C1orf61-induced Hepatocyte Metabolic Reprogramming Is Through The Activation Of C-Myc And AKT

Tumor metabolic disorders have become one of the top ten characteristics of tumor development.Many tumor cells prefer the aerobic glycolysis pathway compared to the oxidative phosphorylation of glucose metabolism in normal cells.On the one hand,the energy generated by aerobic glycolysis can meet the needs of tumor cells themselves,on the other hand aerobic glycolysis pathway can generate a large number of nucleic acids,amino acids,lipids and other biological macromolecular components to meet the demand of rapid proliferation.Along with the changes of cell microenvironment and the development of tumor,oxidative phosphorylation forms a competitive and complementary effect with aerobic glycolysis pathway.In the generation of ATP,it provides a balance between the reduction equivalent and biological macromolecule synthesis.The metabolic pathway transformation is called metabolic reprogramming.C1orf61(chromosome 1 opening reading frame 61)also known as CROC-4 which located in chromosome 1 long arm 22 area,is a frequent site of gene mutation.Peter L.Jeffrey et al first reported and analyzed the C1orf61 mRNA sequence,protein sequence and tissue expression profile and preliminarily studied the role of Clorf61 in brain development.Our previous results show that C1orf61 plays an important role in the development of the liver.During the genesis of hepatocellular carcinoma,C1orf61 promotes the proliferation,invasion and migration of hepatocytes and induces EMT.Therefore Clorf61 is closely related to the malignant transformation of hepatocytes.In order to explore whether C1orf61 can regulate cell metabolism,we constructed a Clorf61 overexpressing cell line(L02-Clorf61-puro,L02-Clorf61-GFP,L02-C lorf61-FLAG)and a control cell line(L02-puro?L02-GFP?L02-FLAG)in normal hepatocytes L02.We also constructed Clorf61 knockout cell line HCCLM9-Clorf61-/-(LM9-KO)and the control cell line HCCLM9-WT(LM9-WT)in the highly metastatic hepatocellular carcinoma cell HCCLM9.We found that Clorf61 significantly promoted the uptake of glucose by L02 cells,and HCCLM9-Clorf61-/-cells was significantly decreased.Detection of mitochondrial membrane potential and cell ATP concentration found that C1orf61 decreased cell mitochondrial membrane potential and ATP concentration,but ROS levels did not change.Detection of lactic acid concentration in the supernatant of cell culture revealed that Clorf61 promoted the production and extracellular transportion of lactic acid,which resulted the decrease of cell microenvironment pH.These results indicate that Clorf61 induces the glucose to be metabolized by glycolysis rather than oxidative phosphorylation.Thus,Clorf61 facilitates cell metabolism reprogramming.The molecular mechanism of metabolic reprogramming can be classified into three categories:the activation of proto-oncogene and inhibition of tumor suppressor genes such as p53 and c-Myc,the expression changes in metabolic enzymes such as PKM2 and LDHA,continuous activation of signal transduction pathways such as PI3K/Akt.In combination of microarray data and GO analysis,we found that Clorf61 regulated differentially expressed genes were associated with glucose metabolism.DAVID pathway analysis also showed that C1orf61 regulates metabolic pathways.Further studies have shown that Clorf61 can significantly promote the transcription of c-Myc,Akt and PKM2.Western blot results show that the protein level is also significantly up-regulated.When knockdown of PKM2 by siRNA,cell glucose uptake and glycolysis process was inhibited.The expression of PKM2 and the uptake of glucose were decreased by Akt inhibitor wortmannin.In addition,Clorf61 could upregulate the expression of NRP2 and interact directly with NRP2.Therefore,we conclude that Clorf61 activates NRP2 and induces cell metabolic reprogramming through Akt,c-Myc and PKM2.In conclusion,our results suggest that Clorf61 can promote the reprogramming of glucose metabolism in liver cells,and its role is accomplished through Akt,c-Myc,PKM2 pathway.Clorf61 activates the transcription and translation of Akt and c-Myc,which then promotes PKM2 expression,inhibits cell oxidative phosphorylation and promotes aerobic glycolysis,and ultimately leads to cell metabolic reprogramming.These results provide a new theoretical basis for tumor metabolic research and tumor therapy.