Reprogramming Of Glucose Metabolism Pathways In Triple-negative Breast Cancer Cells Under Hypoxia

Objective:Physiologically,solid tumor cells are often exposed to hypoxia.The detailed mechanisms of hypoxia remain unclear in the occurrence and development.Many studies have reported that there is a relationship between hypoxia and the glucose metabolism pathway in tumor cells,however,the current reports mainly focus on the aerobic glycolytic pathway mentioned in the Warburg effect or some key enzymes in the glucose metabolism pathway.In this study,two kinds of triple-negative breast cancer（TNBC）cells and two hypoxia cell models were used to construct a cell model of hypoxia.The effects of hypoxia on the expression of all enzymes and their isozymes or subunits,major regulatory enzymes and their isozymes in six glucose metabolism pathways,and the content of products of glucose metabolism pathway in TNBC cells were completely discussed by using RNA-Sequencing（RNA-seq）.We have proven that hypoxia affects metabolism reprogramming in cells.Methods:BT549 and MDA-MB-231 cells were cultured under the conditions of normoxia,2%O₂,and 100μmol/L Co Cl₂.RNA-seq was used for sequencing.This study investigated the RNA-seq data of breast cancer by using bioinformatics analysis.The protein levels of enzyme were detected by western blot.Glucose,lactate,pyruvate dehydrogenase activity,and NADPH assay kit were used to detect glucose intake,lactate production,pyruvate dehydrogenase activity,and NADPH production.Oncomine database was used to analyze the expression of m RNA in the breast cancer tissues.Breast Cancer Gene-Expression Miner v4.8 database was used to analyze the m RNA expression level in TNBC and non-TNBC patients.Results:1.Establishment of physical and chemical hypoxia models in two kinds of TNBC cellsHIF-1αand CA9 expression were increased significantly in physical and chemical hypoxia models of BT549 and MDA-MB-231 cells compared with the normoxia group,indicating that the hypoxia model was successfully constructed.BT549 and MDA-MB-231 cells were sent to BGI for RNA-seq.The company conducted a quality control analysis on 24 samples,and the analysis results were all good.2.Analysis of differentially expressed genes in physical hypoxia models of two kinds of TNBC cellsThere were 249 up-regulated genes and 85 down-regulated genes in BT549cells’physical hypoxia model compared with the normoxia group.There were181 up-regulated genes and 34 down-regulated genes in MDA-MB-231 cells’physical hypoxia model compared with the normoxia group.Venn diagram showed that there were 64 co-expressed genes in the two kinds of cells after the intersection of physical hypoxia model compared with the normoxia group.Most of these co-expressed genes were related to glucose metabolism pathway by GO and KEGG enrichment analysis.3.Analysis of differentially expressed genes in chemical hypoxia models of two kinds of TNBC cellsThere were 650 up-regulated genes and 744 down-regulated genes in the BT549 cells’physical hypoxia model compared with the normoxia group.There were 35 up-regulated genes and 3 down-regulated genes in the MDA-MB-231cells’physical hypoxia model compared with the normoxia group.Venn diagram showed that there were 30 co-expressed genes in the two kinds of TNBC cells after the intersection of the physical hypoxia model compared with the normoxia group.Most of these co-expressed genes also were related to glucose metabolism pathway by GO and KEGG enrichment analysis.4.Analysis of differentially expressed genes in physical and chemical hypoxia models of two kinds of TNBC cellsThe differentially expressed genes of the physical and chemical hypoxia models of two kinds of TNBC cells were intersected.The venn diagram of BT549showed that 103 genes were co-expressed by physical and chemical hypoxia models,while MDA-MB-231 showed only 34 genes.Heatmap showed that most of the co-expressed genes were up-regulated.The enrichment pathways in both TNBC cells were related to glucose metabolism pathway.Protein-protein interaction networks（PPI）analysis of MDA-MB-231 and BT549 cells showed that PDK1,PGK1,CA9,HK2,BNIP3,and PFKFB3 were the most closely connected molecules in MDA-MB-231 cells.In addition to the above molecules,the larger nodes of BT549 