The Investigation Of The Interacted Mechanism Of HCC Specific Molecule GPC3 And Wnt3a

Liver cancer is the sixth most common and the second most lethal malignancy worldwide.Currently,there is no effective treatment for liver cancer.Due to the unique detoxification feature of liver,small molecule drugs like chemo-drugs usually have poor therapeutic effects.Therefore,developing protein drugs such as antibodies and immune cell therapy will be meaningful for liver cancer therapy.More than 90% of patients with hepatocellular carcinoma have pathological activation of the Wnt signaling pathway.Various Wnt co-receptors play essential roles for the activation of Wnt signaling.Therfore,targeting tumor-specific Wnt co-receptor would be an alternative strategy to blolck Wnt signaling.Glypican-3 is a cell surface molecule specifically expressed in hepatocellular carcinoma(HCC).GPC3 functions as a co-receptor of Wnt to regulate HCC growth.GPC3 recruits and enriches Wnt on cell membrane and subsequently promotes the proliferation of HCC.Targeting GPC3 can block the activation of Wnt signaling and inhibit the tumor proliferation.However,the precise interaction between GPC3 and Wnt and the molecular mechanism that GPC3 regulates Wnt signaling are not clear yet,which restricts the development of effective therapeutic strategies by targeting GPC3.The structures of different glypicans are highly conserved,which makes it possible to obtain a reliable GPC3 structure model based on the known GPC1 structure.Moreover,the structure of Wnt/FZD-CRD complex are also resolved.These details would provide us useful information to understand the the molecular mechanism of the interaction between GPC3 and Wnt.Our previous work found a FZD-like cysteine-rich-domain(CRD)in the structural model of GPC3,which might directly mediate Wnt binding.To verify this hypothesis,we identified the Wnt binding site on GPC3 and evaluated its regulatory effect on Wnt activation in vitro and in vivo.The main findings obtained are as follows:1.GPC3 could interact with Wnt through a conserced domain and promotes the activation of Wnt signaling.2.We established structural models of h GPC3 and h Wnt3 a,respectively.By homology analysis,We found a FZD-like cysteine-rich domain(CRD)at the N-terminus of GPC3,which might mediating Wnt binding.3.Two potential Wnt binding sites(site1 and site2)on GPC3-CRD were predicted by protein hydrophobicity analysis.By mutagenesis assay and functional evaluation,we found that site2 is critical for Wnt binding as well as Wnt signal activation.4.The GPC3 knockout Hep3 B cell line was constructed using CRISPR-Cas9 and the GPC3 rescuing cell lines were constructed using lentiviral transfection.Using these HCC cell lines,we found that GPC3 site2 mutation can inhibit Wnt signaling activation in HCC cells,delay tumor proliferation and prolong survival in mice.5.Furthermore,we found that targeting Wnt binding regions of GPC3 with GPC3 antibodie HN3 could blocked Wnt signaling.In conclusion,we identified a Wnt binding site of GPC3 by structural analysis and functional evaluation,which mediates Wnt activation in vitro and regulates HCC tumor growth in mice.Additionaly,blocking the Wnt binding regions of GPC3 could effectively inhibit Wnt signaling.These findings reveal a new mechanism of GPC3 as a Wnt coreceptor to regulate HCC proliferation,help us to further understand the biological functions of GPC3 as well as the mechanisms of liver cancer tumorigenety,and provide a meaningful rational for developing effective strategy for liver cancer therapy.