The Role Of Ankyrin-repeat-containing,SH3-domain-containing,and Proline-rich-region-containing Protein 2 (ASPP2) In Lipid Metabolism In Hepatocellular Carcinoma

Cancer is fundamentally a disorder of cell growth and proliferation,which requires adequate energy and material supply for its own needs.Substance and energy metabolism of cancer cells,such as glucose,lipid and protein metabolism has a degree of abnormal change.The most understood metabolic perturbation in cancer cells is the Warburg effect,an energetically wasteful alteration to glucose metabolism in which cancer cells use carbon from glucose to build other molecules instead of completely oxidizing them to carbon dioxide.However,other metabolic processes,including protein,nucleic acid and lipid biosynthesis,are also enhanced as part of cancer-associated metabolic reprogramming.The majority of adult mammalian tissues satisfy their lipid requirements through the uptake of free fatty acids(FFAs)and lipoproteins,such as low-density lipoprotein(LDL),from the bloodstream.Fatty acid(FA)and cholesterol biosynthesis are restricted to a subset of tissues,including liver,adipose and lactating breast tissues.However,reactivation of lipid biosynthesis is frequently observed in cancer tissue.Over the past few years,increasing attention has been given to the study of the metabolic processes involved in lipid biosynthesis and their regulation in cancer.ASPP(Apoptosis Stimulating Proteins of P53)protein family is a class of proteins that regulate tumor cell apoptosis,including ASPP1,ASPP2 and iASPP.Among them,ASPP2 is an important member of ASPP protein family members.The C-terminus of three ASPP family proteins contains Ankyrin repeats,SH3 domain and Proline-rich region.ASPP2 is a tumor suppressor gene,which can bind to p53 and specifically enhance the binding of p53 to pro-apoptotic gene promoter to promote apoptosis.In the previous studies,our group found that ASPP1 and ASPP2 genes are frequently down-regulated by DNA methylation in HBV-positive HCC,which may play important roles in the development of HCC.With the further study,we found that ASPP2 and liver cancer lipid metabolism is closely related,but the specific mechanism is unclear.Here we will investigate how ASPP2 regulates lipid metabolism in HCC.The study aims to investigate the specific roles and mechanisms of ASPP2 in the lipid droplets and cholesterol metabolic pathways.First,we used lentivirus to down-regulate the expression of ASPP2,then we found that ASPP2 could affect the multiple signaling pathways of HCC-LM3 tumor cells through gene chip analysis.These signaling pathways contain almost all of the pathways of tumor growth,where lipid metabolism changed largely.In these lipid metabolic pathways,we focused on the role of ASPP2 in lipid droplets and cholesterol metabolism pathways.First,we found HCC cells gained lower lipid droplet levels by lentivirus to down-regulate the expression of ASPP2.In order to further explore the specific mechanism of ASPP2 on the effect of lipid droplets,we queried the database and predicting protein binding websites.By intracellular immunofluorescence localization and protein immunoprecipitation,we confirmed that in the nucleus ASPP2 binds to Sirt1 and they have synergistic effects on lipid droplets.Second,we found HCC cells gained higher cholesterol levels and increased cancer stemness by lentivirus to down-regulate the expression of ASPP2.Then,we used simvastatin,which is a cholesterol metabolism pathway inhibitor,to inhibit HCC stemness enhancement.In vivo nude mice tumorigenesis experiments,we used cholesterol metabolism pathway inhibitor simvastatin to effectively inhibit HCC growth caused by reduced ASPP2.We found that the expression of a series of key enzymes in the cholesterol synthase pathway was increased by gene chip analysis.By intracellular immunofluorescence localization and protein immunoprecipitation,we further confirmed that in the nucleus ASPP2 binds to SREBP2,which inhibits the combination of transcription factor SREBP2 and the key enzyme promoter SRE region in the synthesis of cholesterol,thus affecting stem-like properties of hepatocarcinoma cells.This study clarifies the role and the mechanisms of ASPP2 in lipid droplet and cholesterol metabolism in hepatocarcinoma cells.1,ASPP2 influences lipid droplets by combining with Sirt1.2,ASPP2 binds to SREBP2,which inhibits the combination of transcription factor SREBP2 and the key enzyme promoter SRE region in the synthesis of cholesterol,thus affecting stem-like properties of hepatocarcinoma cells.These findings provide new understanding of HCC lipid metabolism,other directions for HCC diagnosis,new insight into study of anti-tumor drugs and potent therapeutic applications.