Abstract 4251: Inhibition of membrane bound and soluble VEGF receptor-2 activity enhances the proliferation of the BON carcinoid cell line
Authors: Silva Scott R. , Valentino Joseph D. , Zaytseva Yekaterina Y. , Townsend Courtney M. , Evers B Mark 
Published: July 2012

Abstract
There is a growing interest in using angiogenesis inhibitors to treat neuroendocrine tumors. Previously, we detected the expression of vascular endothelial growth factor receptor-2 (VEGFR-2) and its soluble isoform (sVEGFR-2) in the epithelial component of carcinoid tumors and in the BON carcinoid cell line. Recent studies have demonstrated that VEGF inhibitors transiently inhibit tumor growth, followed by increased tumor invasion and metastasis. Also, we have reported that shRNA reduction of VEGFR-2 increases BON cell proliferation and invasion. Thus, the purpose of our current study was to: i) assess the effect that the VEGF signaling inhibitors bevacizumab, an antibody against VEGF-A, or sunitinib, a small molecule inhibitor of VEGFR-2, have on BON cell proliferation and, ii) evaluate the effect of knockdown and overexpression of sVEGFR-2 on BON cell proliferation. Methods. i) BON cells were cultured in media supplemented with bevacizumab (50 ng/ml), sunitinib (50 ng/ml), or vehicle control for two weeks. Media was replaced every other day, and cell lysates for western blot and RNA analysis were collected after one and two weeks. After two weeks, cell proliferation was measured using a cell counting kit-8 assay. ii) Knockdown or upregulation of sVEGFR-2 was achieved by transfecting BON cells with shRNA to sVEGFR-2 or a cDNA plasmid expressing the sVegfr2 gene, respectively. Proliferation following sVEGFR-2 knockdown or upregulation was determined and compared to cells transfected with the appropriate controls. Results. i) BON cell proliferation was significantly increased in cells treated with bevacizumab or sunitinib compared to vehicle control. Western blot analysis demonstrated an increase in phospho-ERK and phospho-Akt following treatment with bevacizumab. Interestingly, there were no observed changes in activation of ERK or Akt following treatment with sunitinib. However, sunitinib treatment increased protein and mRNA expression of VEGFR-2, as assessed by western blot and qRT-PCR, respectively. ii) BON cells with reduced expression of sVEGFR-2 demonstrated increased proliferation at 24 h, 48 h, 72 h, 96 h, and 120 h following plating. Conversely, BON cells transfected with plasmid cDNA encoding sVEGFR-2 displayed reduced proliferation at the aforementioned time points compared to cells transfected with empty plasmid. Conclusions. We have demonstrated that the VEGF inhibitors bevacizumab and sunitinib can enhance the proliferation of BON cells in vitro and that sVEGFR-2 inhibits BON cell proliferation, possibly by sequestering VEGF ligands. While VEGF inhibitors have been shown to effectively inhibit angiogenesis, they may also enhance the aggressiveness of surviving tumor cells, suggesting that a multimodal treatment plan is warranted in carcinoid patients.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4251. doi:10.1158/1538-7445.AM2011-4251