The 4P Tool is designed to assist you in gaining a better insight into your personal Parkinson patient population. For general information or help: mail to email@example.com Prof. A. Jeanjean (Cliniques Universitaires Saint Luc Bruxelles) and Dr. A. Flamez (UZ Brussel, Vrije Universiteit Brussel) are acknowledged for their profound expert advise on the content of this program. The 4P Tool is provided for your convenience, without any warranties, representations or guarantees of any kind.
PATHOLOGY ONCOLOGY RESEARCH Vol 7, No 1, 2001 10.1053.paor.2001.0308 available online at http://www.idealibrary.com on The Role of Matrix Metalloproteinases in Tumor Angiogenesis
and Tumor Metastasis
Anitha JOHN,1 George TUSZYNSKI1,2 Medical College of Pennsylvania and Hahnemann University School of Medicine Department of Pathology1 and Surgery2, Philadelphia, USA Although a considerable amount of effort has been
of enzymes that has been shown over the years to
placed on discovering the etiologies of cancer, the
play a role in tumor progression is the matrix met-
majority of the basic cancer research existing today
alloproteinase (MMP) family. The main function
has focused on understanding the molecular mech-
of MMPs, also known as matrixins, is degradation
anism of tumor formation and metastasis. Metasta-
of the extracellular matrix physiologic function
tic spread of tumors continues to be a major obsta-
involving MMPs include wound healing, bone
cle to successful treatment of malignant tumors.
resorption and mammary involution. MMPs, how-
Approximately 30% of those patients diagnosed
ever, also contribute to pathological conditions
with a solid tumor have a clinically detectable
including rheumatoid arthritis, coronary artery
metastasis and for the remaining 70%, metastases
disease, and cancer. Tumor cells are believed to uti-
are continually being formed throughout the life
lize the matrix degrading capability of these
of the tumor. Even after the tumor is excised, the
enzymes to spread to distant sites. In addition,
threat of death is attributable to the metastasis that
MMPs also are thought to promote the growth of
may occur through the remaining tumor cells. In
these tumor cells once they have metastasized.
addition, treating the metastasis often proves futile
This review will discuss the role of MMPs and
since metastasis often vary in size, composition,
their inhibitors in tumor invasion, angiogenesis
and anatomical location. New treatments blocking
and metastasis with special emphasis on the
the formation of metastasis will provide greater
gelatinases, MMP-2 and MMP-9.
chances of survival for cancer patients. One family
logy Research Vol 7, No 1, 14–23, 2001) Keywords: matrix metalloproteinases; metalloproteinase inhibitors; gelatinases; tumor angiogenesis; metastasis Introduction to the MMP family
Received: Febr 1, 2001; accepted: Correspondence: George P. TUSZYNSKI, MCP-Hahnemann The first mammalian MMP, interstitial collagenase, was School of Medicine, MS 435, Broad & Vine, Philadelphia, PA19102, USA; Tel: 215 762 6297/6299; fax: 215 762 8787; discovered over thirty years ago by Gross and Lapiere in amphibian tissue.10 Since then, over 14 members have List of Abbreviations
been added to the family. The MMP family can be subdi- Amino-terminal – NH2-terminal; Carbohydrate Antigen 19-9 – vided into five groups: the collagenases, the stromelysins, CA19-9; Carcinoembryonic Antigen – CEA; Cerebrospinal Fluid – the gelatinases, PUMP-I or matrilysin, and membrane- CSF; Endothelial Cell – EC; Extracellular Matrix – ECM; Human type (MT) MMPs. Although the classification system was Dermal Microvascular Endothelial Cell – HDMVEC; Human developed on the basis of substrate specificity, it is now Umbilical Vein Endothelial Cell – HUVEC, Interleukin-1 Alpha –IL-1α; Interleukin-1 Beta – IL-1β; Interleukin-8 – IL-8; Intravital recognized that there is some overlap between some mem- Videomicroscopy – IVVM; Matrix Metalloproteinase – MMP; bers of the family (see Table 1). For example, MMP-2 has Matrix Metalloproteinase Inhibitor – MMPI; Membrane-Type Met- been reported to have the ability to cleave fibrillar colla- alloproteinase – MT-MMP; Phorbol 12-Myristate 13-Acetate – gen similar to the collagenases.11 PMA; Polymorphonuclear Cells – PMNs; Thrombospondin-1 – This multigene family of metal containing proteases TSP-1; Tissue Inhibitor of Metalloproteinase – TIMP; Transform-ing Growth Factor Beta – TGF- share several common characteristics: (1) each degrade at β; Tumor Necrosis Factor – TNF; Vascular Endothelial Cell Growth Factor – VEGF least one component of the basement membrane; (2) they 2001 W. B. Saunders & Company Ltd on behalf of the Arányi Lajos Foundation 1219-4956/01/010014+10 $ 12.00/0 Matrix Metalloproteinases in Tumor Angiogenesis and Metastasis are active at physiological pH; (3) they require 2 Zn++ transcriptional activation is not fully understood. These ions/molecule in order to be active; (4) they are inhibited factors cause variable patterns of expression in different by metal chelators and tissue inhibitors of metallopro- tissues and have variable effects on the different MMP teinases (TIMPs); and (5) they are secreted as zymogens family members, complicating the understanding of gene and require activation extracellularly.12,13 regulation of MMPs in both physiological and patholog- Comparing the protein and cDNA sequences of cloned ical states. One of the most potent family of inducers are MMP molecules reveal a number of conserved regions phorbol esters such as PMA.8,15 Other inducers include within the family. All latent MMPs contain at least three interleukin (IL)-1α, IL-1β, IL-8, transforming growth domains: (1) a hydrophobic pre-peptide domain that is nec- factor (TGF)β-1, and tumor necrosis factor (TNF).8,9,13 essary to signal secretion, (2) an amino terminal propeptide Other factors implicated in stimulating MMP regulation domain which is removed upon activation, and (3) the Zn++ include basic fibroblast growth factor (bFGF), epidermal containing catalytic domain.8,12,13 This basic 3-part struc- growth factor (EGF), and vascular endothelial cell ture is present in all of the MMPs. Within the family, how- growth factor (VEGF).16-18 Important to remember is that ever, further subdivisions exist based on distinct structural there is often a balancing effect within the corresponding variations. Matrilysin (MMP-7), the smallest member of physiological inhibitors of MMPs, the tissue inhibitor of the MMP family is comprised of only the basic core struc- metalloproteinases (TIMP). To create a favorable state ture. The remaining enzymes all contain a "hemopexin" for invasion, there must be a balance of protease to domain connected to the catalytic domainby a hinge region. The hemopexin domain Table 1. Matrix Metalloproteinase Family*
contains the TIMP binding site and may Molecular Weight (kDa) also be involved in receptor binding. The MMP Name and No ECM Substrate membrane-type metalloproteases (MT-MMPs) are a unique subdivision in that Interstitial Collagenase Fibrillar Collagens they contain a transmembrane domain at the carboxyl terminal anchoring the mole- Neutrophil Collagenase Fibrillar Collagens cule to the cell surface. Another subdivi- Fibrillar Collagens sion, the gelatinases (MMP-2 and MMP- 9) contain a fibronectin-like domain with- Fibrillar Collagens in the catalytic domain responsible forcollagen binding. MMP-9 has an addi- Laminin, fibronectin, tional collagen V-like sequence within the non-fibrillar collagens Laminin, fibronectin, catalytic domain downstream of the Zn++ non-fibrilar collagens Laminin, fibronectin, non-fibrillar collagens Laminin, fibronectin, non-fibrillar collagens Regulation of the MMPs occurs on three levels: alteration of gene expres- sion, activation of latent zymogens, and Gelatinase A, fibrillar inhibition by tissue inhibitors of metallo- proteinases. The cooperative effects of ECM glycoproteins these three factors provides a tightly con- trolled regulation of MMPs in normal physiological states. Alteration of all three levels of control have been associat- Type I, IV, V and fibrillar ed with tumor cell progression.14 Collagens; gelatin Type IV, V collagen, MMPs and TIMPs are thought to be regulated by a variety of cytokines, *The MMP family are listed here in addition with their molecular weights and theirsubstrates.
