Original article

Annals of Oncology 13: 15981604, 2002 DOI: 10.1093/annonc/mdf248

Clinical implications of expression of ETS-1 related to angiogenesis in uterine cervical cancers

J. Fujimoto*, I. Aoki, H. Toyoki, S. Khatun & T. Tamaya
Department of Obstetrics and Gynecology, Gifu University 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

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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 correlated 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 correlated with patient prognosis in the primary tumor of uterine

*Correspondence to: Dr J. Fujimoto, Department of Obstetrics and Gynecology, 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
2002 European Society for Medical Oncology

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 adenocarcinoma 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 angiogenic 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 vascular endothelial cells and the adjacent interstitial cells [8]. Once

1599 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 inducing 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 angiogenesis in uterine cervical cancers. The aim of the study was to formulate an efficient tumor dormancy therapy.
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 l containing 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 products were subsequently extracted with water-saturated [-32P]UTP (New England Nuclear, Boston, MA, USA) as a tracer. The reaction was incubated 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 radioactivity of the transcribed RNA.

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Patients and methods

Patients
Consent for the following studies was obtained from all patients and the Research 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].

Competitive RTPCR Southern blot analysis

Total RNA was isolated from tissues using the acid guanidium thiocyanatephenolchloroform 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 of PCR 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 capillarytransferred to an Immobilon transfer membrane (Millipore Corp., Bedford, 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, pH 7.6, 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 oligonucleotide probe (MIMIC-DT, 5-GCAGATGAGTATCTTGTCCC-3) simultaneously 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

Preparation of internal standard recombinant RNA

Internal standard recombinant RNA (rcRNA) was prepared for competitive reverse transcriptionpolymerase chain reaction (RTPCR) and Southern blot analysis [16] as follows. Deoxynucleic acid construction of the internal standard was originated and synthesized by PCR from a BamHI/ 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-13) in the first PCR reaction were as follows: MIMIC ets-1-5, 5-ATGGAGTCAACCCAGCCTATCGCAAGTGAAATCTCCTCCG-3; MIMIC ets-1-3, 5-CCATGCACATGTTGTCTGGGTCTGTCAATGCAGTTTGTAG-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-TAATACGACTCACTATAGGATGGAGTCAACCCAGCCTAT-3; ets-1-3, 5-CCATGCACATGTTGTCTGGG-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

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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-[-aminoethyl 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 10 000 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), respectively. 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.

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Figure 1. Quantitative analysis of ets-1 mRNA by competitive RTPCR Southern blot analysis. The RTPCR reactions containing ets-1 gene-specific primers were carried out in the presence of total RNA and serially diluted internal standard recombinant RNA (rcRNA) in the range of 1100 fmol for ets-1 mRNA. Southern blots for the competitive RTPCR are shown in (A). In (B), data are plotted to determine ets-1 mRNA levels as the log ratio of rcRNA/ets-1 mRNA total RNA isolated from the samples compared with log rcRNA.

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. Differences were considered significant at P <0.05.

(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.

Results
Immunohistochemical staining for ETS-1 and factor VIIIrelated antigen in a representative case of non-keratinizing squamous cell carcinoma stage IIb is shown in Figure 2. Factor VIII-related antigen was clearly distributed in vascular endothelial cells. ETS-1 was also distributed in vascular endothelial cells and in their adjacent interstitium. There was a significant correlation between microvessel counts (MVC) and ets-1 mRNA levels (P <0.001) in uterine cervical cancers, as shown in Figure 3. Ets-1 mRNA levels increased with increasing uterine cervical cancer disease stage, as shown in Figure 4. The 50 patients who underwent curative resection were arbitrarily divided into two equal groups based on the levels of ets-1 mRNA, with the midpoint being 0.3 pmol rcRNA/g total RNA. The prognosis of the 25 patients with higher levels of ets-1 mRNA (>0.3 pmol rcRNA/g total RNA) in their uterine cervical cancers was extremely poor, while the 24-month survival rate of the other 25 patients with lower ets-1 mRNA levels (<0.3 pmol rcRNA/g total RNA) was 92% (Figure 5).

Immunohistochemistry
Four-micrometre sections of formalin-fixed, paraffin-embedded tissues of uterine endometrial cancers were cut with a microtome and dried overnight at 37C on a silanized-slide (Dako, Carpinteria, CA, USA). Samples were deparaffinized in xylene at room temperature for 80 min and washed with 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-related 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

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Figure 3. Correlation between microvessel counts and ets-1 mRNA levels in uterine cervical cancers.

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

1602 and meningioma cells related to invasive potential and phenotypes [3134]. ETS-1 expression is induced by bFGF in glioma cells related to invasive potential [32], and by VEGF in astrocytoma related to angiogenesis [35]. Furthermore, overexpressed ETS-1 is recognized as an angiogenic mediator in oral squamous cell carcinomas and gastric carcinomas [36, 37]. In the current study, transcription factor ETS-1 was dominantly expressed in vascular endothelial cells and their adjacent interstitium, but not in uterine cervical cancer cells, while ets-1 mRNA levels correlated with microvessel density observed in the immunohistochemical staining for factor VIII-related antigen. Generally, distinct ETS-1 expression in vascular endothelial cells has been recognized as evidence of accelerated angiogenesis [810]. The present data reveal that ets-1 mRNA levels increased with increasing disease stage, and correlated with patient prognosis in uterine cervical cancers. Therefore, ETS-1 might be activated as a direct angiogenic mediator for the initiation and maintenance stages of angiogenesis, and may possibly be an excellent indicator of patient prognosis in uterine cervical cancers. Furthermore, from the results of this study, it appears that ETS-1 expression might be positively regulated by the major angiogenic factors PD-ECGF and IL-8, but not by bFGF or VEGF, notwithstanding the fact that bFGF and VEGF are known to induce ETS-1 expression in vascular endothelial cell lines [11, 12]. Therefore, even if PD-ECGF and IL-8 can be suppressed by some agents, angiogenesis might be suppressed only transiently, which could lead to temporary suppression of tumor growth and secondary spreading. In such a case, other angiogenic factors would be induced and would link to ETS-1 in the search for alternate angiogenic activation, as a kind of tolerance to angiogenic inhibitors. Therefore, suppression of

Figure 5. Survival rate after curative resection for uterine cervical cancers. Patient prognosis was analyzed with a 24-month survival rate. High est-1 mRNA, >0.3 pmol rRNA/g total RNA, n = 25; low ets-1 mRNA, <0.3 pmol rRNA/g total RNA, n = 25.

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There was a significant correlation between ets-1 mRNA levels and PD-ECGF (P <0.001) and IL-8 levels (P <0.001) in uterine cervical cancers, as shown in Figure 6, but not between ets-1 mRNA and bFGF or VEGF levels (data not shown).

Discussion
Ets-1 is expressed in various cancer cells including gastric, pancreatic, esophageal, hepatocellular and cholangiocellular carcinomas, and thyroid and astrocytic tumors, and works on tumor progression as a proto-oncogene [2329]. ETS-1 is up-regulated and involved in the overexpression of MMP-7 in hepatocellular carcinoma cells [30], and positively regulates the expression of uPA in breast cancer, glioma, astrocytoma

Figure 6. Correlation between ets-1 mRNA levels and PD-ECGF or IL-8 levels in uterine cervical cancers.

1603 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].
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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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