Proteases and cancer invasion: from belief to certainty: AACR Meeting on Proteases and Protease...

Meeting report

Proteases and cancer invasion: from belief to certainty

AACR Meeting on Proteases and Protease Inhibitors in Cancer,Nyborg, Denmark, 14^18 June 1998

Francesco Blasi a;*, M. Patrizia Stoppelli b

a DIBIT, H. San Ra¡aele, Milan, Italyb International Institute of Genetics and Biophysics, CNR, Naples, Italy

Received 23 September 1998; accepted 12 November 1998

1. Introduction

A large number of scholars in the ¢eld of proteasesand cancer arrived in the small town of Nyborg onthe island of Fynen (Denmark) on 14 June, the veryday in which the beautiful and impressive bridge thatconnects Sealand to Fynen was o¤cially opened totra¤c. The reason for the gathering was obviouslynot simply to watch the bridge, nor the Royals arriv-ing for the o¤cial ceremony, nor the beautiful col-lection of old cars that took the opportunity to crossthe bridge on the ¢rst day, nor to assist at the lasttrips of the ferries. It was in fact to discuss the role ofproteases in cancer, and to consider possibilities toattack cancer by blocking them. That the phenotypesof multiple human cancers very much depend on thepresence of proteases was ¢rst suggested over 50years ago [1]. In recent years, however, the elucida-tion of the great complexity of this system has re-sulted in a rigorous demonstration of this assertion.The opening of the Fynen bridge, indeed, signaledthe moment of general agreement that proteases rep-resent a truly valid target in future cancer therapy.The meeting covered most of the known proteases,but the main focus was on metalloproteases, plasmi-nogen activators and cathepsins [2,3].

That proteases have functions other than cleaving/clearing unwanted proteins is a fact. The largeamount of biochemical evidence can now be coupledwith in vivo tests of the e¡ects of their absence oroverexpression in various physiological and patho-logical processes, including cancer. In particular, ex-tracellular proteases modulate cellular behavior byaltering cell surface-extracellular matrix interactionsand regulating the processing of growth factors. Inaddition, it is increasingly clear that these enzymescan play non-proteolytic roles through the interac-tion of non-catalytic domains (EGF-like, disintegrin,hemopexin etc.) with other proteins, thereby regulat-ing cell migration and adhesion, egg-sperm interac-tions, angiogenesis, and the cell cycle. A large bodyof evidence supports a role for proteases in prolifer-ation, migration, invasion and apoptosis, likelythrough complex mechanisms which are still underinvestigation.

A major outcome of this meeting was the realiza-tion that proteases are relevant in cancer develop-ment and progression, as well as in stroma-tumorcell interactions.

2. Matrix metalloproteases (MMPs)

The family of MMPs consists of approximately 20members. Some of these enzymes are both secreted

0304-419X / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved.PII: S 0 3 0 4 - 4 1 9 X ( 9 8 ) 0 0 0 3 4 - 1

* E-mail : [email protected]

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and membrane-attached (MT-MMPs). They are allproduced in an inactive form (pro-MMPa) [3]. Amajor question is whether the synthetic process re-quires cell surface binding and, if so, how this isachieved. In ¢broblasts, MMP2 (pro-gelatinase A)is activated at the cell surface (Gillian Murphy, Nor-wich, UK). Membrane-bound MT-MMP1 has animportant role in the activation of pro-MMP2, aprocess in which TIMP-2 is directly involved (Fig.1). The pro-form of MT-MMP1 is activated proteo-lytically, either in Golgi by furin or on the cell sur-face by a serine protease such as plasmin. Activationallows binding of a TIMP-2 molecule to the cell sur-face through its C-terminal domain. The C-terminusof TIMP-2 then acts as an anchor for the C-terminalhemopexin domain of pro-MMP2. The surface MT-MMP1-TIMP-2 complex allows pro-MMP2 process-ing by an adjacent active MT-MMP1 molecule, gen-erating active gelatinase A. So, TIMP-2 has a dualfunction: at low concentrations, it binds MT-MMP1,thereby favoring pro-MMP2 binding to the surface.At higher concentrations, it inhibits activated MT-MMP1.

In the embryo, MMP9 (gelatinase B) has a majorangiogenic e¡ect which resulted in a de¢ciency inosteogenesis when the gene was removed by homol-ogous recombination (Zena Werb, S. Francisco, CA,USA). MMP9 is expressed by trophoblasts duringimplantation (while expression of inhibitors is con-¢ned to adjacent cells), as well as by developing kid-ney and hippocampus, osteoclast, and in£ammatorycells. Werb also reported that the transcription fac-tor, Ets-2, is essential for MMP9 expression, primar-ily in trophoblasts. During embryo implantation,MMP9 appears to be important in determining em-bryo size and decidual vascularization, as re£ected bythe e¡ects of neutralizing antibodies and TIMPs, aswell as placental and embryo behavior in TIMP-1overexpressing mice. In fact, in MMP93=3 mice theplacenta implanted normally despite a profound de-crease in vascularization around the ectoplacentalcone. However, MMP93=3 mice have shorter bones(10% less) because of defective endochondral boneformation. In normal mice, MMP9 is expressed ingrowing bone by osteoclasts and mononuclear cellsat the bone-hypertrophic cartilage interface.MMP93=3 mice show a large increase in hyper-trophic cartilage apparently due to delayed apoptosis

of hypertrophic chondrocytes, decreased or delayeddegradation of hypertrophic cartilage matrix, and amarked reduction of relevant angiogenesis.

