The urokinase plasminogen activator receptor in blood from healthy individuals and patients with cancer

APMIS 107: 160-7, 1999 Printed in Denmark . All rights reserved

Cop,vrixht 0 A P M I S 1999

ISSN 0903-4641

The urokinase plasminogen activator receptor in blood from healthy individuals and patients with cancer

Review article

NILS BRUNNER,' HANS J0RGEN NIELSEN,2 MARIA HAMERS,' IB JARLE CHRISTENSEN,' OLE THORLACIUS-USSING3 and ROSS W. STEPHENS'

Finsen Laboratory, Rigshospitalet, Copenhagen, 2Department of Surgical Gastroenterology, Hvidovre Hospital, Copenhagen, and 3Department of Surgical Gastroenterology, Alborg Hospital,

Alborg, Denmark

Brunner N, Nielsen HJ, Hamers M, Christensen IJ, Thorlacius-Ussing 0 & Stephens RW. The uroki- nase plasminogen activator receptor in blood from healthy individuals and patients with cancer. APMIS 1999; 107: 160-7.

The cell surface plasminogen activation system functions in promoting tumor dissemination, and is facilitated by a glycolipid anchored three domain receptor for urokinase. This receptor can also be found in a soluble form (suPAR) in extracts of tumors, as well as in plasma from both healthy individuals and cancer patients. The suPAR in plasma consists of the intact three domain protein, but neither the precise mechanism of its release from cell surfaces, nor its biological function are understood. Increased levels of plasma suPAR have been found in patients with cancers of the lung, breast, ovary, and colon, and recent data now indicates that the level of the molecule is related to patient prognosis.

Key words: uPAR; suPAR; cancer; prognosis.

Nils Brunner, Finsen Laboratory, Strandboulevarden 49, DK-2 100 Copenhagen, Denmark.

The invasive process by which malignant tumors dis- seminate can now be more accurately described as a process of unregulated tissue remodelling, progress- ively involving normal cells which are recruited into the tumor stroma by the neoplastic cells. In this pro- cess the barriers which normally define the homeo- stasis of differentiated tissue are breached by new sig- nals from the de-differentiating and proliferating neoplastic cells. Recruitment and reorganisation of the normal tissue cells progressively disrupts organ structure, as the neoplastic cells develop and continu- ously remodel a supporting stroma, complete with a new blood capillary network. Tumor tissue remodel- ling is also accompanied by the detachment of tumor cells and their active invasion of blood and lymph vessels, from where they can metastasize to form new tumors at distant sites.

The hallmark of invasive tissue remodelling is the degradation of the existing extracellular matrix which normally maintains the integrity of organised

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tissue structures. In particular, the basement mem- brane is a specialised form of matrix which normally forms a boundary adjacent to the epithelial tissues where carcinomas arise. In order to degrade and tra- verse the first barrier represented by the basement membrane, it is a prerequisite that cancers arising in epithelial tissues either acquire the ability themselves to secrete proteolytic enzymes which can cleave base- ment membrane proteins, or induce the required pro- teases in the normal tissue cells which are progress- ively recruited into the tumor stroma. In addition, proteolysis of the extracellular matrix may in turn stimulate growth of tumors by release and/or acti- vation of growth factors and cytokines and/or by in- ducing neovascularization. Thus, certain proteases are highly expressed in tumor tissue, including mem- bers of both the serine protease family, e.g. uroki- nase-type plasminogen activator and the metallopro- teinase family.

Several publications have demonstrated a prognos-

uPAR IN BLOOD

tic value in cancer patients of molecules involved in the urokinase pathway of plasminogen activation (1- 22). However, almost all these results are based on determination in tumor extracts. The development of more effective diagnostic methods has resulted in earlier detection and thus smaller tumor size at the time of surgery, which is making it increasingly diffi- cult to acquire access to frozen, unfixed tumor samples. Therefore, the conversion of these assays from tests which require tumor extracts to assays that can be performed on plasma or serum would signifi- cantly increase their clinical value. Sample collection would be easier, and the need for careful histological evaluation of the tissue to be analysed as well as the inherent problems of tissue heterogeneity would not apply to blood samples. Even more important, the possibility of obtaining blood samples during clinical follow-up after primary surgery for cancer, would allow sequential measurements of the uPA related an- alytes.

