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Cancer Cell Expression of Urokinase-Type Plasminogen Activator Receptor mRNA in Squamous Cell Carcinomas of the Skin

2001; Elsevier BV; Volume: 116; Issue: 3 Linguagem: Inglês

10.1046/j.1523-1747.2001.01241.x

1523-1747

John Rømer, Charles Pyke, Leif R. Lund, Keld Danø, Elisabeth Ralfkiær,

Skin and Cellular Biology Research

In this study we have used in situ hybridization with radiolabeled antisense RNA probes to examine the expression of mRNA for urokinase-type plasminogen activator and its receptor in histologic samples of squamous cell (n = 7) and basal cell (n = 7) carcinomas of the skin. Messenger RNA for both urokinase-type plasminogen activator and its receptor were expressed in all of the squamous cell carcinomas, but could not be detected in the basal cell carcinomas. In all of the seven squamous cell carcinomas a signal for urokinase-type plasminogen activator receptor mRNA was detected focally in well-differentiated cancer cells surrounding keratinized pearls, and in four specimens urokinase-type plasminogen activator receptor mRNA was in addition expressed by cancer cells at the edge of invasively growing strands of tumor. Urokinase-type plasminogen activator mRNA expression was found in virtually all the cancer cells of the squamous cell carcinomas, and importantly we found, by hybridizations for urokinase-type plasminogen activator and its receptor mRNA on adjacent sections of squamous cell carcinomas, that it was exactly the invading cancer cells that simultaneously expressed both these components required for plasmin-mediated proteolysis at the cell surface. We have previously shown that both urokinase-type plasminogen activator and its receptor mRNA are expressed by the leading-edge keratinocytes in regenerating epidermis during mouse skin wound healing, and that wound healing is impaired in mice made deficient in plasminogen by targeted gene disruption. We propose that there are similarities between the mechanisms of generation and regulation of extracellular proteolysis during skin re-epithelialization and squamous cell carcinoma invasion. The ability of the squamous carcinoma cells to mimic the ‘‘invasive’' phenotype of re-epithelializing keratinocytes may be one of the factors that make squamous cell carcinomas more aggressive tumors than basal cell carcinomas. In this study we have used in situ hybridization with radiolabeled antisense RNA probes to examine the expression of mRNA for urokinase-type plasminogen activator and its receptor in histologic samples of squamous cell (n = 7) and basal cell (n = 7) carcinomas of the skin. Messenger RNA for both urokinase-type plasminogen activator and its receptor were expressed in all of the squamous cell carcinomas, but could not be detected in the basal cell carcinomas. In all of the seven squamous cell carcinomas a signal for urokinase-type plasminogen activator receptor mRNA was detected focally in well-differentiated cancer cells surrounding keratinized pearls, and in four specimens urokinase-type plasminogen activator receptor mRNA was in addition expressed by cancer cells at the edge of invasively growing strands of tumor. Urokinase-type plasminogen activator mRNA expression was found in virtually all the cancer cells of the squamous cell carcinomas, and importantly we found, by hybridizations for urokinase-type plasminogen activator and its receptor mRNA on adjacent sections of squamous cell carcinomas, that it was exactly the invading cancer cells that simultaneously expressed both these components required for plasmin-mediated proteolysis at the cell surface. We have previously shown that both urokinase-type plasminogen activator and its receptor mRNA are expressed by the leading-edge keratinocytes in regenerating epidermis during mouse skin wound healing, and that wound healing is impaired in mice made deficient in plasminogen by targeted gene disruption. We propose that there are similarities between the mechanisms of generation and regulation of extracellular proteolysis during skin re-epithelialization and squamous cell carcinoma invasion. The ability of the squamous carcinoma cells to mimic the ‘‘invasive’' phenotype of re-epithelializing keratinocytes may be one of the factors that make squamous cell carcinomas more aggressive tumors than basal cell carcinomas. urokinase-type plasminogen activator urokinase-type plasminogen activator receptor Squamous cell carcinomas of the skin are generally well-differentiated tumors with invasive infiltrating growth and a significant potential for metastatic spread. Basal cell carcinomas of the skin, on the other hand, are characterized by a tendency to eroding growth and virtually no metastatic capacity. This difference in aggressiveness between the two skin malignancies has been related to the ability of squamous cell carcinomas to generate extracellular proteolysis by the urokinase-type plasminogen activator (uPA) pathway of plasminogen activation (Sappino et al., 1991Sappino A.P. Belin D. Huarte J. Hirschelscholz S. Saurat J.H. Vassalli J.D. Differential protease expression by cutaneous squamous and basal cell carcinomas.J Clin Invest. 