Abstract 1466: The NO donor DETANONOate inhibits the EMT phenotype in human metastatic prostate carcinoma cell lines: pivotal roles of inhibition of NF-κB and Snail activities and induction of RKIP

Abstract

Abstract Nitric oxide (NO) has been shown to mediate contrasting effects on tumorigenesis i.e. at low levels NO is pro-tumorigenic and at high levels NO is anti-tumorigenic. Recent studies revealed that treatment of tumor cell lines with NO donors at high levels significantly inhibited cell growth and survival pathways such as the constitutively activated NF-κB. Tumor cells with activated NF-κB were reported to have the capacity to initiate the metastatic cascade via induction of epithelial to mesenchymal transition (EMT). The EMT phenotype is characterized by decreased expression of epithelial gene products (e.g. E-cadherin, cytokeratin 18) and upregulation of mesenchymal gene products (e.g. vimentin, fibronectin). In addition, the tumor cells exhibit invasive and migratory properties. Based on these above findings, we hypothesized that treatment of metastatic prostate carcinoma cell lines, PC-3 and DU-145 with DETANONOate (500-1000μM) may reverse the EMT phenotype. This hypothesis was tested and treatment of the cells with DETANONOate resulted in inhibition of the EMT phenotype through inhibition of NF-κB activity. Inhibition of NF-κB was shown to be due, in part, to S-nitrosylation of p50. In addition to the inhibition of EMT phenotype, treatment with DETANONOate also inhibited invasive properties. The dual roles of DETANONOate-mediated inhibition of NF-κB and EMT were corroborated by the use of the NF-κB inhibitor DHMEQ. We examined the underlying mechanism by which NF-κB-induced inhibition by DETANONOate resulted in the inhibition of EMT. The EMT-induced transcription factor Snail was highly expressed in PC-3 and DU-145 cells. Treatment with DETANONOate inhibited Snail expression concomitant with inhibition of EMT. The direct role of Snail inhibition by DETANONOate and the inhibition of EMT was corroborated in cells transfected with Snail siRNA and such treatment reversed the EMT phenotype, mimicking DETANONOate. Inhibition of the repressor activity of Snail by DETANONOate resulted in the upregulation of the Raf-1 kinase inhibitory protein (RKIP). The role of RKIP induction in the reversal of EMT was corroborated in cells overexpressing RKIP. The in vitro findings with DETANONOate-induced inhibition of EMT were validated in mice bearing PC-3 xenografts. Treatment with DETANONOate and analysis by IHC of tumor biopsies revealed that the EMT phenotype was reversed. There was an inverse correlation between Snail inhibition and RKIP induction. The present findings demonstrate, for the first time, that NO donors (high concentrations) inhibit the EMT phenotype and suggest their potential therapeutic applications in vivo in patients. These studies also suggest that the NF-κB/Snail/RKIP circuitry is dysregulated in tumor cells and is responsible, in part, for the EMT phenotype. 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 1466.