We recently reported that tumor tissue levels of sol- uble uPAR but not total uPAR were associated with prognosis in breast cancer patients (6). At that time we suspected that this form of soluble uPAR (suPAR: full-length receptor and cleavage products, without the glycolipid anchor) after release from the cell sur- face could find its way into the circulation. In order to study plasma and serum levels of suPAR in cancer patients, we developed a kinetic ELISA specifically evaluated for measurements of suPAR in blood (23). Using this ELISA, we have since shown that in fact patients with various forms of cancer including pa- tients with metastatic breast cancer, Dukes D col- orectal cancer and patients with ovarian cancer (22,

23) have elevated levels of suPAR in their blood, and in two recent studies, one of the patients with colorec- tal cancer (24) and another of the patients with ovarian cancer (22) we found a significant association between elevated suPAR blood levels and shorter sur- vival.

BIOCHEMISTRY OF THE UROKINASE PLASMINOGEN ACTIVATION SYSTEM

Fig. 1 shows the urokinase plasminogen activation system. uPA is a 52 kD serine protease which is se- creted as an inactive single chain proenzyme (pro- uPA) (25, 26). This immature form has only 11250 the activity of the mature enzyme (27). Pro-uPA can be activated to uPA by plasmin, cathepsin B and kallikrein (28, 29), which all cleave the single chain pro-uPA within an intrachain loop held closed by a disulfide bridge. Thus the active enzyme consists of two chains (A+B) held together by this disulfide bridge. Active uPA has high specificity for the Arg560-Val561 bond in plasminogen, and cleavage between these residues gives rise to active plasmin. Plasminogen (28) is present at high concentration (1.5-2.0 pM) in plasma and interstitial fluids, and represents a plentiful potential source of plasmin ac- tivity. Unlike uPA, plasmin is a relatively non-specific protease, cleaving many glycoproteins and proteo- glycans of the extracellular matrix, as well as fibrin (30). In addition, it is important to note that plasmin can activate some of the metalloproteases which also degrade tissue matrix (31, 32), and plasmin is known to activate growth factors, e.g. TGF-P (33), which

Fig. 1. The urokinase plas- minogen activation system. uPA: urokinase plasminogen activator; uPAR: plasmino- gen activator receptor; su- PAR: soluble uPAR; PLG: plasminogen; PL: plasmin; PAI: plasminogen activator inhibitor.

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may further modulate stromal interactions in the ex- pression of enzymes and tumor neo-angiogenesis.

Pro-uPA and uPA bind to a specific cell-surface re- ceptor (uPAR) (34, 35). This receptor consists of a glycolipid-anchored three-domain 60 kD glyco- protein whose N-terminal domain l contains the high affinity binding site for a growth factor domain within the A-chain of uPA. Since uPAR also has an- other important interaction with vitronectin (36), these two interactions explain the observed localiz- ation of pro-uPA and thus potential uPA activity at the focal contacts and junctions formed between cells and with the extracellular matrix (37). Indeed uPA binding to uPAR promotes binding of uPAR to the matrix protein vitronectin (36), and thus adhesion of cells to a vitronectin substratum.

Low affinity, high capacity binding of plasminogen to cell-surface proteins through the lysine binding sites of plasminogen kringles (38) enhances consider- ably the rate of plasminogen activation by uPA (29, 39). This is probably due to the proximity of bound plasminogen to the laterally mobile uPAR (40) on the cell-surface. Furthermore, plasmin formed on the cell surface is able to catalyse the activation of uPAR- bound pro-uPA (29), while remaining protected from plasma proteinase inhibitors, especially a-2-antiplas- min. Cell-surface bound plasmin can then mediate the non-specific matrix proteolysis which facilitates migration of tumor cells through restraining tissue structures and activates or releases growth factors in the matrix.