To further understand the role of MMP9 in vas-cularization, Werb's laboratory performed in vitroangiogenic assays with growth plate fragments incollagen gels using endothelial cells from wild typeand MMP93=3 mice. Also in this system, the strongdelay in neo-angiogenesis associated with a lack ofMMP9 was reversed by addition of the puri¢ed en-zyme. Although the molecular mechanism underlyingthese e¡ects is partly obscure, these and other resultspoint to an angiogenic function of MMP9.

MMP9 plays a direct role in epithelial tumorigen-esis (Werb). Mice transgenic for HPV16 represent amodel of epithelial tumorigenesis, in particular of theskin (K14-HPV16). By 1 month of age, they all de-velop hyperplasia, by 3 months dysplasia (loss ofterminal di¡erentiation), and, by 6 months squamouscell carcinomas of various di¡erentiation levels. Inthese lesions, MMP9 was detected in macrophagesand around the increasingly abundant blood vesselspresent in hyperplastic lesions that precede malignantconversion. At this stage, there is also an enormousincrease in in¢ltrating mast cells (producers of chym-ase, an activator of pro-MMP9). In contrast, inMMP93=3 mice, no progression to epithelial carcino-genesis occurs.

Although overexpressed in human cancers, colla-genase 3 (MMP13) is neither produced by normalbreast nor by epithelial breast cancer cells, but isdetected in the stromal component of breast tumors(C. Lopez-Otin, Oviedo, Spain). Expression ofMMP13 by ¢broblasts can be reproduced in ¢bro-blasts/MCF-7 cancer cell co-cultures, and multiplepolypeptides (e.g. IL-1, TGF-L) released by tumorcells can induce MMP13 in ¢broblasts. Therefore,the induction of this metalloprotease represents an-other example of the complex interactions occurringbetween stromal and cancer cells in tumor progres-sion.

Matrilysin (MMP7) is a small metalloprotease ex-pressed in human colon carcinomas by the tumorepithelium. Matrilysin is expressed focally and at alow level in benign tumors, whereas it is overex-pressed in carcinomas. Lynn Matrisian (Nashville,TN, USA) has employed the Min mouse, which ischaracterized by the presence of multiple intestinal

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neoplasms and a germ line APC mutation, as a mod-el to study the role of MMP7 in tumor development.In tumors generated in these mice, MMP7 was ex-pressed in the tumor epithelium. To test the rele-vance of MMP7 to tumor progression, Matrisiancrossed MMP73=3 mice (which do not exhibit anobvious phenotype) with Min mice and observed a60% reduction in the number of intestinal tumorswhich, in turn, were associated with slower growth.Interestingly, in the Min mice, the metalloproteaseinhibitor, Batimastat, caused a V50% reduction inthe number of developing tumors.

Another aspect of stromal/tumor cells interactionis represented by the studies on stromelysin 3(MMP11) in tumor progression reported by M.

Christine Rio (Strasbourg, France). In invasive car-cinomas, MMP11 is expressed by peritumoral ¢bro-blast-like cells. It is a marker of tumor progression,and its presence correlates with a bad prognosis incolon and breast cancer. MMP113=3 mice are fertile,have no obvious phenotype and show a reduced in-cidence of tumors after DMBA exposure. Stroma/tumor interaction in tumorigenesis was addressedby coinjecting mouse ¢broblasts (either wild type orMMP113=3) and human MCF7 tumor cells intonude mice. When MMP113=3 ¢broblasts wereused, fewer, smaller tumors were observed. Reliabledata show that MMP11 acts during the very earlystages of tumorigenesis, by favoring the implantationof tumor cells. A possible mechanism might be that

Fig. 1. Activation of pro-MMP2, pro-uPA and plasminogen. Role of the cell surface. The receptor for plasminogen is not depicted. Itis in fact always a protein with a carboxy-terminal lysine.

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of releasing growth factors from the extracellularmatrix, hence favoring tumor growth in a paracrinemanner. Indeed, when tumor cells embedded in anarti¢cial extracellular matrix (Matrigel) were injectedinto the mouse, matrix depletion of low molecularweight proteins strongly reduced the tumor incidenceeven in the presence of MMP11 producing ¢bro-blasts. This system may be exploited to identify fac-tors important for tumor cell implantation. The re-construction experiment has not yet been performed,i.e. adding back di¡erent latent growth factors andthen measuring tumor formation. In fact, the role ofthe enzymatic activity of MMP11 has not really beenaddressed: is it really required? MMP11 substratesare not known: might they be unprocessed growthfactors? Is the C-terminal hemopexin domain ofMMP11 involved in non-proteolytic but functionallyrelevant interactions?

