SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 17, NO. 3, 1991
Urokinase-Dependent Proteolysis in Cultured Colon Cancer Is Directed by Its Receptor
High mortality rates associated with many malignancies are, invariably, a consequence of the spread of the disease to distant sites.1 Since, at the present time, there are no effective ways of combating tumor cells that have relocated to secondary sites, the prognosis of those patients afflicted with metastatic lesions is bleak. Because of this, many investigators have argued that therapeutic advances against the disease will depend on a greater knowledge of the mechanisms that underscore tumor dissemination. Although the process of tumor metastases is highly complex, and poorly understood at the present time, it is widely accepted that breach of the basement membrane represents one of the first steps in this direction.2'3 This structure is made up primarily of type IV collagen and the glycoprotein laminin.4 Acquisition of an invasive phenotype is thought to depend, in part, on the ability of the tumor cells to elaborate one or more hydrolases that can solubilize these basement membrane components, thereby removing an impediment to their movement.2'5 In this regard, there is a substantial body of evidence implicating the plasminogen activator (PA) urokinase (UK) in tumor invasion and metastases. UK has been reported to be elevated in several malignancies, including colon cancer.6-8 Moreover, the presence of the PA is most dramatic at the leading edge of these tumors,9 suggesting a role in the invasive process. Perhaps the most compelling evidence implicating UK in tumor dissemination are the findings that UK antibodies inhibit lung colonization by melanoma and HEp3 tumor cells. 10,11 UK probably promotes tumor cell invasion via its ability to convert the inert zymogen plasminogen into the widely acting serine protease plasmin.12 The latter enzyme can degrade laminin and type IV collagen
From the Pharmacology Department, Baylor College of Medicine, Houston, Texas. Reprint requests: Dr. Boyd, Department of Tumor Biology, M.D. Anderson Cancer Center, Houston, TX 77030.
directly13 or indirectly14 via the activation of stromelysin and type IV collagenase, respectively. Recent studies have indicated that tumor cells can bind UK in a saturable manner, this being ascribed to the presence of specific receptors on the surface of the cells.15 The receptor has been purified and cloned.16 Expression of the binding site in receptor-negative cells resulted in the specific and saturable binding of the PA, the latter retaining its activity, as is evident from the conversion of plasminogen into plasmin.16 The role of these binding sites in facilitating the actions of UK is unclear at the present time. The display of these receptors on tumor cells could, arguably, promote unregulated proteolysis by any one of the following mechanisms: Tumor cells equipped with these binding sites could concentrate the PA at the cell surface, this leading to a build-up of plasmin in the immediate vicinity of the cell. Alternatively, receptor binding of UK could alter the kinetics of plasminogen activation in favor of plasmin production.17 Thirdly, the binding site could sequester the UK in a location that is inaccessible to inhibitors, such as plasminogen activator inhibitor type 1 (PAI-1), which might otherwise neutralize the PA. 18 These proposals are, of course, not mutually exclusive. We have chosen to concentrate our efforts on trying to understand the proteolytic mechanisms responsible for basement membrane laminin solubilization, arguing that the breach of this barrier is one of the first steps of tumor cell invasion and, ultimately, metastases. Toward this end, we have made use of a bank of benign and malignant, human colonic cell lines that have been extensively characterized by this laboratory and others 19-21 (Table 1).
LAMININ DEGRADATION AND INVASION BY COLON CANCER CELLS REQUIRES PLASMINOGEN Well (WD) and poorly (PD) differentiated colon cancer cells were compared for their abilities to degrade
Copyright © 1991 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
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SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 17, NO. 3, 1991 TABLE 1. Human Colon Benign and Cancer Cell Lines
Malignant Well differentiated
Cell Line
Properties
CBS
Polarized in monolayer
GEO
Form secretory domes
FET
Manifest tight junctions Form glandular tumors at a low rate of incidence in athymic mice
MOSER
Lack of cell polarization
HCT 116
Do not form secretory domes
HCT 116b
Poor cell-cell communication
RKO
Form anaplastic tumors at high rates of incidence in nude mice Generation time 40 hours
laminin and invade extracellular matrix (ECM) coated filters. Technical problems prohibited these studies being carried out on the benign colonic cells. Both groups of cells were unable to solubilize the basement membrane component under serum-free conditions. However, when supplemented with plasminogen, degradation of the laminin was achieved by all of the cell lines examined, this being more dramatic for the PD cell types.22 Likewise, invasion of an ECM-coated filter was largely dependent on the presence of the zymogen and was more impressive for the PD cell types.23 It would appear that the tumor cell-derived PA responsible for plasmin production and laminin turnover is of the UK type, since a specific polyclonal antibody to this PA (Fig. 1) was capable of titrating up to 80% of plasminogen activation by conditioned medium.
