Skip to main content
PMC home page
British Journal of Cancer logoLink to British Journal of Cancer
. 1999 Dec;81(8):1335–1343. doi: 10.1038/sj.bjc.6692206

Co-expression of vascular endothelial growth factor (VEGF) and its receptors (flk-1 and flt-1) in hormone-induced mammary cancer in the Noble rat

B Xie 1, N N C Tam 1, S W Tsao 1, Y C Wong 1
PMCID: PMC2362981  PMID: 10604730

Abstract

Vascular endothelial growth factor (VEGF) is recognized to play a predominant role in breast cancer prognosis. The action of VEGF is mediated by two high-affinity receptors with ligand-stimulated tyrosine kinase activity: VEGFR-1/flt-1 and VEGFR-2/flk-1, which are expressed mainly in vascular endothelial cells. To the best of our knowledge, no previous studies on the expression of these receptors in breast cancer cells has been made. We have established a new animal model for breast cancer, using a combination of 17β-oestradiol and testosterone as ‘carcinogens’. Taking advantage of the animal model, we have demonstrated that mammary cancer cells expressed not only high levels of VEGF but also, surprisingly, its receptors (flt-1 and flk-1) in mammary cancer cells. Intense reactivities to VEGF, flt-1 and flk-1 were observed in mammary cancer cells, especially in invasive mammary carcinoma. Western blot analysis confirmed the increase in flk-1 and flt-1 proteins in induced mammary cancers. Based on these observations, we hypothesize that in mammary cancer, VEGF regulates, in addition to endothelial proliferation and angiogenesis, also growth of cancer cells by an autocrine mechanism mediated through its receptors. To further verify this hypothesis, we investigated the correlation between cellular proliferation and the expression of VEGF, flt-1 and flk-1. Using double-labelling immunocytochemistry, we have shown a correlation between high VEGF activity and Ki-67 expression. The Ki-67 indices in the areas of strong and weak VEGF reactivities were 58.3% and 3.7% respectively. Similarly, there was also a correlation of strong flk-1 and Ki-67 reactivity. The Ki-67 indices for areas of strong and weak flk-1 reactivities were 53.9% and 3.1% respectively. On the other hand, there was a reverse correlation between flt-1 and Ki-67 activities. These results indicate that overexpression of VEGF and flk-1 is correlated with high Ki-67 index. The data, therefore, suggest that VEGF may act as an autocrine growth factor for mammary cancer cells in vivo and this autocrine regulatory role may be mediated through flk-1. The present study is the first report showing that VEGF may act as a growth stimulator for mammary cancer cells. © 1999 Cancer Research Campaign

Keywords: mammary cancer, angiogenesis, VEGF, VEGF receptors, autocrine regulation

