Suppression of rat breast cancer metastasis and reduction of primary tumour growth by the small synthetic urokinase inhibitor WX-UK1

Buddy Setyono-Han; Jörg Stürzebecher; Wolfgang A. Schmalix; Bernd Muehlenweg; Anieta M. Sieuwerts; Mieke Timmermans; Viktor Magdolen; Manfred Schmitt; Jan G. M. Klijn; John A. Foekens

doi:10.1160/TH04-11-0712

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2005; 93(04): 779-786
DOI: 10.1160/TH04-11-0712

Cellular Proteolysis and Oncology

Schattauer GmbH

Suppression of rat breast cancer metastasis and reduction of primary tumour growth by the small synthetic urokinase inhibitor WX-UK1

Buddy Setyono-Han

¹Department of Medical Oncology, Erasmus Medical Center Rotterdam, The Netherlands

,

Jörg Stürzebecher

²Klinikum der Universität Jena, Zentrum für Vaskuläre Biologie und Medizin, Erfurt, Germany

,

Wolfgang A. Schmalix

³Wilex AG, Munich, Germany

,

Bernd Muehlenweg

³Wilex AG, Munich, Germany

,

Anieta M. Sieuwerts

¹Department of Medical Oncology, Erasmus Medical Center Rotterdam, The Netherlands

,

Mieke Timmermans

¹Department of Medical Oncology, Erasmus Medical Center Rotterdam, The Netherlands

,

Viktor Magdolen

⁴Klinische Forschergruppe, Frauenklinik der Technische Universität München, Klinikum rechts der Isar, Munich, Germany

,

Manfred Schmitt

⁴Klinische Forschergruppe, Frauenklinik der Technische Universität München, Klinikum rechts der Isar, Munich, Germany

,

Jan G. M. Klijn

¹Department of Medical Oncology, Erasmus Medical Center Rotterdam, The Netherlands

,

John A. Foekens

¹Department of Medical Oncology, Erasmus Medical Center Rotterdam, The Netherlands

› Author Affiliations

Abstract

Summary

The serine protease uPA (urokinase-type plasminogen activator) and its receptor uPAR (CD87) are often elevated in malignant tumours, hence, inhibition of this tumour-associated plasminogen activation system provides an attractive target for therapeutic strategies. WX-UK1, a derivative of 3-aminophenylalanine in the L-conformation with inhibitory antiproteolytic properties, was tested for its specificity spectrum using specific chromogenic paranitroanilide peptide substrates. The corresponding D-enantiomer of WX-UK1 was used as a control. The anti-tumour and anti-metastatic (number of lung foci and weight of the axillary lymph nodes) properties were studied by subcutaneous administration of WX-UK1 to Brown Norwegian (BN) rats carrying orthotopically transplanted BN472 rat breast tumours. WX-UK1 selectively inhibited tumour-related proteases from rats and humans such as uPA, plasmin, or thrombin in the sub or low micromolar range. The activity was stereoselective as the D-enantiomer of WX-UK1 inhibited uPA and plas-min at approximately 70-fold higher K_i values than the active L-form. Chronical administration of the L-enantiomer of WXUK1 impaired primary tumour growth and metastasis of BN472 rat breast cancer in a dose-dependent manner. The minimum inhibitory dosage with maximal effect was between 0.15 and 0.3 mg/kg/day. The inactive D-enatiomer of WX-UK1 was not active in this respect. Daily treatment with WX-UK1 for up to 35 days was well tolerated as judged by the unchanged body and organ weight development. In conclusion, our results provide evidence that WX-UK1 as a single agent inhibits breast tumour growth and metastasis in vivo, and thus is a promising candidate drug to treat human cancer.

Keywords

Urokinase-type plasminogen activator (uPA) - synthetic uPA-inhibitor - rat mammary carcinoma - anti-metastatic treatment - serine protease