Plasminogen activation by uPA is regulated by two important inhibitors, PAI-I and PAI-2 (37, 41). PAI- 1, a 52 kD single chain protein, is secreted in an ac- tive but conformationally unstable form, whose in- hibitory activity is stabilized and prolonged by bind- ing to vitronectin (42, 43). PAI-1 is the most abun- dant fast-acting inhibitor of uPA in tumor tissues, and it is also able to inactivate uPA which is bound to uPAR. Formation of PAI-1 or PAI-2 complexes with uPA on the cell-surface leads to internalization of the uPAR/uPA/PAI complex (44) and probably drives uPAR cycling through the endosomal com- partment back to the cell surface (45). uPA activity may also be lost from the cell-surface by plasmin cleavage at Lys135-Lys136 in the A-chain of uPA, leaving only the so-called amino terminal fragment (ATF) bound on the cell. The general plasma protein- ase inhibitor, a2-macroglobulin, inactivates soluble uPA relatively slowly, but uPA bound to uPAR is ap- parently protected by a steric effect (46), thus allowing plasminogen activation to occur on the sur- face of cells even in the presence of serum (29).

The interaction between PAI-1 and the cell-surface uPA system is now also appreciated as a modulator of cell adhesion and migration (47). The basis of this effect is competition by active PAI-1 for uPAR bind-

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ing sites on matrix vitronectin. Thus a high local con- centration of PAL1 can overcome uPAR-mediated adhesion and promote cell detachment, increased motility and migration. This may be most important for transformed epithelial cells which have reduced adhesion interactions with other matrix proteins, e.g. fibronectin (48).

SOLUBLE uPAR

As mentioned above uPAR consists of a three-do- main highly glycosylated protein which in patients with cancer may be expressed on the surface of acti- vated macrophages infiltrating the tumor stroma, and/or on the tumor cells themselves (35). uPAR is anchored to the cell membrane by a glycosylphos- phatidylinositol linkage at its carboxy-terminus (34), and this linkage can be cleaved by e.g. phospholipase C resulting in release of a hydrophilic full-length su- PAR molecule. Between domains 1 and 2, there is a “hinge” region of relatively unstructured backbone, containing cleavage sites for the serine proteases uPA, plasmin, trypsin, chymotrypsin, and probably several other proteases (49). Thus domain 1 may be cleaved off the receptor by proteases either prior to, or after cleavage by a phospholipase. The main forms of uPAR one could expect to find in blood are therefore full length, three-domain soluble uPAR (suPAR), two-domain suPAR consisting of domains (2+3) or free domain 1 (Fig. 2). The two former have both been found in extracts of human tumors xenografted on nude mice (50). However, suPAR is also present in patient tumor extracts (37) as well as in plasma from these patients (33), and one could imagine this consisting of full-length, three domain suPAR, as well as the two domain (2+3) and free domain 1. Free domain 1 is relatively small, comparable in MW to

UROKINASE RECEPTOR CLEAVAGE

PROTEOLYTIC CLEAVAGE

PHOSPHOLIPASE

uPAR IN BLOOD

microglobulin, which is known to be lost from blood through the kidneys. Free domain 1 has not been found in either tumor extracts or plasma. All the evi- dence currently available indicates that suPAR in plasma, both from healthy donors and cancer pa- tients, consists of the full-length, three domain pro- tein (22, 23, 51).

MEASUREMENT OF suPAR IN PLASMA AND SERUM

Various techniques including receptor/ligand assays, immunohisto- (cyto)chemistry, ELISA and Western blot, have been employed to determine uPAR and su- PAR in tumor tissues and blood in order to assess their clinical relevance. For this purpose, a variety of monoclonal antibodies to uPAR have been developed and using these antibodies one can distinguish be- tween different molecular forms of uPAR (e.g. full- length receptor, domains 1, 2, 3 as well as free versus complexed forms) (52).

