FBXW10 Promotes ANXA2 Polyubiquitination To Drive The Malignant Progression In Male Hepatocellular Carcinoma

Background and aimHepatocellular carcinoma(HCC)is a highly aggressive primary malignant tumor and is the third leading cause of cancer death worldwide,with the sixth highest prevalence rate.Hepatocellular carcinoma(HCC),intrahepatic cholangiocarcinoma,and mixed hepatocellular carcinoma are the three main types of liver cancer,of which HCC accounts for 75-85% of all liver cancer.It was investigated that among non-reproductive malignancies,there was a significant gender bias in incidence and mortality rates between male and female patients.In HCC,the incidence and mortality rates were approximately 3 times higher in male patients than in female patients.However,the mechanism about why men are more likely to develop liver cancer than women is still unclear.Early studies assumed that factors associated with poor lifestyle habits in men were the main reason for the high incidence of liver cancer in men.However,recent omics studies have identified that elevated male-specific genes or proteins may be the main factors promoting the development of HCC,but the detailed molecular mechanisms have not been identified clearly yet.Ubiquitinated modifications are a common type of post translational modifications(PTMs)that play an important role in protein degradation,activation and stabilization.The Ubiquitin-Proteasome System(UPS)performs its biological functions through a series of enzymatic reactions,including the initiation of ubiquitin activation by the ubiquitin-activating enzyme E1 and the transfer of ubiquitin to the ubiquitin-binding enzyme E2,which eventually binds to the ubiquitin-protein E3 ligase which can catalyze ubiquitination modifications of specific substrates.FBXW10 belongs to the FBXW subgroup(rich in WD repeat domains),a substrate recognition protein subunit F-box protein in the E3 ubiquitin ligase complex,is involved in substrate recognition and protein ubiquitination degradation or activation.Our recent study discovered that E3 ubiquitin ligase FBXW10 was aberrantly highly expressed in HCC and negatively correlated with prognosis in male patients,independent of female patients.However,the molecular mechanism about how FBXW10 promotes malignant progression in male HCC is unclear.Methods1.We used proteomics and ubiquitination omics to analyze ubiquitinated differential proteins in FBXW10-Tg(+)male and female mouse HCC tissues,combined with immunoprecipitation(Co-IP)experiments to clarify potential proteins that bind to FBXW10 interactions,and screened ubiquitinated upregulated interacting proteins ANXA2(Lys148),FASN and RPL13 A by Venn diagram crossover.The clinical prognosis and CCK-8 assays were used to clarify whether ANXA2 is a key molecule in FBXW10-mediated hepatocarcinogenesis in male.2.In vivo ubiquitination assay was used to clarify the FBXW10-mediated ANXA2 ubiquitination target sites and the type of poly-lysine modification chain.Combined with immunofluorescence,Transwell and CCK-8 assays to clarify the role of ANXA2 and its target sites on FBXW10-mediated proliferation and migration of HCC cells.3.HCC tissues of male FBXW10-Tg(+)mice and wild-type mice were sent for phosphoromic analysis to figure out the phosphorylation sites required for ANXA2 ubiquitination modification.Immunofluorescence,Transwell and CCK-8 assays were used to verify whether ANXA2 phosphorylation sites were associated with functional activity.4.By reviewing the previous reports and GSEA enrichment analysis,we clarified the signaling pathways significantly associated with ANXA2,and combined with Co-IP and Western blot experiments to clarify whether FBXW10 upregulates downstream signaling pathways by activating ANXA2.5.To determine whether ANXA2 inhibition can reverse FBXW10-driven male HCC development and lung metastasis using orthotopic liver transplantation tumor model in nude mice.6.Immunohistochemical staining was used to analyze the expression of FBXW10 and ANXA2 proteins in male and female patients,and statistical analysis was applied to clarify the effect of FBXW10 and ANXA2 on the prognosis of male and female liver cancer patients.Results1.ANXA2 specifically binds to FBXW10 and undergoes K63 type ubiquitination modification at Lys148,thereby improving the membrane localization level of ANXA2.2.Down-regulation of ANXA2 inhibits FBXW10-mediated proliferation and migration of HCC cells(P<0.05).ANXA2 Lys148 can promote the proliferation and migration of HCC cells(P<0.05).FBXW10 cannot activate ANXA2 to promote the proliferation and migration of HCC cells after K148 mutation(P<0.05).3.S6K1 promotes the phosphorylation of ANXA2 at Ser12 and Ser26,which promotes FBXW10 binding,ubiquitination and activation of ANXA2.Activated ANXA2 can bind KRAS to up-regulate p-ERK,and facilitate MEK/ERK and EMT pathways.4.In vivo experiments confirmed that knockdown of ANXA2 significantly inhibited FBXW10-driven HCC development and lung metastasis in male mice(P<0.05).5.Immunohistochemical staining analysis showed that the expression of FBXW10 and ANXA2 was significantly correlated in male HCC(P<0.05),and the co-high expression of FBXW10 and ANXA2 indicated poor prognosis of male HCC patients(P<0.05).ConclusionsFBXW10 and ANXA2 are highly expressed in male HCC tissues,which are positively correlated with poor prognosis.S6K1 promotes the malignant progression of male HCC by promoting FBXW10 to recognize and ubiquitinate ANXA2 to activate KRAS and accelerate MEK/ERK signaling pathways.