IGF binding protein‐6 expression in vascular endothelial cells is induced by hypoxia and plays a negative role in tumor angiogenesis

Abstract

Hypoxia stimulates tumor angiogenesis by inducing the expression of angiogenic molecules. The negative regulators of this process, however, are not well understood. Here we report that hypoxia induced the expression of insulin-like growth factor binding protein-6 (IGFBP-6), a tumor repressor, in human and rodent vascular endothelial cells (VECs) via a HIF-mediated mechanism. Addition of human IGFBP-6 to cultured human VECs inhibited angiogenesis in vitro. An IGFBP-6 mutant with at least 10,000-fold lower binding affinity for IGFs was an equally potent inhibitor of angiogenesis, suggesting that this action of IGFBP-6 is IGF-independent. The functional relationship between IGFBP-6 and VEGF, a major hypoxia-inducible angiogenic molecule, was examined. While VEGF alone increased angiogenesis in vitro, co-incubation with IGFBP-6 abolished VEGF-stimulated angiogenesis. The in vivo role of IGFBP-6 in angiogenesis was tested in flk1:GFP zebrafish embryos, which exhibit green fluorescence protein in developing vascular endothelium, permitting visualization of developing blood vessels. Injection of human IGFBP-6 mRNA reduced the number of embryonic inter-segmental blood vessels by ∼40%. This anti-angiogenic activity is conserved in zebrafish because expression of zebrafish IGFBP-6b had similar effects. To determine the anti-angiogenic effect of IGFBP-6 in a tumor model, human Rh30 rhabdomyosarcoma cells stably transfected with IGFBP-6 were inoculated into athymic BALB/c nude mice. Vessel density was 52% lower in IGFBP-6-transfected xenografts than in vector control xenografts. These results suggest that the expression of IGFBP-6 in VECs is up-regulated by hypoxia and IGFBP-6 inhibits angiogenesis in vitro and in vivo.

Prohibitin-2 Binding Modulates Insulin-like Growth Factor-binding Protein-6 (IGFBP-6)-induced Rhabdomyosarcoma Cell Migration

“…Although PHB2 is indispensable for IGFBP-6-induced RMS cell migration, this action is not dependent on MAPK activation. IGFBP-6 has been shown to have anti-tumorigenic properties by inhibiting IGF-II actions and, more recently, by inhibiting angiogenesis via an IGF-independent mechanism (59). However, IGFBP-6-induced migration may favor cell invasion, and targeting PHB2 may ameliorate this potentially adverse effect of an IGFBP-6-based therapeutic for IGF-II-dependent tumors.…”

40 YEARS OF IGF1: IGF-binding proteins

“…It entered the nucleus by binding to importins via a nuclear 558 localisation sequence in its C-terminal domain, and subsequently modulated cell proliferation, 559 migration and survival (Iosef et al 2008;Iosef, et al 2010; Kuo, et al 2010). IGFBP-6 inhibited 560 basal and VEGF-induced angiogenesis by an IGF-independent mechanism in vitro as well as 561 inhibiting it in rhabdomyosarcoma xenografts and zebrafish embryos in vivo (Zhang, et al 2012). 562 IGFBP-6 may limit the angiogenic response to hypoxia, which slowly increased its expression.…”

Recent insights into the actions of IGFBP-6

“…Intranuclear actions were also implicated in apoptosis induced by IGFBP-6 (Iosef et al 2008(Iosef et al , 2010. As described above, IGFBP-6 overexpression inhibited rhabdomyosarcoma xenograft growth and angiogenesis in vivo Gallicchio et al 2001;Zhang et al 2012), and IGFBP-6 promotes rhabdomyosarcoma cell migration via pathways dependent on PHB2 and MAP kinase signalling (Fu et al 2007.…”

“…Vascular endothelial growth factor (VEGF) pathways are the major mediators of angiogenesis, but IGFs also play a role (Carmeliet and Jain 2000) by stimulating HIF-1α expression (Hoeben, et al 2004), and inducing VEGF synthesis (Hoeben et al 2004;Stearns, et al 2005;Warren, et al 1996) via HIF-1-dependent and -independent pathways (Slomiany and Rosenzweig 2006). The human IGFBP6 promoter contains multiple hypoxia response elements and HIF-1 ancillary sequences, and IGFBP-6 expression was upregulated by hypoxia in endothelial cells via HIF-1α (Zhang, et al 2012). IGFBP-6 overexpression inhibited angiogenesis in rhabdomyosarcoma xenografts and zebrafish embryos, and prolonged hypoxia increased IGFBP-6 expression via HIF-1α (Zhang et al 2012).…”