Department of Obstetrics and Gynecology, Gifu University School of Medicine, Gifu City, Japan
Received 19 February 2002; revised 4 July 2002; accepted 19 July 2002
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Angiogenesis is essential for development, growth and advancement of solid tumors. During angiogenesis, ETS-1 is strongly expressed in vascular endothelial cells and the adjacent interstitial cells, while the inhibition of ETS-1 expression leads to suppression of angiogenesis. This prompted us to study the clinical implications of ETS-1 in relation to angiogenesis in uterine endometrial cancers.
Patients and methods:
Sixty patients underwent resection for uterine endometrial cancers. From the tissues of 60 uterine endometrial 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 RTPCR using recombinant RNA and enzyme immunoassay, and the localization and counts of microvessel were determined by immunohistochemistry.
Results:
There was a significant correlation between microvessel count and ets-1 gene expression levels in uterine endometrial cancers. Immunohistochemical staining revealed that the localization of ETS-1 was similar to that of vascular endothelial cells. The level of ets-1 mRNA tended to increase with increasing disease stage. Furthermore, the level of ets-1 mRNA correlated with levels of VEGF in well-differentiated adenocarcinomas (G1) and of bFGF in moderately differentiated adenocarcinomas (G2) and poorly differentiated adenocarcinomas (G3).
Conclusions:
ETS-1 is a possible angiogenic mediator in uterine endometrial cancers.
Key words: angiogenesis, basic FGF, ets-1, uterine endometrial cancer, VEGF
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During angiogenesis, ETS-1 is strongly expressed in vascular endothelial cells and the adjacent interstitial cells [13]. Once angiogenesis has ended, there is a distinct down-regulation of ETS-1 expression [14, 15]. The representative angiogenic factors VEGF and bFGF immediately induce ETS-1 expression in the early stages of angiogenesis, while the inhibition of ETS-1 expression leads to suppression of angiogenesis [16, 17]. Proteases urokinase type-plasminogen activator (u-PA), matrix metalloprotease (MMP)-1, MMP-3 and MMP-9 conserve an ETS-binding motif, and transcription factor ETS-1 converts vascular endothelial cells to angiogenic phenotypes by inducing proteases u-PA, MMP-1, MMP-3 and MMP-9 and integrin ß3 gene expression [18, 19]. 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 endometrial cancers, with the goal of formulating an efficient tumor dormancy therapy.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of internal standard recombinant RNA (rcRNA) for competitive RTPCR and Southern blot analysis [21]
Deoxynucleic acid construction of the internal standard was originated and synthesized by PCR from a BamH/EcoRI fragment of V-erbB (Clontech, 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 were as follows: MIMIC ets-1-5', 5'-ATGGAGTCAACCCAGCCTATCGCAAGTGAAATCTCCTCCG-3'; MIMIC ets-1-3', 5'-CCATGCACATGTTGTCTGGGTCTGTCAATGCAGTTTGTAG-3' [22, 23]. The sequences for the second set of primers for ets-1 mRNA (MIMIC ets-1-P and ets-1-3') in the secondary PCR were as follows: MIMIC ets-1-P, 5'-TAATACGACTCACTATAGGATGGAGTCAACCCAGC CTAT-3'; ets-1-3', 5'-CCATGCACATGTTGTCTGGG-3'. The first and second PCRs were carried out as previously described [21]. The second PCR products were transcribed using T7 RNA polymerase (Gibco BRL, Gaithersberg, MD, USA) and the amount of transcribed internal marker was calculated as previously described [21].
Competitive RTPCR and Southern blot analysis
Total RNA was isolated from tissues by the acid guanidium thiocyanatephenolchloroform extraction method [24]. To obtain a standard curve each time, total RNA (3 µg) and a series of diluted recombinant RNA for ets-1 mRNA (1100 fmol) were reverse transcribed. Primer sequences used to amplify the ets-1 gene (ets-1-5' and ets-1-3') were as follows: ets-1-5', 5'-ATGGAGTCAACCCAGCCTAT-3' (exon 5); ets-1-3', 5'-CCATGCACATGTTGTCTGGG-3' (exon 6). Competitive PCR was carried out as previously described [21]. In the competitive RTPCR and Southern blot analysis for ets-1 mRNA, only two predicted sizes of DNA fragment were hybridized with the biotinylated ets-1-5' probe to determine quantity as previously described [21]. As a negative control, no ets-1 mRNA was detected without reverse transcription in 30 cycles of PCR. Levels of ets-1 mRNA were determined using a standard curve and a serial dilution of rcRNA in competitive RTPCR and Southern blot analyses, as shown in Figure 1.
|
Vessels were counted in the five highest density areas at 200x magnification (using a combination of 20x objective and 10x ocular, 0.785 mm2 per field). Microvessel counts were expressed as the mean numbers of vessels in these areas [25]. Microvessel density was evaluated by the counting of microvessels.
