2
Department of Gynaecology and Obstetrics, San Raffaele Scientific Institute and Hospital, Milan, Italy
,
Augusto Ferrari
2
Department of Gynaecology and Obstetrics, San Raffaele Scientific Institute and Hospital, Milan, Italy
,
Andrea Lissoni
4
Department of Gynaecology and Obstetrics, S. Gerardo Hospital, Monza, Italy
,
Raffaella Giavazzi
3
Department of Oncology, Mario Negri Institute for Pharmacological Research, Bergamo, Italy
,
Francesco Blasi
1
Department of Molecular Biology and Functional Genomics and FIRC Institute of Molecular Oncology
,
Nicolai Sidenius
1
Department of Molecular Biology and Functional Genomics and FIRC Institute of Molecular Oncology
› Author AffiliationsFinancial support: This work was carried out under the auspices of the Centro di Eccellenza di Fisiopatologia del Differenziamento Cellulare (MIUR).The work was supported by the Italian Association for Cancer Research (AIRC), FIRB (grant numbers RBNE 01 JFFA001 and RBAU 01CHJJ001) and by the European Union (Contract No. QLG1-CT2000-01131). In addition the generous contribution of the Nerina and Mario Mattioli Foundation is gratefully acknowledged.
The urokinase-type plasminogen activator receptor (uPAR) is involved in cell migration and tissue remodelling, as a receptor for pro-uPA, as a cell adhesion component, and in a soluble form as a chemoattractant. We have analyzed the presence and the molecular forms of uPAR and uPAR-fragments in urine of ovarian cancer patients in comparison with tumour tissue, ascites, and serum. Carcinoma tissue contained high levels of uPAR, but more abundantly the D2D3-fragment. Ascitic fluid contained similar ratio’s of suPAR fragments as corresponding tumour tissue, but serum only contained intact suPAR. Interestingly, urine contained predominantly the uPAR-fragments D1 and D2D3, and the pattern of these fragments was different in cancer patients as compared to healthy individuals. To confirm the hypothesis that circulating and urinary suPAR and suPAR-fragments originate from the tumour tissue, the presence of human suPAR (fragments) was analyzed in mice xenografted with human tumours. Indeed, high levels of urinary D1 fragment were found in mice carrying a tumour displaying cleaved uPAR on the cell surface, but little or no D1 was found in the urine from mice carrying a tumour with full-length uPAR. Mouse serum contained only intact suPAR. Our data demonstrate that the enhanced levels of suPAR fragments in the urine of cancer patients is likely to originate from uPAR expressed in the tumour tissue. Considering the biological activities that uPAR fragments display, the level and typing of uPAR fragments in urine might therefore be clinically more relevant than the plain serum uPAR content.
Keywords
uPAR -
cleavage -
fragments -
cancer -
urine
# Present address: Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands
* These authors contributed equally to the study.
References
1
Sidenius N,
Blasi F.
The urokinase plasminogen activator system in cancer: Recent advances and implications for prognosis and therapy. Cancer Metast Rev 2003; 22 (2-3) 205-22.
3
Sier CFM,
Stephens RW,
Bizik J.
et al. The level of urokinase-type plasminogen activator receptor is increased in serum of ovarian cancer patients. Cancer Res 1998; 58 (09) 1843-9.
4
Crowley CW,
Cohen RL,
Lucas BK.
et al. Prevention of metastasis by inhibition of the urokinase receptor. Proc Natl Acad Sci USA 1993; 90 (11) 5021-5.
5
Kobayashi H,
Gotoh J,
Fujie M.
et al. Inhibition of metastasis of Lewis lung carcinoma by a synthetic peptide within growth factor-like domain of urokinase in the experimental and spontaneous metastasis model. Int J Cancer 1994; 57 (05) 727-33.
6
Min HY,
Doyle LV,
Vitt CR.
et al. Urokinase receptor antagonists inhibit angiogenesis and primary tumor growth in syngeneic mice. Cancer Res 1996; 56 (10) 2428-33.