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Dellas A., Schultheiss E., Oberholzer M., Torhorst J., Gudat F. Analysis of proliferative activity using Ki-67 on cervical precancerous lesions and the relationship to p53 expression. Anticancer Res. 1996 Nov-Dec;16(6B):3403–3407. [PubMed] [Google Scholar]
  2. Dvorak H. F., Brown L. F., Detmar M., Dvorak A. M. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol. 1995 May;146(5):1029–1039. [PMC free article] [PubMed] [Google Scholar]
  3. Enomoto T., Okamoto T., Sato J. D. Vascular endothelial growth factor induces the disorganization of actin stress fibers accompanied by protein tyrosine phosphorylation and morphological change in Balb/C3T3 cells. Biochem Biophys Res Commun. 1994 Aug 15;202(3):1716–1723. doi: 10.1006/bbrc.1994.2133. [DOI] [PubMed] [Google Scholar]
  4. Ferrara N., Houck K. A., Jakeman L. B., Winer J., Leung D. W. The vascular endothelial growth factor family of polypeptides. J Cell Biochem. 1991 Nov;47(3):211–218. doi: 10.1002/jcb.240470305. [DOI] [PubMed] [Google Scholar]
  5. Ferrara N. The role of vascular endothelial growth factor in pathological angiogenesis. Breast Cancer Res Treat. 1995;36(2):127–137. doi: 10.1007/BF00666035. [DOI] [PubMed] [Google Scholar]
  6. Fong G. H., Rossant J., Gertsenstein M., Breitman M. L. Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature. 1995 Jul 6;376(6535):66–70. doi: 10.1038/376066a0. [DOI] [PubMed] [Google Scholar]
  7. Fox S. B., Harris A. L. Markers of tumor angiogenesis: clinical applications in prognosis and anti-angiogenic therapy. Invest New Drugs. 1997;15(1):15–28. doi: 10.1023/a:1005714527315. [DOI] [PubMed] [Google Scholar]
  8. Gasparini G., Toi M., Gion M., Verderio P., Dittadi R., Hanatani M., Matsubara I., Vinante O., Bonoldi E., Boracchi P. Prognostic significance of vascular endothelial growth factor protein in node-negative breast carcinoma. J Natl Cancer Inst. 1997 Jan 15;89(2):139–147. doi: 10.1093/jnci/89.2.139. [DOI] [PubMed] [Google Scholar]
  9. Gerdes J., Lelle R. J., Pickartz H., Heidenreich W., Schwarting R., Kurtsiefer L., Stauch G., Stein H. Growth fractions in breast cancers determined in situ with monoclonal antibody Ki-67. J Clin Pathol. 1986 Sep;39(9):977–980. doi: 10.1136/jcp.39.9.977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gitay-Goren H., Halaban R., Neufeld G. Human melanoma cells but not normal melanocytes express vascular endothelial growth factor receptors. Biochem Biophys Res Commun. 1993 Feb 15;190(3):702–708. doi: 10.1006/bbrc.1993.1106. [DOI] [PubMed] [Google Scholar]
  11. Hanahan D., Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996 Aug 9;86(3):353–364. doi: 10.1016/s0092-8674(00)80108-7. [DOI] [PubMed] [Google Scholar]
  12. Hanahan D. Signaling vascular morphogenesis and maintenance. Science. 1997 Jul 4;277(5322):48–50. doi: 10.1126/science.277.5322.48. [DOI] [PubMed] [Google Scholar]
  13. Hendrix M. J., Muschel R. J., Padarathsingh M. Recent advances in breast cancer research: from genes to management. Am J Pathol. 1997 Sep;151(3):883–888. [PMC free article] [PubMed] [Google Scholar]
  14. Martin L., Green B., Renshaw C., Lowe D., Rudland P., Leinster S. J., Winstanley J. Examining the technique of angiogenesis assessment in invasive breast cancer. Br J Cancer. 1997;76(8):1046–1054. doi: 10.1038/bjc.1997.506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Masood R., Cai J., Zheng T., Smith D. L., Naidu Y., Gill P. S. Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi sarcoma. Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):979–984. doi: 10.1073/pnas.94.3.979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Millauer B., Wizigmann-Voos S., Schnürch H., Martinez R., Møller N. P., Risau W., Ullrich A. High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell. 1993 Mar 26;72(6):835–846. doi: 10.1016/0092-8674(93)90573-9. [DOI] [PubMed] [Google Scholar]
  17. Mitola S., Sozzani S., Luini W., Primo L., Borsatti A., Weich H., Bussolino F. Tat-human immunodeficiency virus-1 induces human monocyte chemotaxis by activation of vascular endothelial growth factor receptor-1. Blood. 1997 Aug 15;90(4):1365–1372. [PubMed] [Google Scholar]
  18. Parker S. L., Tong T., Bolden S., Wingo P. A. Cancer statistics, 1997. CA Cancer J Clin. 1997 Jan-Feb;47(1):5–27. doi: 10.3322/canjclin.47.1.5. [DOI] [PubMed] [Google Scholar]
  19. Plate K. H., Breier G., Millauer B., Ullrich A., Risau W. Up-regulation of vascular endothelial growth factor and its cognate receptors in a rat glioma model of tumor angiogenesis. Cancer Res. 1993 Dec 1;53(23):5822–5827. [PubMed] [Google Scholar]
  20. Plate K. H., Breier G., Weich H. A., Mennel H. D., Risau W. Vascular endothelial growth factor and glioma angiogenesis: coordinate induction of VEGF receptors, distribution of VEGF protein and possible in vivo regulatory mechanisms. Int J Cancer. 1994 Nov 15;59(4):520–529. doi: 10.1002/ijc.2910590415. [DOI] [PubMed] [Google Scholar]