1991; 88: 1073-1079Crossref PubMed Scopus (94) Google Scholar;Miller et al., 1992Miller S.J. Jensen P.J. Dzubow L.M. Lazarus G.S. Urokinase plasminogen activator is immunocytochemically detectable in squamous cell but not basal cell carcinomas.J Invest Dermatol. 1992; 98: 351-358Crossref PubMed Scopus (17) Google Scholar). Elevated levels of uPA that may lead to excessive generation of plasmin from plasminogen has been reported in several malignancies (Danø et al., 1999Danø K. Rømer J. Nielsen B.S. Bjørn S. Pyke C. Rygård J. Lund L.R. Cancer invasion and tissue remodeling – cooperation of protease systems and cell types.APMIS. 1999; 107: 120-127Crossref PubMed Scopus (283) Google Scholar;Mignatti and Rifkin, 1993Mignatti P. Rifkin D. Biology and biochemistry of proteinases in tumor invasion.Physiol Rev. 1993; 73: 161-195Crossref PubMed Scopus (1158) Google Scholar). Plasmin can potentially degrade many different proteins in the extracellular matrix, and may also be involved in the activation of other matrix-degrading proteases (Werb et al., 1977Werb Z. Mainardi C. Vater C.A. Harris E.D. Endogeneous activation of latent collagenase by rheumatoid synovial cells.N Engl J Med. 1977; 296: 1017-1023Crossref PubMed Scopus (526) Google Scholar;Okumura et al., 1997Okumura Y. Sato H. Seiki M. Kido H. Proteolytic activation of the precursor of membrane type 1 matrix metalloproteinase by human plasmin. A possible cell surface activator.Febs Lett. 1997; 402: 181-184https://doi.org/10.1016/s0014-5793(96)01523-2Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar) and latent growth factors (Mignatti and Rifkin, 1993Mignatti P. Rifkin D. Biology and biochemistry of proteinases in tumor invasion.Physiol Rev. 1993; 73: 161-195Crossref PubMed Scopus (1158) Google Scholar;Grainger et al., 1994Grainger D.J. Kemp P.R. Liu A.C. Lawn R.M. Metcalfe J.C. Activation of transforming growth factor-beta is inhibited in transgenic apolipoprotein (a) mice.Nature. 1994; 370: 460-462Crossref PubMed Scopus (333) Google Scholar). Increased activity of matrix-degrading proteases such as plasmin and matrix metalloproteinases enables metastasizing cancer cells to penetrate basement membranes and other barriers in the extracellular matrix. An important factor in the generation of plasmin-mediated pericellular proteolysis is the binding of uPA to its specific cell surface receptor (uPAR) (Danø et al., 1994Danø K. Behrendt N. Brünner N. Ellis V. Ploug M. Pyke C. The urokinase receptor: protein structure and role in plasminogen activation and cancer invasion.Fibrinolysis. 1994; 8: 189-203Crossref Scopus (286) Google Scholar;Vassalli, 1994Vassalli J.-D. The urokinase receptor.Fibrinolysis. 1994; 8: 172-181Crossref Scopus (96) Google Scholar). uPAR has been identified in vitro on the surface of many cell types of both normal and malignant origin. It is a highly glycosylated 55–60 kDa single-polypeptide-chain protein consisting of three domains. At the C-terminus uPAR is attached to the cell membrane by a phosphatidyl-inositol glycolipid anchor (Behrendt and Stephens, 1998Behrendt N. Stephens R.W. The urokinase receptor.Fibrinolysis Proteolysis. 1998; 12: 191-204Crossref Scopus (61) Google Scholar). Both active uPA and its virtually inactive proenzyme (pro-uPA) bind to uPAR with high affinity. Pro-uPA can be activated to uPA by plasmin while bound to uPAR, and receptor-bound uPA can activate plasminogen (Blasi et al., 1987Blasi F. Vassalli J.-D. Danø K. Urokinase-type plasminogen activator: proenzyme receptor and inhibitors.J Cell Biology. 1987; 104: 801-804Crossref PubMed Scopus (534) Google Scholar). Concomitant binding of pro-uPA and plasminogen to cell surfaces strongly accelerates plasmin generation (Ellis et al., 1991Ellis V. Behrendt N. Danø K. Plasminogen activation by receptor-bound urokinase: a kinetic study with both cell-associated and isolated receptor.J Biol Chem. 1991; 266: 12752-12758Abstract Full Text PDF PubMed Google Scholar), and surface-bound plasmin is protected from natural plasmin inhibitors. Cell surfaces therefore appear to be preferential sites for the urokinase pathway of plasminogen activation, requiring the concomitant expression of uPA and uPAR (Ellis et al., 1996Ellis V. Ploug M. Plesner T. Danø K. Gene expression and function of the cellular receptor for u-PA (u-PAR).in: Glas-Greenwalt P. Fibrinolysis in Disease. CRC Press, 1996Google Scholar). uPAR may also have functions not directly related to plasminogen activation (Blasi, 1999Blasi F. Proteolysis, cell adhesion, chemotaxis, and invasiveness are regulated by the u-PA-u-PAR-PAI-1 system.Thrombosis Haemostasis. 