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Matrix metalloproteinases and their biological function in human gliomas
Int. J. Devl Neuroscience, Vol. 17, Nos. 5±6, pp. 495±502, 1999 # 1999 ISDN. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0736-5748/99 $20.00 + 0.00
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MATRIX METALLOPROTEINASES AND THEIR BIOLOGICAL FUNCTION IN HUMAN GLIOMAS SHRAVAN K. CHINTALA,$ JORG C. TONN% and JASTI S. RAO$* $Departments of Neurosurgery, The University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030, USA; %Department of Neurosurgery, University of Wurzburg, Wurzburg, Germany
AbstractÐGliomas, a type of devastating primary brain tumors, are distinct from other solid, nonneural primary neoplasms, in that they display extensive in®ltrative invasive behavior but seldom metastasize to distant organs. This invasiveness into the surrounding normal brain tissue makes gliomas a major challenge for clinical intervention. Total surgical resection of gliomas is not possible, and recurrence of tumor growth is common; mean survival time is 8±12 months. Although substantial progress has been made recently toward understanding the behavior of gliomas, the mechanisms that facilitate invasion are still poorly documented. Clues to the invasion process have been ascertained through clari®cation of the key roles played by the extracellular matrix (ECM), cell-adhesion molecules and matrix degrading proteases. Serine proteases and metalloproteinases have been implicated in glioma tumor cell-invasion. Matrix metalloproteinases (MMPs) in particular can degrade almost all known ECM components and seem to play important roles in mediating glioblastoma tumor cell invasion. This review focuses on recent developments concerning the role of MMPs in the invasiveness of human gliomas. # 1999 ISDN. Published by Elsevier Science Ltd All rights reserved Key words: matrix metalloproteases, gelatinase, glioma, TIMP, MT-MMP.
INTRODUCTION Brain tumors are the third most frequent cause of cancer-related death in adults and the second most common cause of cancer-related death in children. Recent estimates indicate that 13,000 people died of brain tumors in 1996 and about 17,000 new cases of brain tumors were expected to be diagnosed in 1997.19 In adults, malignant gliomas are the most common primary brain tumors and account for more than 40% of all central nervous system (CNS) neoplasms. The World Health Organization (WHO) system uses four major grades to describe gliomas, namely pilocytic astrocytoma, low-grade astrocytoma, anaplastic astrocytoma and glioblastoma. Astrocytomas are tumors composed of predominantly neoplastic astrocytes. Pilocytic astrocytomas (WHO grade I) are typically located in midline structures such as the optic nerves, third ventricle, thalamus, medial temporal lobe, brainstem and cerebellum. Pilocytic astrocytomas rarely progress to anaplasia. Low-grade astrocytomas (WHO grade II) tend to diusely in®ltrate the surrounding brain parenchyma. Low-grade astrocytomas can appear anywhere in the CNS, including the spinal cord, but are most common in the cerebral hemispheres. Anaplastic astrocytomas (WHO grade III) are characterized by neoplastic ®brillary or gemistocytic astrocytes and can progress rapidly to glioblastoma. Glioblastoma multiforme (WHO grade IV) is the most frequent malignant brain tumor to aect adults. Glioblastomas are most frequently found in the frontotemporal region, but the parietal lobes can also be aected. Gliomas are thought to be derived from astrocytes, oligodendrocytes or ependymal cells and display a correspondingly broad spectrum of histopathological features. Brain tumors can arise as a result of gradual accumulation of genetic abnormalities in precursor cells, including loss or inactivation of the tumor suppressor gene p162 or overexpression of certain protease receptors *To whom all correspondence should be addressed. Tel.: +1 713 792 3266; Fax: +1 713 794 4950; E-mail: jrao@ notes.mdacc.tmc.edu. 495
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Fig. 1. Expression of MMPs and TIMPs in various grades of glioma tissues and normal brain. Values are based on northern blot analysis and represented as arbitrary values.
such as urokinase-type plasminogen activator (uPA) receptors.14 For reasons that are poorly understood, most primary brain tumors do not metastasize and rarely disseminate through cerebrospinal ¯uid. However, they do invade the surrounding normal brain. This characteristic local invasiveness of gliomas contributes substantially to the inability to achieve total resection by surgery and often results in recurrences at the primary site and at locations on the opposite side of the brain. Glial-cell invasion is a multistep process that is invariably accompanied by the expression of proteases. If cells are to cross an extracellular barrier, they must ®rst attach to the barrier matrix, then create a proteolytic defect in the matrix and ®nally migrate through that defect. Numerous studies have demonstrated a close association between the expression of various proteases, such as serine proteases, the metalloproteinases (MMPs) and the plasminogen activation/plasmin system, and the invasive behavior of gliomas. Although gliomas express other proteases, MMPs seem to be responsible for much of the degradation of a broad range of ECM components, and in particular, elevated levels of MMP-2, MMP-9, MT1-MMP were found in gliomas, compared to normal brain tissue (Fig. 1). The following sections provide a review of MMP expression and function in the invasion and progression of human gliomas.
