William G. Stetler-Stevenson

Cancer metastasis and angiogenesis: An imbalance of positive and negative regulation

Lance A. Liotta, Patricia S. Steeg, and William G. Stetler-Stevenson Laboratory of Pathology National Cancer Institute National Institutes of Health Bethesda, Maryland 20892 The most life-threatening aspects of the oncogenic pro- cess are invasion and metastasis. Even though the clinical significance of such expression of the malignant pheno- type has been well appreciated, advances in understand- ing the molecular mechanisms involved in metastasis have lagged behind other developments in the cancer field. Progress has been hindered by the sheer complexity of this multistep, tumor-host interaction that also encom- passes angiogenic and immunologic mechanisms. To tackle the problem, investigators have separated invasion and metastasis into a series of defined, sequential steps, and focused on one step at a time. For each step, new ex- perimental models had to be developed, and a combined effort using the disciplines of cell biology, protein bio- chemistry, and molecular genetics has now resulted in a surge of new information. General themes are emerging that yield new strategies for prognosis and therapy of hu- man metastatic cancer. A group of coordinated cellular processes is responsi- ble for metastasis. Furthermore, it is now clear that nega- tive regulatory processes may be just as important as positive ones. Some genetic changes result in an im- balance of growth regulation, leading to uncontrolled proliferation. However, unrestrained growth does not, by itself, result in invasion and metastasis. The latter pheno- type may therefore require additional genetic changes. Thus, tumorigenicity and metastatic potential have both overlapping and separate features. Invasion and metasta- sis can be facilitated by proteins that stimulate tumor cell attachment to host cellular or extracellular matrix deter- minants, tumor cell proteolysis of host barriers such as the basement membrane, tumor cell locomotion, and tumor cell colony formation in the target organ for metastasis. Facilitory proteins may act at many levels intracellularly or extracellularly, but are counterbalanced by factors that can block their production, regulation, or action. A com- mon theme has emerged: in addition to loss of growth control, an imbalanced regulation of motility and proteoly- sis appears to be required for invasion and metastasis. Moreover, these same functions are also necessary for angiogenesis. Angiogenesis by normal endothelial cells and metastasis by tumor cells are functionally similar but differ in their regulation. Metastatic Cells Do Not Constitute a Minor Subpopulation The process of metastasis involves a cascade of linked, sequential steps involving multiple host-tumor interac- tions (Fidler and Hart, 1982; Schirrmacher, 1985; Liotta et

Localization of Matrix Metalloproteinase MMP-2 to the Surface of Invasive Cells by Interaction with Integrin αvβ3

Abstract Cellular invasion depends on cooperation between adhesive and proteolytic mechanisms. Evidence is provided that the matrix metalloproteinase MMP-2 can be localized in a proteolytically active form on the surface of invasive cells, based on its ability to bind directly integrin αvβ3. MMP-2 and αvβ3 were specifically colocalized on angiogenic blood vessels and melanoma cells in vivo. Expression of αvβ3 on cultured melanoma cells enabled their binding to MMP-2 in a proteolytically active form, facilitating cell-mediated collagen degradation. In vitro, these proteins formed an SDS-stable complex that depended on the noncatalytic C-terminus of MMP-2, since a truncation mutant lost the ability to bind αvβ3. These findings define a single cell-surface receptor that regulates both matrix degradation and motility, thereby facilitating directed cellular invasion.

Matrix metalloproteinases and metastasis.

Abstract Metastatic disease is responsible for the majority of cancer-related deaths, either directly due to tumor involvement of critical organs or indirectly due to complications of therapy to control tumor growth and spread. An understanding of the mechanisms of tumor cell invasion and metastasis may be important for devising therapies aimed at preventing tumor cell spread. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endoproteinases whose enzymatic activity is directed against components of the extracellular matrix (ECM). In humans, 16 members of this family have been identified by cloning and sequencing. These proteinases are linked by a core of common domain structures and by their relationship to a family of proteinase inhibitors called the tissue inhibitors of metalloproteinases (TIMPs). Four members of the TIMP family have been cloned and sequenced in humans and they inhibit MMPs by forming tight-binding, noncovalent associations with the active site of the MMPs. MMPs facilitate tumor cell invasion and metastasis by at least three distinct mechanisms. First, proteinase action removes physical barriers to invasion through degradation of ECM macromolecules such as collagens, laminins, and proteoglycans. This has been demonstrated in vitro through the use of chemoinvasion assays and in vivo by the presence of active MMPs at the invasive front of tumors. Second, MMPs have the ability to modulate cell adhesion. For cells to move through the ECM, they must be able to form new cell–matrix and cell–cell attachments and break existing ones. Using a cell transfection system that altered the ratio of MMP-2 to TIMP-2 we have demonstrated significant variation in the adhesive phenotype of tumor cells. Finally, MMPs may act on ECM components or other proteins to uncover hidden biologic activities. For example, the angiogenesis inhibitor angiostatin may be produced from plasminogen by MMP action and laminin-5 is specifically degraded by MMP-2 to produce a soluble chemotactic fragment. Thus MMPs play multiple key roles in facilitating the metastasis of tumor cells. Therapies designed to interfere with specific MMP actions may be useful in the control of metastatic disease.