EXPRESSION OF UROKINASE IN BENIGN AND MALIGNANT CULTURED COLON CANCER In view of these data, the colonic cell lines were compared for UK secretion using an enzyme-linked immunosorbent assay, which detects high but not low molecular weight PA. Both benign cell lines (Vaco 235
FIG. 1. Western blotting of conditioned medium derived from cultured colon cancer for urokinase immunoreactivity. Spent medium derived from the indicated cell lines was dialyzed against 10 mM phosphate buffer (pH 7.4) and concentrated 20-fold by freeze drying. The material was electrophoresed in a 12.5% polyacrylamide gel, and the resolved proteins transferred to a nitrocellulose filter. The filter was blocked in a 3% bovine serum albumin solution, and subsequently incubated at 4°C, overnight, with a 1:500 dilution of polyclonal urokinase antibody and, subsequently, with 50,000 dpm radioactive protein A. After, 4 hours at room temperature, the filter was washed extensively and exposed to x-ray film. Molecular weight standards are indicated to the left.
and 330) were low secretors of UK. In contrast, WD and PD colon cancer cell types were avid secretors of the enzyme (Fig. 2). Northern analysis (Fig. 3) indicated that the disparity in UK secretion between the benign and malignant cells could be accounted for by differences in steady-state levels of the UK transcript. The elevated levels of UK transcript seen in the cancer cells was not a consequence of gene amplification or rearrangement. DNA extracted from the cancer cells was digested with either XmnI or Ncol, Southern blotted, and the blot probed with a radioactive cDNA corresponding to part of the UK gene. As a control, placental DNA was subjected to an identical treatment. Figures 4 and 5 show that the fragment sizes of the DNA in the Southern blots are identical to those generated with the placental DNA and, as predicted, from a restriction map of the UK gene. Moreover, the signal intensity of the bands were indistinguishable from the control DNA, ruling out any possibility of gene amplification contributing to the higher levels of steady-state mRNA observed in the cancer cells.
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Doubling time >24 hours Poorly differentiated
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FIG. 2. Urokinase secretion by benign and malignant colonic cells in culture. At approximately 90% confluency, the cultures were replenished with fresh serum-free medium and carried for 3 days. The spent medium was harvested, clarified by centrifugation, and assayed for urokinase by an enzymelinked immunosorbent assay. Cells were enumerated. The data are expressed as average values ±SD. The experiments were carried out at least four times.
LAMININ DEGRADATION AND EXTRACELLULAR MATRIX INVASION BY COLON CANCER CELLS—LACK OF CORRELATION WITH UROKINASE LEVELS AND ITS INHIBITOR, PAI-1 We considered the possibility that the differences in the laminin degrading profiles observed between the two groups of colon cancer cells reflected the selective elaboration of UK inhibitors such as PAI-1 by the WD group, effectively neutralizing the PA. To address this contention, spent medium derived from the cancer cells was analyzed for UK inhibitors by reverse zymography. The PD cells characterized as being active degraders of laminin and capable of invading the ECM coated filter appeared to express more of an inhibitor (Mr approximately 48,000d), than their WD counterparts, the latter regarded as being weakly active in the solubilization and invasions assays.24 Thus, it is unlikely that the selective expression of a UK inhibitor by the WD cells can account for their reduced potential to degrade laminin and invade ECM compared with their PD counterparts.
PLASMINOGEN DEPENDENT PROTEOLYSIS IN CULTURED COLON CANCER IS DIRECTED BY THE UK RECEPTOR There is a growing body of evidence indicating that plasminogen-dependent proteolysis is, in fact, a cell
FIG. 3. Northern blotting of RNA extracted from benign and malignant cultured colon cancer. Near confluent colonic cells were extracted with 5.0 M guanidine isothiocyanate and the RNA purified by centrifugation at 150,000 × g. The purified RNA (10µg)was electrophoresed in a formaldehyde-agarose gel and blotted onto Nytran-modified nylon by capillary action. The immobilized RNA was fixed by baking and the filter probed with a radioactive cDNA corresponding to part of the urokinase message. Stringency washes were performed at 65°C using 0.5 × saline sodium citrate and the blot exposed to x-ray film. Loading equalities were checked by reprobing the filter with a glyceraldehyde-3-phosphate dehydrogenase probe. The data are representative of three separate experiments.
