Skip to main content

Advertisement

SpringerLink
Log in

Overexpression of the urokinase receptor mRNA splice variant uPAR-del4/5 affects tumor-associated processes of breast cancer cells in vitro and in vivo

  • Preclinical study
  • Published:
Breast Cancer Research and Treatment Aims and scope Submit manuscript

Abstract

uPAR, the three-domain membrane receptor of the serine protease urokinase, plays a crucial role in tumor growth and metastasis. Several mRNA splice variants of this receptor have been reported. One of these, uPAR-del4/5, lacking exons 4 and 5, and thus encoding a uPAR form lacking domain DII, is specifically overexpressed in breast cancer and represents a statistically independent prognostic factor for distant metastasis-free survival in breast cancer patients. The aim of the present study was to examine the molecular and cellular properties of the encoded uPAR-del4/5 protein. To investigate the impact of the uPAR-del4/5 overexpression on in vitro and in vivo aspects of tumor progression (e.g., proliferation, adhesion, invasion, metastatic seeding, and/or metastatic growth), we combined the analysis of transfected cancer cell lines with a murine xenograft tumor model. Increased expression of uPAR-del4/5 in human cancer cells led to reduced adhesion to several extracellular matrix proteins and decreased invasion through MatrigelTM, while cell proliferation was not affected in vitro. Moreover, invasion of uPAR-del4/5 overexpressing cells was not altered by addition of urokinase, while that of uPAR-wild-type overexpressing cells was drastically increased. Accordingly, we observed that, in contrast to uPAR-wild-type, uPAR-del4/5 does not interact with urokinase. On the other hand, when overexpressed in human breast cancer cells, uPAR-del4/5 distinctly impaired metastatic dissemination and growth in vivo. We demonstrate that the uPAR-del4/5 mRNA splice variant mediates tumor-relevant biological processes in vitro and in vivo. Our results thus illustrate how tumor-specific alternative splicing can distinctly impact the biology of the tumor.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Blasi F, Sidenius N (2010) The urokinase receptor: focused cell surface proteolysis, cell adhesion and signaling. FEBS Lett 584:1923–1930

    Article  PubMed  CAS  Google Scholar 

  2. Blasi F, Carmeliet P (2002) uPAR: a versatile signalling orchestrator. Nat Rev Mol Cell Biol 3:932–943

    Article  PubMed  CAS  Google Scholar 

  3. Reuning U, Magdolen V, Wilhelm O, Fischer K, Lutz V, Graeff H, Schmitt M (1998) Multifunctional potential of the plasminogen activation system in tumor invasion and metastasis. Int J Oncol 13:893–906

    PubMed  CAS  Google Scholar 

  4. Harbeck N, Kates RE, Gauger K, Willems A, Kiechle M, Magdolen V, Schmitt M (2004) Urokinase-type plasminogen activator (uPA) and its inhibitor PAI-I: novel tumor-derived factors with a high prognostic and predictive impact in breast cancer. Thromb Haemost 91:450–456

    PubMed  CAS  Google Scholar 

  5. Høyer-Hansen G, Lund IK (2007) Urokinase receptor variants in tissue and body fluids. Adv Clin Chem 44:65–102

    Article  PubMed  Google Scholar 

  6. Harris L, Fritsche H, Mennel R, Norton L, Ravdin P, Taube S, Somerfield MR, Hayes DF, Bast RC (2007) American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer. J Clin Oncol 25:5287–5312

    Article  PubMed  CAS  Google Scholar 

  7. Ploug M (2003) Structure-function relationships in the interaction between the urokinase-type plasminogen activator and its receptor. Curr Pharm Des 9:1499–1528

    Article  PubMed  CAS  Google Scholar 

  8. Smith HW, Marshall CJ (2010) Regulation of cell signalling by uPAR. Nat Rev Mol Cell Biol 11:23–36

    Article  PubMed  CAS  Google Scholar 

  9. Reuning U, Magdolen V, Hapke S, Schmitt M (2003) Molecular and functional interdependence of the urokinase-type plasminogen activator system with integrins. Biol Chem 384:1119–1131

    Google Scholar 

  10. Llinas P, Le Du MH, Gårdsvoll H, Danø K, Ploug M, Gilquin B, Stura EA, Ménez A (2005) Crystal structure of the human urokinase plasminogen activator receptor bound to an antagonist peptide. EMBO J 24:1655–1663

    Article  PubMed  CAS  Google Scholar 

  11. Huai Q, Mazar AP, Kuo A, Parry GC, Shaw DE, Callahan J, Li Y, Yuan C, Bian C, Chen L, Furie B, Furie BC, Cines DB, Huang M (2006) Structure of human urokinase plasminogen activator in complex with its receptor. Science 311:656–659

