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Abstract

Cell-cell connections, in part mediated by adherens junctions, play an important role in the formation of cell polarity and tissue homeostasis. Their dysregulation is associated with the development of several diseases, including cancer formation. Exemplary, the function of liver hepatocytes strictly depends on a high degree of spatial organization and disturbance of hepatocellular polarity is a key feature in hepatocarcinogenesis. Indeed, hepatocellular carcinoma (HCC) is characterized by limited treatment options, illustrating the urgent need to identify novel target structures to improve diagnostics and the development of therapeutic strategies for HCC patients. A frequently dysregulated pathway in HCC is the cell-cell contact-sensing and organ size-controlling Hippo/yes-associated protein (YAP) signaling cascade. Regarding Hippo/YAP pathway, it has been demonstrated that junctional proteins like E-cadherin can influence its activity with direct impact on tumor formation. However, it is unknown if Hippo/YAP unspecifically responds to any kind of cell contact alteration or if specific molecular mechanisms connect the aberrant expression of junctional proteins with this pathway. To identify junctional structures which are aberrantly expressed in HCC, and which may control the Hippo/YAP signaling cascade, I systematically screened expression data derived from human HCC patient cohorts. Based on clinical (e.g., patient survival) and molecular (e.g., correlation with YAP target genes) selection parameters, five junctional candidates were identified including the desmoglein 1 (DSG1), desmoglein 2 (DSG2) and vasodilator stimulated phosphoprotein (VASP). Induction (DSG2, VASP) and reduction (DSG1) in HCCs compared to non-malignant livers was confirmed in independent cohorts and by different techniques (e.g., immunofluorescence). While DSG1 does not affect HCC cell biology, silencing of DSG2 and VASP by RNAinterference in HCC cell lines revealed that these proteins support HCC cell viability and proliferation, as well as migration and invasion. Subsequent hydrodynamic gene delivery experiments disclosed that DSG2 and VASP do not act as liver oncogenes in this experimental setup. According to my working hypothesis, YAP abundance and activity is affected by DSG2 and VASP: while DSG2 supports the accumulation of YAP in HCC cells, VASP reduces YAP phosphorylation, which is associated with its activation. To further investigate how dysregulated DSG2 and VASP affect YAP activity, I applied the unbiased BioID approach, followed by mass spectrometry, to identify exclusive DSG2 or VASP interaction partners. Confirmatory, co-immunoprecipitation and proximity ligation experiments demonstrated that VASP directly binds YAP, while DSG2 interacts with the Hippo pathway constituents neurofibromin 2 (NF2, synonym: Merlin) and large tumor suppressor kinase 2 (LATS2). This study shows that altered expression of adherens junction constituents in liver cancer contribute to the aberrant activity of cancer-relevant signaling as illustrated for the Hippo/YAP pathway. Distinct molecular processes control YAP activity after DSG2 and VASP dysregulation with impact of pro-tumorigenic features for HCC cells. My study not only identifies DSG2 and VASP as potential biomarkers for HCC patients that are characterized by YAP activation and poor clinical outcome, but also broadens the view on how cell-cell contact structures transmit information into cell under pathological conditions.