surface phenomenon directed by UK receptors for the activator. We argued that, if this is the case for cultured colon cancer, then the amount of UK recovered from the cell surface should correlate with the abilities of the cells to degrade laminin and invade ECM. A good relationship22 (r2 = 0.9242) was found between laminin turnover and the amount of PA associated with the cell surface. Although less than 1 ng of UK could be dissociated from the surfaces of the WD cell types, between 5 and 13 ng/106 cells of the PA were recovered from their PD counterparts.22 These differences reflected a tenfold disparity in UK receptor numbers, as determined by radioreceptor assay after an acid22 pretreatment to rid the cells of endogenous UK. The binding sites bound the PA with high affinity (0.8 to 2.0 nM) probably via the amino acid sequence 12-32 of the UK A chain. These data, together with the
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FIG. 4. Southern blotting of the urokinase gene using DNA extracted from malignant colonic cells—analysis by digestion with Ncol. The cells were treated with 0.1 mg/ml proteinase K at 50°C and the DNA purified by extraction with a mixture (24:1) of phenol and chloroform/isoamyl alcohol. The purified DNA routinely had an A260/280 ratio in excess of 1.9. Equal amounts of DNA (10 µg) were digested with Ncol at 37°C overnight, and the fragments resolved in a 1% agarose gel. The gel was treated sequentially with acid and base and the DNA transferred to a modified nylon filter. The filter was baked and probed with a radioactive urokinase cDNA as described for Northern blotting.
FIG. 5. Southern blotting of the urokinase gene using DNA extracted from malignant colonic cells—analysis by Xmnl digestion. These were carried out as described for Figure 4 with the exception that Xmnl was substituted for Ncol.
binding sites are occupied are poorly responsive to the receptor antagonist.
BINDING SITES FOR PLASMINOGEN ON COLON CANCER CELLS findings that low molecular weight UK, which lacks part of the A chain, was unable to compete with high molecular weight UK25 for receptor binding, suggested that the receptors displayed on cultured colon cancer are identical to those reported by Stoppelli et al. 15 It is widely thought that these binding sites orient the active site of the PA toward the extracellular target, plasminogen. This provides an ideal mechanism for generating the proteolytic insult at the tumor cell surface where it is required. To substantiate the role of the UK receptors in laminin degradation and invasion further, a peptide (UK 12-32), corresponding to that part of the UK A chain required for the binding,26 was synthesized. The UK 12-32 peptide was effective in displacing radioactive UK from its receptor in a dose-dependent manner.22 These concentrations of UK 12-32 impaired the ability of colon cancer cells equipped with large numbers ( 105/cell) of receptors to degrade laminin22 and invade the extracellular matrix.23 It is worth noting that the sensitivity of the cells to the peptide also reflects the degree of occupation of the binding sites with the endogenous ligand. Thus, cells on which the majority of the receptors are "tagged" with secreted UK appear exquisitively sensitive to the peptide, whereas those on which the minority of the
There have been several reports of plasminogen receptors on the surface of a wide variety of cell types. 27 ' 28 Several investigators have speculated that plasminogen dependent proteolysis could be entirely a cell surface phenomenon governed by the display of receptors for both activator and zymogen. As applied to colon cancer, this concept presumes the presence of specific binding sites for the zymogen on the surface of the colonic cells. Theoretically, these receptors could lead to a build-up of the UK substrate at the cell surface, a situation that, by mass action, could favor the efficient conversion of the zymogen into plasmin, a process presumably mediated by receptor bound UK. A variation of this theme is that the plasminogen binding sites shuttle the zymogen to receptor immobilized UK (or vice versa), thereby juxtaposing both activator and substrate. Our preliminary data suggest the presence of specific binding sites for plasminogen on the surface of cultured colon cancer. These are specific insofar as a variety of competitors, including fibrinogen, transferrin, and myoglobin, did not compete with the radioactive zymogen for the binding to the cells. One WD and two PD cell lines bound 250 ± 60, 280 ± 50, and 210 ± 30 fmol radioactive plasminogen/106 cells, respectively, using an
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input concentration of 25 nM radioligand. Since the concentration of the plasminogen used in our in vitro invasion is approximately 100 nM, we would expect that under these conditions, the colonic tumor cell surface would be charged with a substantial amount of the zymogen.