MATRIX METALLOPROTEINASES (MMPS) MMPs are a group of structurally related enzymes that degrade several components of the ECM, including ®brillar and non®brillar collagens, ®bronectin, laminin and basement membrane proteoglycans (Table 1). At least 20 members of the human MMP family have been identi®ed.3 Most MMPs are secreted as zymogens, and their activity is controlled by activators and inhibitors, the latter termed tissue inhibitors of metalloproteinases (TIMPs). Common characteristics of MMPs include Zn2+ atoms at their active sites, requiring Ca2+ ions for enzyme activity, and having highly conserved N-terminal propeptide and catalytic domains.3 MMPs have been grouped according to their substrate speci®cities, i.e. as collagenases; gelatinases, stromelysins and the membrane-type MMPs (Table 1). One MMP, MMP-2 or gelatinase A, is unusual in its constitutive expression by many cells, its ubiquitous tissue distribution and its mode of activation which diers from that of any other MMP. Moreover, unlike other MMP proenzymes, progelatinases A and progelatinase B (also known as MMP-9) are usually isolated in complex with their endogenous inhibitors, TIMP-2 and TIMP-1 respectively. Both the free enzyme and the enzyme-inhibitor complex can be activated on the cell surface by membrane-type MMPs (MT-MMPs). Commonalities in the structural domains of MMPs are shown in Fig. 1. A 17±20-residue signal peptide, rich in hydrophobic amino acids, directs the translational product to the endoplasmic reticulum in all but MMP-17, which lacks this peptide.20 Other common domanins include a 80-amino acid propeptide domain that contains the highly conserved PRCXXPD
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Table 1. Matrix metalloproteinases (MMPs) Subgroup Collagenases
Enzyme
Substrate
MMP-1 (Interstitial collagenase)
collagen I, II, III, VII, VIII, X, gelatin, proteoglycans same as MMP-1 same as MMP-1 collagen I
MMP-8 (Neutrophil collagenase) MMP-13 (Collagenase-3) MMP-18 (Collagenase-4, xenopus)
Gelatinases
MMP-2 (Gelatinase A) MMP-9 (Gelatinase B)
Stromelysins
MMP-3 (Stromelysin-1) MMP-10 (Stromelysin-2) MMP-11 (Stromelysin-3)
Others
Membrane-type MMPs
MMP-7 (Matrylysin)
collagen I, IV, V, VII, X, gelatin, elastin, ®bronectin proteoglycans same as MMP-2
collagen IV, IX, X, elastin, ®bronectin, laminin, proteoglycans, pro-interstitial collagenase same as MMP-3 alpha-1-antitrypsin
MMP-12 (Metalloelastase) MMP-19 MMP-20 (Enamelysin)
collagen IV, ®bronectin, laminin, gelatin, pro-interstitial collagenase, proteoglycans elastin unknown unknown
MMP-14 (MT1-MMP)
activates MMP-2
MMP-15 (MT2-MMP) MMP-16 (MT3-MMP) MMP-17 (MT4-MMP)
unknown activates MMP-2 unknown
sequence, which is cleaved during activation; a catalytic domain of about 160±170 amino acids, that contains the highly conserved HEXGHXXGXXHS/T region, a thermolysin-type zincbinding region, and a calcium-binding site; a 200-residue carboxy-terminal domain homologous to hemopexin and vitronectin; a hinge region of about 75-amino acids, that connects the catalytic and hemopexin domains. Two MMPs (MMP-2 and MMP-9) have additional three ®bronectin type II gelatin binding domains in the catalytic domain. Membrane-type MMPs also have an additional 80±110-residue transmembrane domain. The presence of the furin cleavage site RXKR/RRKR between the prodomain and catalytic domains in stromelyin-3 and all known MT-MMPs suggest that these MMPs may be activated while they are still in the cell, whereas the other enzymes require proteolytic cleavage after they are secreted as zymogens. Although tumors secrete many dierent proteases, MMP-2 and MMP-9 seem to play major role in tumor progression. The following sections provide a brief review of MMP-2, MMP-9 and MMP-9 and their biological signi®cance, in glioma progression.
GELATINASES The gelatinase subgroup of MMPs has two members, MMP-2 (gelatinase A) and MMP-9 (gelatinase B). MMP-2 perhaps the most widespread of all MMPs; it is produced by a variety of cells and is frequently elevated in malignant tumors. MMP-2 is often secreted as a complex with TIMP-2. The other gelatinase, MMP-9, is expressed by transformed tumor cells, neutrophils, monocytes, and alveolar macrophages,11 and is often secreted as a complex with TIMP-1. MMP-2 has a unique mechanism of activation that involves a trimolecular complex consisting
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of MT-MMP,23 TIMP-2, and the C-terminal domain of MMP-2.24 Details of these two MMPs are given below. MMP-2 (GELATINASE A) MMP-2, a 72-kDa gelatinase (also known as gelatianase A, 72 kDa type IV collagenase and EC3.4.24.24) is constitutively expressed by many cells; MMP-2 not only degrades ECM, but also regulates cell proliferation, adhesion and migration.33 MMP-2 is not transcriptionally activated by TPA or IL-1 and lacks the typical transactivation sequences such as AP-1, PEA-3 or TATA boxes that play pivotal roles in the transcriptional regulation of most promoters. Moreover, unlike other MMPs it lacks an upstream transforming growth factor-beta-inhibitory element (TIE). MMP-2 seems to be posttranscriptionally regulated, but the mechanism has not been studied in detail. MMP activation is also regulated by the TIMPs.29 The N-terminal domains of TIMPs are inhibitory, and the C-terminal domains confer binding speci®city. For example, the N-terminal domains of either TIMP-1 or TIMP-2 can bind to the N-terminal domain of active MMP-2; however, only the C-terminal domain of TIMP-2 binds speci®cally to the C-terminal domain of MMP-2. ProMMP-2 has a unique mechanism of activation at the cell surface that is modulated by both MT1-MMP and TIMP-2. Activation of MMP-2 requires an intact MMP-2 C-terminal domain. MT1-MMP induce activation of MMP-2 by producing ®rst a 64-kDa intermediate, followed by a 62-kDa active enzyme. This MT1-MMP, TIMP-2 complex binds pro-MMP-2 to form a trimeric complex through the carboxyl terminal of proMMP-2.24 Truncation of the transmembrane domain of MT-MMPs abolishes the MMP-2 activation. This ®nding suggests that the transmembrane domain serves to anchor the MT-MMP at a position on the cell surface that facilitates activation of proMMP-2/TIMP-2 complex. However, some tumor cell lines can bind proMMP-2 without activating the enzyme, which suggests the existance of receptor for proMMP-2 that may be distinct from MT-MMP. Integrin aVb3 may also be involved in binding MMP-2 at the cell surface; in one study expression of aVb3 on the surface of cultured
Fig. 2. Cytochalasin D inhibits MMP-9 expression in a human glioma cell line. SNB19 glioma cells were plated in a six-well dish, incubated with cytochalasin-D (5 mM), colchine B (5 mM), PMA (50 ng/ ml). Conditioned medium was collected after 48 h and run on SDS-polyacrylamide gels containing gelatin. Zymographic analysis shows that PMA-induced MMP-9 expression was inhibited when cytochalasin D was added; colchicine B had no eect on MMP-9 expression.