Mammary gland branching morphogenesis is anexcellent system to study epithelial-mesenchymalconversion and associated changes in the pattern ofgene expression, as well as to understand the natureof the changes that occur in breast cancer. The mam-mary gland passes from a virgin state (no milk pro-duction) to a lactating stage (gain of function) toinvolution (loss of function). Mina J. Bissell (Berke-ley, CA, USA) described tools and experimentsaimed at understanding the role of the basementmembrane in mammary gland di¡erentiation. Forthese dramatic changes to occur, the basement mem-brane underlying the acinar structure and productionof stromelysin 1 (MMP3) play an important role.Bissell's laboratory has studied both constitutiveand inducible (tet-regulated) MMP3 transgenicmice. These animals express MMP3 in mammaryepithelial cells. The mammary glands of constitu-tively expressing mice reveal early budding (even inthe virgin state), progression into mammary tumors,metastatic adenocarcinomas and ¢brosarcomas, andgenomic instability. Induced MMP3 expression inmammary epithelial cells resulted in a malignantmammary phenotype, invasion of matrigel by highMMP3 producing cells, upregulation of KGF (in-volved in the growth of mammary cells), desmopla-kin and E-cadherin (substrates of MMP3). Underconditions of MMP3 induction in mammary cells,a marked increase in the stromal level of MMP3was also observed. Interestingly, Bissell's laboratory

has developed an in vitro branching system by mam-mary organ cultures. In this system, growth factorsare required to induce branching, and MMP3, but noother protease, could substitute for them. Thus,MMP3 has a primary role in branching as well asin tumorigenesis. Bissell proposed that understandingthe nature and signi¢cance of the tridimensionality ofbranching will shed light on the molecular alterationsunderlying breast cancer. In her view, one mightimagine the role of di¡erent molecules in promotingdirectionality of branching, which might be gener-ated in situ from precursors activated by speci¢c pro-tease(s).

TIMP-2 (tissue inhibitor of metalloprotease-2) isubiquitously expressed in the adult and restricted tothe maternal trophoblast in the embryo. Overexpres-sion of TIMP-2 in malignant cells following trans-fection results in reduction of malignancy and inva-siveness after subcutaneous injection of these cells inmice. (Yves A. DeClerck, Los Angeles, CA, USA).

The e¡ect of TIMP-1 in oncogene-induced sponta-neous tumorigenesis was studied by Rama Khoka(Toronto, Canada). Transgenic mice expressing theSV40 T antigen (TAg) in the liver produce hepato-carcinomas with a predictable pattern. Progression ofthe process from normal liver, to hyperplasia to hep-atocellular non-metastatic carcinoma can be sequen-tially studied. Overexpression of TIMP-1 driven byan MHC promoter caused a reduction of MMP2activity in liver and delayed tumorigenesis associatedwith a reduction in size and number of tumors. Therewas also inhibition of angiogenesis without e¡ects onextracellular matrix composition or hepatocyte apop-tosis. A possible explanation is that TIMP inhibitorye¡ects on tumor growth are a result of its ability toa¡ect the bioactivity of growth factors. IGF-II is animportant factor in TAg tumorigenesis, signaling viathe IGF-I receptor tyrosine kinase and regulated bythe IGF binding proteins. Overexpression of TIMPhad no e¡ect on TAg, p53 or RB expression. But thebioactivity of IGF-II was reduced, as shown by areduction of IGF-I receptor phosphorylation andof the phosphorylation of certain downstream com-ponents of this pathway. Apparently, TIMP delaysthe MMP-dependent degradation of an IGF-I bind-ing protein, IGFBP-3.

So, TIMP-1 appears to have early e¡ects on tumordevelopment, to act on substrates di¡erent than ex-

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tracellular matrix (ECM) molecules, and to targetgrowth factor activity. In further attempts to testthe validity of this hypothesis, it will be importantto carry out reconstruction experiments with freeIGF and to test tumorigenicity in TAg overexpress-ing mice de¢cient for the IGFBP-33=3 gene.

First generation synthetic inhibitors of MMPs arenow in clinical trials (Peter D. Brown, Oxford, UK).In patients with inoperable gastric cancer, treatmentwith Marimastat resulted in a decrease of tumor sizeand hemorrhage. Preclinical studies combining chem-otherapy with Marimastat are under way in an e¡ortto assess the action spectrum of action of this newagent.

3. Plasminogen, plasminogen activators (PA),plasminogen inhibitors and receptors

Plasminogen activators (urokinase, uPA, and tis-sue plasminogen activator, tPA), the enzymes thatactivate plasminogen (Plg) into plasmin, have beenlong studied in cancer progression and invasion. Ofthe two activators, uPA is important in human can-cer, wound healing, tissue remodeling and in the im-mune response. uPA interacts with a high-a¤nityreceptor (uPAR), but not all uPA functions may re-quire this interaction. Interestingly, in addition toe¡ects mediated by the activation (or inhibition) ofplasminogen, uPA, PAIs (plasminogen activator in-hibitors) and uPAR also display biological e¡ectsthat are not directly attributable to the formationof plasmin. All of the genes of this system havenow been knocked out, and recombinant mice rep-resent important tools for understanding the role ofthese enzymes in cancer.