LACK OF SENSITIVITY OF PLASMINOGENDEPENDENT PROTEOLYSIS DIRECTED BY RECEPTOR-BOUND UK TO PAI-1 The fact that the PD cells elaborated more of an inhibitor than their WD counterparts but in spite of this were more active in degrading laminin and invading ECM-coated filters suggested an insensitivity to the inhibitor. The mass of the inhibitor, as indicated by reverse zymography, was close to if not identical to that of PAI-1. Western blotting experiments and immunoprecipitation/reverse zymography studies revealed this inhibitor to be indistinguishable from PAI-1. 24 If the inhibitor was checking the activity of UK, then neutralizing the PAI-1 would be expected to enhance laminin turnover by PD cells charged with surface bound UK. Colon cancer cells supplemented with plasminogen were cultured on radioactive laminin in the presence or in the absence of neutralizing amounts of PAI-1 antibody. The antibody to PAI-1 did not augment laminin solubilization.24 Control experiments indicated that the antibody was without effect on UK expression. Studies showed, however, that the amount of active PAI-1 was, indeed, small and could, arguably, be of little biologic significance. To address this contention, commercially available PAI-1, activated with guanidine hydrochloride, was added, along with plasminogen, to colon cancer cells, equipped with 105 receptors/cell and grown on radioactive laminin. Although the inhibitor was highly effective in reducing the degradation of the glycoprotein brought about by authentic UK and plasminogen, laminin cleavage by receptor-positive cells cultured in the presence of the zymogen was unaltered.24 We have considered several possibilities that could account for these findings. Firstly, the UK receptor could sequester the PA in a location that is inaccessible to inhibitors, such as PAI-1. 18 Secondly, the receptor might shield the PA from inhibitors, akin to the protection provided to plasmin by its binding site.28 Thirdly, endogenous UK produced by the colon cancer cells could be a poor target for the PAI-1. Our data do not allow us to discriminate between these possibilities at the present time. It should be pointed out, however, that comprehensive studies by other laboratories have indicated that the UK receptor does not protect its PA from inhibitors. We have therefore speculated that these binding sites
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may compartmentalize the inhibitor in a location that is quite separate from that of UK immobilized to cell surface receptors. However, recent studies have indicated a third possible explanation for our data. It now appears that UK produced by some tumor cells can be phosphorylated in the region of the active site, rendering it insensitive to PAI-1. If this is the case for our PD colon cancer cells, it might provide a plausible explanation for the insensitivity of laminin degradation by HCT 116 colon cancer cells to this inhibitor. Clearly, further studies are needed to determine the mechanism responsible for this lack of sensitivity to PAI-1. Thus, in conclusion, it appears that the display of UK receptors by cultured colon cancer is a critical factor in determining their laminin degrading potential and very possibly their invasive phenotype. Acknowledgment. The authors are indebted to Drs. D. Collen and P. Declerck for their generous supply of antibodies.
REFERENCES 1. Fidler IJ, GL Nicolson: The process of cancer invasion and metastasis. Cancer Bull 39:126-131, 1987. 2. Nakajima M, T Irmura, GL Nicolson: Basement membrane degradative enzymes and tumor metastasis. Cancer Bull 39:142149, 1987. 3. Salo T, L Liotta, J Keski-Oja, T Turpeenniemi-Hujanen, K Tryggvason: Secretion of basement membrane collagen degrading enzyme and plasminogen activator by transformed cells—role in metastases. Int J Cancer 30:669-673, 1982. 4. Tryggvason K, M Hoyhtya, T Salo: Proteolytic degradation of extracellular matrix in tumor invasion. Biochim Biophys Acta 907:191-217, 1987. 5. Quian FX, AS Bajkowski, DF Steiner, SJ Chan, A Franfater: Expression of five cathepsins in murine melanomas of varying metastatic potential and normal tissues. Cancer Res 49:48704875, 1989. 6. Markus G, H Takita, S Camiolo, J Corasanti, J Evers, G Hobika: Content and characterization of plasminogen activators in human lung tumors and normal lung tissue. Cancer Res 40:841-848, 1980. 7. Markus G, SM Camiolo, S Kohga, JM Madeja, A Mittelman: Plasminogen activator secretion of human tumors in short-term organ culture, including a comparison of primary and metastatic colon tumors. Cancer Res 43:5517-5525, 1983. 8. de Bruin AF, G Grissioen, HW Verspaget, JH Verheijen, G Dooijewaard, HF van den Ingh, CBHW Lamers: Plasminogen activator profiles in neoplastic tissues of the human colon. Cancer Res 48:4520-4524, 1988. 9. Skriver L, LI Larsson, V Kielberg, LS Nielsen, PB Andresen, P Kristensen, K Dano: Immunocytochemical localization of urokinase-type plasminogen activator in Lewis lung carcinoma. J Cell Biol 99:752-757, 1984. 10. Ossowski L, E Reich: Antibodies to plasminogen activator inhibit human tumor metastasis. Cell 35:611-619, 1983. 11. Hearing V, L Law, A Corti, E Appella, F Blasi: Modulation of metastatic potential by cell surface urokinase of murine melanoma cells. Cancer Res 48:1270-1278, 1988.