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melanoma cells enabled them to bind MMP-2 in a proteolytically active form that allowed cellmediated degradation of collagen.4 According to the `cysteine switch' model proposed by Van Wart et al.,28 the pro-domain of the latent MMP-2 is folded so that the cysteine residue on the conserved PRCGVPD region can form a complex with zinc. The proteinases trypsin, plasmin, chymotrypsin, neutrophil elastase and plasma kalikrein attack a short basic sequence exposed on the surface of the molecule, thereby trigger the activation of proMMPs in vivo. This initial cleavage causes a change in the conformation of the molecule that disrupts the cysteine-zinc interaction and frees the Zinc ion to participate in the proteolytic cleavage. The enzyme can then attack the propeptide sequence downstream of the PRCGVPD in an autocatalytic manner. The pro-enzyme can also be activated with mercurial compound such as aminophenylmercuric acetate (APMA), which disrupts the cystein-zinc interaction and generates free zinc at the catalytic site.28
MMP-9 (GELATINASE B) MMP-9, also known as gelatinase B, type V collagenase and EC3.4.24.25, cleaves native type I collagen, proteoglycans and laminins. MMP-9 was ®rst identi®ed as a neutral protease isolated from human neutrophils that can degrade denatured collagens62. Proteases that degrade type IV and V collagens subsequently identi®ed in human neutrophils were characterized as 90±110-kDa metalloproteinases. A gelatinolytic substance obtained from simian virus 40 (SV40)-transformed human lung ®broblasts shows genetic homology to MMP-2.29 The MMP-9 gene in human is 7.7 kb and contains 13 exons. Unlike MMP-2, MMP-9 has a TATA box, an AP-1 element, an SP1 transcriptional factor, a 12-O-tetradeconyol phorbol 4-acetate responsive element (TRE) and a NF-kB binding site. Activation of MMP-9 is a complex process that diers according to whether it is free or in complex with other molecules. MMP-9 is secreted as a proenzyme, either in complex with TIMP-1 that inhibits MMP-9 activity or in TIMP free form. APMA activates TIMP-free MMP9 by cleaving ®rst at the NH2-terminal peptide at the Ala74-Met75 bond, yielding an 83-kDa intermediate, and then at the COOH terminal, resulting in a 67-kDa enzyme44. TIMP-1 binding to MMP-9 prohibits processing to the 67-kDa form. MMP-9 is also activated by cathepsin G, trypsin, a-chymotrypsin, and stromelysin-1 but not by plasmin, thrombin, or interstitial collagenase. Activation of TIMP-1/MMP-9 by stromelysin-1 produces a 82-kDa cleavage product; if MMP-9 is not complexed with TIMP-1 the product is cleaved further into either an active 67-kDa form with a COOH-terminal, or an inactive 50-kDa form lacking the catalytic domain. Collagenase 1, matrilysin, mast-cell chymase, MMP-2, and trypsin also activate MMP9. The expression of MMP-9 can be in¯uenced by many agents, including growth factors, cytokines, ECM molecules, cell±cell and cell±ECM adhesion molecules and agents that alter cell shape. For example transfecting glioma cells with NCAM-B downregulate their MMP-9 secretion, but transfection with NCAM-C, which is expressed at the cell surface through GPI linkage, had no eect on MMP-9 secretion.9 On the other hand, plating cells on a mixture of tenascin and ®bronectin upregulated their production of MMP-9. Moreover, cytoskeletal reorganization with cytochalasin D, but not with colchicine B decreased MMP-9 expression by human glioma cells (Fig. 2).5 Cytochalasin D also abolished the induction of MMP-9 in HL-60 cell lines treated with TPA.27 Cell-to-cell contact also in¯uences MMP-9 activation. Cocultures of ®broblasts with colon carcinoma cells resulted in MMP-9 induction. MMP-9 is not often expressed in normal cells, but is expressed in melanomas, lung tumors, breast adenocarcinoma, hepatocellular carcinoma and gliomas. Recent studies indicate a dierent mechanism of MMP-9 activation at the cellsurface. A ternary complex consisting of CD44, hyaluronon and MMP-9 is required for cell surface-associated MMP-9 activity, suggesting that CD44 serves to anchor MMP-9 on the tumor cells.32
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EXPRESSION OF MMPS IN GLIOMAS An imbalance between the activities of MMP and TIMPs results in excessive ECM degradation in conditions such as tumor formation and metastasis. MMP-2 expression is increased in many human tumors, including colon carcinoma, pancreas, prostate, bladder, skin, squamous and basal cell carcinomas, breast, ovary; high levels of stromelysin-1 expression has been reported in glioblastomas.12 Gioblastomas express elevated levels of MMPs compared to low-grade gliomas and normal brain both in vitro1,21,22 and in vivo,16,31 in particular MMP-9 activity in surgical specimens was proportional to the grade of glioma.21 Overexpression of MMP-2 and MMP-9 was reported to be associated with the malignancy of gliomas and accompanied by the overexpression of the TIMP-1 gene,17 in contrary, downregulation of TIMP-1 and TIMP-2 has also been attributed to glioma progression.15 We found that MT1MMP expression is correlated with glioma progression and MMP-2 activation.31 Recently Lampert et al.10 also found that amounts of mRNA for MT1-MMP, gelatinase A and B, and TIMP-1 were greater in glioblastomas than in normal brain, and suggested that increased expression of MT1-MMP and gelatinase expression is closely related to malignant progression of gliomas. Other studies have shown that transfection of glioma cells with MT1-MMP cDNA resulted in increased cell-surface activation of proMMP2, increased collagen degradation, and cell migration in a tumor spheroid outgrowth assay.6 Moreover, MMP-2 activity at the cell surface rather than in the soluble form was critical for the glioma cells to remodel the ECM.7 Although increased expression of MMPs has been correlated with glioma tumor progression, the regulatory mechanisms responsible for the expression of these proteases is poorly understood to date. Gliomas express abnormally high levels of