, . 183: 377–379 (1997)

EDITORIAL

CONTROL OF MATRIX METALLOPROTEINASEACTIVITY IN CANCER

. . * . .

Breast Cancer Research Unit, Department of Pathology, University of Leicester, Glenfield General Hospital, Groby Road,Leicester LE3 9QP, U.K.

SUMMARY

Remodelling of extracellular matrix (ECM) and basement membranes is a key component of the process of tumour cell invasion andmetastasis. Matrix metalloproteinases (MMPs) are one of the major classes of enzymes involved in degrading ECM, having differentsubstrate specificities and being inhibited by naturally occurring tissue inhibitors (TIMPs). Elevated levels of MMPs have beenassociated with poor prognosis for a variety of malignancies. However, the expression and effective action of MMPs are influenced bymultiple factors: most are synthesized by stroma rather than tumour cells, suggesting tumour cell–stromal cell co-operation; receptors(MT-MMPs) have to be present on tumour cells for binding and activation of MMP; co-ordination of tissue proteolysis and subsequentintegrin binding will aid cell movement through a matrix; integrin receptors can directly moderate the production of MMP. Thesevarious components need to be considered when trying to determine the key events regulating matrix proteolysis and hence invasion.? 1997 John Wiley & Sons, Ltd.

J. Pathol. 183: 377–379, 1997.No. of Figures: 0. No. of Tables: 0. No. of References: 35.

KEY WORDS—metalloproteinase; MMP; cancer; extracellular matrix; integrins; membrane type-metalloproteinase; MT-MMP

INTRODUCTION

The process of tumour cell invasion and metastasisinvolves extensive remodelling of extracellular matrix(ECM) and of basement membranes (BMS). With thetransition from in situ to invasive malignancy, tumourcells must penetrate their delimiting BM and movethrough surrounding stroma. A metastatically compe-tent cell must gain access to the vascular or lymphaticcompartment and extravasate at a distant site to set upsecondary growth. Another vital requirement fortumour development is a rich vascular network, andangiogenesis also involves extensive matrix remodelling.Because many of the ECM components have specificproteolytic requirements, degradation must involve theaction of a range of enzymes. The major classes ofECM degrading enzymes are the serine proteases,which include the plasminogen activators; the cysteineproteases, which are largely lysosomal and includecathepsin D; and the metalloproteinases. There has beenan explosion of interest in the metalloproteinases inrecent years. This rapidly growing family of proteasesnow comprises at least 17 members which, whilst there issignificant overlap, do display clear substrate specificity:they include the collagenases, which degrade fibrillarinterstitial collagens and include interstitial collagenase(MMP-1), neutrophil collagenase (MMP-8), and

collagenase-3 (MMP-13); the 72 kD and 92 kD gelati-nases (MMP-2 and MMP-9), which cleave denaturedcollagen fibrils and degrade BM type IV collagen; andthe stromelysins (STR), which now include fourmembers, STR-1 (MMP-3), STR-2 (MMP-10), STR-3(MMP-11), and matrilysin (MMP-7 or PUMP-1—putative metalloproteinase-1). With the exception ofSTR-3, the stromelysins have the broadest substratespecificity, being capable of degrading fibronectins,laminin, elastin, proteoglycans, and collagens.1,2All of the MMP members are secreted as inactivezymogens and can be activated in vitro by a widerange of cytokines and growth factors. Their actioncan be further modified by specific inhibition viabinding of the naturally occurring tissue inhibitors ofmetalloproteinases (TIMPs).3Evidence for a role in cancer comes from both in vitro

studies and tissue systems. Elevated levels of a numberof the MMPs have been demonstrated in differentmalignancies and have been related to tumour behav-iour. For example, expression of MMP-1 in colorectalcarcinoma has been shown to be a predictor of poorprognosis4 and high levels of expression of MMP-11(stromelysin-3) mRNA in breast carcinoma have beenassociated with poor patient survival.5 The most consist-ent association with tumour progression, however, hasbeen demonstrated for the gelatinases. Both high levelsand enhanced activity of MMP-2 and MMP-9 have beenreported to correlate with tumour grade in breast andbladder carcinomas,6,7 and other studies have shown arelationship between enhanced MMP-2 production and

*Correspondence to: Dr J. L. Jones, Breast Cancer Research Unit,Department of Pathology, University of Leicester, Glenfield GeneralHospital, Groby Road, Leicester LE3 9QP, U.K.