Enzyme immunoassay for determination of bFGF, VEGF, PD-ECGF and IL-8 antigens
All steps were carried out at 4°C. Uterine endometrial cancers tissue (wet weight 1020 mg) was homogenized in HG buffer (5 mM TrisHCl pH 7.4, 5 mM NaCl, 1 mM CaCl2, 2 mM ethyleneglycol-bis-[ß-aminoethyl ether]-N,N,N',N'-tetraacetic acid, 1 mM MgCl2, 2 mM DTT, 25 µg/ml aprotinin and 25 µg/ml leupeptin) with a Polytron homogenizer (Kinematics, Luzern, Switzerland). This suspension was centrifuged in a microfuge at 12 000 r.p.m. (10 000 g) for 3 min to obtain the supernatant. The protein concentration of samples was measured by the method of Bradford [26] 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 Human bFGF, VEGF and IL-8 Quantikine kits (R&D System, Minneapolis, MN, USA), respectively. PD-ECGF antigen levels were determined by the method of Nishida et al. [27]. The levels of bFGF, VEGF, PD-ECGF and IL-8 were standardized with the corresponding cellular protein concentrations.
Statistics
Levels of ets-1 mRNA, VEGF, bFGF, PD-ECGF and IL-8 were measured from three parts taken from each tissue, and the assay for each sample was carried out in triplicate. The t-test for two independent samples was used to compare the determinations in Figure 4. The sample correlation coefficient was used for the comparisons in Figures 3, 5 and 6. Differences were considered significant for values of P <0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the current study, transcription factor ETS-1 was dominantly expressed in vascular endothelial cells and their adjacent interstitium, but not in cancer cells in uterine endometrial cancers, while ets-1 mRNA levels correlated with microvessel density observed in immunohistochemical staining for factor VIII-related antigen. Generally, distinct ETS-1 expression in vascular endothelial cells has been recognized as evidence of accelerated angiogenesis [1315]. The present data reveal that ets-1 mRNA levels increased with increasing disease stage in uterine endometrial 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 endometrial cancers.
Since levels of ets-1 mRNA in this study correlated with levels of VEGF in G1 and bFGF in G2 and G3, it can be concluded that in all cases VEGF and bFGF act as angiogenic factors in uterine endometrial cancers, especially VEGF in G1 and bFGF in G2 and G3. We previously reported that VEGF expression was down-regulated with dedifferentiation (G1G2
G3) [3] and conversely bFGF expression was up-regulated with dedifferentiation in uterine endometrial cancers [9]. This indicates that ETS-1 is an angiogenic mediator linked to VEGF in G1 and bFGF in G2 and G3, and preserves angiogenic switching in the linkage to angiogenic factors to maintain advancement. Also, it is well known that bFGF and VEGF induce ETS-1 expression in vascular endothelial cell lines [16, 17]. Therefore, even if VEGF or bFGF can be suppressed by some agents, angiogenesis might be suppressed only transiently, which could lead to a temporary suppression of tumor growth and secondary spreading. In such a scenario, other angiogenic factors would be induced, and link to ETS-1 in the recruitment for alternate angiogenic activation, as a kind of tolerance to angiogenic inhibitors. Therefore, suppression of the major angiogenic factors along with suppression of ETS-1 recruitment might be more effective as a tumor dormancy therapy than 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 [43].
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Fujimoto J, Sakaguchi H, Hirose R et al. Expression of vascular endothelial growth factor (VEGF) and its mRNA in uterine cervical cancers. Br J Cancer 1999; 80: 827833.[ISI][Medline]
3. Fujimoto J, Ichigo S, Hori M et al. Expression of basic fibroblast growth factor and its mRNA in advanced uterine cervical cancers. Cancer Lett 1997; 111: 2126.[ISI][Medline]
4. Fujimoto J, Ichigo S, Sakaguchi H et al. The expression of platelet-derived endothelial cell growth factor in uterine cervical cancers. Br J Cancer 1999; 79: 12491254.[ISI][Medline]
5. Fujimoto J, Sakaguchi H, Hirose R et al. Clinical implication of expression of platelet-derived endothelial cell growth factor (PD-ECGF) in metastatic lesions of uterine cervical cancers. Cancer Res 1999; 59: 30413044.
