PO-194 Novel potential targets for hakai oncogene during tumour progression

Abstract

IntroductionThe transition from a benign lesion or adenoma to carcinoma begins with the disruption of cell-cell contacts. The loss of E-cadherin at cell-cell contacts is a hallmark of the epithelial-to-mesenchymal transition (EMT) process. During EMT, cells lose their epithelial phenotype and acquire motility and invasive capacities, due to decreased expression of E-cadherin. Hakai is an E3 ubiquitin-ligase for E-cadherin, which induces its ubiquitination and degradation and it is an oncogene that induces transformation in epithelial cells to a mesenchymal and invasive phenotype both in vitro and in vivo. Moreover, Hakai expression is markedly higher in human colon adenocarcinoma compared to normal tissues. However, not all cellular changes observed when Hakai is overexpressed can be attributed to its action on E-cadherin. Here, we aim to identify new molecular targets for Hakai involved in cancer cell plasticity during tumour progression.Material and methodsWe employed iTRAQ technique followed by LC-MALDI-MS analysis in Hakai overexpresing epithelial cells compared to normal cells. Data obtained from the MS/MS acquisitions were quantified using ProteinPilot software and analysed by bioinformatics. Western blot was performed for validation of several proteins identified. Finally, by confocal analysis it was determined the subcellular localization of specific regulated proteins, such as Galectin-3 and Annexin A1.Results and discussionsOur results show that Hakai influences cytoskeleton-related proteins, extracellular exosome-associated proteins, RNA-related proteins and proteins involved in metabolism. We confirmed the regulation by Hakai of several specific proteins including Galectin-3 and Annexin A1. Confocal images show a total disappearance of Annexin A1 and a colocalization of Hakai and Galectin-3 in the cytoplasm in Hakai overexpressing cells. Interestingly, a profound decreased expression in several proteasome subunits was highlighted.ConclusionHakai influences cellular metabolism, cytoskeleton-related proteins, RNA-related proteins extracellular exosome-associated proteins. It is also evidenced a profound decreased expression in many 26S proteasome subunits during Hakai-driven EMT. Since in clinical trials the use of proteasome inhibitors has failed for solid tumours, our findings suggest that E3 ubiquitin-ligases, such as Hakai, may be a better target for using novel specific inhibitors in tumour subtypes.