Generation of extracellular proteolytic activity fol-lowing plasminogen activation is a major event incancer cell invasion, as shown in Plg3=3 mice (Tho-mas Bugge, Cincinnati, OH, USA). He employedseveral model tumors (MMTV-PymT transgene,Lewis lung carcinoma, B16-BLS melanoma) and ob-served a reduction in tumor size and a generalizeddecrease of metastatic lesions in the Plg3=3 mice. Onthe basis of results obtained with di¡erent tumormodels, Bugge concluded that Plg de¢ciency impairstumor progression, reduces tumor growth and ulcer-ation, and delays the appearance of vicinal and dis-

tant metastases. Interestingly, Bugge did not observeany increase in metastasis after resection of the pri-mary tumor, information that appears to contradictearlier studies on angiostatin (see below).

Activation of plasminogen is not restricted tomammals, but the mechanism has evolved independ-ently in di¡erent organisms. One such example isYersinia pestis, the bubonic plague agent, a highlypathogenic subspecies of Y. tuberculosis. Bugge pre-sented data demonstrating the plasminogen depend-ence of the Y. pestis pathogenic mechanism. Plg3=3

mice are, in fact, highly resistant to Y. pestis infec-tion. In addition, double de¢cient mice for plasmi-nogen and ¢brinogen (Fgn3=3) lost resistance to Y.pestis, suggesting that ¢brin must be degraded toallow bacterial invasion. In infected, Plg3=3 micethere is a massive in¢ltration of neutrophils into cer-tain tissues, an e¡ect which is lacking in Plg3=3-Fgn3=3 mice. These data suggest a chemotactic rolefor ¢brin in the recruitment of neutrophils and pro-vide a possible explanation for the immunode¢cientphenotype of the uPA3=3 mice. These animals didnot display a major de¢cit in ¢brin degradation,since uPA activity could be substituted by that oftPA.

The phenotype associated with the lack of a func-tional plasminogen gene in humans has been re-ported by Anne Marie Mingers (Wu«rzburg, Ger-many). The disease was previously known asligneous conjunctivitis, in which patients develop tis-sue membrane formation causing major mechanicalproblems which hinder or prevent eye opening atbirth and respiratory de¢ciency due to the presenceof pseudomembranes. In homozygotes, in which Plgprotein and activity are missing altogether, there issubepithelial ¢brin deposition and abnormal woundhealing but, surprisingly, there are no abnormalthrombotic events. Treatment with Lys-Plg leads tomembrane regression and re-opening of the eye.Long-term e¡ects of the use of plasminogen concen-trates in plasminogen de¢ciencies were reported byCarl-Erik Demp£e (Mannheim, Germany).

High PAI-1 levels are bad prognostic markers.Jean Michel Foidart (Liege, Belgium) has analyzedthe role of PAI-1 in tumor invasion and angiogenesisusing a silicone transplantation chamber model. Inthis system, DMBA-induced malignant keratinocytesare inoculated onto wounded skin, with a collagen

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gel separating them from the stroma. The collagengel is progressively invaded by capillaries. When thetips of new vessels reached the keratinocyte layer,invasion of the collagen gel by transformed cells oc-curred. In this system, PAI-1 immunostaining wasdetectable in the mesenchymal cells adjacent to ma-lignant cells, at the tip of proliferating blood vesselsand, to a lesser extent, in malignant keratinocytes.

Foidart's group employed PAI-1-de¢cient mice toanalyze the role of PAI-1 in this process. In the ab-sence of host PAI-1, there was no evidence of malig-nant cell invasion. Although the tumor cells survived,they did not invade the collagen gel. This appears tobe due to the inability of the stroma to form newvessels, which were absent from the collagen gel. Insitu zymography showed that in the wild type in-vaded areas there was proteolytic activity, mostlydue to uPA. Interestingly, in this respect, therewere no di¡erences in this parameter between wildtype and PAI-13=3 mice, indicating that di¡erencesin uPA catalytic activity cannot account for the re-duced cell invasiveness observed in the absence ofhost PAI-1. Finally, the non-invasive phenotype ofkeratinocytes in PAI-13=3 mice was rescued by in-fection with an adenovirus vector carrying CMV-PAI-1. In conclusion, in this model system, invasionrequires endothelial cell recruitment for which thepresence of PAI-1 is essential. The independence ofthis function on the ability of PAI-1 to inhibit plas-minogen activation suggests the occurrence of addi-tional mechanisms, possibly those acting on cell ad-hesion and migration. In this respect, it will beimportant to determine whether rescue can be ob-tained with PAI-1 mutants de¢cient in anti-proteo-lytic activity but not in vitronectin binding activity.The dual role of PAI-1 in stimulating both angio-genesis and tumor invasion supports the hypothesisthat these processes share common mediators andmechanisms.