growth factors, steroid hormones and The subdivisions correspond to a combination of structural and substrate homo- phorbol esters.8 Despite a vast array of logy. The newest members including MMP-18, MMP-19, and the MT-MMPs have research in this area of MMP regulation, not yet been fully characterized.
Vol 7, No 1, 2001 JOHN and TUSZYNSKI inhibitor in order for the cell to migrate and invade.
or stromelysin. There seems to be a tighter regulation of Although the exact role of cytokines and growth factors MMP-2 than the other MMPs perhaps in part to the fact is not clear, it appears that they act in conjunction to reg- that MMP-2 is the most commonly expressed MMP in ulate both MMPs and their inhibitors to create the envi- normal tissues.28 The newest subclass of MMPs, the ronment necessary for either physiological or pathologi- MT-MMPs, have the capacity to activate pro-MMP-2.29 The transmembrane domain is essential to MT-MMP's In addition to cytokines and growth factors, MMP pro- capacity to activate MMP-2. Understanding this level of duction can be regulated by environmental factors. For regulation of MMP will provide yet another target for example, extracellular matrix (ECM) component peptides development of anti-cancer therapeutics.
have been observed to have an effect on MMP production.
The laminin-1 peptide, AG-73 has been shown to enhance Inhibitors of Metalloproteases
gelatinase production and increase in vivo B16-F10melanoma cell lung and liver metastasis.19 Vitronectin, The third level of control of MMPs occurs through inhi- collagen, and elastin have also been shown to induce col- bition of enzymatic activity. Various physiological agents lagenases and gelatinases expression in either tumor cells can have an inhibitory effect on MMPs including or fibroblasts.20-22 Our laboratory has done extensive work macroglobulin and TIMPs, tissue inhibitors of metallopro- in identifying thrombospondin-1 (TSP-1), a 450 kDa α2-macroglobulin molecule, a large mole- extracellular matrix protein, as a stimulator of MMP-9 cular weight (780 kDa) serum protein, can inhibit pro- production in endothelial cells, pancreatic cancer cells, teinases, but its size prevents the molecule from entering and breast cancer cells.23,24 The capacity of TSP-1 to up- into tissue spaces. The TIMPs are much smaller molecules regulate MMP-9 is mediated in part by the CSVTCG pep- and are expressed in various tissues and fluids. There are tide sequence present in the type one properdin repeats of four members of the mammalian TIMP family, TIMP-1, TSP-1. Synthetic peptides containing the CSVTCG TIMP-2, TIMP-3, and TIMP-4.31 The amino-terminal sequence probably bind and block a specific TSP-1 recep- domain present in all TIMP molecules is responsible for tor.24 These peptides represent a potential therapeutic the MMP inhibitory activity. The TIMPs form high affin- approach for the treatment of metastasis. These data all ity, non-covalent complexes with all active MMPs in a 1:1 suggest that the cell environment plays a key role in regu- stoichiometric ratio. In addition, TIMP-1 and TIMP-2 can lating MMP production.
block the pro forms of MMP-9 and MMP-2, respectively.
The balance between protease and inhibitor is critical in Activation of Latent MMPs
determining net proteolytic activity.
TIMP-1 is a 28.5 kDa glycoprotein that has a wide vari- Another level of MMP regulation is the activation of ety of functions including growth factor activity, stimulat- the zymogen/proenzyme secreted form of MMPs. All ing cell morphology changes, and inhibiting angiogene- MMPs are secreted as zymogens with the exception of sis.31 Increased TIMP-1 levels have traditionally been the MT-MMPs, the membrane sequestered subdivision.
associated with reduced invasion and metastasis. Some These latent zymogens must be activated in order to controversy has arisen on the role of TIMP-1, however.
degrade matrix components. As mentioned previously, Recent studies have shown that TIMP-1 may increase the these proenzymes remain in an inactive form through an invasive capacity of tumor cells due to its growth factor interaction between a cysteine in the proregion and the like activity.32 TIMP-2, similar to TIMP-1, is associated Zn++ ion in the active site. This interaction blocks access with decreased metastatic potential. TIMP-2, a non-glyco- to the active site and cleavage of this site results in sylated 21 kDa protein, suppresses tumor growth and inva- enzyme activation. Trypsin 2 has the ability to activate sion.31 In contrast to TIMP1 and TIMP-2, which are both free proenzyme and proenzyme:TIMP-1 complexes, secreted, TIMP-3 is associated with the extracellular the major form of MMP in vivo.25 Other agents which matrix.31 A recent report describes melanoma cells trans- have been shown to activate MMPs include cathepsins G, fected with the TIMP-3 gene reduced invasion and B, and L, PMN elastase, and hypochlorous acid.26 In induced cell death in these cells in vitro.33 The most addition, the plasmin/plasminogen system has been recently identified molecule is TIMP-4, a 24 kDa protein.
implicated in tumor cell invasion in part through its abil- Some controversy exists on the roles of TIMP-2 and ity to activate MMPs.27 TIMP-3 as well since several studies report increased MMPs, once activated, are also capable of activating expression of these compounds results in increased themselves. MMP-3 can activate MMP-2 and MMP-9 as metastatic potential. Again, it is important to consider the can MMP-7. The gelatinases, MMP-2 and MMP-9, can ratio of protease to inhibitor which is critical to invasive activate each other. In contrast to other members of the capacity. A small change in the balance could have a pro- family, MMP-2 does not seem to be activated by plasmin found effect on proteolysis.