Extracellular matrix 6: role of matrix metalloproteinases in tumor invasion and metastasis.

Tumor invasion and metastasis formation are major obstacles for successful cancer therapy. Metastasis is a complex multistep process that requires sequential interactions between the invasive cell and the extracellular matrix. A model system for tumor invasion of extracellular matrix barriers has been developed, and application of this model has facilitated our understanding of the molecular mechanisms of metastasis formation. This model consists of three steps: tumor cell adhesion, extracellular matrix proteolysis, and cell migration. The role of the matrix metalloprotease enzymes in tumor cell‐mediated extracellular matrix proteolysis is well established. We review the functional domain structure of the matrix metalloprotease enzymes in general and specifically the interaction of metastasis‐associated gelatinase A (72‐kDa type IV collagenase) with the tissue inhibitor of metalloproteases‐2 (TIMP‐2). We also discuss the physiologic activation of the matrix metalloprotease enzymes and the specific cellular mechanism of action of gelatinase A.— Stetler‐Stevenson, W. G., Liotta, L. A., Kleiner, D. E., Jr. Extracellular matrix 6: role of matrix metalloproteinases in tumor invasion and metastasis. FASEB J. 7: 1434‐1441; 1993.

Type IV collagenases in tumor invasion and metastasis

SummaryThe invasion and metastasis of cancer cells is a complex multistep process involving destruction of basement membranes as an early event in the metastatic cascade. Recent evidence implicates secreted matrix metalloproteinase enzymes, such as type IV collagenases, as playing a central role in this tumor cell mediated extracellular matrix proteolysis. Two distinct type IV collagenase enzymes are now recognized. Immunohistochemical and biochemical studies of several human tumors show correlations between invasive potential and the 72kDa type IV collagenase enzyme. Studies in rodent tumor models suggest that the 92kDa type IV collagenase may play an important role in these models, but data on human tumors and human tumor tissue is lacking. Evidence suggest that the regulation of the 72kDa type IV collagenase enzyme activity may occur at many levels, including transcriptional mechanisms, extracellular activation of latent enzyme and specific inhibitors of active enzyme. Thus the invasion of human tumor cells through basement membranes may be the result of net type IV collagenolytic activity that is the result of a balance of activated enzyme species and inhibitors.

TIMP-2 Mediated Inhibition of Angiogenesis: An MMP-Independent Mechanism

Tissue inhibitors of metalloproteinases (TIMPs) suppress matrix metalloproteinase (MMP) activity critical for extracellular matrix turnover associated with both physiologic and pathologic tissue remodeling. We demonstrate here that TIMP-2 abrogates angiogenic factor-induced endothelial cell proliferation in vitro and angiogenesis in vivo independent of MMP inhibition. These effects require alpha 3 beta 1 integrin-mediated binding of TIMP-2 to endothelial cells. Further, TIMP-2 induces a decrease in total protein tyrosine phosphatase (PTP) activity associated with beta1 integrin subunits as well as dissociation of the phosphatase SHP-1 from beta1. TIMP-2 treatment also results in a concomitant increase in PTP activity associated with tyrosine kinase receptors FGFR-1 and KDR. Our findings establish an unexpected, MMP-independent mechanism for TIMP-2 inhibition of endothelial cell proliferation in vitro and reveal an important component of the antiangiogenic effect of TIMP2 in vivo.

TIMP-2 reduces proteolytic opening of blood-brain barrier by type IV collagenase

Intracerebral hemorrhage occurs in tumors, stroke and head trauma. Proteolysis of the extracellular matrix around cerebral capillaries by naturally occurring mammalian 72-kDa type IV collagenase may initiate this pathologic event. To investigate this hypothesis adult rats underwent intracerebral injection of type IV collagenase purified from human melanoma cells. Histologically, at 4 h there was perivascular cellular infiltration with hemorrhage, and by 24 h there was infarction with necrosis, edema and hemorrhage. Ultrastructurally, the basal lamina of endothelial cells was disrupted at 2 h. Brain uptake of [14C]dextran and [3H]sucrose increased after intracerebral injection of type IV collagenase compared to controls (P less than 0.0001). Tissue inhibitor of metalloproteinase-2 (TIMP-2) reduced the tracer uptake (P less than 0.02). Metalloproteinase inhibitors reduce extracellular matrix proteolysis and protect the blood-brain barrier.

