Metabolomics-based Study Of Metabolic Reprogramming Induced By Oct4 In Lung Adenocarcinoma Cells

Oct4 is the most important transcription factor playing critical roles in maintaining the self-renewal and pluripotence characteristics of pluripotent stem cells,even ables to generate induced pluripotent stem cells(i PSC)from reprogramming differential cell.Normally,Oct4 is exclusively expressed in embryonic stem cells and other stem cells.However,Oct4 is unusually expressed at high levels in the process of tumor development,reprogramming cancer cells into cancer stem cells(CSCs),which plays a momentous role on tumorigenesis,metastasis,drug resistance and disease recurrence.Metabolic reprogramming is the core and driving factor of tumor genesis and development,Oct4 can affect multiple metabolic related genes,which can regulate the key genes of glucose metabolism pathway to change the fate of embryonic stem cells.The current studies primarily focuse on the biological aspects of the metabolic pathway-related genes and proteins,lacking direct evidence at the level of metabolomics.In the previous work,the i PSC technology was used to introduce Oct4 into lung adenocarcinoma cells to obtain the induced lung cancer stem-like cells(i LCSC).The phenotype and surface markers of i LCSC underwent the epithelial-mesenchymal transdifferentiation(EMT),the expression of stemness markers,CD44 and ALDH,were remarkably increased,and the ability of sphere formation as well as tumorigenecity of immunodeficient mice were increased.In this study,based on the above results,our research has developed a ultraperformance liquid chromatography tandem quadrupole time-of-flight mass spectrometry(UPLC-QTOF/MS)method for non-targeted metabolomics analysis and a ultraperformance liquid chromatography tandem triple-quadrupole mass spectrometry(UPLC-QQQ/MS)method for targeted metabolomics analysis of glucose metabolism in order to explore Oct4-induced metabolic reprogramming features.The non-targeted metabolomics analysis showed that ectopic expression of Oct4 induced metabolic changes in lung adenocarcinoma cells,mainly caused the levels of 7 metabolites increased including lactic acid,glutamic acid and argininosuccinate;the level of 4 metabolites decreased including citrulline,phosphocholine and glutathione;mainly affected the urea cycle,arginine acid and proline metabolism,glutathione metabolism,glutamate metabolism and aspartate metabolism.Pathway network analysis showed that these changed metabolic pathway directly or indirectly related with glucose metabolism.The extracellular energy metabolism analysis revealed that Oct4-overexpressed induced glycolysis significantly enhanced,and faintly influenced on mitochondrial respiratory capacity.Stable isotope-resolved metabolomics(SIRM)analysis also indicated that glucose mainly flowed into glycolytic and pentose phosphate pathway,while rarely incorporated into the tricarboxylic acid cycle.In summary,this study developed a comprehensive method combining nontargeted metabolomics analysis with non-labeled and stable isotope-resolved targeted metabolomics analysis to investigate Oct4-overexpressed induced metabolic reprogramming characteristics in lung adenocarcinoma cells.The results suggested Oct4-overexpressed induced the change of metabolism in lung adenocarcinoma cell,and the main flow path of glycolysis and glucose the pentose phosphate pathway.For the first time,the study revealed the metabolic reprogramming caused by Oct4 in metabonomics level,and provide the theoretical basis and evidence for novel and tumor biological effects of stemness factors.