The more objective quantitation of uPAR is ob- tained by ELISA. ELISA quantitation of suPAR was first described by Rsnne et al. (53), who applied an ELISA to measurement of uPAR in human tumor tissue extracts. This ELISA consisted of rabbit anti- uPAR catching antibodies and a mixture of three bi- otinylated murine monoclonal antibodies for detec- tion. The epitopes recognized by these monoclonal antibodies are located in domain 1 and 3, respec- tively, and the assay will thus detect full length recep- tor as well as the cleaved forms. When this assay was used on plasma and sera it was found necessary to modify the detection method to eliminate a source of nonspecific signal that could be traced back to the use of biotinylated reagents. Thus non-biotinylated monoclonal antibodies were used for detection and a rabbit anti-mouse immunoglobulin conjugated with alkaline phosphatase was used to quantitate the bound monoclonals (23). Other changes made in- cluded the use of a rapid blocking agent, a dilution buffer with higher buffering capacity, and alkaline phosphatase as the enzyme conjugate to allow kinetic assay of the bound antigen. These other modifi- cations gave further improvements in the signal-to- noise ratio, and kinetic assay permitted automated fitting of rate curves, which proved considerably more reliable than single-end-point measurements. With this protocol, the assay fulfilled the require- ments of sensitivity, specificity, stability, and good re- covery of an internal calibrator. Fig. 3 shows the composition of the suPAR ELISA (23).

Healthy blood donors Quan!itative studies of suPAR in blood from

healthy donors show that all forms of blood prepara-

Elisa assay for soluble urokinase receptor

B Alkaline Phosphatase

Rabbit anti-mouse IgG

I A A MAbs to huPAR - R2, R3, R5

huPAR 0 Rabbit anti-huPAR

Fig. 3. ELISA for suPAR.

6 0

0

0 ! 1 0 0 7

0 0 0

00

0 1 2 3 4 5 6 suPAR PLASMA

Fig. 4. Correlation between preoperative plasma versus pre- operative serum suPAR in 591 patients with colorectal can- cer. Spearman coefficient of correlation: 0.75; p<O.OOOl.

0

tions appear to be suitable for ELISA determination of suPAR concentrations - serum as well as plasma made with citrate, EDTA, or heparin anticoagulant (23). Similar median values and ranges were found for healthy serum, citrate plasma and heparin plasma (1.2-1.3 ng/ml), while the concentration of suPAR in EDTA plasma was significantly higher (1.5 ng/ml). Thus, if comparisons of donor groups or healthy do-

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nors with patient material are to be valid, the same blood preparation (e.g., serum) should be used con- sistently. By comparing plasma versus serum suPAR levels in healthy blood donors (23) or in patients with colorectal cancer (Fig. 4), a highly significant corre- lation was found.

When comparing serum suPAR values among males and females, a significant elevation was found among females. In contrast, no notable correlation was found between serum suPAR concentration and age of the blood donors (23). uPAR is known to be displayed on the surface of blood monocytes and neutrophils (54), so that senescence of these cells could be one source of suPAR in blood from healthy individuals. The differentiation of megakaryocytes and formation of platelets in the bone marrow may also contribute significant levels of suPAR to the blood compartment (55).

Cancer patients Appreciable amounts of suPAR have been pre-

viously identified and characterized in plasma and as- cites fluid from ovarian cancer patients (22, 51). In the former of these studies serum suPAR was found to be a prognostic marker for overall survival of the patients, high serum suPAR levels predicting shorter survival. An even stronger association to short pa- tient survival was found when both serum suPAR and CA-125 were elevated. In patients with non small cell lung cancer citrate plasma suPAR content was significantly higher than in healthy controls, while no significant increase was found in plasma from pa- tients with small cell lung cancer (18). In a recent

suPAA LEVELS IN EDTA PLASMA FROM 90LON CANCER PATIENTS AND HEALTHY DONORS

7

6

5

f 3 4 n

3 3 3

2

1

0

o Colon Ca Healthy 1

0

8

P

100

Fig. 5. Percentile plot of preoperative plasma suPAR levels in 591 patients with colorectal cancer and in 44 healthy blood donors.