Neuronal Matrix Metalloproteinase-2 Degrades and Inactivates a Neurite-Inhibiting Chondroitin Sulfate Proteoglycan

Chondroitin sulfate proteoglycans (CSPGs) are implicated in the regulation of axonal growth. We previously reported that the neurite-promoting activity of laminin is inhibited by association with a Schwann cell-derived CSPG and that endoneurial laminin may be inhibited by this CSPG as well [Zuo J, Hernandez YJ, Muir D (1998) Chondroitin sulfate proteoglycan with neurite-inhibiting activity is upregulated after peripheral nerve injury. J Neurobiol 34:41–54]. Mechanisms regulating axonal growth were studied by using an in vitro bioassay in which regenerating embryonic dorsal root ganglionic neurons (DRGn) were grown on sections of normal adult nerve. DRGn achieved slow neuritic growth on sections of normal nerve, which was reduced significantly by treatment with metalloproteinase inhibitors. Similar results were obtained on a synthetic substratum composed of laminin and inhibitory CSPG. DRGn expressed the matrix metalloproteinase, MMP-2, which was transported to the growth cone. Recombinant MMP-2 inactivated the neurite-inhibiting CSPG without hindering the neurite-promoting potential of laminin. Similarly, neuritic growth by DRGn cultured on normal nerve sections was increased markedly by first treating the nerve sections with MMP-2. The proteolytic deinhibition by MMP-2 was equivalent to and nonadditive with that achieved by chondroitinase, suggesting that both enzymes inactivated inhibitory CSPG. Additionally, the increases in neuritic growth resulting from treating nerve sections with MMP-2 or chondroitinase were blocked by anti-laminin antibodies. From these results we conclude that MMP-2 provides a mechanism for the deinhibition of laminin in the endoneurial basal lamina and may play an important role in the regeneration of peripheral nerve.

Regulation of Angiostatin Production by Matrix Metalloproteinase-2 in a Model of Concomitant Resistance

We have previously reported the identification of the endogenous angiogenesis inhibitor angiostatin, a specific inhibitor of endothelial cell proliferation in vitro and angiogenesisin vivo. In our original studies, we demonstrated that a Lewis lung carcinoma (LLC-LM) primary tumor could suppress the growth of its metastases by generating angiostatin. Angiostatin, a 38-kDa internal fragment of plasminogen, was purified from the serum and urine of mice bearing LLC-LM, and its discovery provides the first proven mechanism for concomitant resistance (O’Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M. A., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. (1994)Cell 79, 315–328). Subsequently, we have shown that systemic administration of angiostatin can regress a wide variety of malignant tumors in vivo. However, at the time of our initial discovery of angiostatin, the source of the protein was unclear. We hypothesized that the tumor or stromal cells might produce an enzyme that could cleave plasminogen sequestered by the primary tumor into angiostatin. Alternatively, we speculated that the tumor cells might express angiostatin. By Northern analysis, however, we have found no evidence that the tumor cells express angiostatin or other fragments of plasminogen (data not shown). We now report that gelatinase A (matrix metalloproteinase-2), produced directly by the LLC-LM cells, is responsible for the production of angiostatin, which suppresses the growth of metastases in our original model.

Tumor necrosis factor-α-induced gelatinase B causes delayed opening of the blood-brain barrier: an expanded therapeutic window

Proteolytic damage is a late event in the molecular cascade initiated by brain injury. Earlier, we proposed that matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) are important in secondary brain injury. We have shown that intracerebral injection of activated 72-kDa type IV collagenase (gelatinase A) opens the blood-brain barrier, and that during hemorrhagic brain injury there is endogenous production of 92-kDa type IV collagenase (gelatinase B) and uPA. Therefore, to study the functional link between proteolytic enzymes and blood-brain barrier damage, we induced MMP expression by infusing tumor necrosis factor-alpha (TNF) intracerebrally in rats. Initially, the effect on capillary permeability of increasing doses of TNF, using [14C]sucrose uptake, was measured. Then, the time-course of the capillary permeability change was studied at 4, 16, 24 and 72 h. Expression of MMP and uPA was measured by zymography at 24 h after TNF injection and compared to saline-injected controls. A dose-dependent increase in capillary permeability was seen 24 h after TNF injection. Maximal uptake of [14C]sucrose occurred at 24 h compared to saline-injected controls (P < 0.05). Zymography showed production of gelatinase B, which was significantly greater than in saline-injected controls at 24 h (P < 0.05). Batimastat, a synthetic inhibitor to metalloproteinases, reduced sucrose uptake at 24 h (P < 0.0001), and was effective even when given 6 h after TNF (P < 0.01). Thus, gelatinase B is the intermediate substance linking TNF to modulation of capillary permeability. Agents that interfere with transcription of proteolytic enzymes or block their action may reduce delayed capillary injury, extending the therapeutic window.