CEMIP,a Novel Adaptor Protein Of OGT,promotes Colorectal Cancer Metastasis Through Glutamine Metabolic Reprogramming Via Reciprocal Regulation Of β-catenin

Background and aim:Tumor metastasis is not only a difficulty in clinical treatment for colon cancer patients,but also one of the main causes of death.Elucidated molecular mechanism of colon cancer metastasis is the premise of development of prevention and treatment strategies,which has important theoretical and clinical significance.In previous studies.Our research group found that CEMIP(cell migration inducing protein)could promote colon cancer metastasis,but the molecular mechanism was unclear.Methods:In this study,transwell experimentswere carried out in sugar reducing medium with deprivation or addition of glutamine to investigate the relationship between migration and invasion of colon cancer cells and glutamine.PCR(polymerase chain reaction)and Western blot were used to verify the effect of cemip on the expression of GLS1(glutaminase 1),SLC1A5(solution carrier family 1 member 5)and SLC38A2(solution carrier family 38 member 2)in colon cancer cells.The protein expressions of cemip,gls1,SLC1A5 and scl38a2 in colon cancer tissues were evaluated by immunohistochemistry.The correlation analysis was used to explore their expression correlation.The relationship between the expression of cemip in colon cancer cells and the consumption rate of glutamine in culture medium was studied by detecting the change of glutamine content in culture medium.To explore the effect of cemip on TCA cycle and ATP production,ATP(adenosine triphosphate),NAD +(nicotinamide adenine dinucleotide),NADH(nicotinamide adenine dinucleotide(NAD)+ hydrogen(H))and α-KG,a key intermediate metbolite of TCA(tricarboxylic acid)cycle.The metastatic ability of human colon cancer cells was investigated by using the liver metastasismodel derived from intrasplenic injectionof cancer cells in nude mice.The molecular mechanism of CEMIP mediated glutamine metabolic reprogramming was explored by glycosylated protein purification,Co IP(Co-immunoprediction),Western blot,cellular immunofluorescence and proximity ligation assay experiments.Results:Our transwell experiments showed that the migration and invasion of colon cancer cells depended on glutamine,and higher concentration of glutamine could promote the migration and invasion of colon cancer cells.Further experiments found that CEMIP mediated migration and invasion of colon cancer cells also depended on the absence of glutamine.CEMIP could regulate the expression of glutaminase 1(GLS1)and glutamine transporters(SLC1A5 and SLC38A2).Positive correlations between the expression of CEMIP and gluatmine metabolic enzymes(GLS1,SLC1A5 and SLC38A2)were verified in human colon cancer tissues.Silence of CEMIP in colon cancer cells could reduce the consumption velocity of glutamine in the culture medium.CEMIP could promote the TCA cycle and production of ATP to support the migration and invasion of colon cancer cells.Animal experiments confirmed that blockage of glutamine metabolism by inhibition of GLS1 could reverse CEMIP mediated colon cancer metastasis.Further experiments found that CEMIP promotedβ-catenin nuclear translocation and reprogrammed glutamine metabolism via regulation of the expression of c-myc,GLS1,SLC1A5 and SLC38A2.Previous studies and our experiments validated that β-catenin was a transcription factor of CEMIP,thus CEMIP and β-catenin could regulate each other to form a positive feedback loop.CEMIP had no abilities to regulate the ubiquitination and degradation ofβ-catenin,but could regulate shuttles of β-catenin between cell membrane and nucleus.CEMIP could promote O-Glc NAc modification ofβ-catenin and lead to depolymerization of cell membrane β-catenin-cadherins complex.Thereafter,freeβ-catenin derived from β-catenin-cadherins complex shuttle into nucleus.Animal experiments confirmed that CEMIP mediated colon cancer metastasis was dependent on β-catenin.CEMIP could affect neither the expression of O-Glc NAc regulatory enzyme(UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase,OGT andprotein O-Glc NAcase,OGA),nor the concentration of UDP-Glc NAc(UDP-N-acetyl-beta-D-glucosamine).CEMIP could interact with OGT and β-catenin directly.CEMIP acted as a scaffold protein to promote OGT mediated O-Glc NAc modification of β-catenin.Animal experiments confirmed that silence of CEMIP combined with glutamine metabolism blockage could significantly reduce the metastasis rate.Conclusions:In conclusion,CEMIP acts as a scaffold protein of OGT and interacts withβ-catenin to promote O-Glc NAc modification of β-catenin in cell membrane.O-Glc NAc modificated β-catenindepolymerizes from cadherins and shuttles into nucleus.Thereafter,accumulating nuclear β-catenin increases the expression of CEMIP and c-myc.Upregulated GLS1,SLC1A5 and SLC38A2 mediated by c-myc promote glutamine metabolic reprogramming and support colon cancer metastasis.Combined inhibition of CEMIP and glutamine metabolism was thought to be a new strategy for the prevention and treatment of colon cancer metastasis.