1999; 82: 298-304PubMed Google Scholar), such as signal transduction following receptor occupancy (Vassalli, 1994Vassalli J.-D. The urokinase receptor.Fibrinolysis. 1994; 8: 172-181Crossref Scopus (96) Google Scholar), as an adhesion receptor for vitronectin (Wei et al., 1994Wei Y. Waltz D.A. Rao N. Drummond R.J. Rosenberg S. Chapman H.A. Identification of the urokinase receptor as an adhesion receptor for vitronectin.J Biol Chem. 1994; 269: 32380-32388Abstract Full Text PDF PubMed Google Scholar;Høyer-Hansen et al., 1997Høyer-Hansen G. Behrendt N. Ploug M. Danø K. Preissner K.T. The intact urokinase receptor is required for efficient vitronectin binding: receptor cleavage prevents ligand interaction.Febs Lett. 1997; 420: 79-85https://doi.org/10.1016/s0014-5793(97)01491-9Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), and regulation of integrin function (Wei et al., 1996Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Regulation of integrin function by the urokinase receptor.Science. 1996; 273: 1551-1555Crossref PubMed Scopus (683) Google Scholar). In colon adenocarcinomas uPAR has been identified in both cancer cells and macrophages at invasive foci, whereas neighboring fibroblast-like cells express uPA (Grøndahl-Hansen et al., 1991Grøndahl-Hansen J. Ralfkiær E. Kirkeby L.T. Kristensen P. Lund L.R. Danø K. Localization of urokinase-type plasminogen activator in stromal cells in adenocarcinomas of the colon in humans.Am J Pathol. 1991; 138: 111-117PubMed Google Scholar;Pyke et al., 1991Pyke C. Kristensen P. Ralfkiær E. Grøndahl-Hansen J. Eriksen J. Blasi F. Danø K. Urokinase-type plasminogen activator is expressed in stromal cells and its receptor in cancer cells at invasive foci in human colon adenocarcinomas.Am J Pathol. 1991; 138: 1059-1067PubMed Google Scholar,Pyke et al., 1994Pyke C. Ralfkiær E. Rønne E. Høyer-Hansen G. Kirkeby L.T. Danø K. Immunohistochemical detection of the receptor for urokinase plasminogen activator in human colon cancer.Histopathology. 1994; 24: 131-138Crossref PubMed Scopus (106) Google Scholar). In ductal breast carcinomas uPAR is located in tumor-associated macrophages (Pyke et al., 1993Pyke C. Græm N. Ralfkiær E. Rønne E. Høyer-Hansen G. Brünner N. Danø K. Receptor for urokinase is present in tumor-associated macrophages in ductal breast carcinoma.Cancer Res. 1993; 53: 1911-1915PubMed Google Scholar), whereas uPA is expressed by myofibroblasts or in some rare cases by the cancer cells (Danø et al., 1994Danø K. Behrendt N. Brünner N. Ellis V. Ploug M. Pyke C. The urokinase receptor: protein structure and role in plasminogen activation and cancer invasion.Fibrinolysis. 1994; 8: 189-203Crossref Scopus (286) Google Scholar;Nielsen et al., 1996Nielsen B.S. Sehested M. Timshel S. Pyke C. Danø K. Messenger RNA for urokinase plasminogen activator is expressed in myofibroblasts adjacent to cancer cells in human breast cancer.Lab Invest. 1996; 74: 168-177PubMed Google Scholar;Johnsen et al., 1998Johnsen M. Lund L.R. Rømer J. Almholt K. Danø K. Cancer invasion and tissue remodeling: common themes in proteolytic matrix degradation.Curr Opin Cell Biol. 1998; 10: 667-671Crossref PubMed Scopus (321) Google Scholar). These data indicate that in some types of cancer tumor-associated stromal cells may be involved in the generation of extracellular proteolysis during cancer cell invasion (Johnsen et al., 1998Johnsen M. Lund L.R. Rømer J. Almholt K. Danø K. Cancer invasion and tissue remodeling: common themes in proteolytic matrix degradation.Curr Opin Cell Biol. 1998; 10: 667-671Crossref PubMed Scopus (321) Google Scholar). These studies have also shown, however, that the distinctive expression pattern of uPA and its receptor varies between different types of cancer and reflects the expression during certain non-neoplastic tissue remodeling processes in the tissue from which the cancer originates. In order to elucidate the mechanism involved in generation and regulation of plasmin-mediated extracellular proteolysis in skin carcinomas, we have now studied the expression of uPAR mRNA in squamous and basal cell skin carcinomas by in situ hybridization. T7 and T3 polymerase, pBluescript KS(+) plasmid vector and poly(A)quik Oligo dT-columns (Stratagene, CA). RNasin and DNase I (Promega, WI), K5 autoradiographic emulsion (Ilford, Cheshire, U.K.), Tissue-Tek from Miles, IN; [35S]UTPS (Amersham, U.K.); dithiothreitol and restriction endonucleases (Boehringer Mannheim, Germany). Routinely processed, formalin-fixed, and paraffin-embedded specimens from seven squamous cell carcinomas and seven basal cell carcinomas were drawn from the tissue bank at the Departments of Pathology at Gentofte and Herlev Hospitals. Fragments of human uPA cDNA and human uPAR cDNA were used to prepare the following subclones in pBluescript KS(+): pHUPA10, basepair 32-627 corresponding to the A-chain of uPA (EMBO database AQC number K03226); pHUR06, basepair 497-1081 corresponding to uPAR domain D2 (from loop 2) and D3.The specificity of pHUPA10 for uPA mRNA and pHUR06 for uPAR mRNA has previously been verified by use of nonoverlapping probes, which gave identical results in both cases (Pyke et al., 1991Pyke C. Kristensen P. Ralfkiær E. Grøndahl-Hansen J. Eriksen J. Blasi F. Danø K. Urokinase-type plasminogen activator is expressed in stromal cells and its receptor in cancer cells at invasive foci in human colon adenocarcinomas.Am J Pathol. 