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SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 17, NO. 3, 1991
12. Collen D, M Verstraete: Molecular biology of human plasminogen. II. Metabolism in physiological and some pathological conditions in man. Thromb Diath Haemorrh 34:403-408, 1975. 13. Mackay AR, RH Corbitt, JL Hartzler, UP Thorgeirsson: Basement membrane type IV collagen degradation: Evidence for the involvement of a proteolytic cascade independent of metalloproteinases. Cancer Res 50:5997-6001, 1990. 14. Matrisian L, T Bowden: Stromelysin/transin and tumor progression. Semin Cancer Biol 1:107-115, 1990. 15. Stoppelli M, C Tacchetti, M Cubellis, A Corti, V Hearing, G Cassani, E Appella, F Blasi: Autocrine saturation of prourokinase receptors on human A431 cells. Cell 45:675-684, 1986. 16. Roldan A, M Cubellis, M Masucci, N Behrendt, L Lund, K Dano, E Appella, F Blasi: Cloning and expression of the receptor for urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis. EMBO J 9:467-474, 1990. 17. Ellis V, MF Scully, VV Kakkar: Plasminogen activation initiated by single-chain urokinase-type plasminogen activator. J Biol Chem 264:2185-2188, 1989. 18. Hekman CM, DJ Loskutoff: Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants. J Biol Chem 260:11581-11587, 1985. 19. Brattain M, A Levine, S Chakrabarty, L Yeoman, JKV Willson, B Long: Heterogeneity of human colon carcinoma. Cancer Metastasis Rev 3:177-191, 1984. 20. Chantret I, A Barbat, E Dussaulx, M Brattain, A Zweibaum: Epithelial polarity, villin expression and enterocytic differentiation
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of cultured colon carcinoma cells: A survey of twenty cell lines. Cancer Res 48:1936-1942, 1988. Willson J, G Bittner, T Oberley, L Meisner, J Weese: Cell culture of human colon adenomas and carcinomas. Cancer Res 47:27042713, 1987. Schlechte W, G Murano, D Boyd: Examination of the role of the urokinase receptor in human colon cancer mediated laminin degradation. Cancer Res 49:6064-6069, 1989. Schlechte W, M Brattain, D Boyd: Invasion of extracellular matrix by cultured colon cancer cells: Dependence on urokinase receptor display. Cancer Commun 2:173-179, 1990. Schlechte W, DD Boyd: Insensitivity of laminin degradation directed by receptor bound urokinase to PAI-1 in cultured colon cancer. Cancer Commun 2:261-270, 1990. Boyd D, G Florent, P Kim, M Brattain: Determination of the levels of urokinase and its receptor in human colon carcinoma cell lines. Cancer Res 48:3112-3116, 1988. Appella E, E Robinson, S Ullrich, M Stoppelli, A Corti, G Cassani, F Blasi: The receptor-binding sequence of urokinase. J Biol Chem 262:4437-4440, 1987. Miles L, E Levin, J Plescia, D Collen, E Plow: Plasminogen receptors, urokinase receptors, and their modulation on human endothelial cells. Blood 72:628-635, 1988. Plow E, D Freaney, J Plescia, L Miles: The plasminogen system and cell surfaces: Evidence for plasminogen and urokinase receptors on the same cell type. J Cell Biol 103:2411-2420, 1986.
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Urokinase-Dependent Proteolysis in Cultured Colon Cancer Is Directed by Its Receptor
High mortality rates associated with many malignancies are, invariably, a consequence of the spread of the disease to distant sites.1 Since, at the present time, there are no effective ways of combating tumor cells that have relocated to secondary sites, the prognosis of those patients afflicted with metastatic lesions is bleak. Because of this, many investigators have argued that therapeutic advances against the disease will depend on a greater knowledge of the mechanisms that underscore tumor dissemination. Although the process of tumor metastases is highly complex, and poorly understood at the present time, it is widely accepted that breach of the basement membrane represents one of the first steps in this direction.2'3 This structure is made up primarily of type IV collagen and the glycoprotein laminin.4 Acquisition of an invasive phenotype is thought to depend, in part, on the ability of the tumor cells to elaborate one or more hydrolases that can solubilize these basement membrane components, thereby removing an impediment to their movement.2'5 In this regard, there is a substantial body of evidence implicating the plasminogen activator (PA) urokinase (UK) in tumor invasion and metastases. UK has been reported to be elevated in several malignancies, including colon cancer.6-8 Moreover, the presence of the PA is most dramatic at the leading edge of these tumors,9 suggesting a role in the invasive process. Perhaps the most compelling evidence implicating UK in tumor dissemination are the findings that UK antibodies inhibit lung colonization by melanoma and HEp3 tumor cells. 10,11 UK probably promotes tumor cell invasion via its ability to convert the inert zymogen plasminogen into the widely acting serine protease plasmin.12 The latter enzyme can degrade laminin and type IV collagen
From the Pharmacology Department, Baylor College of Medicine, Houston, Texas. Reprint requests: Dr. Boyd, Department of Tumor Biology, M.D. Anderson Cancer Center, Houston, TX 77030.