0 0 20 40 60 80

Percentile

study (23) citrate plasma levels were measured in pa- tients with advanced breast cancer and in patients with advanced (Duke’s D) colorectal cancer. In both groups of patients, a significant increase in plasma suPAR was found. Correlating plasma suPAR con- tent to patient survival in a cohort of 591 patients with colorectal cancer, a highly statistical significant difference was found between patients with high ver- sus low preoperative plasma suPAR levels (24). Fig. 5 shows a percentile plot of plasma suPAR levels in healthy blood donors as compared to preoperative plasma suPAR levels in the 591 patients with colorec- tal cancer.

CHARACTERIZATION OF BLOOD suPAR

The occurrence of suPAR in healthy human plasma has previously been conclusively demonstrated by im- munoaffinity purification and cross-linking to Iz5I- labeled amino-terminal fragment of human uPA (56) . Thus, since the soluble receptor present in plasma was able to efficiently bind its ligand, it therefore most probably consists of full-length receptor. If su- PAR had been proteolytically cleaved to the known products (i.e., domains 2+3, and domain l), then the affinity for uPA would have been reduced by approxi- mately 1500-fold (57). In ascites fluid of ovarian can- cer patients, amino acid analyses have shown good agreement with the expected composition, based on the cDNA sequence of full-length uPAR (22). In ad- dition, while GPI-anchored uPAR reveals the pres- ence of 2% mol/mol phosphoethanolamine, ascitic suPAR had either very little or no phosphoethanlam- ine (22), suggesting that most suPAR in the ascites fluid does not contain a GPI anchor, in agreement with its water solubility. The epitope contributions to total suPAR signal that we have found in ELISA’s of healthy donor plasma and serum samples (23) strongly support that suPAR is present in plasma as the full-length, three-domain receptor protein. Recent Western blotting experiments support this notion.

BIOLOGICAL ROLE OF suPAR

While cell bound uPAR has been ascribed a critical role in uPA mediated plasmin formation (57), the biological role if any of soluble receptor remains to be clarified. However, some recent in vitro experi- ments points to at least four different potential bio- logical roles of suPAR.

1. Chavakis et al. (58) recently showed that vi- tronectin could concentrate suPAR:uPA complexes to cell surfaces and extracellular matrix sites, leading to the accumulation of plasminogen activator ac- tivity.

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uPAR IN BLOOD

2. Resnati et al. (59) found that suPAR was capable of inducing chemotaxis in cells. However, this activity required proteolytic cleavage of suPAR between do- main 1 and domain 2.

3. Through its interaction with uPAR binding to vitronectin (36, 47), PAI-1 can facilitate cell mi- gration. It is tempting to speculate that suPAR might in a similar way interact with cell surface uPAR:vi- tronectin binding and thereby increase cell migration. It should be mentioned that only full-length and not the cleaved forms of suPAR bind vitronectin (60).

4. In an in vitro study, Wilhelm et al. (61) showed that suPAR can serve as a scavenger for uPA thereby inhibiting proliferation and in vitro invasion of hu- man cancer cells.

CONCLUSION

Significant levels of suPAR are present in plasma and serum from healthy blood donors and cancer patients appear to have increased concentrations of suPAR in their blood, which is in agreement with the potentiat- ing of plasmin formation by binding of uPA to cell surface anchored uPAR and concomitant cell surface binding of plasminogen. Recent studies suggest that full-length suPAR without the glycolipid anchor is the predominant form of suPAR in blood. By com- paring plasma or serum suPAR levels with patient survival, high suPAR levels have been implicated as a parameter of shorter survival.

Neither the exact mechanism(s) of suPAR release from cell surfaces nor its biological role if any is yet clearly defined. Future studies should aim at clarify- ing these questions.

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