Abstract 4986: Urokinase plasminogen activator (uPA) interacts with nuclear HOXA5 and inhibits p53 expression in glioma stem cells

Abstract

Abstract Urokinase-type plasminogen activator (uPA) participates in diverse physiological processes, including glioma progression. HOXA5 is a known regulator of p53 expression, and overexpression of HOXA5 has been shown to increase p53 expression. In this study, we have demonstrated that uPA not only translocates to the nucleus but also interacts with the transcription factor HOXA5. We used CD133- and CD44-enriched U87 human glioma cells and 4910 human glioma xenograft cells, which exhibit cancer stem cell properties. Nuclear localization of uPA was determined by western blot analysis of nuclear fractions and by immunocytochemistry. Glioma stem cells showed increased nuclear localization of uPA as compared to normal glioma cells. To determine the in vitro interaction of uPA with HOXA5, we initially used membrane immobilized transcription factors incubated with purified uPA protein and looked for strong protein-protein interactions. We observed that uPA binds preferentially to HOXA5 transcription factor. Subsequently, we immunoprecipitated uPA or HOXA5 from nuclear fractions of U87 and 4910 normal and stem cells and tested for co-precipitation of HOXA5 or uPA respectively. Again, the results showed that uPA binds preferentially to nuclear HOXA5. To determine the effect of uPA or HOXA5 downregulation on these glioma cells, we used RNAi-expressing plasmids targeting uPA or HOXA5. The results showed that downregulation of uPA alone induced overexpression of p53 in both normal and glioma stem cells. Glioma stem cells downregulated for uPA showed at least a 5-fold increase in p53 expression when compared to uPA downregulated normal glioma cells. Downregulation of HOXA5 alone did not show significant change in expression levels of p53; however, simultaneous downregulation of uPA and HOXA5 did not show change in expression levels of p53. To determine whether uPA interacts directly with HOXA5 binding sequences, we performed an electromobility shift assay (EMSA). We observed that uPA does not bind to HOXA5 sequences but rather forms a complex with HOXA5 bound to DNA. In vivo studies showed that subcutaneous tumors injected intratumorally with siRNA-expressing plasmids targeting uPA or HOXA5 either singly or simultaneously showed strong p53 expression in uPA downregulated tumors with no significant change observed in other treatment conditions. Taken together, these results demonstrate that uPA behaves as a negative regulator of p53 expression in glioma cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 4986.