HIF-1α upregulates ANGPTL4 to promote angiogenesis in Uveal Melanoma

Abstract

Purpose: The transcriptional response promoted by hypoxia-inducible factor (HIF)-1α has been associated with angiogenesis and metastatic spread in uveal melanoma (UM). Expression of one HIF-1 target gene, vascular endothelial growth factor (VEGF) correlates with tumor vascularity in UM, as well as other tumors. However, treatment of patients with therapies targeting VEGF has not proven sufficient alone to prevent UM growth or spread. Here we set out to evaluate the potential role of a novel HIF-regulated gene product, angiopoietin-like 4 (ANGPTL4), in the promotion of angiogenesis in UM.

Methods: UM cell lines were examined for expression of HIF-1α, VEGF, and ANGPTL4. Expression of HIF-1α, VEGF, and ANGPTL4 were further assessed by immunohistochemical analysis in UM tissue and quantitated using a UM tissue array. Expression of ANGPTL4 was also examined in vitreous biopsies from patients with uveal melanoma. The role of ANGPTL4 in angiogenesis in UM was assessed using endothelial cell (EC) tubule formation (TF) assays. Inhibition of HIF-1α, VEGF, and ANGPTL4 was performed using RNAi.

Results: Expression of HIF-1α was detected in all UM cell lines tested. Hypoxic stabilization of HIF-1α resulted in the promotion of TF in treated ECs in vitro; this affect was inhibited by blocking HIF-1α translation, but only partially inhibited by blocking VEGF expression, implicating additional HIF-regulated genes in the promotion of angiogenesis in UM. We demonstrate that HIF-1α stabilization results in ANGPTL4 mRNA and protein expression in UM cell lines. These results were corroborated in tissue samples from patients with UM. Using a UM tissue array, we further observed expression of HIF-1α in a majority of tumors in the array. Expression of either ANGPTL4 or VEGF was detected in almost all (99%) of the tumors in the UM tissue array. ANGPTL4 expression was increased more than 50 fold in vitreous biopsies from UM patients at levels that were sufficient to promote angiogenesis in vitro. RNAi targeting ANGPTL4 inhibited the promotion of TF induced by UM cell lines; this effect was additive to RNAi targeting VEGF.

Conclusions: We propose that therapies targeting both VEGF and ANGPTL4 will be necessary to effectively inhibit tumor-induced angiogenesis in patients with UM.