cells compared with the other molecules included SLC2A1,PTGS2,etc.5.Hypoxia increased glucose uptake and promoted the glycolytic pathway of two kinds of TNBC cellsIn BT549 and MDA-MB-231 cells,the m RNA expression of glucose transporter 1（GLUT1）was significantly increased in two kinds of hypoxia model groups,and the protein expression was also increased.The expression of all 11enzymes in the glycolysis pathway was increased to varying degrees,and an isozyme or subunit with elevated expression at the m RNA level was selected for each step of the reaction of the glycolytic pathway for validation at the protein expression level.The results were generally consistent with the m RNA expression level.Compared with the normoxia group,the glucose intake and lactate production in physical and chemical hypoxia groups were increased.In the gluconeogenesis pathway,only the expression of phosphoenolpyruvate carboxykinase（PCK）was increased in the physical and chemical hypoxia group of BT549 cells.In MDA-MB-231 cells,only pyruvate carboxylase（PC）in the chemical hypoxia group was increased significantly.6.Hypoxia inhibited the aerobic oxidation pathway of two kinds of TNBC cellsIn the two hypoxia cell models,the m RNA levels of three enzymes constituting pyruvate dehydrogenase complex（PDC）were decreased,and the activity of pyruvate dehydrogenase was also significantly decreased.Pyruvate dehydrogenation kinase 1（PDK1）was highly expressed at the m RNA level in the two hypoxia model groups of two kinds of TNBC cells,and the protein expression was also consistent.The activity of pyruvate dehydrogenase was increased after treatment with PDK inhibitor（DCA）.After hypoxia treatment of MDA-MB-231 and BT549 cells,the m RNA levels of most enzyme genes that catalyzed the tricarboxylic acid cycle were decreased.7.Hypoxia promoted the pentose phosphate pathway of two kinds of TNBC cellsThe m RNA levels of glucose-6-phosphate dehydrogenase（G6PD）were decreased in BT549 and MDA-MB-231 cells with physical hypoxia,while it was increased significantly in BT549 with chemical hypoxia.However,the protein expression of G6PD in two hypoxia models of two kinds of TNBC cells was increased.NADPH production was increased in two hypoxia cell models.8.Hypoxia promoted the glycogen synthesis pathway of two kinds of TNBC cellsThe m RNA levels of phosphoglucomutase 1（PGM1）,glycogen synthase 1（GYS1）,and glycogen branching enzyme 1（GBE1）were increased significantly in the two hypoxia models of two kinds of TNBC cells.The protein expression of GYS1 was increased significantly in the physical hypoxia model of two kinds of TNBC cells,while it was decreased in the chemical hypoxia model.The m RNA levels of glycogen phosphorylase B（PYGB）were increased in the physical and chemical hypoxia group of BT549 cells,while it was decreased significantly in MDA-MB-231 cells treated with physical hypoxia.9.Glycometabolic enzyme genes were significantly expressed in breast cancer tissuesOncomine database analysis showed that GLUT1,ALDOA,GAPDH,PGK1,ENO2,PKM,LDHA,PCK2,PDK1,G6PD,and GYS1 were significantly increased at m RNA levels in invasive breast cancer tissues.Breast Cancer Gene-Expression Miner v4.8 database showed that the m RNA levels of GLUT1,PFK1,GAPDH,PGK1,PKM,LDHA,PDK1,G6PD,GYS1,and PYGB in the TNBC patients were higher than non-TNBC patients.Conclusion:This study comprehensively analyzed the effects of hypoxia on the expression of all enzyme genes and their isozymes or subunits,major regulatory enzymes and their isozymes in six glucose metabolism pathways,and the content of products in glucose metabolism pathway in TNBC cells.The expression of glucose metabolism enzyme genes in tissues was analyzed through online databases.It was concluded that hypoxia blocked the aerobic oxidation pathway mainly by increasing the enzymatic activity of pyruvate dehydrogenase kinase and promoted the glycolytic pathway by increasing glucose uptake and expression of all enzymes in the glycolytic pathway.It also promoted the pentose phosphate pathway and glycogen synthesis pathway.In summary,the hypoxia environment reprogrammed the glucose metabolic pathway in triple-negative breast cancer cells.In a word,we suggested that the effect of physical hypoxia model was better than chemical hypoxia model.