In addition to increasing the rate of activation ofthe pro-uPA, the pro-enzyme form of uPA, the ur-okinase receptor (uPAR) is capable of direct signal-ing activity, activated by the binding of uPA or of itsamino-terminal non-catalytic domain (ATF) [4,5]. Asreported by Francesco Blasi (Milan, Italy), this re-ceptor, which is composed of three 90-residue do-mains, appears to be highly susceptible to uPA-in-duced conformational changes that elicit di¡erent

biological features. This receptor, in fact, behavesas an activatable, cell-surface chemokine that inducesG-protein dependent chemotaxis, cytoskeletal modi-¢cation, and activation of the Src pathway in a vari-ety of cell types (myeloid, lymphoid, smooth musclecells, ¢broblasts, cancer cells). Binding of uPA ex-poses a speci¢c epitope residing in the region linkingdomains 1 and 2 of uPAR. Since uPAR is a GPI-anchored molecule lacking a transmembrane and cy-toplasmic domain, its signaling must be mediated bya (transmembrane) adaptor. Interestingly, a uPARmitogenic domain can be uncovered in cell cultureby cleavage with uPA at physiological concentra-tions, generating an inactive amino-terminal solublepeptide, and an active carboxy-terminal anchored re-gion. Importantly, both fragments of uPAR can bedetected in growing tumors and recovered from theurine of patients. The importance of this informationis underlined by the immunode¢cient phenotype ofuPA3=3 mice, in which migration of monocytes-mac-rophages and of T cells is strongly delayed [6]. Stud-ies on uPAR3=3 mice are under way, and will beimportant to assess the importance of this moleculein cell migration in vivo.

Recently uPAR has been shown to bind not onlyuPA, but also the extracellular matrix protein, vitro-nectin (VN). By virtue of the latter activity, it actsas an adhesion receptor. Binding of uPA and VNare not mutually exclusive. Indeed, uPA actuallyfavors the binding of VN. On the other hand, VNis also a ligand for PAI-1, the inhibitor of uPA.David Loskuto¡ (La Jolla, CA, USA) reportedthat the high-a¤nity PAI-1 binding site of VN over-laps the uPAR binding site. The former site lies inthe amino-terminal somatomedin B (SB) domain, a44-residue region close to the single RGD sequencemediating VN binding to its integrin receptors.U937 cells, which, upon di¡erentiation, adhere toVN in a uPAR-dependent way, do not adhere toVN in the presence of PAI-1. This is because thelatter prevents uPAR binding to the SB domain ofVN. PAI-1 inhibits U937 cell binding to the SBdomain of VN when this domain is attached toplastic wells in an ELISA-type assay. These ¢ndingsalso suggest that the acquisition of myelomonocyticadherence occurs primarily via uPAR. On the con-trary, constitutively adherent HeLa and MCF-7cells, seeded onto VN, can be removed with PAI-1

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only in the presence of an RGD peptide, suggestingthat combined action of uPAR and integrins sustaintheir adherent state. Thus, the plasminogen activa-tion system regulates adhesion of some cells : it ac-tivates through uPA and uPAR and inhibits throughPAI-1. However, neither the catalytic activity ofuPA nor the inhibitory activity of PAI-1 is directlyinvolved in these e¡ects. Since the PAI-1 bindingsite in VN lies very close to the RGD sequence,PAI-1 can also sterically inhibit adhesion by inhibit-ing the binding of VN to its avb3 integrin receptor,thereby stimulating cell migration [7]. These func-tions of PAI-1 might explain why a high PAI-1 levelin tumor tissue is a bad prognostic marker in breastand other cancers.

Redundancy in proteolytic matrix degradation isre£ected by the minor phenotypes of mice de¢cientfor single plasminogen activators or matrix metallo-proteases. The concept of redundancy and its impor-tance and signi¢cance in cancer were addressed byKeld DanÖ (Copenhagen, Denmark). Dano pointedout the importance of the stromal/cancer cell inter-action in cancer invasion, and of the `orchestration'of the various proteases in this complex process. Inhis view, cancer mimics speci¢c tissue remodelingprocesses with invasion as a form of uncontrolledtissue remodeling. Indeed, the same cell types thatexpress speci¢c components of the proteolytic systemin a remodeling process do so in cancer invasion. Insquamous skin cancer (like in skin wound healing),the epithelial or the cancer cells, in addition to mac-rophages, produce both uPA, gelatinase B anduPAR. In colon cancer, like in the shedding of epi-thelial cells in gastrointestinal tract, uPA or 72Kcollagenase are produced by ¢broblast-type cells,uPAR by cancer (or epithelial) cells, and PAI-1 byendothelial cells. In mammary cancer, like in mam-mary gland involution, uPA is produced by myo¢-broblasts, while uPAR is not produced by cancercells but mostly by macrophages. For this reason,it is important to study the remodeling processes ofnormal tissues in detail.

For example, Plg3=3 mice reveal an extremely de-layed wound healing process. However, even if de-layed, the skin heals; therefore, other factors canpresumably compensate for the absence of Plg. Onehypothesis is that metalloproteases can substitute forPlg. Indeed, galardin, a general inhibitor of metal-

loproteases, which also delays skin wound healing inwild type mice, led to a total block of skin woundhealing in Plg3=3 mice. Thus, the source of the en-zymes does not seem to be important for cancer in-vasion, as long as they are there.