PATHOLOGY ONCOLOGY RESEARCH Matrix Metalloproteinases in Tumor Angiogenesis and Metastasis Tumor progression:
These processes are then repeated upon extravasation of the angiogenic and metastatic cascade
tumor cells from the vessel.38 In vitro and in vivo studies show a definite correlation between gelatinase expression Metastasis, a hallmark of cancer, is the colonization of and metastasis. In addition, MMPs are essential factors in distant sites with cells from the primary tumor.3 The tumor angiogenesis (see Figure 1).39 Proteolytic activity is process of metastasis involves the survival of the primary required during the formation of the capillary bud in order tumor cell through a series of steps including: tumor cell for the endothelial cell to migrate out through the peri-cap- invasion enabling it to break free from the primary tumor, illary membrane and through the ECM. In addition, cap- intravasation, survival and extravasation into and from the illary elongation, lumen formation, and ECM remodeling blood or lymphatic circulation, and finally tumor cell col- all require proteolytic activity. Recent studies have impli- onization and angiogenesis to form the metastatic lesion.3 cated MMPs as an important protease component in angio- Although the majority of those cells that escape from the genesis perhaps as a downstream modulator to known primary tumor will not survive, those capable of invading angiogenesis-related molecules such as VEGF and throm- into and out of the circulatory system will each give rise to a metastatic tumor.
Metastasis can be described clinically as four different The role of MMPs in tumor angiogenesis
patterns: No metastasis is present at the initial diagnosisbut appears within (1) months or (2) years after the prima- Many studies have shown that endothelial cells are ry tumor is removed; (3) metastasis is present at the time capable of differentially expressing and activating MMPs of diagnosis of the primary tumor; and (4) only the metas- and TIMPs. Type I collagen is the predominant constituent tasis is detectable at the time of diagnosis.34 Metastasis that of the perivascular ECM, and as mentioned previously, a appears after several years have been termed "dormant" variety of MMPs are capable of degrading collagen type I metastasis. Considerable research has been done to under- including interstitial collagenase and neutrophil collage- stand the molecular changes a normal cell undergoes to nase.39 Several studies have shown that MMP-1 is a criti- become malignant. However, no single gene has been cal protease in the angiogenic cascade. Immunofluores- implicated as a metastasis-specific gene. There is likely to cent staining revealed that more aggressive skin tumors be a cascade of events that occur to create a malignant cell.
displayed a higher number of collagenase-containing Angiogenesis or neovascularization is a tightly regulated blood vessels.41 This collagen-degrading activity has been system occurring rarely in a normal physiologic environ- shown to be specific through a number of specific MMP ment.34 Reproduction, embryogenesis, and wound healing inhibitors such as TIMP, Batimastat, and Marimastat are among the few situations requiring angiogenesis. The which are capable of suppressing MMPs and therefore formation of these new blood vessels is one of the major capillary tube formation in vivo.42 factors that stimulates tumor growth. Tumors without a A number of angiogenic factors have been shown to reg- blood supply are only able to grow 1-2 mm3, and formation ulate the production of MMPs in endothelial cells. Our of a blood supply is critical to forming metastases.35 Atumor cell cannot escape from the primary tumor until thetumor has been vascularized and once the metastatic cell has reached its target organ, angiogenesis is required forthe metastases to grow.34 Angiogenesis occurs through a series of steps including (1) the release of angiogenic fac- tors (2) the release of proteolytic enzymes to degrade thebasement membrane of the postcapillary venule (3) EC migration toward the tumor (4) EC proliferation and (5)microvessel formation and differentiation.36 Tumors which bear a higher percentage of angiogenic cells have been shown to have increased metastatic potential and are more aggressive tumors.37 Similarly, agents which are capable ofinhibiting angiogenesis, such as anti-bFGF, subsequently reduce growth of malignant cell lines in vivo.37 Figure 1. Schematic of tumor growth dependent on angiogene-
Proteases are required by the malignant cell to invade sis. Tumor cells multiply and grow, eventually requiring a the ECM. The metastatic cell uses proteases to invade blood supply to sustain further growth. Cytokines secreted by through the basement membrane and its underlying con- the tumor cells and adjacent stromal elements stimulate nective tissue and then subsequently through the basement endothelial cells to produce MMPs needed for endothelial cell membrane of the small blood vessels and lymphatics.
migration and tube formation. Vol 7, No 1, 2001 JOHN and TUSZYNSKI laboratory has show that TSP-1 can also stimulate BAECs tor mediated through the lack of MMP-9 expression.
to secrete MMP-9.24 The role of TSP-1 in angiogenesis These data suggest that MMP-9 plays a regulatory role in has been controversial. We have shown that TSP-1 is a angiogenesis not only through proteolytic activity but also bifunctional modulator of angiogenesis acting in part through other downstream angiogenic factors.46 through MMP-9 regulation. In a collagen gel tube forma-tion assay, TSP-1 stimulated endothelial tube formation at Evidence for MMPs in tumor metastasis
low concentration (5 µg/ml) while at higher concentra- µg/ml), TSP-1 inhibited tube formation. The In order for a tumor cell to intravasate and extravasate, ability of TSP-1 to either induce or inhibit tube formation the collagen-rich ECM and basement membrane must be was attributable to the amount of MMP-9 produced. Opti- degraded. This degradative ability can be through either mal MMP-9 levels were induced by lower concentrations enzymatic capacities of the tumor cell or through enzy- of TSP-1. Increasing amounts of MMP-9 caused excessive matic activity of cellular components of the matrix, such proteolysis and inhibited tube formation.