directly13 or indirectly14 via the activation of stromelysin and type IV collagenase, respectively. Recent studies have indicated that tumor cells can bind UK in a saturable manner, this being ascribed to the presence of specific receptors on the surface of the cells.15 The receptor has been purified and cloned.16 Expression of the binding site in receptor-negative cells resulted in the specific and saturable binding of the PA, the latter retaining its activity, as is evident from the conversion of plasminogen into plasmin.16 The role of these binding sites in facilitating the actions of UK is unclear at the present time. The display of these receptors on tumor cells could, arguably, promote unregulated proteolysis by any one of the following mechanisms: Tumor cells equipped with these binding sites could concentrate the PA at the cell surface, this leading to a build-up of plasmin in the immediate vicinity of the cell. Alternatively, receptor binding of UK could alter the kinetics of plasminogen activation in favor of plasmin production.17 Thirdly, the binding site could sequester the UK in a location that is inaccessible to inhibitors, such as plasminogen activator inhibitor type 1 (PAI-1), which might otherwise neutralize the PA. 18 These proposals are, of course, not mutually exclusive. We have chosen to concentrate our efforts on trying to understand the proteolytic mechanisms responsible for basement membrane laminin solubilization, arguing that the breach of this barrier is one of the first steps of tumor cell invasion and, ultimately, metastases. Toward this end, we have made use of a bank of benign and malignant, human colonic cell lines that have been extensively characterized by this laboratory and others 19-21 (Table 1).
LAMININ DEGRADATION AND INVASION BY COLON CANCER CELLS REQUIRES PLASMINOGEN Well (WD) and poorly (PD) differentiated colon cancer cells were compared for their abilities to degrade
Copyright © 1991 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
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SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 17, NO. 3, 1991 TABLE 1. Human Colon Benign and Cancer Cell Lines
Malignant Well differentiated
Cell Line
Properties
CBS
Polarized in monolayer
GEO
Form secretory domes
FET
Manifest tight junctions Form glandular tumors at a low rate of incidence in athymic mice
MOSER
Lack of cell polarization
HCT 116
Do not form secretory domes
HCT 116b
Poor cell-cell communication
RKO
Form anaplastic tumors at high rates of incidence in nude mice Generation time 40 hours
laminin and invade extracellular matrix (ECM) coated filters. Technical problems prohibited these studies being carried out on the benign colonic cells. Both groups of cells were unable to solubilize the basement membrane component under serum-free conditions. However, when supplemented with plasminogen, degradation of the laminin was achieved by all of the cell lines examined, this being more dramatic for the PD cell types.22 Likewise, invasion of an ECM-coated filter was largely dependent on the presence of the zymogen and was more impressive for the PD cell types.23 It would appear that the tumor cell-derived PA responsible for plasmin production and laminin turnover is of the UK type, since a specific polyclonal antibody to this PA (Fig. 1) was capable of titrating up to 80% of plasminogen activation by conditioned medium.
EXPRESSION OF UROKINASE IN BENIGN AND MALIGNANT CULTURED COLON CANCER In view of these data, the colonic cell lines were compared for UK secretion using an enzyme-linked immunosorbent assay, which detects high but not low molecular weight PA. Both benign cell lines (Vaco 235
FIG. 1. Western blotting of conditioned medium derived from cultured colon cancer for urokinase immunoreactivity. Spent medium derived from the indicated cell lines was dialyzed against 10 mM phosphate buffer (pH 7.4) and concentrated 20-fold by freeze drying. The material was electrophoresed in a 12.5% polyacrylamide gel, and the resolved proteins transferred to a nitrocellulose filter. The filter was blocked in a 3% bovine serum albumin solution, and subsequently incubated at 4°C, overnight, with a 1:500 dilution of polyclonal urokinase antibody and, subsequently, with 50,000 dpm radioactive protein A. After, 4 hours at room temperature, the filter was washed extensively and exposed to x-ray film. Molecular weight standards are indicated to the left.
and 330) were low secretors of UK. In contrast, WD and PD colon cancer cell types were avid secretors of the enzyme (Fig. 2). Northern analysis (Fig. 3) indicated that the disparity in UK secretion between the benign and malignant cells could be accounted for by differences in steady-state levels of the UK transcript. The elevated levels of UK transcript seen in the cancer cells was not a consequence of gene amplification or rearrangement. DNA extracted from the cancer cells was digested with either XmnI or Ncol, Southern blotted, and the blot probed with a radioactive cDNA corresponding to part of the UK gene. As a control, placental DNA was subjected to an identical treatment. Figures 4 and 5 show that the fragment sizes of the DNA in the Southern blots are identical to those generated with the placental DNA and, as predicted, from a restriction map of the UK gene. Moreover, the signal intensity of the bands were indistinguishable from the control DNA, ruling out any possibility of gene amplification contributing to the higher levels of steady-state mRNA observed in the cancer cells.
