Abstract 81: Endogenous ADAMTS-13 regulates vasculogenic functions in glioblastoma cells

Abstract

Abstract ADAMTS-13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13, is a zinc-containing metalloprotease that cleaves von Willebrand factor. Previous publications by our laboratory have shown that either endogenous or exogenous ADAMTS-13 may regulate angiogenesis in endothelial cells; for example, siRNA knockdown of endogenous endothelial cell ADAMTS-13 reduced angiogenic activities including cell migration, proliferation, and tube formation (Tang et al. Microvascular Res 2017; 113:85-70). Many tumor cells have the ability to develop a vascular phenotype, a process known as vasculogenic mimicry (VM). There is a multitude of evidence demonstrating that tumor cell-mediated VM may play a vital role in tumor development, independent of endothelial cell angiogenesis. For this study, we report that decreasing ADAMTS-13 levels inhibit vasculogenic functions in a glioblastoma cell line (LN-229), while increasing ADAMTS-13 levels had no effect. Incubating up to 30ng/ml of exogenous ADAMTS-13 did not promote cell proliferation and up to 200ng/ml did not change tube formation in LN-299 brain tumor cells. One possible explanation for this observation is that LN-299 brain tumor cells have almost twice the endogenous ADAMTS-13 expression than endothelial cells (~200pg/ml vs ~100pg/ml), so this cell line is not sensitive to exogenous ADAMTS-13. Using siRNA, we were able to successfully knockdown endogenous ADAMTS-13 in human brain tumor LN-229 cells. 50nM of ADAMTS-13 siRNA in a six-well plate inhibited HUVEC ADAMTS-13 expression levels by 75% after 48hr incubation, whereas control siRNA did not affect endogenous ADAMTS-13 levels. Additionally, expression of ADAMTS-1, a related family member of ADAMTS-13 was not affected by the knockdown. The effects of reduced endogenous ADAMTS-13 on glioblastoma vasculogenic functions were studied. Transfection of HUVEC with 50nM of ADAMTS-13 siRNA in a 24-well plate resulted in about 20% decrease in proliferation after 24hr and 48hr incubation. We demonstrated that decreased expression of endogenous ADAMTS-13 also affected vasculogenic mimicry as measured by glioblastoma cell tube formation using a Matrigel matrix method. The tube lengths, sizes and junction numbers of the ADAMTS-13 knockdown cells were all significantly lower compared to control cells by about 30-40%, respectively. Moreover, the expression of VEGF, a well-known regulator of angiogenesis, was significantly decreased by 25% after knockdown of endogenous LN-229 ADAMTS-13. Overall, these data suggest that endogenous ADAMTS-13 may regulate glioblastoma cell growth and function. Citation Format: Huiyuan Tang, Manfai Lee, Eun Ho Kim, Daniel Bishop, George M. Rodgers. Endogenous ADAMTS-13 regulates vasculogenic functions in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 81.