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Abstract

Polarization of hepatocytes is important for the liver to carry out its functions such as bile secretion and detoxification. In this context, cell polarity complexes, which are spatially segregated at specific membranous domains, define the 3-dimensional orientation of hepatocytes. The disturbance of cell polarity in early phases of tumorigenesis is associated with the activation of intracellular oncogenic signaling pathways affecting tumor initiation and progression. However, if the disturbance of hepatocellular polarity participates in the tumor formation and how the dysregulation of polarity factors affect hepatocarcinogenesis is poorly understood. In order to define relevant cell polarity factors, which may support hepatocellular carcinoma (HCC) development, high-dimensional expression array data derived from HCC patients were screened for significantly dysregulated cell polarity genes. A panel of significantly dysregulated factors, including the baso-lateral complex protein Scribble (Scrib), correlated with poor patient outcome and tumor recurrence. Scrib overexpression in HCC tissues associated with genomics gains of the Scribble gene locus on chromosome 8q24.3. The loss of membranous Scrib expression or its cytoplasmic enrichment was frequently observed in HCC tissues and HCC cell lines but not in normal liver tissues. In order to analyze the biological impact of cytoplasmic Scrib accumulation, polarized HCC cell lines (HepG2) stably expressing wildtype Scrib (ScribWT, with membrane binding) and a mutated Scrib isoform (ScribP305L, with enforced cytoplasmic enrichment) were generated. In vitro, cytoplasmic ScribP305L reduced the formation of canalicular structures and moderately supported cell proliferation. In vivo, hydrodynamic gene delivery of ScribP305L and c-MYC induced elevated liver tumor formation compared to ScribWT and c-MYC co-injection. Importantly, ScribP305L stimulated HCC cell invasiveness, reorganization of actin filaments and overexpression of Rho GTPases. A comparative analysis of oncogenic pathways revealed an activation of AKT by ScribP305L, which was mediated through the destabilization of negative AKT regulators leucine rich repeat protein phosphatase 1 (PHLPP1) and phosphatase and tensin homolog (PTEN). Transcriptome analysis detected ScribP305L-dependent upregulation of genes, which were associated with cell motility, epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) remodeling. Among these genes, secreted protein acidic and cysteine rich (SPARC) was highly induced by cytoplasmic Scrib and facilitates cell invasion depending on the AP-1 transcription factor subunits ATF2 and JunB. Lastly a significant association between cytoplasmic Scrib, AKT and ATF2 activation/phosphorylation and loss of E-cadherin was confirmed in human HCC samples and ScribP305L-induced gene signatures were detected in patient subgroups with worse overall survival. Taken together, this study illustrates that the overexpression and cytoplasmic enrichment of the cell polarity factor Scrib supports HCC development and tumor cell dissemination through the induction of distinct molecular mechanisms. These mechanisms include activation of the AKT signaling axis and stimulation of AP1-target genes, which are critical for the migratory phenotype of HCC cells.