    Article  PubMed  CAS  Google Scholar 

  12. Gårdsvoll H, Ploug M (2007) Mapping of the vitronectin-binding site on the urokinase receptor: involvement of a coherent receptor interface consisting of residues from both domain I and the flanking interdomain linker region. J Biol Chem 282:13561–13572

    Article  PubMed  Google Scholar 

  13. Huai Q, Zhou A, Lin L, Mazar AP, Parry GC, Callahan J, Shaw DE, Furie B, Furie BC, Huang M (2008) Crystal structures of two human vitronectin, urokinase and urokinase receptor complexes. Nat Struct Mol Biol 15:422–423

    Article  PubMed  CAS  Google Scholar 

  14. Stewart CE, Sayers I (2009) Characterisation of urokinase plasminogen activator receptor variants in human airway and peripheral cells. BMC Mol Biol 10:75

    Article  PubMed  Google Scholar 

  15. Luther T, Kotzsch M, Meye A, Langerholc T, Fussel S, Olbricht N, Albrecht S, Ockert D, Muehlenweg B, Friedrich K, Grosser M, Schmitt M, Baretton G, Magdolen V (2003) Identification of a novel urokinase receptor splice variant and its prognostic relevance in breast cancer. Thromb Haemost 89:705–717

    PubMed  CAS  Google Scholar 

  16. Pyke C, Eriksen J, Solberg H, Nielsen BS, Kristensen P, Lund LR, Danø K (1993) An alternatively spliced variant of mRNA for the human receptor for urokinase plasminogen activator. FEBS Lett 326:69–74

    Article  PubMed  CAS  Google Scholar 

  17. Kotzsch M, Farthmann J, Meye A, Fuessel S, Baretton G, Tjan-Heijnen VC, Schmitt M, Luther T, Sweep FC, Magdolen V, Span PN (2005) Prognostic relevance of uPAR-del4/5 and TIMP-3 mRNA expression levels in breast cancer. Eur J Cancer 41:2760–2768

    Article  PubMed  CAS  Google Scholar 

  18. Kotzsch M, Sieuwerts AM, Grosser M, Meye A, Fuessel S, Meijer-van Gelder ME, Smid M, Schmitt M, Baretton G, Luther T, Magdolen V, Foekens JA (2008) Urokinase receptor splice variant uPAR-del4/5-associated gene expression in breast cancer: identification of rab31 as an independent prognostic factor. Breast Cancer Res Treat 111:229–240

    Article  PubMed  CAS  Google Scholar 

  19. Möbus V, Gerharz CD, Press U, Moll R, Beck T, Mellin W, Pollow K, Knapstein PG, Kreienberg R (1992) Morphological, immunohistochemical and biochemical characterization of 6 newly established human ovarian carcinoma cell lines. Int J Cancer 52:76–84

    Article  PubMed  Google Scholar 

  20. Luther T, Magdolen V, Albrecht S, Kasper M, Riemer C, Kessler H, Graeff H, Müller M, Schmitt M (1997) Epitope-mapped monoclonal antibodies as tools for functional and morphological analyses of the human urokinase receptor in tumor tissue. Am J Pathol 150:1231–1244

    PubMed  CAS  Google Scholar 

  21. Goretzki L, Bognacki J, Koppitz M, Rettenberger P, Magdolen V, Creutzburg S, Hammelburger J, Weidle UH, Wilhelm O, Kessler H, Graeff H, Schmitt M (1997) Quantitative assessment of interaction of urokinase-type plasminogen activator and its receptor (CD87) by use of a solid-phase uPA-ligand binding assay. Fibrinol Proteol 11:11–19

    Article  CAS  Google Scholar 

  22. Krüger A, Soeltl R, Sopov I, Kopitz C, Arlt M, Magdolen V, Harbeck N, Gänsbacher B, Schmitt M (2001) Hydroxamate-type matrix metalloproteinase inhibitor batimastat promotes liver metastasis. Cancer Res 61:1272–1275

    PubMed  Google Scholar 

  23. Kopitz C, Anton M, Gänsbacher B, Krüger A (2005) Reduction of experimental human fibrosarcoma lung metastasis in mice by adenovirus-mediated cystatin C overexpression in the host. Cancer Res 65:8608–8612

    Article  PubMed  CAS  Google Scholar 

  24. Krüger A, Schirrmacher V, Khokha R (1998) The bacterial lacZ gene: an important tool for metastasis research and evaluation of new cancer therapies. Cancer Metastasis Rev 17:285–294

    Article  PubMed  Google Scholar 

  25. Beaufort N, Leduc D, Eguchi H, Mengele K, Hellmann D, Masegi T, Kamimura T, Yasuoka S, Fend F, Chignard M, Pidard D (2007) The human airway trypsin-like protease modulates the urokinase receptor (uPAR, CD87) structure and functions. Am J Physiol Lung Cell Mol Physiol 292:1263–1272

