prognosis should be remarkably improved

without the severe side effects of chemotherapy. Because

chemotherapy is often not so specific to cancer cells, it

produces severe side effects even on normal cells, especially

bone marrow cells. On the other hand, tumor dormancy

therapy is specific to the rapidly growing vascular endothelial

cells in tumors, without an effect on slow-growing vascular

endothelial cells and other normal cells. However, if an angio-

genic factor is suppressed by tumor dormancy therapy for a

long period, another angiogenic factor might be induced by an

alternatively linked angiogenic pathway; this is recognized as

tolerance.

During angiogenesis, ETS-1 is strongly expressed in vascu-

lar endothelial cells and the adjacent interstitial cells [8]. Once

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angiogenesis has finished, ETS-1 expression is distinctly

down-regulated [9, 10]. The representative angiogenic factors

VEGF and bFGF immediately induce ETS-1 expression in the

early stage of angiogenesis, while the inhibition of ETS-1

expression leads to suppression of angiogenesis [11, 12]. The

proteases urokinase type-plasminogen activator (u-PA), and

matrix metalloprotease (MMP)-1, -3 and -9 conserve an

ETS-binding motif, and transcription factor ETS-1 converts

vascular endothelial cells to angiogenic phenotypes by induc-

ing u-PA, MMP-1, MMP-3 and MMP-9 and integrin 3 gene

expression [13, 14]. This status prompted us to study whether

transcription factor ETS-1 works as an angiogenic mediator,

and, if so, which angiogenic factors link to ETS-1 for angio-

genesis in uterine cervical cancers. The aim of the study was to

formulate an efficient tumor dormancy therapy.

Patients and methods

Patients

Consent for the following studies was obtained from all patients and theResearch Committee for Human Subjects, Gifu University School of

Medicine. Of 60 patients (stage Ib, 15 cases; stage II, 20 cases; stage III,

20 cases; and stage IVa, 5 cases) ranging from 31 to 87 years of age, 50

underwent curative resection at the Department of Obstetrics and

Gynecology, Gifu University School of Medicine, between October 1995

and December 1998; this resulted in macroscopic disease-free status. The

prognosis for these 50 patients was analyzed with a 24-month survival

rate. None of the patients had received any therapy before the cervical

cancer tissue was taken. A section of each uterine cervical cancer tissue

was snap-frozen in liquid nitrogen to determine levels of ets-1 mRNA,

VEGF, bFGF, PD-ECGF and IL-8, and a neighboring piece of tissue was

submitted for histopathological analysis, including immunohistochemical

staining for ets-1 and factor VIII-related antigen. The clinical stage of the

uterine cervical cancers was determined according to the International

Federation of Obstetrics and Gynecology (FIGO) classification [15].

Preparation of internal standard recombinant RNA

Internal standard recombinant RNA (rcRNA) was prepared for competit-

ive reverse transcriptionpolymerase chain reaction (RTPCR) and South-

ern blot analysis [16] as follows. Deoxynucleic acid construction of the

internal standard was originated and synthesized by PCR from aBamHI/

EcoRI-ligated fragment of V-erbB (Clontech Laboratories, Palo Alto, CA,

USA) with two sets of oligonucleotide primer sequences. The sequences for

the first set of primers for ets-1 mRNA (MIMIC ets-1-5 and MIMIC ets-1-

3) in the first PCR reaction were as follows: MIMIC ets-1-5, 5-ATGG-

AGTCAACCCAGCCTATCGCAAGTGAAATCTCCTCCG-3; MIMIC

ets-1-3, 5-CCATGCACATGTTGTCTGGGTCTGTCAATGCAGTTT-GTAG-3 [17, 18]. The sequences for the second set of primers for ets-1

mRNA (MIMIC ets-1-P and ets-1-3) in the secondary PCR reaction were

as follows: MIMIC ets-1-P, 5-TAATACGACTCACTATAGGATGG-

AGTCAACCCAGCCTAT-3 ; ets-1-3, 5-CCATGCACATGTTGTCT-

GGG-3. The first PCR cycle was conducted in a final volume of 50 l

containing PCR buffer (50 mM KCl, 10 mM TrisHCl pH 8.3, 1.5 mM

MgCl2), 0.2 mM deoxyribonucleoside triphosphates (dNTPs), 2 ng of

BamHI/EcoRI-ligated DNA fragment of V-erbB, 10 pmol each of the first

set of PCR primers and 2.5 U Amplitaq DNA polymerase (Perkin-Elmer

Cetus, Norwalk, CT, USA). The second PCR cycle was conducted in a

final volume of 100 l containing PCR buffer, 0.2 mM dNTPs, 50 pg of

the first PCR products, 20 pmol each of the second set of PCR primers and

5 U Amplitaq DNA polymerase. The mixture was amplified for 28 cycles

of PCR for 45 s at 94C for denaturing, 45 s at 55C for annealing and 90 s

at 72C for extension in a DNA Thermal Cycler (Perkin-Elmer Cetus).