CCC 0022–3417/97/120377–03 $17.50 Received 19 May 1997? 1997 John Wiley & Sons, Ltd. Accepted 5 June 1997

activation with the metastatic phenotype.8–10 In modelsystems, inhibition of type IV collagenase activityreduces tumour invasive and metastatic potential.11 It isproposed that the gelatinases may be pivotal in tumourprogression, because their action bestows on tumoursthe ability to penetrate BM. However, it is now recog-nized that in the majority of cases, most MMPs aresynthesized by the peri-tumoural stroma rather than thetumour cells themselves.12,13 This suggests aco-operation between tumour cell and stromal cell in thedevelopment of a pro-invasive micro-environment. Onemechanism for this action is via the tumour cell-derivedcollagenase stimulatory factor (TCSF), also known asEMMPRIN (extracellular MMP inducer), a cell surfacereceptor with homology to the immunoglobulin super-family, which is expressed by some tumour cells andstimulates MMP expression in fibroblasts.14For effective proteolysis, tumour cells must be able to

localize the enzyme activity at the site of invasion.Studies using antibodies that detect newly exposedepitopes on ECM substrates following proteolytic cleav-age have shown that staining is concentrated in thepericellular environment.15 Chen16 has demonstratedlocalization of MMP on tumour cell membraneprojections called invadopodia and has shown thatdegradative activity mediated by these invadopodia isinhibited by tyrosine kinase inhibitors, indicating that anactive process is occurring at these sites of cell–matrixinteraction. This implies a receptor mechanism ontumour cells for the binding and potential activation ofMMP. Support for such a mechanism came from thecloning and sequencing of the membrane type MMP,now termed MT1-MMP, using degenerate primers withhomology to conserved regions in MMP genes.17Following transfection of COS-1 cells with MT1-MMPplasmid, the protein was immunolocalized to the cellsurface and resulted in specific activation of MMP-2zymogen. It has since been demonstrated that activationof pro-MMP-2 requires the formation of a ternarycomplex, initiated by binding of TIMP-2 (tissue inhibi-tor of metalloproteinase-2) to MT1-MMP, althoughhigher concentrations of TIMP-2 inhibit MMP-2 activa-tion.18 Three further MT-MMPs have now beendescribed: MT2-MMP, isolated from a placenta cDNAlibrary;19 MT3-MMP, which appears to be highlyexpressed in tissues showing low levels of MT1-MMPsuch as the heart and brain;20 and MT4-MMP, whichwas isolated from, and found to be highly expressed in,a series of breast cancer cell lines.21 As is the case withthe other MMPs, the MT-MMPs are also synthesized asinactive precursors, but in contrast to the majority ofMMPs which are activated following secretion, theMT-MMPs are activated intracellularly, prior to trans-port to the cell surface. In common with stromelysin-3(MMP-11), the MT-MMPs possess a decapeptide inser-tion between pro- and catalytic domains.22,23 Thisinsertion comprises a triad of basic residues recognizableby a family of precursor-processing endopeptidases, alsoknown as pro-protein convertases, of which furin ismost widely expressed. Furin is concentrated in thetrans-Golgi network, is capable of activating a range ofgrowth factors and membrane receptors, and has been

shown to activate both stromelysin-3 and MTI-MMP.23This activation step may be an important regulator ofMMP activity, and given the potential role for MT1-MMP in tumour progression, manipulation of this acti-vation step could provide a useful therapeutic target.Pro-protein convertase inhibitors have been devel-oped,24 although their use as potential anti-canceragents has not yet been described. A recent report hasindicated that MT1-MMP may also be activated at thecell surface in a plasmin-dependent mechanism.25The capacity to invade depends not only on proteo-