4. Components of the matrix protease system asprognostic indicators

Established prognostic factors may allow one toidentify high- and low-risk cancer patients, facilitatethe follow-up, and educate doctors regardingwhether or not to employ certain adjuvant therapies.John Foekens (Rotterdam, The Netherlands) re-ported data on 2780 patients with recurrent breastcancer, showing that the evaluation of uPA, PAI-1and PAI-2 levels will be of genuine value in decidingwhether or not to employ chemotherapy. In addition,the analysis of cErbB2, uPA and p53 levels are usefulin predicting response to tamoxifen. These assayswere mostly performed on extracts of the surgicallyexcised tumors. Niels Brunner (Copenhagen, Den-mark) and Kees F.M. Sier (Milan, Italy) reportedon the use of plasma and serum measurements ofsoluble uPAR (suPAR) as prognostic markers inbreast, colon, kidney and ovarian cancers.

5. uPAR antagonists and cancer therapy

There is much evidence pointing to an importantrole of uPAR in cancer progression and invasiveness.Steven Rosenberg (Emoryville, CA, USA), presentedexciting data on the therapeutic consequences of in-hibiting urokinase/urokinase receptor interaction.Rosenberg has produced species-speci¢c uPAR an-tagonists that allow one to compare the role ofuPAR synthesized by human tumor cells to thatpresent on host (mouse) ¢broblast-like stromal cells.The antagonists employed by Rosenberg (aa 1^48 ofhuman or mouse pro-uPA) inhibit uPA surface ac-tivity, by virtue of their ability to bind uPAR withhigh a¤nity. In well-controlled experiments per-formed in NK3 nude mice, Rosenberg detected acytostatic e¡ect on tumor growth, leaving tumor cellsin a state of dormancy, in agreement with previousdata [8]. Under these conditions, tumor growth was

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totally inhibited at the highest doses. However, uponremoval of the drug, tumors began to grow. Interest-ingly, no toxicity of this therapy has been observedto date. The possibility that the cytostatic e¡ect isnot simply due to the inhibition of the uPA/uPARinteraction is suggested by the ¢nding that the R3anti-uPAR blocking antibody had no cytostatic ef-fect. Rosenberg also reported that his antagonistspromoted adhesion to vitronectin better than full-size uPA, thereby raising the possibility that theyblock motility by increasing cell adhesion. AuPAR-dependent growth factor e¡ect of the amino-terminal moiety of uPA was described in the past [9],but its nature and mechanism have not yet beenclari¢ed: do these antagonists act on tumor growth?Also, the e¡ects on angiogenesis have not been sys-tematically de¢ned, since they appear to be di¡erentin di¡erent tumor models. One wonders about theoutcome of combining anti-uPAR antagonists withanti-MMP drugs.

6. PA and the vascular system

The role of plasminogen activators in vascular dis-eases was assessed in nullizygous mice (Peter Carme-liet, Louvain, Belgium). Models of atheroscleroticaneurysm, neointima thickening, transplant athero-sclerosis, and myocardial ischemia with ventricularrupture or scar formation were studied in mice de¢-cient for speci¢c genes of the plasminogen activationsystem. The basic message was that the establishmentof these lesions depends particularly on uPA, whichappears to allow monocyte recruitment by stimulat-ing their migration and di¡erentiation into macro-phages. De¢ciency of uPA protects against athero-sclerotic aneurysm by reducing plasmin-dependentactivation of pro-MMPs. These ¢ndings may haveimplications for the design of therapeutic strategiesto prevent aortic aneurysm, which is currently a ma-jor cause of death.

Angiostatin, a proteolytic fragment of plasmino-gen [10], has had wide publicity recently as a possi-ble anti-cancer drug, speci¢cally blocking angiogen-esis. The role of proteolytic balance in angiogenesiswas addressed by Michael Pepper (Geneva, Switzer-land). Development of polyoma virus endotheliomasis highly dependent on the proteolytic state of the

host. In particular, uPA de¢ciency leads to a majorreduction in the tumorigenic potential of the derivedendothelioma lines. Pepper also addressed the mech-anism of action of native, human angiostatin, theproteolytic fragment of plasminogen containingkringles 1^3 or 1^4, or of recombinant murine andhuman angiostatin. He demonstrated apoptotice¡ects speci¢c for endothelial cells, with blockadeof growth and DNA fragmentation. He suggestedthat angiostatin induces dormancy of metastasesby shifting the balance between proliferation andapoptosis.