PKC activity and inhibition of PKC in glioblastomas inhibit MMP-2 activity and invasion of glioma cells. MEMBRANE-TYPE MMPS (MT-MMPS) A dierent type of MMP, one having a membrane-binding domain rather than being secreted in proenzyme form, was ®rst described by Sato et al.23 MT-MMPs are distributed widely, and have been found in colon, breast and head and neck carcinomas. The MT-MMP family has at least four members, MMP-14,23 MMP-15,30 MMP-1625 and MMP-17.20 MMP-14 and MMP-16 speci®cally activate MMP-2.33 Structurally all MT-MMPs resemble the other MMPs, but have three additional inserts: an 11-amino acid furin recognition site, located between the propeptide and the N-terminal catalytic domains; an 8-amino acid insertion in the N-terminus; and a 72amino acid hydrophobic insert in the C-terminus that can pass through the plasma membrane and thus acts as a potential transmembrane domain. All MT-MMPs share 30±50% sequence homology and a common multidomain structure.23,20 The potential furin/prohormone cleavage site at the end of the propeptide domain, common to all MT-MMPs is also conserved in stromelyin-3, a soluble MMP. The function of an 8-amino acid insertion within the catalytic domain of MT-MMPs is not clear but may be related to substrate speci®city or impaired TIMP-1 binding of MT1-MMP and MT2-MMP. The cytoplasmic tail at the carboxyl end of three of the MT-MMPs contains an additional conserved cysteine residue that is ¯anked by tyrosine and serine residues, which may be a potential phosphorylation sites. However these tyrosine and serine residues are absent in MT4-MMP.20 MT-MMP expression is tightly regulated at the transcriptional level by growth factors and cytokines. MMP-14 expression is upregulated in synovial ®broblasts tumor necrosis factor-alpha (TNF-a), interleukin-1B (IL-1B), epidermal growth factor (EGF), and by basic ®broblast growth factor (bFGF).13 Little is known about the regulatory elements of the MT1-MMP promoter sequence. The lectin concanavalin A (ConA) induces MT1-MMP activity in a c-Rasdependent manner26 and usually induces proMMP-2 activation as well. However, ConA could also be responsible for cross-linking of cell surface MT1-MMP or concentrating the enzyme at the surface, thereby facilitating rapid activation of proMMP-2. In some cell types, MT1-MMP is modulated by phorbol esters and proMMP-2 processing. MT1-MMP synthesis is also in¯uenced by cytoskeleton; treatment of ®broblasts with cytochalasin-D, which disrupts stress ®bers, lead to increase in MT1-MMP mRNA levels and proMMP-2 activation.27 In addition to
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their ability to activate proMMP-2, MT-MMPs can degrade denatured interstitial collagenase, cartilage aggrecan, perlecan, ®bulins 1 and 2, ®bronectin (FN), vitronectin (VN), nidogen and large tenascin-C.8,18 We recently showed that MT1-MMP mRNA was expressed at high levels in human gliomas both in vitro and in vivo.31 CONCLUSIONS AND FUTURE PERSPECTIVES Although the expression of MMPs has been demonstrated in gliomas, the role and activation mechanisms of the two critical MMPs (MMP-2 and MMP-9) that play a pivotal role in glioma invasion is not clearly understood and still a subject of investigation. The activation mechanism of MMP-2 is entirely dierent from the other MMPs; moreover, MMP-2 is constitutively expressed in all glioma grades in vitro and in vivo, unlike MMP-9. One therapeutic strategy being proposed to inhibit gliomas is to use chemotherapy with MMP inhibitors in addition to surgery and radiotherapy. These agents, still in the early stages of development, show promise for inhibiting not only glioma invasion but also growth and angiogenesis. Although MMP inhibitors may not cure gliomas, they may prolong patient survival by inhibiting tumor invasion process. Also, preliminary studies by us and others have shown that gene therapy may be a feasible approach for the inhibition of invasion in gliomas. AcknowledgementsÐWe thank Christine F. Wogan for her suggestions on the manuscript. This work was supported in part by CA56792, CA75557 and CA76350 from the NCI (JSR).
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16. Nakagawa, T., Kubota, T., Kabuto, M., Sato, K., Kawano, H., Hayakawa, T. and Okada, Y., Production of matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 by human brain tumors. J. Neurosurg., 1994, 81(1), 69±77. 17. Nakano, A., Tani, E., Miyazaki, K., Yamamoto, Y. and Furuyama, J., Matrix metalloproteinases and tissue inhibitors of metalloproteinases in human gliomas. J. Neurosurg., 1995, 83, 298±307. 18. Ohuchi, E., Imai, K., Fujii, Y., Sato, H., Seiki, M. and Okada, Y., Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. J. Biol. Chem., 1997, 272(4), 2446±2451. 19. Parker, S. L., Tong, T., Bolden, S. and Wingo, P. A., Cancer statistics. CA Cancer J. Clin., 1997, 47, 5±27. 20. Puente, X. S., Pendas, A. M., Llano, E., Velasco, G. and Lopez-Otin, C., Molecular cloning of a novel membranetype matrix metalloproteinase from a human breast carcinoma. Cancer Res., 1996, 56(5), 944±949. 21. Rao, J. S., Steck, P. A., Mohanam, S., Stetler-Stevenson, W. G., Liotta, L. A. and Sawaya, R., Elevated levels of M(r) 92,000 type IV collagenase in human brain tumors. Cancer Res., 1993, 53(10 Suppl.), 2208±2211. 22. Rutka, J. T., Hubbard, S. L., Fukuyama, K., Matsuzawa, K., Becker, L. E., Stetler-Stevenson, W. and Dirks, P. B., Expression of TIMP-1, TIMP-2, 72-kDa and 92-kDa type IV collagenase transcript in human astrocytoma cell lines: correlation with astrocytoma cell invasiveness. Int. J. Oncol., 1995, 6, 877±884. 23. Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E. and Seiki, M., A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature, 1994, 370(6484), 61±65. 24. Strongin, A. Y., Collier, I., Bannikov, G., Marmer, B. L., Grant, G. A. and Goldberg, G. I., Mechanism of cell surface activation of 72-kDa type IV collagenase: isolation of the activated form of the membrane metalloprotease. J. Biol. Chem., 1995, 270(10), 5331±5338. 