lysis, but also on adhesion and migration; for effectivedirected invasion, these processes must be inter-linked. The integrin family of adhesion molecules areheterodimeric transmembrane proteins composed ofdissimilar á and â subunits which mediate cell–matrixinteractions.26 As well as providing adhesion, integrin–matrix interactions are essential for motility. Theregulation of integrin function is complex, beinginfluenced by both external factors such as growthfactors and internal signals—so-called ‘inside-out’ regu-lation.26 Proteolytic changes in ECM can alter adhesiveinteractions with integrins; so, for example, whilst theintegrin receptor ávâ3 fails to bind to native collagen,it does interact with proteolysed collagen, owing toexposure of a cryptic RGD site–the binding motif ofmany integrins.27 This ability to bind to modified matrixcould provide cells expressing ávâ3 with an invasiveadvantage. In support of this, melanoma cells expressingthis integrin are highly invasive, compared with thosethat lack ávâ3.28 It is evident that co-ordination of tissueproteolysis and subsequent integrin binding would pro-vide the most efficient means of cell movement througha matrix. Brooks et al.29 have recently demonstratedthis link by showing that cell surface ávâ3 can bindactive MMP-2, so focusing enzyme activity at the cellmembrane, in conjunction with a molecule capable ofbinding the cleaved matrix.In addition to the spatial control of MMP, integrin

receptors can directly modulate the production ofMMP. In osteogenic cell lines, overexpression of thecollagen receptor á2â1 results in enhanced synthesis ofMMP-1 and this is inhibited following transfection withanti-sense á2 integrin.30 A role for integrin receptors insignal transduction is well established and the ability ofa cell to directly control metalloproteinase expression inresponse to changes in matrix could be a powerfulmechanism of orchestrating tumour cell invasion. Someindication of the complexity of matrix–integrin signal-ling has been demonstrated in studies plating rabbitsynovial fibroblasts (RSFs) on different fragments offibronectin.31 When grown on intact fibronectin, RSFsexpress low levels of MMP, but when grown on thecentral cell-binding fragment of fibronectin, MMPexpression is enhanced in a mechanism dependent onbinding of the á5â1 integrin receptor. The low-levelexpression of MMP appears to depend on interaction ofá4â1 integrin and the CS-1 region of fibronectin. It isinteresting to consider ways in which these processescould be involved in tumour cell behaviour. The stromaaround a tumour is different from the normal organstroma,32 and whereas fibronectin fragments have not

378 J. L. JONES AND R. A. WALKER

? 1997 John Wiley & Sons, Ltd. , . 183: 377–379 (1997)

been identified, there are other mechanisms by which theadhesive and signalling properties of fibronectin may bealtered. Tenascin (TN) is an extracellular matrix glyco-protein which is typically highly expressed in situationsassociated with cell motility such as during embryogen-esis, in tissue remodelling, and in tumour-associatedstroma. TN has been shown to have an anti-adhesiveaction on a number of cell types by directly inhibitingadhesion to fibronectin33 and has also been shown toenhance MMP expression in certain situations. MMPexpression is up-regulated when RSF cells are grown ona combination of TN together with fibronectin, com-pared with growth on fibronectin alone.34 It is proposedthat TN directly blocks the fibronectin-mediated signalsthat normally maintain low-level MMP expression. Thesituation is certainly even more complex, since at leasteight different isoforms of TN exist, with potentiallydistinct effects on cell adhesion and matrix signalling,Furthermore, different isoforms exhibit differing suscep-tibility to degradation by MMP, which would alsoinfluence its pro-migratory action.35In summary, the expression and effective action of

MMP are influenced by multiple environmental factorsincluding growth factors; specific membrane activators,which are themselves subject to control mechanisms;integrin receptor expression; and matrix composition.Whilst early studies on current synthetic inhibitors ofMMP reveal a clinical response in some tumours, dis-section of the key events regulating matrix proteolysismay identify pivotal targets for future therapeutic use.

