Netrin-1 promotes retinoblastoma-associated angiogenesis

Retinoblastoma (Rb) is the most common intraocular cancer of infancy and childhood, with an incidence of nearly 0.006% in all live births. Although a functional loss or inactivation of both alleles of the retinoblastoma 1 (RB1) gene during retinal development appears to be the predominant etiology for Rb, genes associated with tumor angiogenesis are also likely to be involved in the development of this condition. Netrin-1 is a factor that regulates pathological angiogenesis, while its role in Rb is largely unknown. The present study examined the role of netrin-1 in Rb.The expression of netrin-1 in Rb was assessed using public databases and using clinical specimens by RT-qPCR for mRNA and by ELISA for protein. The expression of netrin-1 was suppressed in Rb by siRNA and the effects on cell growth were determined by a CCK-8 assay, while the effects on angiogenesis were examined in vitro using human umbilical vein endothelial cell (HUVEC) assays and in vivo by quantification of tumor vessel density.Analysis of published databases revealed that the netrin-1 gene is significantly upregulated in Rb, which was confirmed by immunohistochemistry on clinical specimens. Inhibition of netrin-1 in Rb cell lines significantly reduced their effects on angiogenesis in vitro using a HUVEC co-culture assay without affecting cell growth. Inhibition of netrin-1 expression in vivo suppressed the growth of grafted Rb, and this effect could be abolished by co-expression of vascular endothelial growth factor A (VEGF-A).This data demonstrated a novel role for netrin-1 in the regulation of Rb-associated cancer vascularization and may represent a novel therapeutic target for patients with Rb.