25. Takino, T., Sato, H., Shinagawa, A. and Seiki, M., Identi®cation of the second membrane-type matrix metalloproteinase (MT-MMP-2) gene from a human placenta cDNA library. MT-MMPs form a unique membrane-type subclass in the MMP family. J. Biol. Chem., 1995, 270(39), 23013±23020. 26. Thant, A. A., Serbulea, M., Kikkawa, F., Liu, E., Tomoda, Y. and Hamaguchi, M., c-Ras is required for the activation of the matrix metalloproteinases by concanavalin A in 3Y1 cells. FEBS Lett., 1997, 406(1-2), 28±30. 27. Tomasek, J. J., Halliday, N. L., Updike, D. L., Ahern-Moore, J. S., Vu, T. K., Liu, R. W. and Howard, E. W., Gelatinase A activation is regulated by the organization of the polymerized actin cytoskeleton. J. Biol. Chem., 1997, 272(11), 7482±7487. 28. Van Wart, H. E. and Birkedal-Hansen, H., The cysteine switch: a principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family Proc. Natl. Acad. Sci. U.S.A., 1990, 87(14), 5578±5582. 29. Wilhelm, S. M., Collier, I. E., Marmer, B. L., Eisen, A. Z., Grant, G. A. and Goldberg, G. I., SV40-transformed human lung ®broblasts secrete a 92-kDa type IV collagenase which is identical to that secreted by normal human macrophages. J. Biol. Chem., 1989, 264(29), 17213±17221. 30. Will, H. and Hinzmann, B., cDNA sequence and mRNA tissue distribution of a novel human matrix metalloproteinase with a potential transmembrane segment. Eur. J. Biochem., 1995, 231(3), 602±608. 31. Yamamoto, M., Mohanam, S., Sawaya, R., Fuller, G. N., Seiki, M., Sato, H., Gokaslan, Z. L., Liotta, L. A., Nicolson, G. L. and Rao, J. S., Dierential expression of membrane-type matrix metalloproteinase and its correlation with gelatinase A activation in human malignant brain tumors in vivo and in vitro. Cancer Res., 1996, 56(2), 384±392. 32. Yu, Q., and Stamenkovic, I. Abstract. 2.2 VII international congress of the metastatic research society, San Diego, CA, 1998. 33. Yu, A. E., Hewitt, R. E., Kleiner, D. E. and Stetler-Stevenson, W. G., Molecular regulation of cellular invasion-role of gelatinase A and TIMP-2. Biochem. Cell Biol., 1996, 74(6), 823±831.
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MATRIX METALLOPROTEINASES AND THEIR BIOLOGICAL FUNCTION IN HUMAN GLIOMAS SHRAVAN K. CHINTALA,$ JORG C. TONN% and JASTI S. RAO$* $Departments of Neurosurgery, The University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030, USA; %Department of Neurosurgery, University of Wurzburg, Wurzburg, Germany
AbstractÐGliomas, a type of devastating primary brain tumors, are distinct from other solid, nonneural primary neoplasms, in that they display extensive in®ltrative invasive behavior but seldom metastasize to distant organs. This invasiveness into the surrounding normal brain tissue makes gliomas a major challenge for clinical intervention. Total surgical resection of gliomas is not possible, and recurrence of tumor growth is common; mean survival time is 8±12 months. Although substantial progress has been made recently toward understanding the behavior of gliomas, the mechanisms that facilitate invasion are still poorly documented. Clues to the invasion process have been ascertained through clari®cation of the key roles played by the extracellular matrix (ECM), cell-adhesion molecules and matrix degrading proteases. Serine proteases and metalloproteinases have been implicated in glioma tumor cell-invasion. Matrix metalloproteinases (MMPs) in particular can degrade almost all known ECM components and seem to play important roles in mediating glioblastoma tumor cell invasion. This review focuses on recent developments concerning the role of MMPs in the invasiveness of human gliomas. # 1999 ISDN. Published by Elsevier Science Ltd All rights reserved Key words: matrix metalloproteases, gelatinase, glioma, TIMP, MT-MMP.
INTRODUCTION Brain tumors are the third most frequent cause of cancer-related death in adults and the second most common cause of cancer-related death in children. Recent estimates indicate that 13,000 people died of brain tumors in 1996 and about 17,000 new cases of brain tumors were expected to be diagnosed in 1997.19 In adults, malignant gliomas are the most common primary brain tumors and account for more than 40% of all central nervous system (CNS) neoplasms. The World Health Organization (WHO) system uses four major grades to describe gliomas, namely pilocytic astrocytoma, low-grade astrocytoma, anaplastic astrocytoma and glioblastoma. Astrocytomas are tumors composed of predominantly neoplastic astrocytes. Pilocytic astrocytomas (WHO grade I) are typically located in midline structures such as the optic nerves, third ventricle, thalamus, medial temporal lobe, brainstem and cerebellum. Pilocytic astrocytomas rarely progress to anaplasia. Low-grade astrocytomas (WHO grade II) tend to diusely in®ltrate the surrounding brain parenchyma. Low-grade astrocytomas can appear anywhere in the CNS, including the spinal cord, but are most common in the cerebral hemispheres. Anaplastic astrocytomas (WHO grade III) are characterized by neoplastic ®brillary or gemistocytic astrocytes and can progress rapidly to glioblastoma. Glioblastoma multiforme (WHO grade IV) is the most frequent malignant brain tumor to aect adults. Glioblastomas are most frequently found in the frontotemporal region, but the parietal lobes can also be aected. Gliomas are thought to be derived from astrocytes, oligodendrocytes or ependymal cells and display a correspondingly broad spectrum of histopathological features. Brain tumors can arise as a result of gradual accumulation of genetic abnormalities in precursor cells, including loss or inactivation of the tumor suppressor gene p162 or overexpression of certain protease receptors *To whom all correspondence should be addressed. Tel.: +1 713 792 3266; Fax: +1 713 794 4950; E-mail: jrao@ notes.mdacc.tmc.edu. 495
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Fig. 1. Expression of MMPs and TIMPs in various grades of glioma tissues and normal brain. Values are based on northern blot analysis and represented as arbitrary values.
