Aminopeptidase N Promotes Hepatocellular Carcinoma Metastasis And Proliferation Via Activating The BCKDK-ERK Axis

Objectives:Hepatocellular carcinoma(HCC)is one of the most prevalent human malignancies worldwide and has high morbidity and mortality.The high proportion of liver cancer in the incidence of malignant tumors in China is due to its strong ability to migrate and invade,and surgical resection and liver transplantation are not the most practical methods for treating the disease.Therefore,finding molecular mechanisms that regulate HCC metastasis is critical to identifying new therapeutic targets.Aminopeptidase N(APN,also known as CD13)is a multifunctional exo-peptidase,and studies have shown that the protein is related to the metastasis of many cancers,but its function in the mechanism of liver cancer metastasis and proliferation is not clear.This study aimed to determine the roles of aminopeptidase N(APN,also known as CD13)in HCC proliferation and metastasis and its underlying mechanisms.Methods:1.Through immunohistochemistry assays of liver cancer tissue microarray,analyze the expression difference of APN in liver cancer tissue and adjacent tissues,and the relationship between clinical indicators and APN expression level.The relationship between APN content and metastatic ability in liver cancer cell lines was detected by techniques such as flow cytometry,enzyme activity detection,quantitative PCR(q PCR),and transwell migration assay.2.Construct a series of hepatocellular carcinoma cell lines that knocked out or overexpressed APN,and compared the effects of APN on the ability of liver cancer cells to metastasize in vitro through transwell migration and invasion assays.An animal model of liver metastasis in NOD/SCID mice was established to analyze the effect of APN on liver cancer metastasis in vivo.The survival curve was determined by a liver orthotopic xenograft model in NOD/SCID mice.3.To determine the colony formation and anti-apoptotic ability of liver cancer cells in vitro after intervention of intracellular APN levels.A subcutaneous xenograft model of NOD/SCID mice was established to analyze the effect of APN level on the proliferation of liver cancer in experimental animals.4.Using RNA sequencing technology to analyze downstream gene transcription levels,pathway activation and changes in biological functions after APN knockout.The results of RNA-seq analysis were verified by in vitro experiments such as immunoblotting,Transwell migration and invasion,and colony formation.5.We use phosphorylation proteomics to screen for proteins that change phosphorylation levels in APN-knocked HCC cells.A series of point mutation and western blot assays were performed to verify the function of Branched-chain alpha-ketoacid dehydrogenase kinase(BCKDK)phosphorylation site.Analysis the interaction between BCKDK and ERK1/2 using co-immunoprecipitation(Co-IP)and proximity ligation assay(PLA,also known as Duolink).Finally,the role of APN-mediated BCKDK phosphorylation in liver cancer proliferation and metastasis was observed through an liver orthotopic xenograft model.Result:We found that APN was frequently upregulated in HCC tumor tissues and high metastatic cell lines.Knockout of APN inhibited HCC cell metastasis and proliferation in vitro and in vivo.Mechanism studies have shown that the loss of APN inhibits the activation of ERK signaling pathway in HCC cells.APN can mediate the phosphorylation of serine at position 31 of BCKDK,promote the interaction between BCKDK and ERK1/2,and increase the level of ERK1/2 phosphorylation,thereby activating ERK signaling pathway and promoting HCC metastasis and proliferation.Conclusions:Our findings indicate that APN mediates the phosphorylation of BCKDK^S31 and activates its downstream phosphor-ERK pathway to promote HCC proliferation and metastasis.Therefore,the APN/BCKDK/ERK axis may serve as a new therapeutic target for HCC,and help to identify new biomarkers in HCC progression.