  21. Relf M., LeJeune S., Scott P. A., Fox S., Smith K., Leek R., Moghaddam A., Whitehouse R., Bicknell R., Harris A. L. Expression of the angiogenic factors vascular endothelial cell growth factor, acidic and basic fibroblast growth factor, tumor growth factor beta-1, platelet-derived endothelial cell growth factor, placenta growth factor, and pleiotrophin in human primary breast cancer and its relation to angiogenesis. Cancer Res. 1997 Mar 1;57(5):963–969. [PubMed] [Google Scholar]
  22. Rose D. S., Maddox P. H., Brown D. C. Which proliferation markers for routine immunohistology? A comparison of five antibodies. J Clin Pathol. 1994 Nov;47(11):1010–1014. doi: 10.1136/jcp.47.11.1010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ross W., Hall P. A. Ki67: from antibody to molecule to understanding? Clin Mol Pathol. 1995 Jun;48(3):M113–M117. doi: 10.1136/mp.48.3.m113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Russo J., Russo I. H., Rogers A. E., van Zwieten M. J., Gusterson B. Pathology of tumours in laboratory animals. Tumours of the rat. Tumours of the mammary gland. IARC Sci Publ. 1990;(99):47–78. [PubMed] [Google Scholar]
  25. Seetharam L., Gotoh N., Maru Y., Neufeld G., Yamaguchi S., Shibuya M. A unique signal transduction from FLT tyrosine kinase, a receptor for vascular endothelial growth factor VEGF. Oncogene. 1995 Jan 5;10(1):135–147. [PubMed] [Google Scholar]
  26. Senger D. R., Galli S. J., Dvorak A. M., Perruzzi C. A., Harvey V. S., Dvorak H. F. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science. 1983 Feb 25;219(4587):983–985. doi: 10.1126/science.6823562. [DOI] [PubMed] [Google Scholar]
  27. Senger D. R., Van de Water L., Brown L. F., Nagy J. A., Yeo K. T., Yeo T. K., Berse B., Jackman R. W., Dvorak A. M., Dvorak H. F. Vascular permeability factor (VPF, VEGF) in tumor biology. Cancer Metastasis Rev. 1993 Sep;12(3-4):303–324. doi: 10.1007/BF00665960. [DOI] [PubMed] [Google Scholar]
  28. Shibuya M. Role of VEGF-flt receptor system in normal and tumor angiogenesis. Adv Cancer Res. 1995;67:281–316. doi: 10.1016/s0065-230x(08)60716-2. [DOI] [PubMed] [Google Scholar]
  29. Shim J. W., Koh Y. C., Ahn H. K., Park Y. E., Hwang D. Y., Chi J. G. Expression of bFGF and VEGF in brain astrocytoma. J Korean Med Sci. 1996 Apr;11(2):149–157. doi: 10.3346/jkms.1996.11.2.149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Takahashi T., Shibuya M. The 230 kDa mature form of KDR/Flk-1 (VEGF receptor-2) activates the PLC-gamma pathway and partially induces mitotic signals in NIH3T3 fibroblasts. Oncogene. 1997 May 1;14(17):2079–2089. doi: 10.1038/sj.onc.1201047. [DOI] [PubMed] [Google Scholar]
  31. Takahashi Y., Kitadai Y., Bucana C. D., Cleary K. R., Ellis L. M. Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer. Cancer Res. 1995 Sep 15;55(18):3964–3968. [PubMed] [Google Scholar]
  32. Toi M., Hoshina S., Takayanagi T., Tominaga T. Association of vascular endothelial growth factor expression with tumor angiogenesis and with early relapse in primary breast cancer. Jpn J Cancer Res. 1994 Oct;85(10):1045–1049. doi: 10.1111/j.1349-7006.1994.tb02904.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Waltenberger J., Claesson-Welsh L., Siegbahn A., Shibuya M., Heldin C. H. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J Biol Chem. 1994 Oct 28;269(43):26988–26995. [PubMed] [Google Scholar]
  34. Wintzer H. O., Zipfel I., Schulte-Mönting J., Hellerich U., von Kleist S. Ki-67 immunostaining in human breast tumors and its relationship to prognosis. Cancer. 1991 Jan 15;67(2):421–428. doi: 10.1002/1097-0142(19910115)67:2<421::aid-cncr2820670217>3.0.co;2-q. [DOI] [PubMed] [Google Scholar]
  35. Xie B., Tsao S. W., Wong Y. C. Induction of high incidence of mammary tumour in female Noble rats with a combination of 17beta-oestradiol and testosterone. Carcinogenesis. 1999 Jun;20(6):1069–1078. doi: 10.1093/carcin/20.6.1069. [DOI] [PubMed] [Google Scholar]
  36. Yamane A., Seetharam L., Yamaguchi S., Gotoh N., Takahashi T., Neufeld G., Shibuya M. A new communication system between hepatocytes and sinusoidal endothelial cells in liver through vascular endothelial growth factor and Flt tyrosine kinase receptor family (Flt-1 and KDR/Flk-1). Oncogene. 1994 Sep;9(9):2683–2690. [PubMed] [Google Scholar]
  37. Yang X., Cepko C. L. Flk-1, a receptor for vascular endothelial growth factor (VEGF), is expressed by retinal progenitor cells. J Neurosci. 1996 Oct 1;16(19):6089–6099. doi: 10.1523/JNEUROSCI.16-19-06089.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from British Journal of Cancer are provided here courtesy of Cancer Research UK

RESOURCES