A role for nucleotides in support of breast cancer angiogenesis: Heterologous receptor signalling

Abstract

Background:Human breast carcinoma cells secrete an adenosine 5′-diphosphate transphosphorylase (sNDPK) known to induce endothelial cell tubulogenesis in a P2Y receptor-dependent manner. We examined sNDPK secretion and its effects on human endothelial cells.Methods:Nucleoside diphosphate kinase (NDPK) secretion was measured by western blot and enzyme-linked immunosorbent assay, while transphosphorylase activity was measured using the luciferin-luciferase ATP assay. Activation of MAPK was determined by western blot analysis, immunofluorescence and endothelial cell proliferation and migration.Results:A panel of breast cancer cell lines with origin as ductal carcinoma, adenocarcinoma or medullary carcinoma, secrete sNDPK-A/B. Addition of purified NDPK-B to endothelial cultures activated VEGFR-2 and Erk 1/2, both of which were blocked by inhibitors of NDPK and P2Y receptors. Activation of VEGFR-2 and ErK 1/2 by 2-methylthio-ATP (2MeS-ATP) was blocked by pretreatment with the P2Y 1-specific antagonist MRS2179, the proto-oncogene non-receptor tyrosine kinase (Src) inhibitor PP2 or the VEGFR-2 antagonist SU1498. Nucleoside diphosphate kinase-B stimulates cell growth and migration in a concentration-dependent manner comparable to the effect of vascular endothelial growth factor. Treatment of endothelial cells with either NDPK-B or 2MeS-ATP induced migration, blocked by P2Y 1, Src or VEGFR-2 antagonists.Conclusion:sNDPK supports angiogenesis. Understanding the mechanism of action of sNDPK and P2Y 1 nucleotide signalling in metastasis and angiogenesis represent new therapeutic targets for anti-angiogenic therapies to benefit patients. © 2011 Cancer Research UK All rights reserved.