Abstract 1282: Notch and VEGF regulate tumor endothelial cell survival

Abstract

Abstract Notch proteins function in the specification of endothelial cell (EC) identity during physiologic angiogenesis, in part through interaction with VEGF signaling. However, the role of Notch in tumor EC (TEC) survival and apoptosis is less well defined. In EC, Notch signaling regulates survival pathways such as AKT, and anti-apoptotic proteins such as Bcl-2, with feedback loops modulating the VEGF and Notch pathways. We therefore asked whether targeting the Notch pathway affects EC apoptosis in tumors, specifically by affecting EC dependence on VEGF, a key endothelial survival factor. We used a Notch decoy (ND) construct composed of the extracellular domain of the Notch1 receptor, which we have reported blocks Notch activation by multiple ligands. Cultured NGP neuroblastoma cells were engineered to secrete ND construct (NGP-ND). 10[6] NGP-ND and NGP-Lacz cells were implanted intrarenally in nude mice, and treated with either placebo or the anti-VEGF antibody bevacizumab (BV). Tumors were harvested at 5 weeks. We have previously reported that Notch blockade disrupts NGP xenograft blood vessels. Here, we show that combining BV and ND further destabilizes tumor angiogenesis and architecture. Both tumor hypoxia and tumor cell apoptosis (quantified by pimonidazole and TUNEL stain, respectively) increased two-fold (p<0.05). Quantification of the EC marker PECAM-1 demonstrated a 25% reduction in the EC coverage in the presence of either BV or ND, while there was a 75% decrease in BV + ND tumors (p<0.05 vs BV and ND alone). TEC death also increased in BV + ND tumors as measured by the number of triple-immunopositive PECAM-1/TUNEL/DAPI+ cells. Regressing vessels can leave empty “sleeves” of vascular basement membrane. Therefore, we quantified type IV collagen on serial sections. In contrast to the decrease in TEC, none of the treatments altered the type IV collagen deposition (p=ns), consistent with loss of pre-existing endothelial vessels. In cultured human umbilical vein endothelial cells (HUVEC) incubated with conditioned media (CM) containing ND, cell death increased twofold as compared to HUVEC cultured with control CM (p<0.0001). VEGF could rescue this effect, as addition of rhVEGF (20ng/ml) to the CM reduced the cell death of both control and ND cells by 40% as compared to HUVEC in control CM (p=0.01). This striking reduction of cell death in ND-treated EC in response to VEGF is consistent with the ability of Notch to repress transcription of VEGFR2. Thus, when Notch signaling is blocked, endothelial VEGFR2 expression is derepressed, allowing VEGF/VEGFR2 survival signaling to rescue apoptosis. In conclusion, we show that blocking the VEGF and Notch pathways increases TEC death in an additive manner. These results strongly suggest that VEGF signals from tumor cells can partially compensate for the apoptotic effects of Notch blockade, and provide a rationale for combined targeting of VEGF and Notch as a therapeutic approach. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1282.