REFERENCES1. Mignatti P, Rifkin D. Biology and biochemistry of proteinases in tumour

invasion. Physiol Rev 1993; 73: 161–195.2. Birkedal Hansen H. Proteolytic remodeling of extracellular matrix. Curr

Opin Cell Biol 1995; 7: 728–735.3. Matrisian LM. Metalloproteinases and their inhibitors in matrix re-

modeling. Trends Genet 1990; 6: 121–125.4. Murray GI, Duncan ME, O’Neil P, Melvin WT, Fothergill JE. Matrix

metalloproteinase-1 is associated with poor prognosis in colorectal cancer.Nature Med 1996; 2: 461–462.

5. Engel G, Heseemeyer K, Auger G, Backdahl M, Eriksson E, Linder S.Correlation between stromelysin-3 level and outcome of human breastcancer. Int J Cancer 1994; 58: 1–7.

6. Davies B, Miles DW, Happerfield LC, et al. Activity of type IV collagenasesin benign and malignant breast disease. Br J Cancer 1993; 67: 1126–1131.

7. Davies B, Waxman J, Wasan H, et al. Levels of matrix metalloproteases inbladder cancer correlate with tumour grade and invasion. Cancer Res 1993;53: 5365–5369.

8. Iwata H, Kobayashi S, Iwase H, Masaoka A, Fujimoto N, Okada Y.Production of matrix metalloproteinases and tissue inhibitors of metallo-proteinases in human breast carcinomas. Jpn J Cancer Res 1996; 87:602–611.

9. Tokuraku M, Sato H, Murakami S, Okada Y, Watanabe Y, Seiki M.Activation of the precursor of gelatinase A/72 kDa type IV collagenase/MMP-2 in lung carcinomas correlates with the expression of membrane-type matrix metalloproteinase (MT-MMP) and with lymph node metastasis.Int J Cancer 1995; 64: 355–359.

10. Brown PD, Bloxidge RE, Stuart NS, Gatter KC, Carmichael J. Associationbetween expression of activated 72-kilodalton gelatinase and tumour spreadin non-small-cell lung carcinoma. J Natl Cancer Inst 1993; 85: 574–578.

11. Alvarez OA, Carmichael DF, DeClerck YA. Inhibition of collagenolyticactivity and metastasis of tumour cells by a recombinant human tissueinhibitor of metalloproteinases. J Natl Cancer Inst 1990; 82: 589–595.

12. Heppner KJ, Matrisian LM, Jensen RA, Rodgers WH. Expression of mostmatrix metalloproteinase family members in breast cancer represents atumour-induced host response. Am J Pathol 1996; 149: 273–282.

13. Poulsom R, Pignatelli M, Stetler Stevenson WG, et al. Stromal expressionof 72 kDa type IV collagenase (MMP-2) and TIMP-2 mRNAs in colorectalneoplasia. Am J Pathol 1992; 141: 389–396.

14. Biswas C, Zhang Y, DeCastro R, et al. The human tumour cell-derivedcollagenase stimulatory factor (renamed EMMPRIN) is a member of theimmunoglobulin superfamily. Cancer Res 1995; 55: 434–439.

15. Singer II, Kawka DW, Bayne EK, et al. VDIPEN, a metalloproteinase-generated neoepitope, is induced and immunolocalized in articular cartilageduring inflammatory arthritic. J Clin Invest 1995; 95: 2178–2186.

16. Chen WT. Proteases associated with invadopodia, and their role in degra-dation of extracellular matrix. Enzyme Protein 1996; 49: 59–71.

17. Sato H, Takino T, Okada Y, et al. A matrix metalloproteinase expressed onthe surface of invasive tumour cells. Nature 1994; 370: 61–65.

18. Strongin AY, Collier I, Bannikov G, Marmer BL, Grant GA, Goldberg GI.Mechanism of cell surface activation of 72-kDa type IV collagenase.Isolation of the activated form of the membrane metalloprotease. J BiolChem 1995; 270: 5331–5338.