as fibroblasts. Likely there is a cooperation between the The roles of the type IV collagenases, MMP-2 and two components enabling the tumor cell to reach its target MMP-9, in angiogenesis have been explored in a variety organ and survive. Although all five major classes (serine, of studies. In a tube formation assay model, MMP-2 was aspartic, cysteine, threonine, and metalloproteinases) are produced by HUVECs as they formed tube like structures involved in metastasis, a great deal of emphasis has been and this formation could be inhibited by adding TIMP-1 or placed on the type IV collagenases, MMP-2 and MMP-9.38 TIMP-2 to the cultures.43 A recent study examining the Type IV collagen is a major structural protein in the base- progression of mycosis fungoides, a hematological tumor ment membrane and ECM. A number of studies have of T-cell lineage, revealed that the more advanced the linked elevated MMP-2 and MMP-9 levels with an tumor, the microvessel density increases as does MMP-2 increased metastasis. The conclusions which can be drawn and MMP-9 expression.44 The MMP-2 was expressed thus far are that the number and the relative levels of mostly by the microvascular cells of the blood vessels MMPs increase with tumor progression.
within and surrounding the tumor in addition to fibroblasts Several recent studies have been done to try and charac- adjacent to the tumor stroma. MMP-9 was present in the terize the phenotypic and enzymatic profiles of more tissue macrophages located close to the tumor nodules. In aggressive tumor cell lines. Selection of progressively contrast, both MMP-2 and MMP-9 were only weakly more invasive human lung carcinoma cells from an estab- expressed in normal tissue.
lished CL1cell line, revealed that the more invasive cells Perhaps the greatest evidence associating MMP-2 in the had a higher expression of MMP-9.47 These cells had a 4 angiogenic process is the recent MMP-2 knock-out model to 6 fold increase in invasive activity over the parentals examining angiogenesis and tumor progression.45 Gelati- and had an increased metastatic potential in vivo. MMP-9 nase A deficient mice displayed reduced tumor induced has also been shown to be overexpressed in advanced angiogenesis as measured by the dorsal air sac assay. In stage melanoma cells.48 Cell lines from early stage addition tumor volumes of B16-BL6 melanoma cells and melanoma lesions revealed no MMP-9 expression while Lewis lung cancer cells when injected intradermally those cells from advanced lesions not only expressed decreased by 39% and 24%, respectively. The number of MMP-9 but were also capable of being induced to secrete lung colonies also decreased by 54% for the B16-BL6 more MMP-9 by TGF, IL-1 and TPA.48 Other tumor mod- melanoma cells and 77% for the Lewis lung cancer. There- els involving MMP-9 in their invasive phenotype include fore, host derived gelatinase A is necessary to promote human non-hodgkins lymphoma cells and human giant tumor angiogenesis and tumor progression.
cell tumors.49,50 Examining MMP-9 production in human Similarly, the MMP-9 knock-out model also provides giant cell tumors, tumors characterized by frequent vascu- key evidence to the role of gelatinase B in angiogenesis.
lar invasion, revealed that MMP-9 is highly expressed by MMP-9 knock-out mice exhibit an abnormal pattern of the giant cells and the absence of collagen and laminin in skeletal growth plate vascularization and ossification. This the basement membrane correlated with those regions of aberrant pattern of vascularization returns to normal after high MMP-9 expression. MMP-9 through all of these data transplanting wild-type bone marrow cells indicating that is certainly one of the key enzymes involved in invasion MMP-9 expression in cells of bone marrow origin is one and metastasis.
factor that regulates normal vascularization in the skeletal MMP-2 has also been observed to be overexpressed in growth plate. Important to note, however, is that vascular- more aggressive tumor cells. Comparing a highly metasta- ization, apoptosis, and ossification compensates to pro- tic mouse mammary tumor cell line to its parental poorly duce a normal growth plate after three weeks postnatal in metastatic cell line, show the highly metastatic cells these animals. The control on angiogenesis is believed to express more MMP-2.51 The level of pro-MMP-2 vs.
be mediated by a delayed release of an angiogenic activa- active MMP-2 also plays a role in determining invasive PATHOLOGY ONCOLOGY RESEARCH Matrix Metalloproteinases in Tumor Angiogenesis and Metastasis and metastatic capacity. The MMP-2 activation ratio in localization of the MMPs in tumor tissue is similar to that tumor tissue was also higher in pancreatic carcinoma of normal tissue. Although there are several studies show- patients with positive regional lymph nodes than those ing MMP-2 and MMP-9 production to be localized to the without metastasis.52 The increase in MMP-2 activity lev- tumor cells, the majority show MMP-2 and MMP-9 to be els correlates with the metastatic potential of the two car- produced by the tumor stromal elements. Immunohisto- cinoma types. Elevated levels of MT-MMPs have been chemical studies of several types of carcinomas including shown to increase activated levels of MMP-2 in breast, ovarian, thyroid, hepatocellular, and gastrointestinal show cervical, and lung carcinomas producing higher levels of MMP expression in stromal fibroblasts.56-59 In both ovari- invasiveness and metastasis. Transfection of several cell an and hepatocellular cancers, MMP-2 was localized to lines (HT-1080 fibrosarcoma, MCF-7 breast carcinoma, the tumor cell membrane at both the invasive fronts and at and U251.3 glioma cell lines) with the MT-MMP gene sites of vascular invasion.58,59 In breast carcinoma, tumor caused an increase in tumor cell invasion and migration.53 cells themselves were capable of modulating MMP-2 Cell lines displaying an intermediate level of activation binding via MT1-MMPs as well as via TIMP-2 and Vβ3 were the most invasive while those cells with a high level integrins.60 An explanation as to why both the tumor cells of activation were the least invasive. This is probably due and the adjacent stromal cells express MT1-MMP, MMP-2, to the balance required between MMPs and TIMP to cre- and MMP-9 may be that both cellular components con- ate a controlled proteolytic system.
tribute to a different part of the metastatic cascade. The Although the major role of MMPs in metastasis has tumor cell MMPs may contribute to the invasive growth of been inferred from the in vivo and in vitro data presented the tumor while the stromal elements contribute to the above to be breakdown of the ECM, recent studies have remodeling process and the desmoplastic reaction that proposed additional roles for the MMP family. A recent occurs in the tissue adjacent to the tumor.
review summarized these new roles for MMPs in invasionand metastasis.38 Most of the in vivo and in vitro assays MMPs and clinical therapeutics
designed to examine the role of MMPs on tumor invasion MMPs as diagnostic markers
measure the end results, as in the number of micrometas-tases formed. The mechanism, however, remains Numerous studies have shown the higher the MMP unknown. Intravital videomicroscopy (IVVM) allows for expression in the tumor, the more aggressive the cancer.
