Glypican-3 Interacts With Atypical Cadherin FAT1 And Promotes The Migration Of Hepatocellular Carcinoma Cells

Primary liver cancer is the fourth most prevalent malignancy and the second most common cause of cancer related deaths in China.It remains one of the major threats to human life and health.Primary liver cancer can be classified to hepatocellular carcinoma(HCC),cholangiocarcinoma(CCA),and mixed liver cancer.Among them,HCC is the most common form,accounting for 90% of the incidence.Glypican-3(GPC3)is related to the occurrence and development of liver cancer.In recent years,GPC3 is emerging as a tumor marker of HCC and being used as a target for HCC diagnosis and immunotherapy.GPC3 is a member of the heparan sulfate proteoglycans(HSPGs)family,and attached to the cell surface by glycosylphosphatidylinositol(GPI)anchor.GPC3 is able to bind a varity of extracellular proteins(such as Wnt,FGF,IGF-2)and cell surface receptors(such as Frizzled,IGFR)to regulate different signaling pathway(such as Wnt,Hippo,Hedgehog,ERK),and finally influence cell proliferation and migration.Atypical cadherin FAT1 encodes a 506 KD peptide,containing 34 cadherin repeats,a Laminin G(LG)-like domain,5 EGF-like domains,a hydrophobic transmembrane(TM)region,and an intracellular domain(ICD).FAT1 is predominantly expressed in the embryonic stage and is crucial for embryonic development and organ formation.Currently,it has been found that the abnormal expression of FAT1 is related to multiple cancers.Moreover,FAT1 has divergent roles in different malignancies,functioning either as tumor suppressor or oncogenic gene.In HCC,FAT1 expression is upregulated and associated with advanced tumor staging.In the current study,we identified that atypical cadherin FAT1 is a new GPC3 interacting protein in HCC cells by using Co-IP/MS method.We further carried out a series of studies on the function of GPC3 and FAT1 in the growth and metastasis of HCC cells.The main results are as follows:1.The interacting proteins of GPC3 were screened by immunocoprecipitation and mass spectrometry,and one of the candidates,FAT1,was confirmed by CO-IP,ELISA and flow cytometry.The binding region of GPC3 on FAT1 was mapped;2.By using Co-IP,the GPC3 binding region on FAT1 was partially mapped to the C-terminus of the extracellular domain(Q14517,a.a.3662-4181),which covered a putative receptor tyrosine phosphatase(RTP)-like domain that has not been previously determined,a Laminin G(LG)-like domain,and five EGF-like domains;3.To precisely map the GPC3 binding region,we purified 4 progressively truncated fragments(FAT1A,B,C,D)of FAT1 extracellular C-terminal region(Q14517,a.a.3662-4181)in HEK293 F expression system.The protein binding ELISA results showed that FAT1B(LG-EGFL2 domain,a.a.3830-4050)and FAT1D(RTP-Like domain,a.a.3662-3788)had strong GPC3 binding,which were 10.1 fold and 8.58 fold normalized with the negative control(h Ig G);FAT1A(EGFL2-5,a.a.4013-4181)had weaker GPC3 binding which was 4.19 fold;while FAT1C(EGFL1,a.a.3789-3829)had no GPC3 binding.4.The flow cytometry was used to determine whether FAT1 able to bind cell surface GPC3.The results showed that FAT1 A had strong specific GPC3 binding,which were 4.5 fold normalized with GPC3 negative control(A431);FAT1B,FAT1 C and FAT1 D had non-specific GPC3 binding.In gernal,the ELISA and flow cytometry results suggested that the last four EGF-like domains(residues 4013-4181)were the major GPC3 binding regions.5.We knocked down GPC3 and FAT1 by small hairpin RNA(sh RNA)in Hep3B-Luciferase and Hep G2-Luciferase cell lines that are overexpressing Luciferase reporter gene.The impact of GPC3 and FAT1 on cell proliferation was determined by Luciferase reporter assay.Compared with control sh RNA,the cell proliferation of Hep3B-Luciferase and Hep G2-Luciferase were decreased by 36% and 20% after GPC3 knockdown,and 52% and 31% after FAT1 knockdown.These results indicate that GPC3 and FAT1 promote HCC cell proliferation.6.We knocked down GPC3 and FAT1 by sh RNA in Hep3 B,and determined cell migration by Transwell assay.Compared with control sh RNA,GPC3 knockdown decreased cell migration by 60%,and FAT1 knockdown decreased cell migration by 57%.More importantly,double knockdown of GPC3 and FAT1 decreased cell migration by 84%,suggesting that GPC3 and FAT1 had an additive effect in promote cell migration.7.Under the 2,2-dipyridyl(DP)simulated hypoxia conditions,GPC3,FAT1,and HIF1? had up-regulated expression in a time-dependent manner.In terms of m RNA and protein level,GPC3 was up-regulated by a maximum of 2.74 and 1.97-fold,FAT1 by a maximum of 1.98 and 2.27-fold,and HIF1? by a maximum of 1.97 and 3.06-fold,respectively.8.We further elucidated the relationship among GPC3,FAT1,and HIF1? under DP simulated hypoxia condition.The results showed that HIF1? plays a regulatory role on GPC3 expression,but not FAT1.9.We detected the expression of tumor metastasis related genes(SNAI1,VIM,CDH1)in DP treated Hep G2 cells.The expression of SNAI1 and VIM was up-regulated and CDH1 was down-regulated over time.Knockdown of GPC3 and FAT1 separately or simultaneously abolished the DP-treatment induced up-regulation of SNAI1 and VIM and down-regulation of CDH1.10.We immunized BALB/C mice with FAT1 A protein and obtained murine serum contain polyclonal anti-targeting FAT1.Taken together,our results identified a new GPC3 interacting protein FAT1.The GPC3 binding domain on FAT1 was mapped.The biological significance of the GPC3-FAT1 interaction was explored in terms of cancer proliferation and metastsis.The results of the current study expanded the current knowledge on GPC3 function and the mechanisms of tumor metastasis mediated by GPC3.