Glypican 3 (GPC3)‐CD81 axis regulates spleen tyrosine kinase(Syk)‐Ezrin mediated Hippo pathway via cross talking with HGF/c‐Met axis in hepatocytes and hepatocellular carcinoma (HCC)
Authors: Xue Yuhua , Bowen William , Koral Kelly , Orr Anne , Mars Wendy , Michalopoulos George 
Published: November 2023

The experiments that integrate different pathways in normal and abnormal circumstances will help us to develop better targeted therapies because of multiple pathways involve in the solid tumor carcinogenesis. GPC3 is over‐expressed in HCC. Previous studies have shown that GPC3 is associated with inhibition of hepatocyte growth and GPC3 transgenic mice with targeted expression in hepatocytes have decreased liver regeneration. We have also shown that GPC3 binds to the membrane tetraspanin CD81, which is one of the portals of entry of hepatitis C virus (HCV). Studies elsewhere have shown that activation of CD81 is associated with phosphorylation of Ezrin. The latter has been shown to regulate the Hippo pathway by modulation activity of tumor suppressor gene NF2 (Merlin), responsible for regulating nuclear levels of the protein known as Yes‐associated protein (Yap). It also has been shown that HGF/Met axis which promotes growth in HCC can phosphorylate Ezrin as well by unknown mechanisms. Expression of Yap in the nucleus enhances hepatocyte growth. Mice over‐expressing GPC3 have decreased levels of nuclear Yap. Since we have demonstrated that GPC3 binds to CD81, the purpose of this study is to explore the role of GPC3 in regulation of Yap via CD81 and Hippo pathway and to investigate the possible mechanisms. CD81 expression could be detected in membrane and organelle fraction in human hepatoma cell lines HepG2, Huh7 and Hep3B, but immunoprecipitation experiment suggests that CD81 from latter two human hepatoma cell lines has lost association with GPC3. Human hepatoma cell line had dramatically up regulated Ezrin expression and Yap activity. We found that treatment of primary rat hepatocytes with CD81 agonist antibody or HGF led to higher levels of phosphorylated Ezrin and lower Hippo activity in a Syk dependent manner. While mice over‐expressing GPC3 decreased p‐Ezrin but increased Hippo activity. Transgenic Met knock out mice showed less Yap expression at baseline level, and abrogated up regulated Yap expression which was observed in wild type mice after 2d PHx and increased subsequent apoptosis. Rat hepatoma cell lines JM1 and JM2 had dramatically down regulated CD81 expression and Hippo activity, and up regulated Ezrin activity in vitro. Force expression of Syk expression in JM2 rat hepatoma cells downregulated Hippo activity while upregulated Ezrin activity in vitro.

Conclusions

Both Met‐phosphoEzrin and CD81‐phosphoEzrin signaling enhances nuclear Yap levels. These pathways in normal hepatocytes are inhibited by GPC3 and low HGF/Met activity. In human HCC, loss of CD81 expression and highly activity of HGF/Met promote the CD81‐phosphoEzrin effect on Hippo pathway and cause a increase in nuclear Yap. The different distribution pattern of HGF/Met and GPC3/CD81 between normal hepatocytes and HCC cells provides the chance to develop precise anti‐HCC therapies.