Expression of ecto-5′-nucleotidase (eN, CD73) in cell lines from various stages of human melanoma

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Ecto-5′-nucleotidase is a GPI-anchored enzyme localized in cell membrane lipid rafts. Although it is highly expressed in many tumour cells, its specific function during tumorigenesis is unclear. We have found that, among different melanoma cells, upregulated expression of ecto-5′-nucleotidase is associated with a highly invasive phenotype. Analysis of other cell membrane proteins involved in melanoma adhesion and metastasis demonstrated that expression of α5, β1, β3-integrin subunits and CD44 was elevated gradually in accordance with increasing metastatic potential. Expression of αv-integrin and caveolin-1 was seen mostly in cells derived from metastatic melanomas. Furthermore, in contrast to N-cadherin, which was unaltered in all lines, we could not detect E-cadherin in any cell type. Functional assays demonstrated that highly expressed ecto-5′-nucleotidase is a catalytically competent protein that is very sensitive to inhibition by concanavalin A. The interaction with concanavalin A also caused increased association of ecto-5′-nucleotidase-rich lipid rafts with much heavier cytoskeletal complexes as determined by density gradient centrifugation. A similar shift towards heavier cytoskeletal fractions also took place with other proteins coexpressed with ecto-5′-nucleotidase, such as αv, α5, β1 and β3-integrins, caveolin-1 and CD44. As ConA-induced clustering may reflect the interactions of membrane proteins with extracellular matrix, we also analysed the effect of several extracellular matrix proteins on the in-situ activity of ecto-5′-nucleotidase in WM9 cells and found that tenascin C strongly inhibited ecto-5′-nucleotidase activity and adenosine generation from AMP. We also developed WM9 cells with reduced ecto-5′-nucleotidase expression and tested differences in cell adhesion on various extracellular matrix proteins. WM9(eN−) cells attached significantly weaker to tenascin C layer. These observations indicate that expression of ecto-5′-nucleotidase correlates with a number of metastasis-related markers and thus may have a function in this process. Furthermore, our data suggest that, in addition to generating adenosine, ecto-5′-nucleotidase may have independent roles in adhesion and interaction with extracellular matrix components in melanoma.

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