1991; 138: 1059-1067PubMed Google Scholar). Generation of 35S-UTP labeled antisense and sense probes from these plasmids by in vitro transcription using the relevant polymerase (T3 or T7) was performed as described byPyke et al., 1991Pyke C. Kristensen P. Ralfkiær E. Grøndahl-Hansen J. Eriksen J. Blasi F. Danø K. Urokinase-type plasminogen activator is expressed in stromal cells and its receptor in cancer cells at invasive foci in human colon adenocarcinomas.Am J Pathol. 1991; 138: 1059-1067PubMed Google Scholar. Before transcription the plasmids were linearized using the following restriction endonucleases: pHUPA10, SpeI or HindIII; pHUR06, SpeI or EcoRI. All probe preparations including both sense and antisense probes were adjusted to 2 × 106 cpm per μl. Probes were stored at -20°C until use. In situ hybridization was performed using the method described byPyke et al., 1991Pyke C. Kristensen P. Ralfkiær E. Grøndahl-Hansen J. Eriksen J. Blasi F. Danø K. Urokinase-type plasminogen activator is expressed in stromal cells and its receptor in cancer cells at invasive foci in human colon adenocarcinomas.Am J Pathol. 1991; 138: 1059-1067PubMed Google Scholar. Briefly, paraffin sections mounted on chrome-alum gelatine coated slides were heated to 60°C for 30 min, deparaffinized in xylene, and rehydrated. The slides were acid treated in 0.2 mol per l HCl, Proteinase K (5 μg per ml) treated, and subsequently fixed in 4% paraformaldehyde, followed by dehydration before the 35S-UTP labeled antisense or sense probes were added. The sections were then incubated overnight at 47°C in a hybridization solution containing radiolabeled RNA probe (80 pg per μl) in a solution of 50% deionized formamide, 10% dextran sulfate, tRNA (1 μg per ml), Ficoll 400 (0.02% wt/vol), 0.02% polyvinylpyrrolidone (wt/vol), 0.2% bovine serum albumin fraction V (wt/vol), 10 mM dithiothreitol (DTT), 0.3 M NaCl, 0.5 mM ethylenediamine tetraacetic acid, 10 mM Tris-Cl, and 10 mM NaPO4 (pH 6.8). After hybridization, slides were washed twice for 1 h at 50°C in a washing mix similar to the hybridization solution except that probe, dextran sulfate, DTT, and tRNA were omitted. The sections were then treated with RNase A (20 μg per ml), dehydrated, and air dried. Autoradiographic emulsion was applied and the sections were developed after 10–14 d of exposure. Analysis of archival paraffin-embedded tissue samples from cutaneous squamous and basal cell carcinomas by in situ hybridization showed uPAR mRNA and uPA mRNA expression in all the squamous cell carcinomas investigated (seven of seven). In contrast neither uPAR nor uPA mRNA could be detected in any of the basal cell carcinomas (seven of seven). The detection of uPAR and uPA mRNA was done with radiolabeled antisense RNA probes transcribed from plasmids containing either uPAR or uPA cDNA fragments, respectively. The corresponding sense RNA probes were used as a control of the specificity of these results and all of these control hybridizations were negative (Figure 1d). In situ hybridization analysis of the squamous cell carcinomas showed that the most abundant signal for uPAR mRNA is in cancer cells located centrally around keratinized pearls in well-differentiated areas of the lesions (arrows in Figure 1a, b, e, f). In a few cases weak uPAR mRNA expression was also detected in singly located cancer cells dispersed among inflammatory and stromal elements. In addition, in four of the specimens, strong uPAR mRNA expression was detected focally in cancer cells located at the tip of invading strands of tumor (Figure 2c-f). In addition to the signal for uPAR mRNA in cancer cells, weak hybridization was also detected in scattered macrophage-like stromal cells immediately surrounding the cancer cells or in the vicinity of the invasive front (results not shown). The hybridization signal for uPAR mRNA in these stromal cells was always weaker than that seen in the cancer cells in the same sections. uPAR mRNA was in some cases also detected in normal keratinocytes bordering minor ulcerations adjacent to or overlying the malignant lesions, but otherwise uPAR mRNA was not detected in any cells distant from the malignant lesions. In all