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Doubling time >24 hours Poorly differentiated
227
FIG. 2. Urokinase secretion by benign and malignant colonic cells in culture. At approximately 90% confluency, the cultures were replenished with fresh serum-free medium and carried for 3 days. The spent medium was harvested, clarified by centrifugation, and assayed for urokinase by an enzymelinked immunosorbent assay. Cells were enumerated. The data are expressed as average values ±SD. The experiments were carried out at least four times.
LAMININ DEGRADATION AND EXTRACELLULAR MATRIX INVASION BY COLON CANCER CELLS—LACK OF CORRELATION WITH UROKINASE LEVELS AND ITS INHIBITOR, PAI-1 We considered the possibility that the differences in the laminin degrading profiles observed between the two groups of colon cancer cells reflected the selective elaboration of UK inhibitors such as PAI-1 by the WD group, effectively neutralizing the PA. To address this contention, spent medium derived from the cancer cells was analyzed for UK inhibitors by reverse zymography. The PD cells characterized as being active degraders of laminin and capable of invading the ECM coated filter appeared to express more of an inhibitor (Mr approximately 48,000d), than their WD counterparts, the latter regarded as being weakly active in the solubilization and invasions assays.24 Thus, it is unlikely that the selective expression of a UK inhibitor by the WD cells can account for their reduced potential to degrade laminin and invade ECM compared with their PD counterparts.
PLASMINOGEN DEPENDENT PROTEOLYSIS IN CULTURED COLON CANCER IS DIRECTED BY THE UK RECEPTOR There is a growing body of evidence indicating that plasminogen-dependent proteolysis is, in fact, a cell
FIG. 3. Northern blotting of RNA extracted from benign and malignant cultured colon cancer. Near confluent colonic cells were extracted with 5.0 M guanidine isothiocyanate and the RNA purified by centrifugation at 150,000 × g. The purified RNA (10µg)was electrophoresed in a formaldehyde-agarose gel and blotted onto Nytran-modified nylon by capillary action. The immobilized RNA was fixed by baking and the filter probed with a radioactive cDNA corresponding to part of the urokinase message. Stringency washes were performed at 65°C using 0.5 × saline sodium citrate and the blot exposed to x-ray film. Loading equalities were checked by reprobing the filter with a glyceraldehyde-3-phosphate dehydrogenase probe. The data are representative of three separate experiments.
surface phenomenon directed by UK receptors for the activator. We argued that, if this is the case for cultured colon cancer, then the amount of UK recovered from the cell surface should correlate with the abilities of the cells to degrade laminin and invade ECM. A good relationship22 (r2 = 0.9242) was found between laminin turnover and the amount of PA associated with the cell surface. Although less than 1 ng of UK could be dissociated from the surfaces of the WD cell types, between 5 and 13 ng/106 cells of the PA were recovered from their PD counterparts.22 These differences reflected a tenfold disparity in UK receptor numbers, as determined by radioreceptor assay after an acid22 pretreatment to rid the cells of endogenous UK. The binding sites bound the PA with high affinity (0.8 to 2.0 nM) probably via the amino acid sequence 12-32 of the UK A chain. These data, together with the
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FIG. 4. Southern blotting of the urokinase gene using DNA extracted from malignant colonic cells—analysis by digestion with Ncol. The cells were treated with 0.1 mg/ml proteinase K at 50°C and the DNA purified by extraction with a mixture (24:1) of phenol and chloroform/isoamyl alcohol. The purified DNA routinely had an A260/280 ratio in excess of 1.9. Equal amounts of DNA (10 µg) were digested with Ncol at 37°C overnight, and the fragments resolved in a 1% agarose gel. The gel was treated sequentially with acid and base and the DNA transferred to a modified nylon filter. The filter was baked and probed with a radioactive urokinase cDNA as described for Northern blotting.
FIG. 5. Southern blotting of the urokinase gene using DNA extracted from malignant colonic cells—analysis by Xmnl digestion. These were carried out as described for Figure 4 with the exception that Xmnl was substituted for Ncol.
binding sites are occupied are poorly responsive to the receptor antagonist.