the observation of the metastatic cascade by following the Measuring the MMP level in the serum, plasma, or CSF has tumor cell through the microcirculation. The results fromthese experiments suggest that the destruction of tumorcells in the circulation and during extravasation do not contribute as much as previously thought to the inefficien-cy of metastasis. Rather, the growth of the individualtumor cell once in the target organ appears to be the rate limiting step.38 Tumor cells engineered to overexpress TIMP-1 were shown to extravasate at rates equal to wild- Angiogenic Stimuli type cells but were unable to form proliferative colonies within the target organ.54 Although these data suggest that MMPs may play a role in tumor cell growth, the studies ofMMPs involvement in ECM degradation and basementmembrane invasion still support the core role of MMPs inmetastatic invasion. The steps in which MMPs play a roleare summarized in MMP expression in various tissue types has been exam- Figure 2. Roles of MMPs in tumor cell metastasis. The roles of
MMPs in metastasis are multifold. The steps where MMPs are
ined through immunohistochemical analysis and in situ involved are depicted in this schematic. 1) MMPs are involved hybridization. MMPs are expressed in a variety of tissues in primary tumor growth and angiogenesis; 2) MMPs are and are a necessary component of the normal tissue secreted by both tumor cells and stromal cells upon cytokine remodeling process that occurs during wound healing, stimuli thus enabling the tumor cell to invade and intravasate; pregnancy, and bone resorption. MMP-2 seems to be the 3) Extravasation of the tumor cell; 4) Tumor cell migration in most common enzyme expressed in normal tissues, with ECM of distant site; 5) Growth and angiogenesis of distant expression being the highest in stromal elements.55 The Vol 7, No 1, 2001 JOHN and TUSZYNSKI received some attention as a possible pre- Table 2. Metalloproteinase Inhibitors and Their Clinical Uses*
dictor of tumor stage, metastasis, and recurrence. A study examining the serum Class Name Clinical Use levels of MMP-2 and MMP-9 in gastriccarcinoma patients revealed a higher level of both pro-enzymes in the cancer patients TIMP-1 (28.5 kDa) Inhibits all activated MMPs; Possible target for vs. healthy volunteers.61 In addition, the inhibits pro-MMP9 gene therapy; basis MMP level may be used in conjunction for synthetic MMPI development with other markers, such as CEA for colon Inhibits all activated MMPs; carcinoma, to better determine the stage of inhibits pro-MMP2 the tumor. Other studies show that there isa correlation with MMP levels and tumor metastasis. Similar results were observed Substrate analog to all Used to generate in prostate cancer where serum MMP-2 preclinical data formarimastat levels were higher in the carcinomapatients vs. healthy and benign prostatic Substrate analog to all Used in clinical tri- hyperplasia patients, and among the can- als in colon, pancre- cer cases, there was a higher level in those atic, ovarian cancer, patients with metastasis.62 In addition to correlating with metastasis, MMP levels Weak MMP inhibitor Used in rheumatoid can also predict the recurrence of tumors.
arthritis and peri- In a study examining the recurrence of urothelial cancer after resection showed (clinical Trials) that those patients with recurrence had a Chelates zinc, inhibiting In vitro data sug- higher MMP-2:TIMP-2 serum level than gests use in malig- those without recurrences.62 Also, an inde- pendent study showed a higher MMP-2/TIMP-2/MT1-MMP level in bladder * A sample of MMPIs are listed here along with their potential clinical uses. MMPIsare still in clinical trials. They constitute a new class of emerging drugs targeting cancer patients is associated with angiogenesis and metastasis formation.
decreased survival.63 Metalloproteinase inhibitors: chemotherapeutic agents
melanoma cells, and a human astrocytoma cell line trans-fected with the TIMP-1 gene showed metastasis with a As explained previously, the TIMPs are one mechanism reduced growth when injected into nude mice.65-67 Addi- of control used physiologically to regulate MMP function.
tional evidence includes the ability of monoclonal anti- A number of studies have shown that overexpression of bodies against gelatinases to inhibit invasion in TIMPs produce a less metastatic phenotype presumably by assays.68,69 These data all suggest a role for MMPIs as a reducing the amount of active metalloproteinases.
possible anti-cancer therapy. By altering the MMP-TIMP Although the initial evidence for use as possible chemo- ratio, the invasive and metastatic ability of tumor cells can therapeutic agents came from the studies involving natur- al TIMPs, the inability to mass produce and orally formu-late natural TIMP cost-effectively have led to the develop- Synthetic MMPIs ment of synthetic MMPs. Some more common MMPIs arelisted in Two of the prototype MMPIs today are batimastat and marimastat. Batimastat, one of the first synthetic MMPIs, Evidence for MMPIs as effective anti-cancer agents has a potent activity against most MMPs with an IC50 inthe low nanomolar range. Batimastat acts by competitive, Studies with the TIMP molecules provide the basis for reversible inhibition by mimicing the substrate of MMPs.
developing synthetic MMPIs as anticancer agents. Exper- The long half-life and the route of administration of bati- iments with recombinant TIMP-1 have shown that rTIMP- mastat is convenient for animal models. Marimastat is 1 inhibits the invasion of tumor cells through amniotic another MMPI with similar inhibitory functions as bati- membranes.64 Administering rTIMP-1 to mice injected mastat. Marimastat is almost completely absorbed after with metastatic B16 melanoma cells also inhibits the for- oral administration and has a high bioavailability. The half mation of lung metastasis.64 Gastric cancer cells, B16F10 life of marimastat is approximately 15 hours providing a PATHOLOGY ONCOLOGY RESEARCH Matrix Metalloproteinases in Tumor Angiogenesis and Metastasis convenient twice a day dosing schedule. Marimastat is bospondin-1 to up-regulate MMP production in both metabolized quickly in rodents, therefore, it has been used endothelial cells and tumor cells.24 Thrombospondin-1, a in clinical trials while batimastat has been used to generate 450 kDa protein, has multiple domains with a variety of preclinical data.
functions. TSP-1 has been shown to promote invasion in Preclinical data seem to strongly support the ability of vitro and in vivo. One of the mechanisms involved is MMPIs to reduce invasion and spontaneous metastases.
through the up-regulation of MMP-9. Specifically, the Studies with the B16 murine melanoma model show a type-1 repeat peptide of TSP-1 has been shown to block decrease in growth of subcutaneously implanted tumors the production of MMP-9 in endothelial cells by 86%. We and reduced spontaneous metastasis formation after surgi- interpret this result to mean that the peptide alone binds cal removal of the tumor. The number of lung colonies and blocks the receptor through which TSP-1 acts. The formed after IV injection of cells was also decreased by type I repeat peptides have been shown in mice to block 68%.70 More recently, in another colon cancer model, bati- the formation of B16 melanoma colonies in the lung. Pep- mastat treated animals had decreased peritoneal carcino- tides of matrix components such as the type I repeat of matosis development and liver metastasis development.