7
Pedersen N,
Schmitt M,
Rønne E.
et al. A ligand-free, soluble urokinase receptor is present in the ascitic fluid from patients with ovarian cancer. J Clin Invest 1993; 92 (05) 2160-7.
8
Riisbro R,
Stephens RW,
Brünner N.
et al. Soluble urokinase plasminogen activator receptor in preoperatively obtained plasma from patients with gynecological cancer or benign gynecological diseases. Gynecol Oncol 2001; 82 (03) 523-31.
9
Stephens RW,
Pedersen AN,
Nielsen HJ.
et al. ELISA determination of soluble urokinase receptor in blood from healthy donors and cancer patients. Clin Chem 1997; 43 (10) 1868-76.
10
Pappot H,
Høyer-Hansen G,
Rønne E.
et al. Elevated plasma levels of urokinase plasminogen activator receptor in non-small cell lung cancer patients. Eur J Cancer 1997; 33 (06) 867-72.
13
Sier CF,
Sidenius N,
Mariani A.
et al. Presence of urokinase-type plasminogen activator receptor in urine of cancer patients and its possible clinical relevance. Lab Invest 1999; 79 (06) 717-22.
14
Resnati M,
Guttinger M,
Valcamonica S.
et al. Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactic effect. EMBO J 1996; 15 (07) 1572-82.
16
Resnati M,
Pallavicini I,
Wang JM.
et al. The fibrinolytic receptor for urokinase activates the G protein-coupled chemotactic receptor FPRL1/LXA4R. Proc Natl Acad Sci USA 2002; 99 (03) 1359-64.
17
Massazza G,
Lucchini V,
Tomasoni A.
et al. Malignant behavior and resistance to cisplatin of human ovarian carcinoma xenografts established from the same patient at different stages of the disease. Cancer Res 1991; 51 (23 Pt 1): 6358-62.
18
Brünner N,
Thompson EW,
Spang-Thomsen M.
et al. lacZ transduced human breast cancer xenografts as an in vivo model for the study of invasion and metastasis. Eur J Cancer 1992; 28A (12) 1989-95.
19
Holst-Hansen C,
Hamers MJ,
Johannessen BE.
et al. Soluble urokinase receptor released from human carcinoma cells: a plasma parameter for xenograft tumour studies. Br J Cancer 1999; 81 (02) 203-11.
20
Verheijen JH,
Mullaart E,
Chang GT.
et al. A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma. Thromb Haemost 1982; 48 (03) 266-9.
21
Sidenius N,
Sier CFM,
Blasi F.
Shedding and cleavage of the urokinase receptor (uPAR): identification and characterisation of uPAR fragments in vitro and in vivo. FEBS Lett 2000; 475 (01) 52-6.
22
Giavazzi R,
Garofalo A,
Ferri C.
et al. Batimastat, a synthetic inhibitor of matrix metalloproteinases, potentiates the antitumor activity of cisplatin in ovarian carcinoma xenografts. Clin Cancer Res 1998; 04 (04) 985-92.
24
Sidenius N,
Blasi F.
Domain 1 of the urokinase receptor (uPAR) is required for uPARmediated cell binding to vitronectin. FEBS Lett 2000; 470 (01) 40-46.
25
Koolwijk P,
Sidenius N,
Peters E.
et al. Proteolysis of the urokinase-type plasminogen activator receptor by metalloproteinase-12: implication for angiogenesis in fibrin matrices. Blood 2001; 97 (10) 3123-31.
27
Andreasen PA,
Kjøller L,
Christensen L.
et al. The urokinase-type plasminogen activator system in cancer metastasis: a review. Int J Cancer 1997; 72 (01) 1-22.
28
Casslèn B,
Bossmar T,
Lecander I.
et al. Plasminogen activators and plasminogen activator inhibitors in blood and tumour fluids of patients with ovarian cancer. Eur J Cancer 1994; 30A (09) 1302-9.
29
Pedersen H,
Brünner N,
Francis D.
et al. Prognostic impact of urokinase, urokinase receptor, and type 1 plasminogen activator inhibitor in squamous and large cell lung cancer tissue. Cancer Res 1994; 54 (17) 4671-5.