the squamous cell carcinomas investigated, uPA mRNA was detected in virtually all of the cancer cells. The strongest hybridization signal for uPA mRNA was found in cancer cells located at the invading edge of the tumor (Figure 1c). A weak hybridization for uPA mRNA was also found in fibroblast-like cells located in the tumor stroma at the border between normal and malignant tissue (not shown). uPA mRNA was detected in nonmalignant keratinocytes at ulcerations adjacent to the tumor, where uPAR mRNA was also found, but otherwise uPA mRNA was not detected in any cells distant from the malignant lesions. Examination of adjacent sections from squamous cell carcinomas showed that the uPAR mRNA-expressing cancer cells at the invasive front also express uPA mRNA (Figure 2a-d). This coexpression of mRNAs for uPA and its receptor was found in all of the four specimens in which uPAR expression was detected in the invading cancer cells. uPA or uPAR mRNA were not detected in either cancer cells or stromal cells inside the lesions in any of the seven basal cell carcinomas (Figure 3). Two of the specimens showed signals for both uPA and uPAR mRNA in nonmalignant keratinocytes at the edge of cutaneous ulcerations above the tumor area. In one specimen uPA and uPAR mRNA were found focally in inflammatory cells beneath the lesion (not shown). uPA or uPAR mRNA could not be detected in cells in any other neighboring normal tissue by in situ hybridization analysis. By in situ hybridization we have shown that uPAR mRNA is expressed in squamous cell carcinomas of the skin. In all the squamous cell carcinomas investigated the signal for uPAR mRNA was expressed focally in well-differentiated cancer cells. Furthermore in four out of seven squamous cell carcinomas uPAR mRNA could also be detected in cancer cells at the edge of invasively growing strands of the tumors. In accordance with previous reports, uPA mRNA was found in virtually all neoplastic cells in the squamous cell carcinomas (Sappino et al., 1991Sappino A.P. Belin D. Huarte J. Hirschelscholz S. Saurat J.H. Vassalli J.D. Differential protease expression by cutaneous squamous and basal cell carcinomas.J Clin Invest. 1991; 88: 1073-1079Crossref PubMed Scopus (94) Google Scholar;Miller et al., 1992Miller S.J. Jensen P.J. Dzubow L.M. Lazarus G.S. Urokinase plasminogen activator is immunocytochemically detectable in squamous cell but not basal cell carcinomas.J Invest Dermatol. 1992; 98: 351-358Crossref PubMed Scopus (17) Google Scholar). Importantly, hybridization for uPA and uPAR mRNA on adjacent sections showed simultaneous expression of both these components in cancer cells located at the edge of invading squamous cell strands. In contrast to the expression in squamous cell carcinomas, we did not detect expression of either uPA or uPAR mRNA in any of the seven basal cell carcinoma specimens we investigated. There are no previous reports on histologic studies of uPAR expression in either squamous or basal cell carcinomas. With respect to uPA our findings are in agreement with a study bySappino et al., 1991Sappino A.P. Belin D. Huarte J. Hirschelscholz S. Saurat J.H. Vassalli J.D. Differential protease expression by cutaneous squamous and basal cell carcinomas.J Clin Invest. 1991; 88: 1073-1079Crossref PubMed Scopus (94) Google Scholar, who did not detect uPA mRNA in basal cell carcinomas, but they are in contrast to a study bySpiers et al., 1996Spiers E.M. Lazarus G.S. Lyons-Giordano B. Expression of plasminogen activators in basal cell carcinoma.J Pathol. 1996; 178: 290-296https://doi.org/10.1002/(sici)1096-9896(199603)178:3<290::aid-path472>3.3.co;2-wCrossref PubMed Scopus (0) Google Scholar reporting expression of uPA mRNA in basal cell carcinomas. We do not know the reason for the discrepancy between the latter study and ours. The direct comparisons done both bySappino et al., 1991Sappino A.P. Belin D. Huarte J. Hirschelscholz S. Saurat J.H. Vassalli J.D. Differential protease expression by cutaneous squamous and basal cell carcinomas.J Clin Invest. 1991; 88: 1073-1079Crossref PubMed Scopus (94) Google Scholar and in this study do, however, show that the level of uPA mRNA expression is much higher in squamous cell carcinomas than in basal cell carcinomas of the skin. This conclusion is also supported by an immuno-histochemical study showing the presence of uPA protein in squamous cell carcinomas but not detectable amounts in basal cell carcinomas (Miller et al., 1992Miller S.J. Jensen P.J. Dzubow L.M. Lazarus G.S. Urokinase plasminogen activator is immunocytochemically detectable in squamous cell but not basal cell carcinomas.J Invest Dermatol. 