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Abstract

Phosphoinositide dynamics play a crucial role in a diverse range of cellular functions and in the development of diseases. Among them, accumulating evidence suggests significant contributions of phosphatidylinositol (PI) 4-phosphate (PI4P) in numerous important signaling pathways. The conversion to PI4P from PI requires enzymes of the phosphatidylinositol 4-kinase (PI4K) family in which phosphatidylinositol 4-kinase III alpha (PI4KA) is an important member. A recent study has suggested a connection between high expression of PI4KA and undifferentiated status and poor prognosis of hepatocellular carcinoma (HCC), although the mechanism has yet to be discovered. Hepatitis C virus (HCV) is one of the main etiological factors associated with HCC. In addition, HCV activates PI4KA via interactions with nonstructural (NS) proteins NS3-NS5B to provide sufficient PI4P for efficient genome replication. The aim of this work was therefore to mechanistically understand the impact of PI4KA signaling pathways on liver pathogenesis and the potential contributions of HCV in this process. In this study, by using shRNA mediated gene silencing and a specific inhibitor, I found that PI4KA via its product PI4P positively regulated levels of phosphorylation of two important cytoskeletal proteins regulating actin dynamics, paxillin (PXN) and cofilin (CFL1), in HCC derived cell lines with high expression of the phospholipid kinase. Increased PXN and CFL1 phosphorylation was associated with morphological differences including cell spreading, increased numbers of focal adhesions (FAs) and higher cell motility as compared to cells with lower expression and/or activity of PI4KA. In agreement with these results, the activation of PI4KA upon HCV infection or expression of NS3-5B further increased p-PXN and p-CFL1, resulted in elevated numbers of FAs and enhanced cell invasiveness, suggesting PI4P concentration as the driving force. These phenotypes are hallmarks of epithelial–mesenchymal transition, implying important roles of PI4KA in cancer progression. To understand the mechanism how PI4KA regulated cytoskeletal dynamics, I evaluated PIP synthesis pathways by targeting different phospholipid kinases which are connected to PI4P. This approach revealed that silencing of Phosphatidylinositol 3-kinase C2 domain-containing subunit gamma (PIK3C2G), a lipid kinase responsible for producing phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2] from PI4P, led to similar phenotypes observed in PI4KA-knockdown cells on cytoskeleton-related cellular pathways, leading to less spreading morphology, downregulation of p-PXN/p-CFL1 and dampening of cell invasiveness. Furthermore, knockdown of PI4KA or PIK3C2G reduced the abundance of PI(3,4)P2 specialized-enriched structures at the cell plasma membrane. These PI(3,4)P2 containing structures had been linked to invadopodia which are utilized by cancer cells for their invasiveness, suggesting a strong connection of the two lipid kinases with cell invasion. To indentify the protein kinase connecting increased PI(3,4)P2 levels with cytoskeletal changes, I applied a human kinase array in combination with western blot analysis. To this end, p-AKT2 levels were found to be correlated with PI4KA and PIK3C2G abundances. Furthermore, AKT2 was enriched at the invadopodia-like structures. AKT2 activity was correlated with the phosphorylation of PXN and CFL1 and changes in cell morphology. HCV infection or expression in contrast stimulated AKT2 phosphorylation and enhanced PI(3,4)P2 at the cell plasma membrane. These data suggested AKT2 as the predominant protein kinase linking PI4KA/PIK3C2G with the downstream targets PXN and CFL1. To verify the key phenotypes observed in HCC-derived cell lines, I applied more physiological relevant models, including expression of HCV proteins in primary human hepatocytes and several mouse models. These approaches supported the concept of PI4KA abundance and activity as key regulators of cell morphology and motility via stimulating phosphorylation of the cytoskeletal proteins PXN and CFL1. In conclusion, my data demonstrate that PI4KA functions on cellular pathways governing cell morphology and actin cytoskeleton dynamics favorable for cancer progression via the signaling molecule PI(3,4)P2 and its downstream mediator AKT2. HCV, therefore, contributes to liver pathogenesis via activating PI4KA, mediating similar phenotypes as increased PI4KA expression observed in HCC derived cells.