Lars Holmgren (Stockholm, Sweden) discussed themechanism of angiostatin action, starting from earlyexperiment with Lewis lung cancer growth in mice, inwhich removal of the primary tumor led to a massivemetastatic burst in the lung. The primary tumor,through its production of angiostatin and inhibitionof angiogenesis, keeps tumor cells dormant and in-capable of invading. Those early studies led to thediscovery of angiostatin and its production as a po-tential anti-cancer drug. At 1 mg/kg mouse/day, an-giostatin inhibited the proliferation of endothelialcells in vivo. An obvious control in the early experi-ment might have been the use of plasminogen-de¢-cient mice. Importantly, the experiments presentedby Thomas Bugge on Plg-de¢cient mice and Lewislung tumorigenesis (see above) were performed dif-ferently, and those results cannot be compared withthe aforementioned Lewis lung experiment. The useof these mice will likely increase overall understand-ing of the mechanisms leading to angiostatin forma-tion and function.

Although the data on regression of primary tu-mors into microscopic dormant foci are impressive,the molecular details of angiostatin action are stillobscure. Holmgren reported on the search for anangiostatin receptor. Angiostatin was found to spe-ci¢cally bind to and be internalized by endothelialcells, not ¢broblasts or HeLa cells, and not to com-pete with other angiogenic factors (like bFGF orVEGF). Since angiostatin contains plasminogenkringles, which contain binding sites for carboxy-ter-minal lysines, the e¡ects of O-aminocaproic acid onangiostatin cell binding should be tested.

Using a two-hybrid system, an angiostatin bindingmolecule has been identi¢ed (ABP-1). The structureof the encoded protein was not disclosed, but it con-

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tains a transmembrane domain and is present on thecell surface. However, it does not appear to be ex-pressed solely in endothelial cells.

In vitro and in vivo regulation of TGF-L (anotherfactor important in angiogenesis) activation was dis-cussed by Dan Rifkin (New York, NY, USA). TGF-L is released from cells as a latent complex (incapableof interacting with the receptor) in which the 25-kDaactive homodimer is still associated with its propep-tide and deposited in the ECM. To elicit a biologicalresponse, the mature growth factor must be releasedfrom a latency complex which includes latency TGF-L binding protein or LTBP, which is structurallysimilar to the micro¢brillar protein, ¢brillin. The as-sociation between LTBP-1 and ECM is transgluta-minase (TGase)-dependent. The conversion of thelatent complex into active TGF-L and its releasefrom ECM represent key regulatory steps. Conver-sion requires uPA, uPAR, plasmin, TGase andLTBP. Plasmin converts latent TGF-L into matureTGF-L, but only after cross-linking of the complexto the ECM by tissue TGase. The TGase-sensitivesite is in the LTBP moiety of the complex, and trans-glutamination of a speci¢c glutamine of the EGF-likedomain of LTBP is required for TGF-L activation.Subsequently, the ECM-incorporated TGF-L-LTBPcomplex is attacked by plasmin at the C-terminalside of the modi¢ed glutamine. Production of a trun-cated recombinant LTBP, containing exclusively itsTGase-sensitive region, provides a reagent able tosquelch TGase, thereby preventing ECM incorpora-tion and leading to direct channeling of TGF-L toactive receptor sites. Transgenic mice expressing wildtype LTBP or its 8-cysteine containing, TGase-re-sponsive EGF domain under the K14 promoterhave been generated and are perfectly normal. Ani-mals transgenic for truncated LTBP have less fur,and their hair length is 20% shorter than wild type.Detailed investigation reveals decreased DNA syn-thesis in the hair follicle cells (less than 50% ofwild type). The importance of LTBP in the regula-tion of TGF-L biological activity is underscored byits structural similarity to ¢brillin, mutations ofwhich cause Marfan's syndrome. Indeed, mutationsin LTBP-2, one of four members of the LTBP fam-ily, have now also been found in patients with Mar-fan's syndrome.

7. Cathepsins

Cathepsins (CTS) are endo/lysosomal proteinasesthat include enzymes belonging to the pepsin familyof aspartyl proteases (at least two members, CTS-Dand CTS-E) or to the papain family of cysteinylproteases (at least seven members identi¢ed so far).While some cathepsins are ubiquitously expressed(CTS-B, CTS-L and CTS-H), others (CTS-S, CTS-K) exhibit a restricted pattern of expression. Chris-toph W.B. Peters (Freiburg, Germany) investigatedthe role of cathepsins D (aspartyl), B and L (cys-teinyl) by producing mice de¢cient in the respectivegenes. Available evidence suggests that lysosomalproteinases are involved in bulk proteolysis, antigenpresentation, processing of the invariant chain of themajor histocompatibility complex (MHC), bone re-sorption, chronic in£ammation, tumor formationand metastases. Deletion of the individual genescan shed light on this and other functions of cathep-sins. In all three cases, the recombinant mice areviable, but develop di¡erent phenotypes. CTS-B3=3

mice appear to be perfectly normal and show nor-mal MHC expression. CTS-D3=3 mice are normalat birth but, in the third week after birth, developan anorexic phenotype and die at about day 26.Their MHC expression is normal, and overall anti-gen presentation capacity is una¡ected. However,these mice present atrophic intestinal mucosa, ne-crosis, an abrupt loss of T and B cells with destruc-tion of lymphoid organs, although bulk proteolysisappears una¡ected. Hence, CTS-D might be in-volved in the degradation of speci¢c proteins thatare presently unknown. CTS-L3=3 mice display de-layed hair follicle morphogenesis, skin thickening,and higher mitotic rate of the basal epidermal cells,eventually leading to a furless phenotype. Peters re-ported that these mice are allelic to the previouslyknown spontaneous furless mouse which, indeed,has a catalytically inactivated CTS-L. In CTS-L3=3 mice, expression of MHC and antigen presen-tation in peripheral cells is normal. However, unlikeCTS-B3=3 or CTS-D3=3 mice, CTS-L3=3 miceshow an impaired degradation of the invariant chainof MHC class II in cortical epithelium of the thy-mus and reduction of the positive selection of CD4�

cells.