such as urokinase-type plasminogen activator (uPA) receptors.14 For reasons that are poorly understood, most primary brain tumors do not metastasize and rarely disseminate through cerebrospinal ¯uid. However, they do invade the surrounding normal brain. This characteristic local invasiveness of gliomas contributes substantially to the inability to achieve total resection by surgery and often results in recurrences at the primary site and at locations on the opposite side of the brain. Glial-cell invasion is a multistep process that is invariably accompanied by the expression of proteases. If cells are to cross an extracellular barrier, they must ®rst attach to the barrier matrix, then create a proteolytic defect in the matrix and ®nally migrate through that defect. Numerous studies have demonstrated a close association between the expression of various proteases, such as serine proteases, the metalloproteinases (MMPs) and the plasminogen activation/plasmin system, and the invasive behavior of gliomas. Although gliomas express other proteases, MMPs seem to be responsible for much of the degradation of a broad range of ECM components, and in particular, elevated levels of MMP-2, MMP-9, MT1-MMP were found in gliomas, compared to normal brain tissue (Fig. 1). The following sections provide a review of MMP expression and function in the invasion and progression of human gliomas.
MATRIX METALLOPROTEINASES (MMPS) MMPs are a group of structurally related enzymes that degrade several components of the ECM, including ®brillar and non®brillar collagens, ®bronectin, laminin and basement membrane proteoglycans (Table 1). At least 20 members of the human MMP family have been identi®ed.3 Most MMPs are secreted as zymogens, and their activity is controlled by activators and inhibitors, the latter termed tissue inhibitors of metalloproteinases (TIMPs). Common characteristics of MMPs include Zn2+ atoms at their active sites, requiring Ca2+ ions for enzyme activity, and having highly conserved N-terminal propeptide and catalytic domains.3 MMPs have been grouped according to their substrate speci®cities, i.e. as collagenases; gelatinases, stromelysins and the membrane-type MMPs (Table 1). One MMP, MMP-2 or gelatinase A, is unusual in its constitutive expression by many cells, its ubiquitous tissue distribution and its mode of activation which diers from that of any other MMP. Moreover, unlike other MMP proenzymes, progelatinases A and progelatinase B (also known as MMP-9) are usually isolated in complex with their endogenous inhibitors, TIMP-2 and TIMP-1 respectively. Both the free enzyme and the enzyme-inhibitor complex can be activated on the cell surface by membrane-type MMPs (MT-MMPs). Commonalities in the structural domains of MMPs are shown in Fig. 1. A 17±20-residue signal peptide, rich in hydrophobic amino acids, directs the translational product to the endoplasmic reticulum in all but MMP-17, which lacks this peptide.20 Other common domanins include a 80-amino acid propeptide domain that contains the highly conserved PRCXXPD
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Table 1. Matrix metalloproteinases (MMPs) Subgroup Collagenases
Enzyme
Substrate
MMP-1 (Interstitial collagenase)
collagen I, II, III, VII, VIII, X, gelatin, proteoglycans same as MMP-1 same as MMP-1 collagen I
MMP-8 (Neutrophil collagenase) MMP-13 (Collagenase-3) MMP-18 (Collagenase-4, xenopus)
Gelatinases
MMP-2 (Gelatinase A) MMP-9 (Gelatinase B)
Stromelysins
MMP-3 (Stromelysin-1) MMP-10 (Stromelysin-2) MMP-11 (Stromelysin-3)
Others
Membrane-type MMPs
MMP-7 (Matrylysin)
collagen I, IV, V, VII, X, gelatin, elastin, ®bronectin proteoglycans same as MMP-2
collagen IV, IX, X, elastin, ®bronectin, laminin, proteoglycans, pro-interstitial collagenase same as MMP-3 alpha-1-antitrypsin
MMP-12 (Metalloelastase) MMP-19 MMP-20 (Enamelysin)
collagen IV, ®bronectin, laminin, gelatin, pro-interstitial collagenase, proteoglycans elastin unknown unknown
MMP-14 (MT1-MMP)
activates MMP-2
MMP-15 (MT2-MMP) MMP-16 (MT3-MMP) MMP-17 (MT4-MMP)
unknown activates MMP-2 unknown
sequence, which is cleaved during activation; a catalytic domain of about 160±170 amino acids, that contains the highly conserved HEXGHXXGXXHS/T region, a thermolysin-type zincbinding region, and a calcium-binding site; a 200-residue carboxy-terminal domain homologous to hemopexin and vitronectin; a hinge region of about 75-amino acids, that connects the catalytic and hemopexin domains. Two MMPs (MMP-2 and MMP-9) have additional three ®bronectin type II gelatin binding domains in the catalytic domain. Membrane-type MMPs also have an additional 80±110-residue transmembrane domain. The presence of the furin cleavage site RXKR/RRKR between the prodomain and catalytic domains in stromelyin-3 and all known MT-MMPs suggest that these MMPs may be activated while they are still in the cell, whereas the other enzymes require proteolytic cleavage after they are secreted as zymogens. Although tumors secrete many dierent proteases, MMP-2 and MMP-9 seem to play major role in tumor progression. The following sections provide a brief review of MMP-2, MMP-9 and MMP-9 and their biological signi®cance, in glioma progression.