19. Takino T, Sato H, Shinagawa A, Seiki M. Identification of the secondmembrane-type matrix metalloproteinase (MT-MMP-2) gene from a humanplacenta cDNA library. MT-MMPs form a unique membrane-type subclassin the MMP family. J Biol Chem 1995; 270: 23013–23020.

20. Will H, Hinzmann B. cDNA sequence and mRNA tissue distribution of anovel human matrix metalloproteinase with a potential transmembranesegment. Eur J Biochem 1995; 231: 602–608.

21. Puente XS, Pendas AM, Llano E, Velasco G, Lopez Otin C. Molecularcloning of a novel membrane-type matrix metalloproteinase from a humanbreast carcinoma. Cancer Res 1996; 56: 944–949.

22. Pei D, Weiss SJ. Furin-dependent intracellular activation of the humanstromelysin-3 zymogen. Nature 1995; 375: 244–247.

23. Sato H, Kinoshita T, Takino T, Nakayama K, Seiki M. Activation of arecombinant membrane type 1-matrix metalloproteinase (MT1-MMP) byfurin and its interaction with tissue inhibitor of metalloproteinases(TIMP)-2. FEBS Lett 1996; 393: 101–104.

24. Anderson ED, Thomas L, Hayflick JS, Thomas G. Inhibition of HIV-1gp160-dependent membrane fusion by a furin-directed á 1-antitrypsinvariant. J Biol Chem 1993; 33: 24887–24891.

25. Okumura Y, Sato H, Seiki M, Kido H. Proteolytic activation of theprecursor of membrane type 1 matrix metalloproteinase by human plasmin.A possible cell surface activator. FEBS Lett 1997; 402: 181–184.

26. Hynes RO. Integrins: versatility, modulation and signalling in cell adhesion.Cell 1992; 69: 11–25.

27. Davis GE. Affinity of integrins for damaged extracellular matrix: alpha vbeta 3 binds to denatured collagen type I through RGD sites. BiochemBiophys Res Commun 1992; 182: 1025–1031.

28. Cheresh DA. Structure, function and biological properties of integrin alphav beta 3 on human melanoma cells. Cancer Metastasis Rev 1991; 10: 3–10.

29. Brooks PC, Stromblad S, Sanders LC, et al. Localization of matrixmetalloproteinase MMP-2 to the surface of invasive cells by interaction withintegrin alpha v beta 3. Cell 1996; 85: 683–693.

30. Riikonen T, Westermarck J, Koivisto L, Broberg A, Kahari VM, Heino J.Integrin alpha 2 beta 1 is a positive regulator of collagenase (MMP-1) andcollagen alpha 1(I) gene expression. J Biol Chem 1995; 270: 13548–13552.

31. Huhtala P, Humphries MJ, McCarthy JB, Tremble PM, Werb Z, DamskyCH. Cooperative signalling by alpha 5 beta 1 and alpha 4 beta 1 integrinsregulates metalloproteinase gene expression in fibroblasts adhering tofibronectin. J Cell Biol 1995; 129: 867–879.

32. van den Hooff A. Stromal involvement in malignant growth. Adv CancerRes 1988; 50: 159–196.

33. Chiquet Ehrismann R, Kalla P, Pearson CA, Beck K, Chiquet M. Tenascininterferes with fibronectin action. Cell 1988; 53: 383–390.

34. Tremble P, Chiquet Ehrismann R, Werb Z. The extracellular matrix ligandsfibronectin and tenascin collaborate in regulating collagenase gene expres-sion in fibroblasts. Mol Biol Cell 1994; 5: 439–453.

35. Siri A, Knauper V, Veriana N, Caocci F, Murphy G, Zardi L. Differentsusceptibility of small and large human tenascin-C isoforms to degradationby matrix metalloproteinases. J Biol Chem 1995; 270: 8650–8654.

379CONTROL OF MMP ACTIVITY IN CANCER

? 1997 John Wiley & Sons, Ltd. , . 183: 377–379 (1997)