6. Fujimoto J, Sakaguchi H, Aoki I, Tamaya T. The value of platelet-derived endothelial cell growth factor as a novel predictor of advancement of uterine cervical cancers. Cancer Res 2000; 60: 36623665.
7. Fujimoto J, Ichigo S, Sakaguchi H et al. Expression of platelet-derived endothelial cell growth factor (PD-ECGF) and its mRNA in uterine endometrial cancers. Cancer Lett 1998; 130: 115120.[ISI][Medline]
8. Fujimoto J, Ichigo S, Hori M, et al. Expression of basic fibroblast growth factor and its mRNA in advanced uterine cervical cancers. Cancer Lett 1997; 111: 2126.[ISI][Medline]
9. Fujimoto J, Hori M, Ichigo S, Tamaya T. Expression of basic fibroblast growth factor and its mRNA in uterine endometrial cancers. Invasion Metastasis 1995; 15: 203210.[ISI][Medline]
10. Fujimoto J, Sakaguchi H, Aoki I, Tamaya T. Clinical implications of expression of interleukin 8 related to angiogenesis in uterine cervical cancers. Cancer Res 2000; 60: 26322635.
11. Fujimoto J, Sakaguchi H, Hirose R et al. Progestins suppress estrogen-induced expression of vascular endothelial growth factor (VEGF) subtypes in uterine endometrial cancer cells. Cancer Lett 1999; 141: 6371.[ISI][Medline]
12. Fujimoto J, Ichigo S, Sakaguchi H et al. Expression of platelet-derived endothelial cell growth factor in uterine endometrium during the menstrual cycle. Mol Hum Reprod 1998; 4: 509513.[Abstract]
13. Wernert N, Raes MB, Lassalle P et al. c-ets proto-oncogene is a transcription factor expressed in endothelial cells during tumor vascularization and other forms of angiogenesis in humans. Am J Pathol 1992; 140: 119127.[Abstract]
14. Kola I, Brppkes S, Green AR et al. The Ets-1 transcription factor is widely expressed during murine embryo development and is associated with mesodermal cells involved in morphogenic process such as organ formation. Proc Natl Acad Sci USA 1993; 90: 75887592.
15. Maroulakou IG, Papas TS, Green JE. Differential expression of ets-1 and ets-2 proto-oncogenes during murine embryogenesis. Oncogene 1994; 9: 15111565.
16. Iwasaka C, Tanaka K, Abe M et al. Ets-1 regulates angiogenesis by inducing the expression of urokinase-type plasminogen activator and matrix metalloproteinase-1 and the migration of vascular endothelial cells. J Cell Physiol 1996; 169: 522531.[ISI][Medline]
17. Tanaka K, Abe M, Sato Y. Roles of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase in the signal transduction of basic fibroblast growth factor in endothelial cells during angiogenesis. Jpn J Cancer Res 1999; 90: 647654.[ISI][Medline]
18. Oda N, Abe M, Sato Y. ETS-1 converts endothelial cells to the angiogenic phenotype by inducing the expression of matrix metalloproteinases and integrin ß3. J Cell Physiol 1999; 178: 121132.[ISI][Medline]
19. Sato Y, Abe M, Tanaka K et al. Signal transduction and transcriptional regulation of angiogenesis. Adv Exp Med Biol 2000; 476: 109115.[ISI][Medline]
20. International Federation of Obstetrics and Gynecology (FIGO) News. Int J Gynecol Obstet 1989; 28: 189193.
21. Fujimoto J, Hirose R, Sakaguchi H, Tamaya T. Expression of oestrogen receptor- and -ß in ovarian endometriomata. Mol Hum Reprod 1999; 5: 742747.
22. Vanden Hevvel JP, Tyson FL, Bell DA. Construction of recombinant RNA templates for use as internal standards in quantitative RTPCR. Biotechnology 1993; 14: 395398.