GELATINASES The gelatinase subgroup of MMPs has two members, MMP-2 (gelatinase A) and MMP-9 (gelatinase B). MMP-2 perhaps the most widespread of all MMPs; it is produced by a variety of cells and is frequently elevated in malignant tumors. MMP-2 is often secreted as a complex with TIMP-2. The other gelatinase, MMP-9, is expressed by transformed tumor cells, neutrophils, monocytes, and alveolar macrophages,11 and is often secreted as a complex with TIMP-1. MMP-2 has a unique mechanism of activation that involves a trimolecular complex consisting
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of MT-MMP,23 TIMP-2, and the C-terminal domain of MMP-2.24 Details of these two MMPs are given below. MMP-2 (GELATINASE A) MMP-2, a 72-kDa gelatinase (also known as gelatianase A, 72 kDa type IV collagenase and EC3.4.24.24) is constitutively expressed by many cells; MMP-2 not only degrades ECM, but also regulates cell proliferation, adhesion and migration.33 MMP-2 is not transcriptionally activated by TPA or IL-1 and lacks the typical transactivation sequences such as AP-1, PEA-3 or TATA boxes that play pivotal roles in the transcriptional regulation of most promoters. Moreover, unlike other MMPs it lacks an upstream transforming growth factor-beta-inhibitory element (TIE). MMP-2 seems to be posttranscriptionally regulated, but the mechanism has not been studied in detail. MMP activation is also regulated by the TIMPs.29 The N-terminal domains of TIMPs are inhibitory, and the C-terminal domains confer binding speci®city. For example, the N-terminal domains of either TIMP-1 or TIMP-2 can bind to the N-terminal domain of active MMP-2; however, only the C-terminal domain of TIMP-2 binds speci®cally to the C-terminal domain of MMP-2. ProMMP-2 has a unique mechanism of activation at the cell surface that is modulated by both MT1-MMP and TIMP-2. Activation of MMP-2 requires an intact MMP-2 C-terminal domain. MT1-MMP induce activation of MMP-2 by producing ®rst a 64-kDa intermediate, followed by a 62-kDa active enzyme. This MT1-MMP, TIMP-2 complex binds pro-MMP-2 to form a trimeric complex through the carboxyl terminal of proMMP-2.24 Truncation of the transmembrane domain of MT-MMPs abolishes the MMP-2 activation. This ®nding suggests that the transmembrane domain serves to anchor the MT-MMP at a position on the cell surface that facilitates activation of proMMP-2/TIMP-2 complex. However, some tumor cell lines can bind proMMP-2 without activating the enzyme, which suggests the existance of receptor for proMMP-2 that may be distinct from MT-MMP. Integrin aVb3 may also be involved in binding MMP-2 at the cell surface; in one study expression of aVb3 on the surface of cultured
Fig. 2. Cytochalasin D inhibits MMP-9 expression in a human glioma cell line. SNB19 glioma cells were plated in a six-well dish, incubated with cytochalasin-D (5 mM), colchine B (5 mM), PMA (50 ng/ ml). Conditioned medium was collected after 48 h and run on SDS-polyacrylamide gels containing gelatin. Zymographic analysis shows that PMA-induced MMP-9 expression was inhibited when cytochalasin D was added; colchicine B had no eect on MMP-9 expression.
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melanoma cells enabled them to bind MMP-2 in a proteolytically active form that allowed cellmediated degradation of collagen.4 According to the `cysteine switch' model proposed by Van Wart et al.,28 the pro-domain of the latent MMP-2 is folded so that the cysteine residue on the conserved PRCGVPD region can form a complex with zinc. The proteinases trypsin, plasmin, chymotrypsin, neutrophil elastase and plasma kalikrein attack a short basic sequence exposed on the surface of the molecule, thereby trigger the activation of proMMPs in vivo. This initial cleavage causes a change in the conformation of the molecule that disrupts the cysteine-zinc interaction and frees the Zinc ion to participate in the proteolytic cleavage. The enzyme can then attack the propeptide sequence downstream of the PRCGVPD in an autocatalytic manner. The pro-enzyme can also be activated with mercurial compound such as aminophenylmercuric acetate (APMA), which disrupts the cystein-zinc interaction and generates free zinc at the catalytic site.28
MMP-9 (GELATINASE B) MMP-9, also known as gelatinase B, type V collagenase and EC3.4.24.25, cleaves native type I collagen, proteoglycans and laminins. MMP-9 was ®rst identi®ed as a neutral protease isolated from human neutrophils that can degrade denatured collagens62. Proteases that degrade type IV and V collagens subsequently identi®ed in human neutrophils were characterized as 90±110-kDa metalloproteinases. A gelatinolytic substance obtained from simian virus 40 (SV40)-transformed human lung ®broblasts shows genetic homology to MMP-2.29 The MMP-9 gene in human is 7.7 kb and contains 13 exons. Unlike MMP-2, MMP-9 has a TATA box, an AP-1 element, an SP1 transcriptional factor, a 12-O-tetradeconyol phorbol 4-acetate responsive element (TRE) and a NF-kB binding site. Activation of MMP-9 is a complex process that diers according to whether it is free or in complex with other molecules. MMP-9 is secreted as a proenzyme, either in complex with TIMP-1 that inhibits MMP-9 activity or in TIMP free form. APMA activates TIMP-free MMP9 by cleaving ®rst at the NH2-terminal peptide at the Ala74-Met75 bond, yielding an 83-kDa intermediate, and then at the COOH terminal, resulting in a 67-kDa enzyme44. TIMP-1 binding to MMP-9 prohibits processing to the 67-kDa form. MMP-9 is also activated by cathepsin G, trypsin, a-chymotrypsin, and stromelysin-1 but not by plasmin, thrombin, or interstitial collagenase. Activation of TIMP-1/MMP-9 by stromelysin-1 produces a 82-kDa cleavage product; if MMP-9 is not complexed with TIMP-1 the product is cleaved further into either an active 67-kDa form with a COOH-terminal, or an inactive 50-kDa form lacking the catalytic domain. Collagenase 1, matrilysin, mast-cell chymase, MMP-2, and trypsin also activate MMP9. The expression of MMP-9 can be in¯uenced by many agents, including growth factors, cytokines, ECM molecules, cell±cell and cell±ECM adhesion molecules and agents that alter cell shape. For example transfecting glioma cells with NCAM-B downregulate their MMP-9 secretion, but transfection with NCAM-C, which is expressed at the cell surface through GPI linkage, had no eect on MMP-9 secretion.9 On the other hand, plating cells on a mixture of tenascin and ®bronectin upregulated their production of MMP-9. Moreover, cytoskeletal reorganization with cytochalasin D, but not with colchicine B decreased MMP-9 expression by human glioma cells (Fig. 2).5 Cytochalasin D also abolished the induction of MMP-9 in HL-60 cell lines treated with TPA.27 Cell-to-cell contact also in¯uences MMP-9 activation. Cocultures of ®broblasts with colon carcinoma cells resulted in MMP-9 induction. MMP-9 is not often expressed in normal cells, but is expressed in melanomas, lung tumors, breast adenocarcinoma, hepatocellular carcinoma and gliomas. Recent studies indicate a dierent mechanism of MMP-9 activation at the cellsurface. A ternary complex consisting of CD44, hyaluronon and MMP-9 is required for cell surface-associated MMP-9 activity, suggesting that CD44 serves to anchor MMP-9 on the tumor cells.32