1992; 98: 351-358Crossref PubMed Scopus (17) Google Scholar). Furthermore, in a recent study of skin tumor extracts by enzyme-linked immunosorbent assay it was shown that both uPA and uPAR are present at significantly lower levels in basal cell carcinomas compared with squamous cell carcinomas and malignant melanomas (Maguire et al., 2000Maguire T. Chin D. Soutar D. Duffy M.J. Low levels of urokinase plasminogen activator components in basal cell carcinoma of the skin.Int J Cancer. 2000; 85: 457-459https://doi.org/10.1002/(sici)1097-0215(20000215)85:4<457::aid-ijc2>3.3.co;2-yCrossref PubMed Google Scholar). Both uPA and uPAR expression thus appears to be much more abundant in squamous cell carcinomas than in basal cell carcinomas. A correlation between high expression of both uPA and uPAR in tumors and their aggressiveness has been demonstrated in several experimental models of invasive growth (Ossowski et al., 1991Ossowski L. Clunie G. Masucci M.T. Blasi F. In vivo paracrine interaction between urokinase and its receptor – effect on tumour cell invasion.J Cell Biol. 1991; 115: 1107-1112Crossref PubMed Scopus (198) Google Scholar;Crowley et al., 1993Crowley W.C. Cohen R.L. Lucas B.K. Liu G. Shuman M.A. Levinson A.D. Prevention of metastasis by inhibition of the urokinase receptor.Proc Natl Acad Sci USA. 1993; 90: 5021-5025Crossref PubMed Scopus (360) Google Scholar;Kook et al., 1994Kook Y.H. Adamski J. Zelent A. Ossowski L. The effect of antisense inhibition of urokinase receptor in human squamous cell carcinoma on malignancy.EMBO J. 1994; 13: 3983-3991Crossref PubMed Scopus (174) Google Scholar;Shapiro et al., 1996Shapiro R.L. Duquette J.G. Roses D.F. et al.Induction of primary cutaneous melanocytic neoplasms in urokinase-type plasminogen activator (uPA) -deficient and wild-type mice: cellular blue nevi invade but do not progress to malignant melanoma in uPA-deficient animals.Cancer Res. 1996; 56: 3597-3604PubMed Google Scholar;Kim et al., 1998Kim J. Yu W. Kovalski K. Ossowski L. Requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay.Cell. 1998; 94: 353-362Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). On this background we suggest that the higher metastatic potential of squamous cell carcinomas compared with basal cell carcinomas may be related to the coexpression of the two components required for cell surface plasminogen activation in invading squamous carcinoma cells. The coexpression of mRNAs for uPA and uPAR in invading squamous carcinoma cells means that uPA released from the cancer cells subsequently is bound to the receptor in an autocrine manner. Assembly of the components required for generation of uPA-mediated cell surface proteolysis in this way is apparently different from the situation during invasive growth of colon and breast cancer. In colon adenocarcinomas both cancer cells and macrophages express uPAR, whereas neighboring fibroblast-like cells express uPA (Grøndahl-Hansen et al., 1991Grøndahl-Hansen J. Ralfkiær E. Kirkeby L.T. Kristensen P. Lund L.R. Danø K. Localization of urokinase-type plasminogen activator in stromal cells in adenocarcinomas of the colon in humans.Am J Pathol. 1991; 138: 111-117PubMed Google Scholar;Pyke et al., 1991Pyke C. Kristensen P. Ralfkiær E. Grøndahl-Hansen J. Eriksen J. Blasi F. Danø K. Urokinase-type plasminogen activator is expressed in stromal cells and its receptor in cancer cells at invasive foci in human colon adenocarcinomas.Am J Pathol. 1991; 138: 1059-1067PubMed Google Scholar,Pyke et al., 1994Pyke C. Ralfkiær E. Rønne E. Høyer-Hansen G. Kirkeby L.T. Danø K. Immunohistochemical detection of the receptor for urokinase plasminogen activator in human colon cancer.Histopathology. 1994; 24: 131-138Crossref PubMed Scopus (106) Google Scholar). In ductal breast carcinomas uPAR is located in tumor-associated macrophages (Pyke et al., 1993Pyke C. Græm N. Ralfkiær E. Rønne E. Høyer-Hansen G. Brünner N. Danø K. Receptor for urokinase is present in tumor-associated macrophages in ductal breast carcinoma.Cancer Res. 1993; 53: 1911-1915PubMed Google Scholar), whereas uPA is expressed by myofibroblasts or in some rare cases by the cancer cells (Nielsen et al., 1996Nielsen B.S. Sehested M. Timshel S. Pyke C. Danø K. Messenger RNA for urokinase plasminogen activator is expressed in myofibroblasts adjacent to cancer cells in human breast cancer.Lab Invest. 1996; 74: 168-177PubMed Google Scholar). In the last two types of cancer, secreted uPA therefore binds to uPAR in a paracrine fashion. These observations illustrate that different cancer types have different expression patterns of the molecules needed for localized generation of plasmin activity. This is also the case for the expression of several matrix-degrading metalloproteases, and there is increasing evidence for similarities in the pattern of expression of molecules generating or regulating proteolysis between certain types of cancers and certain tissue remodeling processes (Johnsen et al., 1998Johnsen M. Lund L.R. Rømer J. Almholt K. Danø K. Cancer invasion and tissue remodeling: common themes in proteolytic matrix degradation.Curr Opin Cell Biol. 1998; 10: 667-671Crossref PubMed Scopus (321) Google Scholar). Examples are ductal mammary carcinoma and postlactational mammary gland involution (Lund et al., 1996Lund L.R. Rømer J. Thomasset N. et al.Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways.Development. 1996; 122: 181-193Crossref PubMed Google Scholar,Lund et al., 2000Lund L.R. Bjørn S.F. Sternlicht M.D. et al.Lactational competence and involution of the mouse mammary gland require plasminogen.Development. 2000; 127: 4481-4492PubMed Google Scholar;Nielsen et al., 1996Nielsen B.S. Sehested M. Timshel S. Pyke C. Danø K. Messenger RNA for urokinase plasminogen activator is expressed in myofibroblasts adjacent to cancer cells in human breast cancer.Lab Invest. 1996; 74: 168-177PubMed Google Scholar), colon adenocarcinoma and epithelial cell shedding into the gastro-intestinal tract (Larsson et al., 1984Larsson L.I. Skriver L. Nielsen L.S. Grøndahl-Hansen J. Kristensen P. Danø K. Distribution of urokinase-type plasminogen activator immunoreactivity in the mouse.J Cell Biol. 1984; 98: 894-903Crossref PubMed Scopus (138) Google Scholar;Pyke et al., 1991Pyke C. Kristensen P. Ralfkiær E. Grøndahl-Hansen J. Eriksen J. Blasi F. Danø K. Urokinase-type plasminogen activator is expressed in stromal cells and its receptor in cancer cells at invasive foci in human colon adenocarcinomas.Am J Pathol. 1991; 138: 1059-1067PubMed Google Scholar), and squamous cell skin cancer and skin wound healing as discussed below. The distinctive and simultaneous expression of both uPA and uPAR mRNA in cancer cells at invasive foci in squamous cell skin cancer is thus very similar to the expression of uPA and uPAR mRNA in leading-edge keratinocytes during re-epithelialization of skin wounds (Grøndahl-Hansen et al., 1988Grøndahl-Hansen J. Lund L.R. Ralfkiær E. Ottevanger V. Danø K. Urokinase- and tissue-type plasminogen activators in keratinocytes during wound reepithelialization in vivo.J Invest Dermatol. 1988; 90: 790-795Abstract Full Text PDF PubMed Google Scholar;Rømer et al., 1991Rømer J. Lund L.R. Eriksen J. et al.Differential expression of urokinase-type plasminogen activator and its type-1 inhibitor during healing of mouse skin wounds.J Invest Dermatol. 1991; 97: 803-811Abstract Full Text PDF PubMed Google Scholar,Rømer et al., 1994Rømer J. Lund L.R. Eriksen J. Pyke C. Kristensen P. Danø K. The receptor for urokinase-type plasminogen activator is expressed by keratinocytes at the leading edge during re-epithelialization of mouse skin wounds.J Invest Dermatol. 1994; 102: 519-522Abstract Full Text PDF PubMed Google Scholar). Furthermore the matrix metalloproteases interstitial collagenase, stromelysin-1, collagenase-3, and gelatinase B in both squamous cell skin cancer (Polette et al., 1991Polette M. Clavel C. Muller D. Abecassis J. Binninger P. Birembaut P. Detection of mRNAs encoding collagenase I and stromelysin-2 in carcinomas of the head and neck by in situ hybridization.Invasion Metastasis. 1991; 11: 76-83PubMed Google Scholar;Pyke et al., 1992Pyke C. Ralfkiær E. 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Cell–matrix interactions modulate interstitial collagenase expression by human keratinocytes actively involved in wound healing.J Clin Invest. 1993; 92: 2858-2866Crossref PubMed Scopus (266) Google Scholar,Saarialho-Kere et al., 1994Saarialho-Kere U.K. Pentland A.P. Birkedal-Hansen H. Parks W.C. Welgus H.G. Distinct populations of basal keratinocytes express stromelysin-1 and stromelysin-2 in chronic wounds.J Clin Invest. 1994; 94: 79-88Crossref PubMed Scopus (197) Google Scholar;Salo et al., 1994Salo T. Makela M. Kylmaniemi M. Autio-Harmainen H. Larjava H. Expression of matrix metalloproteinase-2 and -9 during early human wound healing.Lab Invest. 1994; 70: 176-182PubMed Google Scholar;Madlener et al., 1998Madlener M. Parks W.C. Werner S. Matrix metalloproteinases (MMPs) and their physiological inhibitors (TIMPs) are differentially expressed during excisional skin wound repair.Exp Cell Res. 1998; 242: 201-210https://doi.org/10.1006/excr.1998.4049Crossref PubMed Scopus (273) Google Scholar;Lund et al., 1999Lund L.R. Rømer J. Bugge T.H. et al.Functional overlap between two classes of matrix degrading proteases in wound healing.EMBO J. 1999; 18: 4645-4656https://doi.org/10.1093/emboj/18.17.4645Crossref PubMed Scopus (221) Google Scholar) are expressed by the epithelial cells, whereas gelatinase A and stromelysin-3 in both cases are expressed in fibroblastic stromal cells (Wolf et al., 1992Wolf C. Chenard M.P. Durand de Grossouvre P. Bellocq J.P. Chambon P. Basset P. Breast-cancer-associated stromelysin-3 gene is expressed in basal cell carcinoma and during cutaneous wound healing.J Invest Dermatol. 