TSP-1 are possible anti-invasive therapies for the treat- The treated animals also had significantly prolonged sur- ment of metastases formation. Other MMP inhibitors vival.71 In addition, a hemangioma model using virus being studied in clinic trials include AG3340, COL-3, transformed endothelial cells measured the antiangiogenic Neovastat, and BMS-275291.
ability of MMPIs through measuring new vessel forma-tion. Batimastat treatment resulted in a decrease in vessel formation as assessed through hemoglobin content.42 Clinical trials examining the efficacy of marimastat also The MMPs are a key family of enzymes used by tumor yielded favorable results. Clinical trials involving MMPIs cells to invade and metastasize. The up-regulation of these must involve different parameters than just cytotoxic enzymes during the invasive state can be caused by a vari- responses alone because MMPIs are not toxic to the tumor ety of factors including increased production through cell. The measure of effectiveness is the actual reduction cytokines and growth factors, increased activation through of tumor growth along with the measure of survival time.
mechanisms such as uPa, and decreased inhibition by The studies involving batimastat included the treatment of reduced levels of TIMP-1. Developing orally active syn- malignant ascites and malignant pleural effusion. These thetic inhibitors of MMPs is a possible treatment for con- studies seemed to show favorable results; however, the trolling the metastatic potential of many tumors. While poor bioavailability and the need to inject the drug direct- this therapy alone will not destroy the tumor, used in com- ly into a body cavity led to the clinical trials with marima- bination with other therapies, these MMPIs could halt the stat. In a recent review by Steward, an overview of the tri- disease progression and slow the spread of the tumor.
als with marimastat are provided. Phase I studies revealed Research still continues in this field and exciting new ther- the severe joint and muscle pain that occurred with Mari- apeutic opportunities will ultimately emerge.
mastat at doses of 50 mg twice daily. Symptoms weredecreased by decreasing the dose to 10 mg twice daily.72 Phase II studies have shown a decrease in tumor specif- ic antigens, a measure of tumor activity, after treatment 1.²Sugarbaker EV: Patterns of metastasis in human malignancies.
with marimastat. Studies done on pancreatic cancer Cancer Biol Rev 2:235, 1981.
patients show that there was a decrease in disease progres- 2.²Weiss L Gilbert HA, Bone Metastasis: 1981, Boston: G.K. Hall.
3.²Liotta LA Stetler-Stevenson WG, Principles of molecular cell sion (as assessed through CT scan) and an increase in biology of cancer: Cancer Metastasis., in Cancer, Principles & overall survival.73 A more recent study with pancreatic Practice of Oncology, VT DeVita, S Hellman, and S.A. Rosen- cancer patients show optimal doses of batimastat to be 5, berg, Editors. 1993, Lippincott Co.: Philadelphia. p. 134-149.
10, and 25 mg twice daily.74 Phase III trials are currently 4.²Delaisse J-M Vaes G: Mechanism of mineral solubilisation and exploring the use of marimastat in conjunction with other matrix degradation in osteoclastic bone resorption, in Biologyand Physiology of the Osteoclast., B.R. Rifkin and C.V. Gay, chemotherapeutics.75 Several impediments exist in the use Editors. 1992, CRC Press: Raton, Florida. p. 290-314.
of MMPIs, however. The side effect profile of marimas- 5.²Talhouk RS, Bissell MJWerb Z: Coordinated expression of tat and batimastat require reduced dosing of the drugs in ECM-degrading proteinases and their inhibitors regulates mam- order to reduce toxicity. In addition, study end points are mary epithelial function during involution. J Cell Biol difficult to establish as MMPIs are not cytotoxic to tumor cells, making some studies difficult to interpret. 6.²Agren MS, Jorgensen LN, Andersen M, et al: Matrix metallo- proteinase 9 level predicts optimal collagen deposition during Other therapeutic possibilities include blocking the sig- early wound repair in humans. Brit J Surgery 85:68-71, 1998.
naling agents which can up-regulate MMP production.
7.²Gruber BL, Sorbi D, French DL, et al: Markedly elevated Our laboratory has discovered the ability of throm- serum MMP-9 (gelatinase B) levels in rheumatoid arthritis: a Vol 7, No 1, 2001 JOHN and TUSZYNSKI potentially useful laboratory marker. Clinical Immunol Immu- heparin and cholesterol in fibroblast cells. J Mol Cell Cardio- nopathol 78:161-171, 1996.
logy 29:391-404, 1997.
8.²Parsons SL, Watson SA, Brown PD, et al: Matrix Metallopro- 27.²Mazzieri R, Masiero L, Zanetta L, et al: Control of type IV col- teinases. Brit J Surgery 84:160-166, 1997.
lagenase activity by components of the urokinase-plasmin sys- 9.²Tyagi SC: Proteinases and myocardial extracellular matrix tem: a regulatory mechanism with cell-bound reactants. EMBO turnover. Molecular and Cellular Biochemistry 168:1-12, 1997.
J 16:2319-2332, 1997.
10.²Gross J Lapiere CM: Collagenolytic activity in amphibian tis- 28.²Okada Y, Morodomi T, Enghild JJ, et al: Matrix metallopro- sues: a tissue culture assay. Proc Natl Acad Sci USA 48:1014- teinase 2 from human rheumatoid synovial fibroblast: purifica- tion and activation of the precursor and enzymatic properties.
11.²Aimes RT, Quigley JP: Matrix metalloproteinase-2 is an inter- Eur J Biochem 194:721-730, 1990.
stitial collagenase. Inhibitor-free enzyme catalyzes the cleav- 29.²Sato H, Seiki M: Membrane-type matrix metalloproteinases age of collagen fibrils and soluble native type I collagen gener- (MT-MMPs) in tumor metastasis. J Biochem (Tokyo) 119:209- ating the specific 3/4- and *-length fragments. J Biol Chem 30.²Cawston TE, Inhibitors of metalloproteinases, in 12.²Matrisian L: Metalloproteinases and their inhibitors in matrix inhibitors, A.J. Barret and G. Salveson, Editors. 1986: Amers- remodeling. Trends Genet 6:121-125, 1990.
terdam. p. 589-610.