Full Text

References

References
1 Andreasen PA, Egelund R, Petersen HH. The plasminogen activation system in tumour growth, invasion and metastasis. Cell Mol Life Sci 2000; 57: 25-40.
2 Schmitt M, Wilhelm OG. et al. The urokinase plasminogen activator system as a novel target for tumour therapy. Fibrinol Proteol 2000; 14: 114-32.
3 Chapman HA, Wei Y. Protease crosstalk with integrins: the urokinase receptor paradigm. Thromb Haemost 2001; 86: 124-9.
4 Preissner KT, Kanse SM, May AE. Urokinase receptor: a molecular organizer in cellular communication. Curr Opin Cell Biol 2000; 12: 621-8.
5 Duffy MJ, Maguire TM. et al. Urokinase plasminogen activator: a prognostic marker in multiple types of cancer. J Surg Oncol 1999; 71: 130-5.
6 Sidenus N, Blasi F. The urokinase plasminogen activator system in cancer: recent advances and implication for prognosis and therapy. Cancer Metast Rev 2002; 22: 201-22.
7 Jänicke F, Prechtl A, Thomssen C. et al. for the German Chemo N o Study Group. Randomized adjuvant therapy trial in high-risk lymph node-negative breast cancer patients identified by urokinase-type plasminogen activator and plasminogen activator inhibitor type 1. J Natl Cancer Inst 2001; 93: 913-20.
8 Look MP, Van Putten WLJ. et al. Pooled analysis of prognostic impact of uPA and PAI-1 in 8,377 breast cancer patients. J Natl Cancer Inst 2002; 94: 116-28.
9 Foekens JA, Look MP, Peters HA. et al. Urokinasetype plasminogen activator and its inhibitor PAI-1: predictors of poor response to tamoxifen therapy in recurrent breast cancer. J Natl Cancer Inst 1995; 87: 751-6.
10 Harbeck N, Kates RE. et al. Enhanced benefit from adjuvant chemotherapy in breast cancer patients classified high-risk according to urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 (n =3424). Cancer Res 2000; 62: 4617-22.
11 Harbeck N, Kates RE, Schmitt M. Clinical relevance of invasion factors urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 for individualized therapy decisions in primary breast cancer is greatest when used in combination. J Clin Oncol 2002; 20: 1000-7.
12 Meijer-van Gelder ME. et al. Urokinase-type plasminogen activator system in breast cancer: association with tamoxifen therapy. Cancer Res 2004; 64: 4563-8.
13 Carmeliet PF. Physiological consequences of loss of plasminogen activator gene function in mice. Nature 1994; 368: 419-24.
14 Frandsen TL, Holst-Hansen C, Nielsen BS. et al. Direct evidence of the importance of stromal urokinase plasminogen activator (uPA) in the growth of an experimental human breast cancer using a combined uPA gene-disrupted and immunodeficient xenograft model. Cancer Res 2001; 61: 532-7.
15 Towle MJ, Lee A. et al. Inhibition of urokinase by 4-substituted benzo[b]thiopene-2-carboxamidines: an important new class of selective synthetic urokinase inhibitor. Cancer Res 1993; 53: 2553-9.
16 Schmitt M, Harbeck N, Thomssen C. et al. Clinical impact of the plasminogen activator system in tumour invasion and metastasis: prognostic relevance and target for therapy. Thromb Haemost 1997; 78: 285-96.
17 Bürgle M, Koppitz MM, Riemer C. et al. Inhibition of the interaction of urokinase-type plasminogen activator (uPA) with its receptor (uPAR) by synthetic peptides. Biol Chem 1997; 378: 231-7.
18 Stürzebecher J, Vieweg H, Steinmetzer T. et al. 3-Aminophenylalanine-based inhibitors of urokinase. Bioorg Med Chem Lett 1999; 9: 3147-52.
19 Muehlenweg B, Assfalg-Machleidt I, Parrado SG. et al. A novel type of bifunctional inhibitor directed against proteolytic activity and receptor/ligand interaction. J Biol Chem 2000; 276: 33562-6.
20 Rosenberg S. Modulators of the urokinase-type plasminogen activator system for cancer. Exp Opin Ther Patents 2000; 10: 1834-52.
21 Muehlenweg B, Sperl S, Magdolen V. et al. Interference with the urokinase plasminogen activation system: a promising therapy concept for solid tumours. Exp Opin Biol Ther 2001; 1: 683-91.
22 Sperl S, Mueller MM, Wilhelm OG. et al. The uPA/ uPAR system as a target for tumour therapy. Drug News Perspect 2001; 14: 401-11.
23 Rabbani SA, Gladu J. Urokinase receptor antibody can reduce tumour volume and detect the presence of occult tumour metastases in vivo. Cancer Res 2002; 62: 2390-7.
24 Ploug M, Gårdsvoll H, Jorgensen TJ. et al. Structural analysis of the interaction between urokinase-type plasminogen activator and its receptor: a potential target for anti-invasive cancer therapy. Biochem Soc Trans 2002; 30: 177-83.
25 Ertongur S, Lang S. et al. Inhibition of the invasion capacity of carcinoma cells by WX-UK1, a novel synthetic inhibitor of the urokinase-type plasminogen activator system. Int J Cancer 2004; 110: 815-24.
26 Kort WJ, Zondervan PE, Hulsman LOM. et al. Incidence of spontaneous tumours in a group of retired breeder female Brown Norway rats. J Natl Cancer Inst 1984; 72: 709-13.
27 Albrecht S, Magdolen V, Herzog U. et al. Soluble tissue factor interferes with angiostatin-mediated inhibition of endothelial cell proliferation by lysine-specific interaction with plasminogen kringle domains. Thromb Haemost 2002; 88: 1054-9.
28 Martens JWM, Sieuwerts AM, Bolt-de Vries J. et al. Ageing of stromal-derived human breast fibroblasts might contribute to breast cancer progression. Thromb Haemost 2003; 89: 393-404.
29 Stürzebecher J, Prasa D, Hauptmann J. et al. Synthesis and structure-activity relationships of potent thrombin inhibitors: piperazides of 3-amidinophenylalanine. J Med Chem 1997; 40: 3091-9.
30 Royston P. Approximating the Shapiro-Wilk W-test for non-normality. Statistics and Computing 1992; 2: 117-9.
31 Slamon DJ, Leyland-Jones B, Shak S. et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001; 344: 783-92.
32 Tripathy D, Slamon DJ. et al. Safety of treatment of metastatic breast cancer with trastuzumab beyond disease progression. J Clin Oncol 2004; 22: 1063-70.
33 Druker BJ, Talpaz M, Resta DJ. et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001; 344: 1031-7.
34 Demetri GD, Von Meheren M. et al. Efficay and safety of imatinib mesylate in advanced gastrointestinal stromal tumours. N Engl J Med 2002; 347: 472-80.
35 Yang JC, Haworth L, Sherry RM. et al. A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med 2003; 349: 427-34.
36 Willett CG, Boucher Y, Di Tomaso E. et al. Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nat Med 2004; 10: 145-7.
37 Harris M. Monoclonal antibodies as therapeutic agents for cancer. Lancet Oncol 2004; 5: 292-302.