Abstract 4160: SPARC overexpression combined with irradiation reduces VEGF-A dependent angiogenesis in vitro and in vivo via up regulation of miR410 in neuroblastoma cells

Abstract

Abstract Neuroblastoma (NBL) is the third most common malignancy in children and accounts for more than 15% of cancer-related deaths in children in the US. Approximately 50% of patients present with advanced-stage and/or high risk disease. Despite significant intensification of conventional chemotherapy and the addition of immunotherapy to treatment regimens, the long-term survival rates for these children remain less than 40%. Secreted protein, acidic and rich in cysteine (SPARC), also known as osteonectin or BM-40, belongs to the matricellular family of secreted proteins. The biological functions of SPARC are known to be variable in human cancers. In neuroblastoma however, SPARC expression has been found to be associated with impaired tumor growth and decreased tumor related angiogenesis. In the present study we have attempted to elucidate the mechanisms of this angiogenesis reduction. We overexpressed SPARC adenovirus (human) with and without ionizing radiation (5Gy) in two neuroblastoma cell lines SK-N-BE2 (p53mt) and NB1691 (p53wt) and observed suppression of Vascular Endothelial Growth factor A (VEGFA) in both NB1691 (5-fold) and SK-N-BE(2) (2-fold) cells. To determine angiogenic potential of these neuroblastoma cells after SPARC overexpression, we used the dorsal skin fold model assay from which we observed that controls developed tumor cell induced vasculature (TV) which was clearly differentiated from pre-existing vasculature (PV). Radiation treatment alone of neuroblastoma cells caused the development of smaller and leaky capillaries. Overexpression of SPARC reduced the tumor cell induced vasculature to 18±6% of controls in NB1691 cells and to 48±5% of controls in SKNBE2 cells. Addition of radiation treatment to SPARC overexpressed cells further reduced tumor induced vasculature to 7±2% in NB1691 and to 15±3% of controls in SKNBE2 cells. Interestingly irradiation treatment alone increased the number of new vessel formation in both NB1691 (110±7% of controls) and SKNBE2 (136±10% of controls) cells. Previously, we observed that SPARC overexpression decreased VEGF-A expression in both NB1691 and SKNBE2 cells in vitro. To further determine the mechanism by which VEGF-A is suppressed via SPARC overexpression we determined the expression levels of VEGF-A targeting microRNAs. From the microRNA analysis we observed that miR410 was consistently overexpressed in SPARC overexpressed cells both in vitro and in vivo. We also observed that miR410 targets VEGF-A mRNA at 4 different sites, more than any other microRNA that targets VEGF-A. Our results indicate that: 1. SPARC overexpression can sensitize neuroblastoma cells to irradiation even in a varied p53 environment and; 2. Overexpression of SPARC suppresses angiogenesis by specifically inducing the expression of VEGF-A targeting microRNA miR410. Citation Format: Smita Tanpure, Jerusha Boyineni, Reuben Antony, Karen Fernández, Julian Lin, David Pinson, Christopher S. Gondi. SPARC overexpression combined with irradiation reduces VEGF-A dependent angiogenesis in vitro and in vivo via up regulation of miR410 in neuroblastoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4160. doi:10.1158/1538-7445.AM2015-4160