Abstract 5283: Autophagy regulation of angiogenesis in human neuroblastoma

Abstract

Abstract Neuroblastoma is a progressive solid tumor in infants and children that originates from neural crest and it remains a clinical challenge to treat. It is a highly malignant tumor, in part, because angiogenesis is a key factor in tumor progression of neuroblastoma. We have previously identified that gastrin-releasing peptide (GRP)-receptor-mediated cell signaling is critical to its tumorigenicity. In this study, we hypothesized that increased autophagy via blockage of GRP receptor could potentially inhibit angiogenesis in human neuroblastomas. METHODS. Autophagic key molecular markers were detected with ATG5, Beclin-1 and LC3 antibodies. Phosphorylation of Akt, mTOR and S6R were detected by Western blotting. Angiogenesis were measured using tubule formation of human umbilical vein endothelial cells (HUVECs) grown on top of Matrigel Matrix Basement Membrane in vitro. They were also determined using a mouse model of the cornea pocket assay in vivo. Autophagy was determined by punctuate localization of GFP-LC3 fusion protein, and observed under a confocal fluorescence microscope in vitro. RESULTS. Levels of phospho-Akt, -mTOR and -S6R were increased in BE(2)-C cells treated with GRP in both time- as well as dose-dependent manner. However, the PI3K/Akt/mTOR pathway was inactivated in short-hairpin (sh) silenced targeted at GRP receptor in BE(2)-C cells when compared to control shLac cells, indicating that angiogenesis may be reduced. We also observed increased levels of ATG5, Beclin-1 and LC3 proteins, key autophagic mediators, in shGRP receptor-BE(2)-C cells when compared to controls (shLac cells). In particular, overexpression of ATG5 and Beclin-1 also significantly decreased tube formation on HUVECs when treated with exogenous GRP (100 nM). Consistent with the in vitro findings, treatment with GRP receptor antagonist (RC3095) showed a dramatically decrease in angiogenesis in a mouse model of the cornea pocket assay in vivo. CONCLUSIONS. Our results show that enhanced autophagy was associated with a downregulation of angiogenesis signaling pathways, and an increase in characteristic punctuate localization of GFP-LC3 as a marker for autophagy. A better understanding of how autophagic proteins can regulate the angiogenic process may lead to novel therapeutic strategies in the treatment of neuroblastomas or its vascular disorders. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5283. doi:1538-7445.AM2012-5283