    Article  Google Scholar 

  26. Wei Y, Lukashev M, Simon DI, Bodary SC, Rosenberg S, Doyle MV, Chapman HA (1996) Regulation of integrin function by the urokinase receptor. Science 273:1551–1555

    Article  PubMed  CAS  Google Scholar 

  27. Xue W, Mizukami I, Todd RF, Petty HR (1997) Urokinase-type plasminogen activator receptors associate with beta1 and beta3 integrins of fibrosarcoma cells: dependence on extracellular matrix components. Cancer Res 57:1682–1689

    PubMed  CAS  Google Scholar 

  28. Krüger A, Soeltl R, Lutz V, Wilhelm OG, Magdolen V, Rojo EE, Hantzopoulos PA, Graeff H, Gänsbacher B, Schmitt M (2000) Reduction of breast carcinoma tumor growth and lung colonization by overexpression of the soluble urokinase-type plasminogen activator receptor (CD87). Cancer Gene Ther 7:292–299

    Article  PubMed  Google Scholar 

  29. Setyono-Han B, Sturzebecher J, Schmalix WA, Muehlenweg B, Sieuwerts AM, Timmermans M, Magdolen V, Schmitt M, Klijn JG, Foekens JA (2005) Suppression of rat breast cancer metastasis and reduction of primary tumour growth by the small synthetic urokinase inhibitor WX-UK1. Thromb Haemost 93:779–786

    PubMed  CAS  Google Scholar 

  30. Ward AJ, Cooper TA (2010) The pathobiology of splicing. J Pathol 220:152–163

    PubMed  CAS  Google Scholar 

  31. Fackenthal JD, Godley LA (2008) Aberrant RNA splicing and its functional consequences in cancer cells. Dis Model Mech 1:37–42

    Article  PubMed  CAS  Google Scholar 

  32. Bjarnadóttir TK, Geirardsdóttir K, Ingemansson M, Mirza MA, Fredriksson R, Schiöth HB (2007) Identification of novel splice variants of Adhesion G protein-coupled receptors. Gene 387:38–48

    Article  PubMed  Google Scholar 

  33. Alfano D, Franco P, Vocca I, Gambi N, Pisa V, Mancini A, Caputi M, Carriero MV, Iaccarino I, Stoppelli MP (2005) The urokinase plasminogen activator and its receptor: role in cell growth and apoptosis. Thromb Haemost 93:205–211

    PubMed  CAS  Google Scholar 

  34. Durand MK, Bodker JS, Christensen A, Dupont DM, Hansen M, Jensen JK, Kjelgaard S, Mathiasen L, Pedersen KE, Skeldal S, Wind T, Andreasen PA (2004) Plasminogen activator inhibitor-I and tumour growth, invasion, and metastasis. Thromb Haemost 91:438–449

    PubMed  CAS  Google Scholar 

  35. Steeg PS (2003) Metastasis suppressors alter the signal transduction of cancer cells. Nat Rev Cancer 3:55–63

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This study was supported in part by grants provided by the Deutsche Krebshilfe e.V., Germany (Grant No. 106 162) to MK and VM, and by the Framework Programme 7 project HEALTH-2007-201279, Microenvimet to AK. We are grateful to Sabine Creutzburg, Katja Richter, and Antje Zobjack for excellent technical assistance.

Conflicts of interest statement

None.

Author information

Authors and Affiliations

  1. Clinical Research Unit, Department of Obstetrics and Gynecology, Technical University Munich, Ismaninger Str. 22, 81675, Munich, Germany

    Sumito Sato, Bettina Grismayer, Nathalie Beaufort, Ute Reuning, Manfred Schmitt & Viktor Magdolen

  2. Department of Surgery, Yokohama Asahi Central and General Hospital, 4-20-1 Wakabadai Asahi-ku, Yokohama, Kanagawa, 241-0801, Japan

    Sumito Sato

  3. Institute of Experimental Oncology and Therapy Research, Technical University Munich, Ismaninger Str. 22, 81675, Munich, Germany

    Charlotte Kopitz & Achim Krüger

  4. Institute of Pathology, Dresden University of Technology, Fetscherstr. 74, 01307, Dresden, Germany

    Thomas Luther & Matthias Kotzsch

Authors
  1. Sumito Sato
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Charlotte Kopitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bettina Grismayer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nathalie Beaufort
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ute Reuning
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Manfred Schmitt
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Thomas Luther
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Matthias Kotzsch
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Achim Krüger
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Viktor Magdolen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Viktor Magdolen.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sato, S., Kopitz, C., Grismayer, B. et al. Overexpression of the urokinase receptor mRNA splice variant uPAR-del4/5 affects tumor-associated processes of breast cancer cells in vitro and in vivo. Breast Cancer Res Treat 127, 649–657 (2011). https://doi.org/10.1007/s10549-010-1042-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10549-010-1042-5

Keywords

Search

Navigation