30
Kuhn W,
Pache L,
Schmalfeldt B.
et al. Urokinase (uPA) and PAI-1 predict survival in advanced ovarian cancer patients (FIGO III) after radical surgery and platinum-based chemotherapy. Gynecol Oncol 1994; 55 (3 Pt 1): 401-9.
31
Verspaget HW,
Sier CFM,
Ganesh S.
et al. Prognostic value of plasminogen activators and their inhibitors in colorectal cancer. Eur J Cancer 1995; 31A 7-8 1105-9.
32
Schmitt M,
Harbeck N,
Thomssen C.
et al. Clinical impact of the plasminogen activation system in tumor invasion and metastasis: prognostic relevance and target for therapy. Thromb Haemost 1997; 78 (01) 285-96.
33
Chambers SK,
Ivins CM,
Carcangiu ML.
Plasminogen activator inhibitor-1 is an independent poor prognostic factor for survival in advanced stage epithelial ovarian cancer patients. Int J Cancer 1998; 79 (05) 449-54.
34
Nielsen HJ,
Pappot H,
Christensen IJ.
et al. Association between plasma concentrations of plasminogen activator inhibitor-1 and survival in patients with colorectal cancer [see comments]. Br Med J 1998; 316 7134 829-30.
36
Young TN,
Rodriguez GC,
Moser TL.
et al. Coordinate expression of urinary-type plasminogen activator and its receptor accompanies malignant transformation of the ovarian surface epithelium. Am J Obstet Gynecol 1994; 170 (5 Pt 1): 1285-96.
37
Schmalfeldt B,
Kuhn W,
Reuning U.
et al. Primary tumor and metastasis in ovarian cancer differ in their content of urokinase-type plasminogen activator, its receptor, and inhibitors types 1 and 2. Cancer Res 1995; 55 (18) 3958-63.
38
Solberg H,
Rømer J,
Brünner N.
et al. A cleaved form of the receptor for urokinase-type plasminogen activator in invasive transplanted human and murine tumors. Int J Cancer 1994; 58 (06) 877-81.
39
Mustjoki S,
Sidenius N,
Sier CF.
et al. Soluble urokinase receptor levels correlate with number of circulating tumor cells in acute myeloid leukemia and decrease rapidly during chemotherapy. Cancer Res 2000; 60 (24) 7126-32.
40
Ploug M,
Ellis V,
Danø K.
Ligand interaction between urokinase-type plasminogen activator and its receptor probed with 8-anilino-1-naphthalenesulfonate. Evidence for a hydrophobic binding site exposed only on the intact receptor. Biochemistry 1994; 33 (30) 8991-7.
41
Høyer-Hansen G,
Behrendt N,
Ploug M.
et al. The intact urokinase receptor is required for efficient vitronectin binding: receptor cleavage prevents ligand interaction. FEBS Lett 1997; 420 (01) 79-85.
42
Wagner SN,
Atkinson MJ,
Wagner C.
et al. Sites of urokinase-type plasminogen activator expression and distribution of its receptor in the normal human kidney. Histochem Cell Biol 1996; 105 (01) 53-60.
43
Walker PD,
Kaushal GP,
Shah SV.
Meprin A, the major matrix degrading enzyme in renal tubules, produces a novel nidogen fragment in vitro and in vivo. Kidney Int 1998; 53 (06) 1673-80.
45
O’Reilly MS,
Holmgren L,
Shing Y.
et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma [see comments]. Cell 1994; 79 (02) 315-28.
46
Taraboletti G,
Belotti D,
Borsotti P.
et al. The 140-kilodalton antiangiogenic fragment of thrombospondin- 1 binds to basic fibroblast growth factor. Cell Growth Differ 1997; 08 (04) 471-9.
48
Fazioli F,
Resnati M,
Sidenius N.
et al. A urokinase-sensitive region of the human urokinase receptor is responsible for its chemotactic activity. EMBO J 1997; 16 (24) 7279-86.
