Neurotrophin stimulation of human melanoma cell invasion: selected enhancement of heparanase activity and heparanase degradation of specific heparan sulfate subpopulations.
Heparanase is an endo-beta-D-glucuronidase whose enzymatic targets are the glycosaminoglycan chains of heparan sulfate proteoglycans (50). Elevated levels of heparanase are associated with the metastatic potential of melanoma cells, and treatment of murine and human melanoma cells with the prototypic neurotrophin nerve growth factor (NGF) increases the production of heparanase by melanoma cells. We previously reported that physiological concentrations of NGF increased invasion of early passage human brain-metastatic 70W melanoma cells but not melanoma cells metastatic to other sites or nonmetastatic melanoma cells as measured in Matrigel invasion assays. Here we found that treatment of 70W melanoma cells with neurotrophin-3 (NT-3) increased Matrigel invasion, whereas treatment with neurotrophins other than NGF or NT-3 did not influence invasion. Mutants of NGF that do not bind to the neurotrophin receptor p75NTR or other nonneuronal growth factors were not able to enhance the invasion of 70W melanoma cells. When 70W cells were exposed to antisense oligonucleotides directed against p75NTR mRNA, there was a reduction in NGF and NT-3 binding, and the neurotrophins failed to enhance Matrigel invasion. To study the properties of heparanase in neurotrophin-regulated malignant melanoma invasive processes, we developed a sensitive heparanase assay consisting of purified [35S]HS subpopulations separated by agarose gel electrophoresis. Incubation of 70W cells with NGF or NT-3 but not brain-derived neurotrophic factor, neurotrophin-4/5 or mutant NGF resulted in increased release of heparanase activity that was capable of degrading a subpopulation of heparan sulfate molecules.
Marchetti
D
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Department of Tumor Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.
Nicolson
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GL
D004587:Electrophoresis, Agar Gel; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005966:Glucuronidase; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D008545:Melanoma / Q000201:enzymology / Q000473:pathology*; D009361:Neoplasm Invasiveness; D009414:Nerve Growth Factors / Q000378:metabolism / Q000494:pharmacology*; D020933:Neurotrophin 3; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D020800:Receptor, Nerve Growth Factor; D017475:Receptors, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D000099175:Heparanase
D004815:Epidermal Growth Factor; D005966:Glucuronidase; D006026:Glycoside Hydrolases; D006497:Heparan Sulfate; D009414:Nerve Growth Factors; D020933:Neurotrophin 3; D016376:Oligonucleotides, Antisense; D020800:Receptor, Nerve Growth Factor; D017475:Receptors, Nerve Growth Factor; D000099175:Heparanase