4907

NT5E

5'-nucleotidase, ecto (CD73);NT5E;5'-nucleotidase, ecto (CD73)

5'-NT|5'-nucleotidase|Purine 5-Prime-Nucleotidase|ecto-5'-nucleotidase

6q14-q21

protein-coding

Medulloblastoma (MB) is the most common malignant brain tumor in children and occurs mainly in the cerebellum. Important intracellular signaling molecules, such those present in the Sonic Hedgehog and Wnt pathways, are involved in its development and can also be employed to determine tumor grade and prognosis. Ectonucleotidases, particularly ecto-5'NT/CD73, are important enzymes in the malignant process of different tumor types regulating extracellular ATP and adenosine levels. Here, we investigated the activity of ectonucleotidases in three malignant human cell lines: Daoy and ONS76, being representative of primary MB, and the D283 cell line, derived from a metastatic MB. All cell lines secreted ATP into the extracellular medium while hydrolyze poorly this nucleotide, which is in agreement with the low expression and activity of pyrophosphate/phosphodiesterase, NTPDases and alkaline phosphatase. The analysis of AMP hydrolysis showed that Daoy and ONS76 completely hydrolyzed AMP, with parallel adenosine production (Daoy) and inosine accumulation (ONS76). On the other hand, D283 cell line did not hydrolyze AMP. Moreover, primary MB tumor cells, Daoy and ONS76 express the ecto-5'NT/CD73 while D283 representative of a metastatic tumor, revealed poor expression of this enzyme, while the ecto-adenosine deaminase showed higher expression in D283 compared to Daoy and ONS76 cells. Nuclear beta-catenin has been suggested as a marker for MB prognosis. Further it can promotes expression of ecto-5'NT/CD73 and suppression of adenosine deaminase. It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity. In summary, the absence of ecto-5'NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

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 alpha5, beta1, beta3-integrin subunits and CD44 was elevated gradually in accordance with increasing metastatic potential. expression of alphav-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 alphav, alpha5, beta1 and beta3-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 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.

Upregulated expression of eN has been found in the highly invasive human melanoma cell lines but neither in melanocytes nor in primary tumor cells. Membrane proteins associated with cell adhesion and metastasis: alpha5-, beta1-, beta3-integrins, and CD44 were elevated gradually in accordance with increasing metastatic potential. alphav-integrin was seen mostly in aggressive melanomas. The expression of eN correlated with a number of metastasis-related markers and thus may have a function in the process. eN activity went parallel with its amount in all cells. Concanavalin A strongly inhibited the enzyme in a noncompetitive way. Clustering of eN protein in overexpressing cells by ConA-treatment increased the enzyme association with the heavy cytoskeletal complexes. A similar shift towards cytoskeletal fractions took also place with other membrane proteins coexpressed with eN. This ConA-induced association may reflect a putative interaction of eN with physiological ligand, that upon interaction, aggregates protein components of lipid rafts and triggers signaling pathway that may be intrinsically involved in cell-stroma adhesion.

Angiogenesis is essential for tumor growth, progression and metastasis. Studies indicate that expression and activity of ecto-5'-nucleotidase (CD73) are elevated in metastatic carcinomas. Our previous studies found that angiogenesis of tumor xenografts was decreased when the activity of CD73 in cancer cells was inhibited, implying that this enzyme is involved in tumor angiogenesis. To elucidate the mechanism, we investigated CD73 influence on tumor angiogenesis in both in vitro assays and in tumor bearing mice. We found that capillary-like structures were formed more in CD73(+/+) pulmonary microvascular endothelial cells (PMECs) than CD73(-/-) PMECs, and this was more pronounced when the cells were cultured in cancer-conditioned medium. Meanwhile, CD73 decreased endothelial cells adhesion to collagen IV and promoted migration. Additionally, the extent of tumor angiogenesis and the size of tumors were greater in CD73(+/+) mice than in CD73(-/-) mice. Thus, we concluded that CD73 can promote endothelial cells forming new vessels in cancer condition, facilitating tumor growth and hematogenous metastasis.