Balance of pro‐ versus anti‐angiogenic splice isoforms of vascular endothelial growth factor as a regulator of neuroblastoma growth

Abstract

Neuroblastoma (NB) is the second most common extracranial tumour of childhood. Angiogenesis plays a crucial role in the growth and development of NB and vascular endothelial growth factor (VEGF), one of the most potent stimuli of angiogenesis, has been studied extensively in vitro. VEGF(165) has been shown to be the predominant angiogenic isoform expressed in NB cell lines and tumours. In this study, we investigated the anti-angiogenic isoform of VEGF-A, generated from distal splice site selection in the terminal exon of VEGF (VEGF(165)b) and shown to be down-regulated in epithelial malignancies. The expression of both the pro- (VEGF(xxx)) and the anti-angiogenic (VEGF(xxx)b) isoforms was compared in a range of NB and ganglioneuroma (GN) tumours. Whereas VEGF(xxx)b and VEGF(xxx) were both expressed in GN, specific up-regulation of the VEGF(xxx) isoforms was seen in NB at RNA and protein levels. Highly tumourigenic NB cell lines also showed up-regulation of the angiogenic isoforms relative to VEGF(xxx)b compared to less tumourigenic cell lines, and the isoforms were differentially secreted. These results indicate that VEGF(165) is up-regulated in NB and that there is a difference in the balance of isoform expression from anti-angiogenic VEGF(165)b to angiogenic VEGF(165). Treatment with recombinant human VEGF(165)b significantly reduced the growth rate of established xenografts of SK-N-BE(2)-C cells (4.24 +/- 1.01 fold increase in volume) compared with those treated with saline (9.76 +/- 3.58, p < 0.01). Microvascular density (MVD) was significantly decreased in rhVEGF(165)b-treated tumours (19.4 +/- 1.9 vessels/mm(3)) in contrast to the saline-treated tumours (45.5 +/- 8.6 vessels/mm(3)). VEGF(165)b had no significant effect on the proliferative or apoptotic activity, viability or cytotoxicity of SK-N-BE(2)-C cells after 48 h. In conclusion, VEGF(165)b is an effective inhibitor of NB growth. These findings provide the rationale for further investigation of VEGF(165)b in NB and other paediatric malignancies.