13.²Rooprai HK, McCormick D: Proteases and their inhibitors in 31.²Gomez DE, Alonso DF, Yoshiji H, et al: Tissue Inhibitors of human brain tumours: a review. Anticancer Res 17:4151-4162, metalloproteinases: structure, regulation and biological func- tions. Europ J Cell Biol 74:111-122,1997.
14.²Celentano DC, Frishman WH: Matrix metalloproteinases and 32.²Kossakowska AE, Urbanski SJ, Edwards DR: Tissue inhibitor coronary artery disease: a novel therapeutic target. J Clin Phar- of metalloproteinases (TIMP-1) RNA is expresses at elevated macol 37:991-1000, 1997.
levels in malignant non-Hodgkin's lymphomas. Blood 15.²Toth M, Gervasi DC, Fridman R: Phorbol ester-induced cell sur- face association of matrix metalloproteinase-9 in human 33.²Brian J, Wang Y, Smith MR, et al: Suppression of in vivo tumor MCF10A breast epithelial cells. Cancer Res 57:3159-3167, 1997.
growth and induction of suspension cell death by tissue 16.²Miyake H, Yoshimura K, Hara I, et al: Basic fibroblast growth inhibitor of metalloproteinases (TIMP-3). Carcinogenesis factor regulates matrix metalloproteinases production and in 9:1805-1811, 1996.
vitro invasiveness in human bladder cancer cell lines. J Urology 34.²Folkman J: Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature Medicine 1:27-31, 1995.
17.²Kanno N, Nonomura N, Miki T, et al: Effects of epidermal 35.²Folkman J: Tumor angiogenesis: therapeutic implications.
growth factor on the invasion activity of the bladder cancer cell [Review]. New Engl J Med 285:1182-1186, 1971.
line. J Urology 159:586-590, 1998.
36.²Fox SB, Gatter KC, Harris AL: Tumour angiogenesis. J Patho- 18.²Lamoreaux WJ, Fitzgerald MEC, Reiner A, et al: Vascular logy 179:232-237, 1996.
endothelial growth factor increases release of gelatinase A and 37.²Pluda JM: Tumor-associated angiogenesis: mechanisms, clini- decreases release of tissue inhibitor of metalloproteinases by cal implications, and therapeutic strategies. Seminars in Onco- microvascular endothelial cells in vitro. Microvascular Res logy 24:203-218, 1997.
38.²Chambers AF, Matrisian LM: Changing views of the role of 19.²Song SY, Nomizu M, Yamada Y, et al: Liver metastasis formation matrix metalloproteinases in metastasis. J Natl Cancer Inst by laminin-1 peptide (LQVQLSIR)-adhesion selected B16 - 89:1260-1270, 1997.
F10 melanoma cells. Int J Cancer 71:436-441, 1997.
39.²Moses MA: The regulation of neovascularization by matrix met- 20.²Tyagi SC, Kumar GS, Glover G: Induction of tissue inhibitor alloproteinases and their inhibitors. Stem Cells 15:180-189, and matrix metalloproteinase by serum in human heart-derived fibroblast and endomyocardial endothelial cells. J Cell 40.²Zucker S, Mirza H, Conner CE, et al: Vascular endothelial Biochem 58:360-371, 1995.
growth factor induces tissue factor and matrix metallopro- 21.²Bafetti LM, Young TN, Itoh Y, et al: Intact vitronectin induces teinase production in endothelial cells – conversion of pro- matrix metalloproteinase-2 and tissue inhibitor of metallopro- thrombin to thrombin results in progelatinase A activation and teinases-2 expression and enhanced cellular invasion by cell proliferation. Internat J Cancer 75:780-786, 1998.
melanoma cells. J Biol Chem 273:143-149, 1998.
41.²Karelina TV, Goldberg GI, Eisen AZ: Matrix metalloproteinases 22.²Haas TL, Davis SJ, Madri JA: Three-dimensional type I colla- in blood vessel development in human fetal skin and in cuta- gen lattices induce coordinate expression of matrix metallopro- neous tumors. J Invest Dermatol 105:411-417, 1995.
teinases MT1-MMP and MMP-2 in microvascular endothelial 42.²Taraboletti G, Garofalo A, Belotti D, et al: Inhibition of angio- cells. J Biol Chem 273:3604-3610, 1998.
genesis and murine hemangioma growth by batimastat, a syn- 23.²Qian X, Tuszynski GP: Expression of thrombospondin-1 in can- thetic inhibitor of matrix metalloproteinases. J Natl Cancer Inst cer: a role in tumor progression. Proc Soc Exp Biol Med 212:199-207, 1996.
43.²Braunhut SJ, Moses MA: Retinoids modulate endothelial cell pro- 24.²Qian X, Wang TN, Rothman VL, et al: Thrombospondin-1 mod- duction of matrix-degrading proteases and tissue inhibitors of met- ulates angiogenesis in vitro by up-regulation of matrix metallo- alloproteinases (TIMP). J Biol Chem 269:13472-13479, 1994.
proteinase-9 in endothelial cells. ExpCell Res 235:403-412, 44.²Vacca A, Moretti S, Ribatti D, et al: Progression of mycosis fun- goides is associated with changes in angiogenesis and expres- 25.²Sorsa T, Salo T, Koivunen E, et al.: Activation of type IV pro- sion of the matrix metalloproteinases 2 and 9. Eur J Cancer collagenases by human tumor-associated trypsin-2. J Biol 33:1685-1692, 1997.
Chem 272:21067-21074, 1997.
45.²Itoh T, Tanioka M, Yoshida H, et al: Reduced angiogenesis and 26.²Tyagi SC, Kumar S, Katwa L: Differential regulation of extra- tumor progression in gelatinase A-deficient mice. Cancer Res cellular matrix metalloproteinase and tissue inhibitor by 58:1048-1051, 1998.
PATHOLOGY ONCOLOGY RESEARCH Matrix Metalloproteinases in Tumor Angiogenesis and Metastasis 46.²Vu TH, Shipley JM, Bergers G, et al: MMP-9/Gelatinase B is a 61.²Endo K, Maehara Y, Baba H, et al: Elevated levels of serum and Key Regulator of Growth Plate Angiogenesis and Apoptosis if plasma metalloproteinases in patients with gastric cancer. Anti- Hypertropic Chondrocytes. Cell 93:411-422, 1998.
cancer Res 17:2253-2258, 1997.