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EXPRESSION OF MMPS IN GLIOMAS An imbalance between the activities of MMP and TIMPs results in excessive ECM degradation in conditions such as tumor formation and metastasis. MMP-2 expression is increased in many human tumors, including colon carcinoma, pancreas, prostate, bladder, skin, squamous and basal cell carcinomas, breast, ovary; high levels of stromelysin-1 expression has been reported in glioblastomas.12 Gioblastomas express elevated levels of MMPs compared to low-grade gliomas and normal brain both in vitro1,21,22 and in vivo,16,31 in particular MMP-9 activity in surgical specimens was proportional to the grade of glioma.21 Overexpression of MMP-2 and MMP-9 was reported to be associated with the malignancy of gliomas and accompanied by the overexpression of the TIMP-1 gene,17 in contrary, downregulation of TIMP-1 and TIMP-2 has also been attributed to glioma progression.15 We found that MT1MMP expression is correlated with glioma progression and MMP-2 activation.31 Recently Lampert et al.10 also found that amounts of mRNA for MT1-MMP, gelatinase A and B, and TIMP-1 were greater in glioblastomas than in normal brain, and suggested that increased expression of MT1-MMP and gelatinase expression is closely related to malignant progression of gliomas. Other studies have shown that transfection of glioma cells with MT1-MMP cDNA resulted in increased cell-surface activation of proMMP2, increased collagen degradation, and cell migration in a tumor spheroid outgrowth assay.6 Moreover, MMP-2 activity at the cell surface rather than in the soluble form was critical for the glioma cells to remodel the ECM.7 Although increased expression of MMPs has been correlated with glioma tumor progression, the regulatory mechanisms responsible for the expression of these proteases is poorly understood to date. Gliomas express abnormally high levels of PKC activity and inhibition of PKC in glioblastomas inhibit MMP-2 activity and invasion of glioma cells. MEMBRANE-TYPE MMPS (MT-MMPS) A dierent type of MMP, one having a membrane-binding domain rather than being secreted in proenzyme form, was ®rst described by Sato et al.23 MT-MMPs are distributed widely, and have been found in colon, breast and head and neck carcinomas. The MT-MMP family has at least four members, MMP-14,23 MMP-15,30 MMP-1625 and MMP-17.20 MMP-14 and MMP-16 speci®cally activate MMP-2.33 Structurally all MT-MMPs resemble the other MMPs, but have three additional inserts: an 11-amino acid furin recognition site, located between the propeptide and the N-terminal catalytic domains; an 8-amino acid insertion in the N-terminus; and a 72amino acid hydrophobic insert in the C-terminus that can pass through the plasma membrane and thus acts as a potential transmembrane domain. All MT-MMPs share 30±50% sequence homology and a common multidomain structure.23,20 The potential furin/prohormone cleavage site at the end of the propeptide domain, common to all MT-MMPs is also conserved in stromelyin-3, a soluble MMP. The function of an 8-amino acid insertion within the catalytic domain of MT-MMPs is not clear but may be related to substrate speci®city or impaired TIMP-1 binding of MT1-MMP and MT2-MMP. The cytoplasmic tail at the carboxyl end of three of the MT-MMPs contains an additional conserved cysteine residue that is ¯anked by tyrosine and serine residues, which may be a potential phosphorylation sites. However these tyrosine and serine residues are absent in MT4-MMP.20 MT-MMP expression is tightly regulated at the transcriptional level by growth factors and cytokines. MMP-14 expression is upregulated in synovial ®broblasts tumor necrosis factor-alpha (TNF-a), interleukin-1B (IL-1B), epidermal growth factor (EGF), and by basic ®broblast growth factor (bFGF).13 Little is known about the regulatory elements of the MT1-MMP promoter sequence. The lectin concanavalin A (ConA) induces MT1-MMP activity in a c-Rasdependent manner26 and usually induces proMMP-2 activation as well. However, ConA could also be responsible for cross-linking of cell surface MT1-MMP or concentrating the enzyme at the surface, thereby facilitating rapid activation of proMMP-2. In some cell types, MT1-MMP is modulated by phorbol esters and proMMP-2 processing. MT1-MMP synthesis is also in¯uenced by cytoskeleton; treatment of ®broblasts with cytochalasin-D, which disrupts stress ®bers, lead to increase in MT1-MMP mRNA levels and proMMP-2 activation.27 In addition to
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their ability to activate proMMP-2, MT-MMPs can degrade denatured interstitial collagenase, cartilage aggrecan, perlecan, ®bulins 1 and 2, ®bronectin (FN), vitronectin (VN), nidogen and large tenascin-C.8,18 We recently showed that MT1-MMP mRNA was expressed at high levels in human gliomas both in vitro and in vivo.31 CONCLUSIONS AND FUTURE PERSPECTIVES Although the expression of MMPs has been demonstrated in gliomas, the role and activation mechanisms of the two critical MMPs (MMP-2 and MMP-9) that play a pivotal role in glioma invasion is not clearly understood and still a subject of investigation. The activation mechanism of MMP-2 is entirely dierent from the other MMPs; moreover, MMP-2 is constitutively expressed in all glioma grades in vitro and in vivo, unlike MMP-9. One therapeutic strategy being proposed to inhibit gliomas is to use chemotherapy with MMP inhibitors in addition to surgery and radiotherapy. These agents, still in the early stages of development, show promise for inhibiting not only glioma invasion but also growth and angiogenesis. Although MMP inhibitors may not cure gliomas, they may prolong patient survival by inhibiting tumor invasion process. Also, preliminary studies by us and others have shown that gene therapy may be a feasible approach for the inhibition of invasion in gliomas. AcknowledgementsÐWe thank Christine F. Wogan for her suggestions on the manuscript. This work was supported in part by CA56792, CA75557 and CA76350 from the NCI (JSR).
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