Netrin-1 promotes retinoblastoma-associated angiogenesis

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.