IDH1 Mutation Tumors Compensatively Upregulate IDH2 To Participate In Cell Proliferation In Hypoxia

Gliomas are the most common primary tumors of the central nervous system and classifies into four grades by the World Health Organization(WHO)according to clinical and histopathological criteria.Most gliomas grow invasively,causing high recurrence,disability and mortality rate,thereby seriously threatenig the human health.Over the past few years,although treatments of gliomas have largely improved,the effects are not very satisfactory.In 2008,Parsons et al firstly repored that IDH1 mutation was found in 12% of glioblastoma.Subsequently,more studies found that IDH1/2 mutations as good prognostic markers were prevalent in grade Ⅱ/Ⅲ gliomas and secondary glioblastoma.The following and accumulation studies demonstrated that IDH1/2 mutations play important roles in tumor initiation and development.IDH1/2 are homodimeric NADP+-dependent enzymes which catalyze the reversible oxidative decarboxylation of isocitrate to produce a-KG,NADPH,and CO2.IDH1,which is located in the cytoplasm and peroxisomes,plays a crucial role in facilitating the activity of the numerous nuclear and cytoplasmic dioxygenases that require a-KG as a cosubstrate.Another important function of IDH1 is the generation of NADPH,that provides critical reducing equivalent which is needed for lipid biosynthesis.Besides NADPH is also an essential antioxidant which protects cells from oxidative damage.The mutant IDH1/2 enzymes lose the oxidate activity of catalyzing isocitrate to produce a-KG and instead reduce a-KG to 2-HG,leading to its extreme accumulation.2-HG,as a oncometabolite,plays an crucial role in the tumor initiation and development.So far there are mainly three mechanisms of 2HG,including HIF-1α carcinogenic theory,genome hypermethylation and insulator dysfunction caused by 2-HG,which was published most recently showing that IDH1/2 mutation disrupt chromosomal topology and induce oncogene expression.Lipids,as important components of human tissue,play important roles in supplying energy and maintaining cell structure and function.Under normoxia,glucose generate pyruvate through the glycolysis pathway.Afterwards,the pyruvate enters in mitochondria and converts to AcCoA via pyruvate dehydrogenase complex.AcCoA produced in mitochondria relies on citrate shuttle to transfer into the cytoplama,whereby it serves as a biosynthetic precursor and is converted to fatty acid by fatty acid synthase.However,under hypoxia,the pyruvate dehydrogenase complex is inhibited by HIF-1α,causing impaired generation of the fatty acid precursor AcCoA.In order to support rapid proliferation,the cell have to alter its metabolic routes to accommodate the need for precursor AcCo A for the fatty acid synthesis under hypoxia.Like glucose,glutamine has been recognized as one of the most important nutrients to provide precursors including carbon and nitrogen for biosynthetic reactions in mammalian cells.Glutamine can supply carbon to AcCoA through two major routes.One pathway is described as glutaminolysis where glutamine enters the TAC as α-KG and traverses in the forward direction to maintain oxidative phosphorylation.The other pathway is called reductive carboxylation in which glutamine enters the TAC as α-KG and is converted to isocitrate and citrate by reverse flux through IDH1/2.Thus,under hypoxia,tumor cells rely on IDH1/2 to convert α-KG to isocitrate,producing AcCo A to maintain lipid synthesis and cell survival.IDH1 mutation not only inhibit the oxidative decarboxylation reaction,but also inhibit the reduction of carboxylation reaction.While tumor cells need to synthesize a mass of lipids to support constant proliferation,in this state IDH1 R132 H mutant tumor cells was lipid-synthesis defective under hypoxia,leading to depressed proliferation.Because both IDH1 and IDH2 are involved in cell lipid synthesis under hypoxia,we hypothesize that IDH1 R132 H mutant tumor cells would compensate for impaired lipid sythesis by overexpressing IDH2.Three experimental cells were used in the experiment.Firstly,we successfully cultivated IDH mutant astrocytoma primary cell line through tissue-culture.Second,IDH1 WT and IDH1 R132H-overexpressing glioma cell line U251 was successfully established by virus infection and drug screening.The third group was HCT116 colon cancer cell lines that carry either wild type IDH1(HCT116 IDH1 WT)or only one allele of R132H(HCT116 IDH1 R132H);Firstly,CCK-8 assay was carried out to compare cell prolifereation and fatty acid content in WT and IDH1 R132 H tumor cells under normoxia and hypoxia.We validated that IDH1 R132 H mutant tumor cells are depressed in the proliferation in hypoxia.Secondly,Western blot was performed to determine the expression level of IDH2 in WT and IDH1 R132 H tumor cells under normoxia and hypoxia.The results showed that IDH2 was upregulated in IDH1 R132 H mutant tumor cells under hypoxia;Finally,we knocked down IDH2 in IDH1 R132 H mutant tumor cells and examined the resultant effect on cell prolifereain.We demonstrated that IDH2 silence can significantly inhibit proliferation of IDH1 R132 H mutant cells under hypoxia.Our study first proposed that IDH1 R132 H mutant cells upregulate IDH2 to compensate cell proliferation under hypoxia.This study suggested that IDH2 could serve as a potential anti-tumor target for IDH1 R132 H harboring tumors,which may provide a new clue for the treatment.