BINDING SITES FOR PLASMINOGEN ON COLON CANCER CELLS findings that low molecular weight UK, which lacks part of the A chain, was unable to compete with high molecular weight UK25 for receptor binding, suggested that the receptors displayed on cultured colon cancer are identical to those reported by Stoppelli et al. 15 It is widely thought that these binding sites orient the active site of the PA toward the extracellular target, plasminogen. This provides an ideal mechanism for generating the proteolytic insult at the tumor cell surface where it is required. To substantiate the role of the UK receptors in laminin degradation and invasion further, a peptide (UK 12-32), corresponding to that part of the UK A chain required for the binding,26 was synthesized. The UK 12-32 peptide was effective in displacing radioactive UK from its receptor in a dose-dependent manner.22 These concentrations of UK 12-32 impaired the ability of colon cancer cells equipped with large numbers ( 105/cell) of receptors to degrade laminin22 and invade the extracellular matrix.23 It is worth noting that the sensitivity of the cells to the peptide also reflects the degree of occupation of the binding sites with the endogenous ligand. Thus, cells on which the majority of the receptors are "tagged" with secreted UK appear exquisitively sensitive to the peptide, whereas those on which the minority of the
There have been several reports of plasminogen receptors on the surface of a wide variety of cell types. 27 ' 28 Several investigators have speculated that plasminogen dependent proteolysis could be entirely a cell surface phenomenon governed by the display of receptors for both activator and zymogen. As applied to colon cancer, this concept presumes the presence of specific binding sites for the zymogen on the surface of the colonic cells. Theoretically, these receptors could lead to a build-up of the UK substrate at the cell surface, a situation that, by mass action, could favor the efficient conversion of the zymogen into plasmin, a process presumably mediated by receptor bound UK. A variation of this theme is that the plasminogen binding sites shuttle the zymogen to receptor immobilized UK (or vice versa), thereby juxtaposing both activator and substrate. Our preliminary data suggest the presence of specific binding sites for plasminogen on the surface of cultured colon cancer. These are specific insofar as a variety of competitors, including fibrinogen, transferrin, and myoglobin, did not compete with the radioactive zymogen for the binding to the cells. One WD and two PD cell lines bound 250 ± 60, 280 ± 50, and 210 ± 30 fmol radioactive plasminogen/106 cells, respectively, using an
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input concentration of 25 nM radioligand. Since the concentration of the plasminogen used in our in vitro invasion is approximately 100 nM, we would expect that under these conditions, the colonic tumor cell surface would be charged with a substantial amount of the zymogen.
LACK OF SENSITIVITY OF PLASMINOGENDEPENDENT PROTEOLYSIS DIRECTED BY RECEPTOR-BOUND UK TO PAI-1 The fact that the PD cells elaborated more of an inhibitor than their WD counterparts but in spite of this were more active in degrading laminin and invading ECM-coated filters suggested an insensitivity to the inhibitor. The mass of the inhibitor, as indicated by reverse zymography, was close to if not identical to that of PAI-1. Western blotting experiments and immunoprecipitation/reverse zymography studies revealed this inhibitor to be indistinguishable from PAI-1. 24 If the inhibitor was checking the activity of UK, then neutralizing the PAI-1 would be expected to enhance laminin turnover by PD cells charged with surface bound UK. Colon cancer cells supplemented with plasminogen were cultured on radioactive laminin in the presence or in the absence of neutralizing amounts of PAI-1 antibody. The antibody to PAI-1 did not augment laminin solubilization.24 Control experiments indicated that the antibody was without effect on UK expression. Studies showed, however, that the amount of active PAI-1 was, indeed, small and could, arguably, be of little biologic significance. To address this contention, commercially available PAI-1, activated with guanidine hydrochloride, was added, along with plasminogen, to colon cancer cells, equipped with 105 receptors/cell and grown on radioactive laminin. Although the inhibitor was highly effective in reducing the degradation of the glycoprotein brought about by authentic UK and plasminogen, laminin cleavage by receptor-positive cells cultured in the presence of the zymogen was unaltered.24 We have considered several possibilities that could account for these findings. Firstly, the UK receptor could sequester the PA in a location that is inaccessible to inhibitors, such as PAI-1. 18 Secondly, the receptor might shield the PA from inhibitors, akin to the protection provided to plasmin by its binding site.28 Thirdly, endogenous UK produced by the colon cancer cells could be a poor target for the PAI-1. Our data do not allow us to discriminate between these possibilities at the present time. It should be pointed out, however, that comprehensive studies by other laboratories have indicated that the UK receptor does not protect its PA from inhibitors. We have therefore speculated that these binding sites
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may compartmentalize the inhibitor in a location that is quite separate from that of UK immobilized to cell surface receptors. However, recent studies have indicated a third possible explanation for our data. It now appears that UK produced by some tumor cells can be phosphorylated in the region of the active site, rendering it insensitive to PAI-1. If this is the case for our PD colon cancer cells, it might provide a plausible explanation for the insensitivity of laminin degradation by HCT 116 colon cancer cells to this inhibitor. Clearly, further studies are needed to determine the mechanism responsible for this lack of sensitivity to PAI-1. Thus, in conclusion, it appears that the display of UK receptors by cultured colon cancer is a critical factor in determining their laminin degrading potential and very possibly their invasive phenotype. Acknowledgment. The authors are indebted to Drs. D. Collen and P. Declerck for their generous supply of antibodies.
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