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8. Conclusions

Four messages can be extracted from this meeting,which rather well summarized the state of the art inthe ¢eld.

(1) The redundancy of proteases and their intricateinterrelationships which range from the mechanismsof activation (i.e. plasmin function in pro-MMP ac-tivation) to substrate speci¢city. While speci¢c func-tions are being discovered for individual proteases(uPA-dependent migration and MMP9 osteogeneticfunction), many of them appear to play individuallyimportant roles in cancer. Hence, blocking of theaction of individual species appears to have a majorimpact on tumor growth and spread. This certainlyoccurs in mice. However, the prognostic marker datasuggest that the same may be true in human cancer.

(2) In cancer, expression of a given protease gen-erally occurs in the same cell type that would pro-duce it in the homologous tissue remodeling process:e.g. uPA and uPAR are produced by keratinocytes inskin wound healing and by the epithelial cancer cellsin skin carcinomas; uPA and MMPs in myo¢bro-blasts (and macrophages) and by mammary cancercells, etc.

(3) Proteases in cancer appear to respond to a wellorganized scheme of stromal-cancer cell interactions.In this regard, the cellular source of the proteasedoes not appear to be essential since cancer cellscan orchestrate the various proteases, receptors,and polypeptide inhibitors, channelling all of theminto the invasion process.

(4) The mechanisms involved in protease functionin cancer are not limited to their proteolytic activ-ities. Other mechanisms are beginning to be eluci-dated, which in some cases may be just as relevant.Certain proteases and their receptors are endowedwith direct chemotactic, proliferative, and apoptoticproperties. Their de¢nition will be a future challenge.

(5) The importance of proteases, their inhibitors,and receptors has triggered a search for anti-proteasedrugs for use in cancer therapy. This is already oc-

curring for MMPs, uPA, and its receptor, and theseinitial attempts provide hope of an expansion of ouranti-cancer weaponry. In these very early attempts, itis clear that major synergistic e¡ects can be obtainedcombining anti-PA with anti-MMP drugs. There is apossible, future breakthrough to be made here, sincethe speci¢c anti-protease e¡ect would not be ex-pected to cause major toxicity.

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[2] P.A. Andreasen, L. KjÖller, L. Christensen, M.J. Du¡y, Theurokinase-type plasminogen activator system in cancer meta-stasis : a review, Int. J. Cancer 72 (1997) 1^22.

[3] Z. Werb, ECM and cell surface proteolysis : regulating cel-lular ecology, Cell 91 (1997) 439^442.

[4] F. Blasi, uPAR-uPA-PAI-1: a key intersection in proteoly-sis, adhesion and chemotaxis, Immunol. Today 18 (1997)415^417.

[5] H.A. Chapman, Plasminogen activators, integrins and thecoordinated regulation of cell adhesion and migration,Curr. Opin. Cell Biol. 9 (1997) 714^724.

[6] M.R. Gyetko, G.-H. Chen, R.A. McDonald, R. Goodman,G.B. Hu¡nagle, C.C. Wilkinson, J.A. Fuller, G.B. Toews,Urokinase is required for the pulmonary in£ammatory re-sponse to Cryptococcus neoformans, J. Clin. Invest. 97 (1996)1818^1826.

[7] S. Stefansson, D.A. Lawrence, The serpin PAI-1 inhibits cellmigration by blocking integrin alpha V beta 3 binding tovitronectin, Nature 383 (1996) 441^443.

[8] Y.H. Kook, J. Adamski, A. Zelent, L. Ossowski, The e¡ectof antisense inhibition of urokinase receptor in human squa-mous cell carcinoma on malignancy, EMBO J. 13 (1994)3983^3991.

[9] S.A. Rabbani, J. Desjardins, A.W. Bell, D. Banville, A. Ma-zar, J. Henkin, D. Goltzman, An amino-terminal fragmentof urokinase isolated from a prostate cancer cell line (PC-3)is mitogenic for osteoblast-like cells, Biochem. Biophys. Res.Commun. 173 (1990) 1058^1064.

[10] M.S. O'Reilly, L. Holmgren, Y. Shing, C. Chen, R.A.Rosenthal, M. Moses, W.S. Lane, Y. Cao, E.H. Sage, J.Folkman, Angiostatin: a novel angiogenesis inhibitor thatmediates suppression of metastasis by a Lewis lung carcino-ma, Cell 79 (1994) 315^328.

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Proteases and cancer invasion: from belief to certainty: AACR Meeting on Proteases and Protease...

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