47.²Chu YW, Yang PC, Yang SC, et al: Selection of Invasive and 62.²Gohji K, Fujimoto N, Hara I, et al: Serum matrix metallopro- Metastatic Subpopulation from a Human Lung Adenocarcino- teinase-2 and its density in men with prostate cancer as a new ma Cell Line. Amer J Resp Cell Mol Biol 17:353-360, 1997.
predictor of disease extension. Int J Cancer 79:96-101, 1998.
48.²MacDougall JR, Bani MR, Lin Y, et al: The 92-kDa gelatinase 63.²Kanayama H, Yokota K, Kurokawa Y, et al: Prognostic Values of B is expressed by advanced stage melanoma cells: Suppression matrix metalloproteinase-2 and tissue inhibitor of metalloprotei- by somatic cell hybridization with early stage melanoma cells.
nase-2 expression in bladder cancer. Cancer 82:1359-1366, 1998.
Cancer Res 55:4174-4181, 1995.
64.²Alvarez OA, Carmichael DF, DeClerck YA: Inhibition of col- 49.²Ueda Y, Imai K, Tsuchiya H, et al: Matrix metalloproteinase 9 lagenolytic activity and metastasis of tumor cells by a recombi- (gelatinase B) is expressed in multinucleated giant cells of nant human tissue inhibitor of metalloproteinases. J Natl Can- human giant cell tumor of bone and is associated with vascular cer Inst 82:589-595, 1990.
invasion. Amer J Pathol 148:611-622, 1996.
65.²DeClerck YA, Perez N, Shimada H, et al: Inhibition of invasion 50.²Kossakowska AE, Hinek A, Edwards DR, et al: Proteolytic and metastasis in cell transfected with an inhibitor of metallo- activity of human non-Hodgkin's lymphomas. Amer J Pathol proteinases. Cancer Res 52:701-708, 1992.
66.²Tsuchiya Y, Sato H, Endo Y, et al: Tissue inhibitor of metallo- 51.²Llorens A, Vinyals A, Alia P, et al: Metastatic Ability of MXT proteinase 1 is a negative regulator of the metastatic ability of a Mouse Mammary Subpopulations Coorelates with Clonal human gastric cancer cell line, KKLS, in the chick embryo.
Expression and/or Membrane-Association of Gelatinase A.
Cancer Res 53:1397-1402, 1993.
Molecular Carcinogenesis 19: 54-56, 1997.
67.²Matsuzawa K, Fukuyama K, Hubbard SL, et al: Transfection of 52. Koshiba T, Hosotani R, Wada M, et al.: Involvement of matrix an invasive human astrocytoma cell line with a TIMP-1 cDNA: metalloproteinase-2 activity in invasion and metastasis of pan- modulation of astrocytoma invasive potential. J Neuropathol creatic carcinoma. Cancer 82:642-50, 1998.
Exp Neurol 55:88-96, 1996.
53.²Deryugina EI, Luo GX, Reisfeld RA, et al: Tumor cell invasion 68.²Hoyhtya M, Hujanen E, Turpeenniemi-Hujanen T, et al: Modu- through matrigel is regulated by activated matrix metallopro- lation of type-IV collagenase activity and invasive behavior of teinase-2. Anticancer Res 17:3201-3210, 1997.
metastatic human melanoma (A2058) cells in vitro by mono- 54.²Koop S, Khokha R, Schmidt EE, et al.: Overexpression of met- clonal antibodies to type-IV collagenase. Int J Cancer 46:282- alloproteinase inhibitor in B16F10 cells does not affect extrava- sation but reduces tumor growth. Cancer Res 54:4791-4797, 69.²French DL, Ramos-Desimone N, Rozinski K, et al: Matrix met- alloproteinase-9 in tumor cell invasion. Ann NY Acad Sci 55.²Matrisian LM: Matrix metalloproteinase gene expression. Ann 732:324-334, 1994.
NY Acad Sci 732:42-50, 1993.
70.²Chirivi RGS, Garofalo A, Crimmin MJ, et al: Inhibition of the 56.²Zeng ZS, Guillem JG: Distinct pattern of matrix metallopro- metastatic spread and growth of B16-BL6 murine melanoma by teinase 9 and tissue inhibitor of metalloproteinase 1 mRNA a synthetic matrix metalloproteinase inhibitor. Int J Cancer expression in human colorectal cancer and liver metastases. Bri 58:460-464, 1994.
J Cancer 72:575-582, 1995.
71.²Aparicio T, Kermorgant S, Dessirier V, et al: Matrix Metallopro- 57.²Tomita T: Matrix metalloproteinases and tissue inhibitors of teinase inhibition prevents peritoneal carcinomatosis development metalloproteinases in thyroid C-cells and medullary thyroid and prolongs survival in rats. Carcinogenesis 20:1445-1451, 1999.
carcinomas. Histopathology 31:150-6, 1997.
72.²Steward WP: Marimastat (BB2516): current status of develop- 58.²Afzal S, Lalani EN, Poulsom R, et al: MT1-MMP and MMP-2 ment. Cancer Chemotherapy and Pharmacology 43 Suppl.: mRNA expression in human ovarian tumors: possible implica- 556-560, 1999.
tions for the role of desmoplastic fibroblasts. Human Pathol 73.²Rosemurgy A, Harris J, Langleben A, et al: Marimastat, a novel 29:155-165, 1998.
metalloproteinase inhibitor in patients with advanced carcino- 59.²Harada T, Arii S, Mise M, et al: Membrane-type matrix metal- ma of the pancreas. 1996. Philadelphia.
loproteinase-1 (MT1-MMP) gene is overexpressed in highly 74.²Rosemurgy A, Harris J, Langleben A, et al: Marimastat in invasive hepatocellular carcinomas. J Hepatology 28:231-239, patients with advanced pancreatic cancer: a dose finding study.
Amer J Clin Oncol 22:247-252, 1999.
60.²Menashi S, Dehem M, Souliac I, et al: Density-dependent regu- 75.²Kroep JR, Pinedo HM, VanGroeningen CJ, et al: Experimental lation of cell-surface association of matrix metalloproteinase-2 drugs and drug combinations in pancreatic cancer. Ann Oncol (MMP-2) in breast-carcinoma cells. Internat J Cancer 75:259- 10 Suppl:234-238, 1999.
Vol 7, No 1, 2001
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