The second PCR products were purified with a Gene Clean kit (Bio 101

Inc, La Jolla, CA, USA) and transcribed with 100 U T7 RNA polymerase

(Gibco-BRL, Gaithersberg, MD, USA) in a final volume of 100 lcontaining T3/T7 buffer (40 mM TrisHCl, pH 8.0, 8 mM MgCl2, 2 mM

spermidine-(HCl)3, 25 mM NaCl), 0.1 M dithiothreitol (DTT), 10 mM

ribonucleotide triphosphate, 40 U RNase inhibitor (Promega, Madison,

WI, USA), 20 mM template DNA and 10 Ci of DNase (Takara Shuzo,

Kyoto, Japan) at 37C for 5 min to remove the DNA template. The pro-

ducts were subsequently extracted with water-saturated [-32P]UTP (New

England Nuclear, Boston, MA, USA) as a tracer. The reaction was incu-

bated at 37C for 1 h, and then treated with 70 U of RNase-free phenol/

chloroform and passed through a Sephadex G50 II column (Boehringer

Mannheim, Mannheim, Germany). The amount of transcribed internal

marker was calculated from the total radioact ivity of the transcribed RNA.

Competitive RTPCR Southern blot analysis

Total RNA was isolated from tissues using the acid guanidium thio-

cyanatephenolchloroform extraction method [19].

To obtain a standard curve each time, the total RNA (3 g) and a series

of diluted recombinant RNA for ets-1 mRNA (1100 fmol) were reverse

transcribed for 1 h at 37C in a 20 l volume with a mixture of 200 U

Moloney murine leukemia virus reverse transcriptase (MMLV-RTase;

Gibco-BRL) and the following reagents: 50 mM TrisHCl, pH 8.3,

75 mM KCl, 3 mM MgCl2, 40 U RNAsin (Toyobo, Osaka, Japan), 10 mM

DTT, 0.5 mM dNTPs and 30 pmol 3-end specific primer as detailed

below. The reaction was incubated for 5 min at 94C to inactivate the

MMLV-RTase.

The sequences of primers to amplify the genes of ets-1 (ets-1-5 and

ets-1-3) were: ets-1-5, 5-ATGGAGTCAACCCAGCCTAT-3 (exon 5);

ets-1-3, 5-CCATGCACATGTTGTCTGGG-3 (exon 6). The sizes ofPCR products for ets-1 mRNA and its internal standard rcRNA were

288 bp and 440 bp, respectively. PCR with reverse-transcribed RNAs as

templates (1 l) and 5 pmol of each specific primer was carried out using

a DNA Thermal Cycler with 0.5 U Amplitaq DNA polymerase in a buffer

containing 50 mM KCl, 10 mM TrisHCl, pH 8.3, 1.5 mM MgCl2 and

0.2 mM dNTPs in a 20 l volume. Thirty cycles of amplification for ets-1

mRNA were performed at 94C for 45 s for denaturing, 55C for 45 s for

annealing and 72C for 90 s for extension.

Amplified PCR products (8 l) were electrophoresed with 1.2% agarose

gel in a 100 V constant voltage field. PCR products were capillary-

transferred to an Immobilon transfer membrane (Millipore Corp., Bed-

ford, MA, USA) for 16 h. The membrane was dried at 80C for 30 min,

then ultra violet (UV)-irradiated to fix PCR products tightly. These PCR

products were prehybridized in buffer (1 M NaCl, 50 mM TrisHCl, pH7.6, 1% sodium dodecyl sulfate) at 42C for 1 h, and hybridized in the

same solution with the biotinylated oligodeoxynucleotide probe (ets-1-DT,

5-TGGTATTGAGCATGCCCAGT-3 for the ets-1 gene), synthesized

from the sequences of ets-1 cDNA between the specific primers, and the

corresponding biotinylated internal standard gene-specific oligonucleo-

tide probe (MIMIC-DT, 5-GCAGATGAGTATCTTGTCCC-3 ) simul-

taneously to check gene specificity at 65C overnight. They were also

hybridized with the biotinylated ets-1-5 probe (10 pmol/ml) to determine

the signal intensity under the same conditions. Specific bands hybridized

with the biotinylated probes were detected with Plex Luminescent Kits

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(Millipore Corp.), and X-ray film was exposed on the membrane at room

temperature for 10 min. The quantification of Southern blot was carried

out with Bio Image (Millipore, Ann Arbor, MI, USA).

In the competitive RTPCR Southern blot analysis for ets-1 mRNA,

only two predicted sizes of DNA fragment were detected with ets-1-DT

and ets-1-5 simultaneously to check specificity and determine quantity,

respectively. As a negative control, no ets-1 mRNA was detected without

reverse transcription in 30 cycles of PCR. The levels of ets-1 mRNA were

determined using a standard curve and a serial dilution of rcRNA in

competitive RTPCR Southern blot analyses, as shown in Figure 1.

