Abstract 1130: Subcellular localization of the cell polarity protein Scribble defines its oncogenic activity in hepatocellular carcinoma

Abstract

Abstract Background: Disturbance of hepatocyte polarity is frequently observed in liver cancer tissues; however, it is still controversially discussed if this phenotype represents the cause or the result of tumorigenesis. Due to their multi-functional properties, hepatocytes are characterized by distinct luminal, lateral and basal domains that are defined by specific protein complex. These cell protein complexes such as the apical Crumbs- (CRB3, Pals1, Patj), the lateral Par- (Par3, Par6, aPKC) and the basolateral Scribble-complex (Scrib, Dlg, Lgl) are essential for the formation of hepatocellular polarity. So far, it is unknown if and which protein complex constituents are involved in liver tumor development and how these factors induce possible downstream effector mechanisms. Methods: To identify complex proteins that affect hepatocellular polarity, a siRNA screen targeting all relevant polarity factors was performed in HCC cells that form bile canaliculi in vitro (HepG2). Localization of Scribble in HCC cell lines and human HCC tissues was analyzed by western immunoblotting and immunofluorescence. Expression vectors containing wildtype Scribble and an isoform lacking polarity complex binding capacity were cloned (Scrib-wt, Scrib-P305L) and stably transfected in HCC cells. The impact of these isoforms on PI3K/AKT signaling pathway activity and cell viability were analyzed. In addition, the interaction of Scrib-wt, Scrib-P305L with phosphatases regulating the PI3K/AKT pathway was tested by Co-IP analysis. Results: The siRNA screen revealed that Scribble strongly affects hepatocellular polarity. Scribble located near the membrane in polarized cells (e.g., HepG2, HuH-1) but in the cytoplasm in non-polarized cells (HLE, HLF). Scribble overexpression and cytoplasmic accumulation correlates with poor overall survival of HCC patients. In vitro, the inhibition of Scribble reduced and overexpression of cytoplasmic Scribble (Scrib-P305L) induced AKT phosphorylation. In addition, Scrib-P305L supported HCC cell viability in an AKT-dependent manner. Immunofluorescence revealed co-localization of Scribble with the AKT phosphatases PTEN and PHLPP1. Co-IP studies revealed a physical interaction between PHLPP1 and both Scribbles isoforms (wildtype and P305L mutant). Conclusion/Outlook: Our data demonstrate that the cytoplasmic accumulation of Scribble in HCC cells promotes tumor cell growth in an AKT-dependent manner probably through the interaction with AKT phosphatases. These results indicate that the disturbance of cell polarity (e.g. by Scribble overexpression) can foster oncogenic effects and therefore must be regarded as initiating event in hepatocarcinogenesis. Citation Format: Shan Wan, Anne-Sophie Meyer, Sofia Weiler, Teresa Lutz, Stephanie Roessler, Peter Schirmacher, Federico Pinna, Kai Breuhahn. Subcellular localization of the cell polarity protein Scribble defines its oncogenic activity in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1130.