Human heparanase: a molecular determinant of brain metastasis
Introduction
Mechanisms responsible for the progression of malignant melanoma to highly aggressive brain-metastatic disease remain largely unknown (1). We have reported that neurotrophins (NT) can affect melanoma cell invasion and production of heparanase 2., 3.. This enzyme is an endo-β-d-glucuronidase (4) that degrades the heparan sulfate chains of heparan sulfate proteoglycans (HSPG), essential and ubiquitous macromolecules associated with the cell surface and the extracellular matrix (ECM) of a wide range of cells and tissues (5). Production of heparanase has been associated with invasion and metastasis 6., 7.. Of particular interest, human heparanase has been found to be a potential target for antimetastasis drugs because of its relevant roles in angiogenic and invasive processes (8). The sequence, genomic organization and chromosome localization of the human heparanase gene have been recently described 4., 9., 10., 11., improving our understanding of the contribution of this gene to normal physiology as well as disease states.
We have postulated that heparanase is relevant for the extravasation of blood-borne cancer cells like melanoma into secondary organs by degrading ECM-associated HSPG. The brain is an ideal environment for melanoma cell invasion because of the presence of high levels of heparanase-regulatory neurotrophins (NT). NT have been found to induce the synthesis and secretion of melanoma heparanase, with brain-metastatic melanoma cells expressing the highest levels of heparanase (2).
Astrocytes are the first brain cells encountered by extravasating melanoma cells that have breached the two layers of the blood–brain barrier (BBB), the brain microvessel endothelium and its HSPG-enriched ECM. Injury-reacting astrocytes are frequently found in areas surrounding brain melanoma metastases, and these brain cells can produce NGF, the prototypic NT (12). Furthermore, astrocytes are capable of binding NT since they express members of the trk receptor family as well as the low-affinity p75 NT receptor (p75NTR; 12).
In this study we have examined interactions between astrocytes and selected melanoma cell lines capable of brain colonization in vivo 13., 14.. We report that astrocytes produce heparanase and potentiate melanoma brain invasive properties, in part because of the concerted action of heparanases produced by neoplastic and astrocytic cells. We also provide direct evidence for a role of heparanase in metastatic invasion mechanisms. Transfection of the heparanase gene into nonmetastatic melanoma cells resulted in augmented expression and functional heparanase activity, resulting in significantly increased in vitro invasion. Finally, heparanase RNA was also found to be present in greater amounts in melanoma specimens at later stages of tumor progression, with the highest expression found in highly invasive melanoma tissues. These data indicate that heparanase expression may be part of the cellular switch from noninvasive to invasive melanoma phenotypes.
Section snippets
Reagents and cell culture
Human MeWo melanoma cell lines consisting of parental MeWo and two WGA-derived variants, poorly metastatic 3S5 and highly metastatic 70W cells were obtained as described (13). Murine brain-metastatic melanoma B16-F1/B16-B15b cells were selected previously for their ability to colonize the brain (14). Purified astrocytes were prepared from newborn rat or mouse cerebral cortices using a modification of the method of McCarthy and de Vellis (15). Briefly, cerebral cortices were isolated, meningeal
Heparanase Expression and its Functional Activity in Astrocytes
We investigated the presence of heparanase in astrocyte cultures. Primary glial cells were obtained from newborn rat cerebra. The majority of astrocytes appeared as large and flat cells with fibroblast-like morphology. Their identification as astrocytes was established by positive immunoreactivity with an antibody against the astrocyte-specific intermediate filament GFAP (Fig. 1A). Neither vimentin, myelin basic protein, or ganglioside recognized by MAb A2B5 was expressed by the astrocyte
Discussion
Malignant melanoma is one of the most rapidly increasing cancers among young adults, and it is a malignancy with one the highest frequencies of brain metastasis formation. Approximately 13% of all cancer patients will present clinically with symptomatic complications related to brain metastases, but almost 40% of melanoma patients will be treated for complications due to CNS lesions, reaching 70–80% of cases at autopsy (1). Since the brain microvasculature represents an exceedingly small
Summary
Mechanisms responsible for the progression of malignant melanoma to highly aggressive brain-metastatic disease remain largely unknown. Brain neurotrophins (NT) can modulate brain invasion of melanoma cells and the activity of an ECM degradative enzyme, heparanase. This enzyme is an endo-β-d-glucuronidase that degrades the heparan sulfate chains of HSPG, essential and ubiquitous macromolecules associated with the cell surface and ECM of a wide range of cells and tissues. Human heparanase has
Acknowledgements
We would like to thank Dr. Philip Tofilon, M.D. Anderson Cancer Center, Houston, TX, for his help in the initial stages of our studies, and Dr. Robert L. Heinrikson, Research Operations, Pharmacia and Upjohn, Inc., Kalamazoo, MI, for making available antibodies against human heparanase.
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Abbreviations: NT, neurotrophins; NGF, nerve growth factor; HSPG, heparan sulfate proteoglycans; BBB, blood-brain barrier; ECM, extracellular matrix; ACM, astrocyte conditioned medium; GFAP, glial fibrillary acidic protein; PCR, polymerase chain reaction; bp, base pairs; kb, kilobases; TBE, tris-borate-EDTA; CNS, central nervous system; DMEM, Dulbecco's modified Eagle medium; FBS, fetal bovine serum; HPLC, high-pressure liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; MAb, monoclonal antibody; EST, expressed sequence tags; WGA, wheat germ agglutinin.