1992; 99: 870-872Abstract Full Text PDF PubMed Google Scholar;Pyke et al., 1992Pyke C. Ralfkiær E. Huhtala P. Hurskainen T. Danø K. Tryggvason K. Localization of messenger RNA for Mr 72,000 and 92, 000 type IV collagenases in human skin cancers by in situ hybridization.Cancer Res. 1992; 52: 1336-1341PubMed Google Scholar;Madlener et al., 1998Madlener M. Parks W.C. Werner S. Matrix metalloproteinases (MMPs) and their physiological inhibitors (TIMPs) are differentially expressed during excisional skin wound repair.Exp Cell Res. 1998; 242: 201-210https://doi.org/10.1006/excr.1998.4049Crossref PubMed Scopus (273) Google Scholar;Asch et al., 1999Asch P.H. Basset P. Roos M. Grosshans E. Bellocq J.P. Cribier B. Expression of stromelysin 3 in keratoacanthoma and squamous cell carcinoma.Am J Dermatopathol. 1999; 21: 146-150Crossref PubMed Scopus (35) Google Scholar;Lund et al., 1999Lund L.R. Rømer J. Bugge T.H. et al.Functional overlap between two classes of matrix degrading proteases in wound healing.EMBO J. 1999; 18: 4645-4656https://doi.org/10.1093/emboj/18.17.4645Crossref PubMed Scopus (221) Google Scholar;Tsukifuji et al., 1999Tsukifuji R. Tagawa K. Hatamochi A. Shinkai H. Expression of matrix metalloproteinase-1-2 and -3 in squamous cell carcinoma and actinic keratosis.Br J Cancer. 1999; 80: 1087-1091Crossref PubMed Scopus (60) Google Scholar). A crucial difference between skin repair and malignancy, however, is that the expression of uPA, uPAR, and the matrix metalloproteases in the migrating keratinocytes is downregulated when the skin wound is healed, whereas a similar downregulation appears to be lacking in the squamous cell carcinoma cells. This is in accordance with the proposal by Dvorak, based on observations of stromal–epithelial cell interactions, that cancer can be considered as wounds that do not heal (Dvorak, 1986Dvorak H.F. Tumors. Wounds that do not heal.N Engl J Med. 1986; 315: 1650-1659Crossref PubMed Scopus (3214) Google Scholar). Studies on skin wound healing are therefore likely to contribute to our understanding of squamous cell skin cancer. We recently found that skin wound repair is severely impaired in mice made deficient in plasminogen by targeted gene disruption (Rømer et al., 1996Rømer J. Bugge T.H. Pyke C. Lund L.R. Flick M.J. Degen J. Danø K. Impaired wound healing in mice with a disrupted plasminogen gene.Nature Med. 1996; 2: 287-292Crossref PubMed Scopus (466) Google Scholar), and completely arrested when the plasminogen-deficient mice were treated with the metalloprotease inhibitor Galardin (Lund et al., 1999Lund L.R. Rømer J. Bugge T.H. et al.Functional overlap between two classes of matrix degrading proteases in wound healing.EMBO J. 1999; 18: 4645-4656https://doi.org/10.1093/emboj/18.17.4645Crossref PubMed Scopus (221) Google Scholar). These results definitively prove that both plasminogen and certain matrix metalloproteases participate in the restoration of an intact epidermal layer following acute skin injury. This is probably reflecting that keratinocytes in the epithelial outgrowth use plasmin and certain metalloproteases to proteolytically dissect their way through the extracellular matrix (Bugge et al., 1996Bugge T.H. Kombrinck K.W. Flick M.J. Daugherty C.C. Danton M.J.S. Degen J.L. Loss of fibrinogen rescues mice from the pleiotropic effects of plasminogen deficiency.Cell. 1996; 87: 709-719Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar;Rømer et al., 1996Rømer J. Bugge T.H. Pyke C. Lund L.R. Flick M.J. Degen J. Danø K. Impaired wound healing in mice with a disrupted plasminogen gene.Nature Med. 1996; 2: 287-292Crossref PubMed Scopus (466) Google Scholar;Lund et al., 1999Lund L.R. Rømer J. Bugge T.H. et al.Functional overlap between two classes of matrix degrading proteases in wound healing.EMBO J. 1999; 18: 4645-4656https://doi.org/10.1093/emboj/18.17.4645Crossref PubMed Scopus (221) Google Scholar). A similar function of the plasminogen activation system is likely in squamous skin cancer. The ability of the squamous cell carcinomas to simultaneously produce uPA and uPAR may therefore be crucial for their more aggressive and metastatic behavior. Further support for such a role of plasminogen activation in metastasis comes from studies showing that metastasis of experimental mammary tumors is impaired in plasminogen-deficient mice (Bugge et al., 1998Bugge T.H. Lund L.R. Kombrinck K.W. et al.Reduced metastasis of polyoma virus middle T antigen-induced mammary cancer in plasminogen-deficient mice.Oncogene. 1998; 16: 3097-3104Crossref PubMed Scopus (136) Google Scholar). We are grateful to Dr. R. Miskin for the gift of uPA cDNA. The excellent technical assistance of Jette Mandelbaum, Kirsten Lund Jacobsen, and Britt Nagel Epstrup is gratefully acknowledged. The Danish Biotechnology Program, Center for Medical Biotechnology, and the Danish Cancer Society supported this project financially.