UBE2O-mediated Ubiquitination And Degradation Of UPF1 Promotes Hepatocellular Carcinoma Proliferation By Enhancing Cholesterol Biosynthesis

Objective:Hepatocellular carcinoma（HCC）is one of the most common malignancies in the world and the third leading cause of cancer-related death worldwide.Currently,due to the lack of effective treatment strategies,the 5-year survival rate of HCC patients is very low.Metabolic reprogramming is recognized as a key event during tumor initiation and progression,and the change of cholesterol biosynthesis is an important hallmark of tumor metabolic reprogramming.Cholesterol biosynthesis plays a key role in the rapid proliferation of tumor cells.The liver is an important organ for cholesterol biosynthesis,so the role of cholesterol metabolism in liver cancer deserves more attention.More and more studies have revealed that ubiquitination is involved in the occurrence and development of tumors by controlling the degradation of oncoproteins or tumor suppressor proteins.Ubiquitin-conjugating enzyme E2O（UBE2O）is related to the occurrence and development of multiple tumors such as head and neck squamous cell carcinoma,lung cancer,breast cancer and colon cancer.It promotes the proliferation of colon cancer cells by enhancing glycolysis.However,the role of UBE2O in HCC is not completely understood.The present study aimed to examine the role of UBE2O in the regulation of HCC and cholesterol biosynthesis and provide new targets and treatment strategies for HCC patients.Methods:TCGA and GEO databases,RT-q PCR,Western blot and tissue microarray immunohistochemical staining were used to analyze the m RNA and protein expression levels of UBE2O in HCC.Clone formation assays,Cell Count Kit-8（CCK-8）assays,5-Ethynyl-2’-deoxyuridine（Ed U）experiments and mice xenograft experiments were used to observe the effect of UBE2O on HCC proliferation.Total cholesterol content assay kit and Filipin staining were used to test the change of cholesterol level in HCC cells.Immunoprecipitation-Mass Spectrometry（IP-MS）,co-immunoprecipitation,RNA Immunoprecipitation,immunofluorescence,luciferase reporter assays and polyubiquitination experiments were used to explore the molecular mechanisms.Results:UBE2O was upregulated in HCC tissues and HCC cells.The high expression of UBE2O was correlated with poor prognosis of HCC patients.UBE2O knockdown led to the decrease in proliferation,cholesterol biosynthesis of HCC cells and tumor growth,while its overexpression showed opposite results.Further exploration found that UBE2O promoted the proliferation of HCC through the SREBP2-dependent cholesterol biosynthesis pathway.Mechanistically,UBE2O catalyzed K48-linked polyubiquitination modification and degradation of up-frameshift protein 1（UPF1）.UPF1 induced SREBP2 m RNA degradation through binding to the 3’UTR of SREBP.UPF1 abolished UBE2O-mediated cholesterol biosynthesis and proliferation of HCC cells.In addition,cell and animal experiments showed that UBE2O inhibitor arsenic trioxide（ATO;As₂O₃）in combination with cholesterol biosynthesis inhibitor simvastatin could markedly inhibit UBE2O-mediated HCC proliferation compared with ATO and simvastatin alone.Conclusions:Our study revealed that UBE2O promotes the proliferation of HCC cells through enhancing cholesterol biosynthesis.We subsequently show that the UBE2O/UPF1/SREBP2 axis is crucial for enhancing cholesterol biosynthesis and tumorigenesis in HCC cells.Hence,UBE2O might be a promising therapeutic for anti-tumor therapeutic strategies that focus on cancer metabolism in HCC.