23. Watson DK, McWilliams MJ, Lapis P et al. Mammalian ets-1 and ets-2 genes encode highly conserved proteins. Proc Natl Acad Sci USA 1988; 85: 78627866.[Abstract]
24. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanatephenolchloroform extraction. Anal Biochem 1987; 162: 156159.[ISI][Medline]
25. Maeda K, Chung Y, Ogawa Y et al. Thymidine phosphorylase/platelet-derived endothelial cell growth factor expression associated with hepatic metastasis in gastric carcinoma. Br J Cancer 1996; 73: 884888.[ISI][Medline]
26. Bradford MA. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 315323.[ISI][Medline]
27. Nishida M, Hino A, Mori K et al. Preparation of anti-human thymidine phosphorylase monoclonal antibodies useful for detecting the enzyme levels in tumor tissues. Biol Pharm Bull 1996; 19: 14071411.[ISI][Medline]
28. Nakayama T, Ito M, Ohtsuru A et al. Expression of the ets-1 proto-oncogene in human gastric carcinoma: correlation with tumor invasion. Am J Pathol 1996; 149: 19311939.[Abstract]
29. Ito T, Nakayama T, Ito M et al. Expression of the ets-1 proto-oncogene in human pancreatic carcinoma. Mod Pathol 1998; 11: 209215.[ISI][Medline]
30. Nakayama T, Ito M, Ohtsuru A et al. Expression of the ets-1 proto-oncogene in human thyroid tumor. Mod Pathol 1999; 12: 6168.[ISI][Medline]
31. Kitange G, Kishikawa M, Nakayama T et al. Expression of the Ets-1 proto-oncogene correlates with malignant potential in human astrocytic tumors. Mod Pathol 1999; 12: 618626.[ISI][Medline]
32. Saeki H, Kuwano H, Kawaguchi H et al. Expression of ets-1 transcription factor is correlated with penetrating tumor progression in patients with squamous cell carcinoma of the esophagus. Cancer 2000; 89: 16701676.[ISI][Medline]
33. Ito Y, Miyoshi E, Takeda T et al. Expression and possible role of ets-1 in hepatocellular carcinoma. Am J Clin Pathol 2000; 114: 719725.[ISI][Medline]
34. Ito Y, Miyoshi E, Takeda T et al. Ets-1 expression in extrahepatic bile duct carcinoma and cholangiocellular carcinoma. Oncology 2000; 58: 248252.[ISI][Medline]
35. Ozaki I, Mizuta T, Zhao G et al. Involvement of the ets-1 gene in overexpression of matrilysin in human hepatocellular carcinoma. Cancer Res 2000; 60: 65196525.
36. Watabe T, Yoshida K, Shindoh M et al. The Ets-1 and Ets-2 transcription factors activate the promoters for invasion-associated urokinase and collagenase genes in response to epidermal growth factor. Int J Cancer 1998; 77: 128137.[ISI][Medline]
37. Kitange G, Shibata S, Tokunaga Y et al. Ets-1 transcription factor-mediated urokinase-type plasminogen activator expression and invasion in glioma cells stimulated by serum and basic fibroblast growth factors. Lab Invest 1999; 79: 407416.[ISI][Medline]
38. Nakada M, Yamashita J, Okada Y et al. Ets-1 positively regulates expression of urokinase-type plasminogen activator (uPA) and invasiveness of astrocytic tumors. J Neuropathol Exp Neurol 1999; 58: 329334.[ISI][Medline]
39. Kitange G, Tsunoda K, Anda T et al. Immunohistochemical expression of Ets-1 transcription factor and the urokinase-type plasminogen activator is correlated with the malignant and invasive potential in meningiomas. Cancer 2000; 89: 22922300.[ISI][Medline]
40. Valter MM, Hugel A, Huang HJ et al. Expression of the ets-1 transcription factor in human astrocytomas is associated with fms-like tyrosine kinase-1 (Flt-1)/vascular endothelial growth factor receptor-1 synthesis and neoangiogenesis. Cancer Res 1999; 59: 56085614.
41. Pande P, Mathur M, Shukla NK et al. Ets-1: a plausible marker of invasive potential and lymph node metastasis in human oral squamous cell carcinomas. J Pathol 1999; 189: 4045.[ISI][Medline]
42. Tsutsumi S, Kuwano H, Asao T et al. Expression of ets-1 angiogenesis-related protein in gastric cancer. Cancer Lett 2000; 60: 4550.
43. Nakano T, Abe M, Tanaka K et al. Angiogenesis inhibition by transdominant mutant Ets-1. J Cell Physiol 2000; 184: 255262.[ISI][Medline]