Immunohistochemistry

Four-micrometre sections of formalin-fixed, paraffin-embedded tissues

of uterine endometrial cancers were cut with a microtome and dried over-

night at 37C on a silanized-slide (Dako, Carpinteria, CA, USA). Samples

were deparaffinized in xylene at room temperature for 80 min and washedwith a graded ethanol/water mixture and then with distilled water. The

samples for ETS-1 were soaked in a citrate buffer, and then microwaved at

100C for 10 min, and those for factor VIII-related antigen were treated

with 0.3 g/ml trypsin in phosphate buffer at room temperature for

20 min. The protocol for a DAKO LSAB2 Kit, Peroxidase (Dako), for

immunohistochemical staining was followed for each sample. In this

protocol, rabbit anti-human ETS-1 (C-20; Santa Cruz Biotechnology,

Santa Cruz, CA, USA) and rabbit anti-factor VIII-rela ted antigen (Zymed,

San Francisco, CA, USA) were used as the first antibodies at dilutions of

1:2000 and 1:2, respectively. The addition of the first antibody, rabbit

anti-human ETS-1 or rabbit anti-factor VIII-related antigen, was omitted

in the protocols for negative controls of ETS-1 or factor VIII-related

antigen, respectively.

Vessels were counted in the five highest density areas at magnification

200 (0.785 mm2 per field). Microvessel counts were expressed as the

mean numbers of vessels in these areas [20]. Microvessel density was

evaluated by counting microvessels.

Enzyme immunoassay for determination of bFGF, VEGF,PD-ECGF and IL-8 antigens

All steps were carried out at 4C. Tissues of uterine cervical cancers

(wet weight 1020 mg) were homogenized in HG buffer (5 mM TrisHCl,

pH 7.4, 5 mM NaCl, 1 mM CaCl2, 2 mM ethyleneglycol-bis-[-amino-

ethyl ether]-N,N,N,N- tetraacetic acid, 1 mM MgCl2, 2 mM DTT,

25 g/ml aprotinin, and 25 g/ml leupeptin) with a Polytron homogenizer

(Kinematics, Luzern, Switzerland). This suspension was centrifuged in a

microfuge at 10000 g for 3 min to obtain the supernatant. The protein

concentration of samples was measured by the method of Bradford [21] to

standardize VEGF, bFGF, PD-ECGF and IL-8 antigen levels.

Basic FGF, VEGF and IL-8 antigen levels in the samples were

determined by a sandwich enzyme immunoassay using a Human bFGF

Quantikine kit, a Human VEGF Quantikine kit and a Human IL-8

Quantikine kit (all from R&D Systems, Minneapolis, MN, USA), respect-

ively. PD-ECGF antigen levels were determined by the method described

by Nishida et al. [22]. The levels of bFGF, VEGF, PD-ECGF and IL-8

were standardized with the corresponding cellular protein concentrations.

Statistics

Survival curves were calculated using the KaplanMeier method and

analyzed by the log-rank test. The levels of ets-1 mRNA, VEGF, bFGF,

PD-ECGF and IL-8 were measured from three samples taken from each

tissue, and the assay for each sample was carried out in triplicate. Differ-

ences were considered significant at P

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Figure 2. Immunohistochemical staining for ETS-1 and factor VIII-

related antigen in uterine cervical cancers. A case of uterine cervical

cancer. Rabbit anti-human ETS-1 (Santa Cruz Biotechnology) and mouse

anti-human factor VIII-related antigen (Zymed) were each used at

dilutions of 1:2000 and 1:2, respectively, as the first antibodies. Original

magnification: 200.

Figure 3. Correlation between microvessel counts and ets-1 mRNA

levels in uterine cervical cancers.

Figure 4. Levels of ets-1 mRNA in uterine cervical cancers, classified

according to clinical stage (FIGO). Each level is the mean of nine

determinations. Alive and deceased cases are shown as open and closed

circles, respectively.

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There was a significant correlation between ets-1 mRNA

levels and PD-ECGF (P

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the major angiogenic factors along with suppression of ETS-1

recruitment might be more effective as a tumor dormancy

therapy than as mere suppression of major angiogenic factors.

A specific inhibitor for ETS-1, transdominant mutant ETS-1,

has already been shown to act as a dominant negative

molecule, and can be used as an efficient tool for angiogenic

inhibition [38].

Acknowledgements

This study was supported in part by funds from the following

Ministry of Health and Welfare programs of the Japanese

Government: Grant for Scientific Research Expenses for

Health and Welfare Programs, Foundation for Promotion of

Cancer Research, and Grant-in-Aid for the Second Term

Comprehensive 10-year Strategy for Cancer Control. The

authors wish to thank Mr John Cole for proofreading this

manuscript.

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