Knockout Of PKM2 Mediates The Effect Of ACADVL Upregulation On Proliferation And Metastasis Of Triple-negative Breast Cancer Cells

Background:Breast cancer(breast cancer)is the most common malignancy in women.Breast cancer is divided into four molecular subtypes,of which triple negative breast cancer(TNBC)accounts for about 15% and is characterized by high metastasis and poor prognosis.Compared to normal tissue cells,cancer cells exhibit a high rate of inefficient energy production-aerobic glycolysis,i.e.Warburg effect.Pyruvate kinase type M2(PKM2)is the third step of glycolysis and the key protein of the Warburg effect,which is highly expressed in a variety of tumor tissues.Numerous studies have shown that tumor cells have a strong energy compensatory capacity among glycans,lipids and amino acids,but the exact mechanism remains to be elucidated.Our preliminary study found that PKM2 knockdown only partially inhibited tumor cell proliferation and migration,tumor cells were partially induced to undergo apoptosis and autophagy,and tumor cells did not undergo severe energy deficiency.So,is this related to the energy compensation of cells? What is its specific molecular mechanism?Objective:1.To investigate the effect of knockout of PKM2 in MDA-MB-231 of triple negative breast cancer cells on cellular substance metabolism after blocking the Warburg effect.2.To investigate the possible molecular mechanisms underlying the alteration of cellular substance metabolism after knockout of PKM2 in MDA-MB-231 of triple-negative breast cancer cells to block the Warburg effect.Methods:1.Based on the UALCAN/TCGA database,we analyzed the correlation between PKM2 and breast carcinogenesis,the transcript levels of PKM2 in various tumor tissues and survival prognosis analysis,and the expression levels of PKM2 in various breast cancer subtypes and various stages of breast cancer development.2.The PKM2 knockout triple-negative breast cancer cell line MDA-MB-231 was constructed based on CRISPR/Cas9 technology;Western blot was used to verify the knockout effect of PKM2;the PK activity,ATP content,glucose content and lactate production of PKM2 knockout cells were detected using kits;CCK8 method and clone formation assay were used to detect the proliferation ability of PKM2 knockdout cells;Transwell and trauma healing assay were used to detect the migration ability of PKM2 knockout cells.The proliferation ability of PKM2 knockdout cells was detected by CCK8 method and clone formation assay;the migration ability of PKM2 knockout cells was detected by Transwell and wound healing assay.3.The metabolism of MDA-MB-231 cells after PKM2 knockout was examined by metabolomics;the transcriptome expression of cells after PKM2 knockout was examined by RNA-seq and the possible molecular mechanisms underlying the altered substance metabolism after PKM2 knockout were first explored.4.To detect changes in lipid metabolism levels in PKM2 knockout MDA-MB-231 cells: lipi Dye staining and Oil Red O staining to detect lipid droplets and neutral lipid content;FAO Blue to detect fatty acid oxidation(FAO)levels;total cholesterol and triglyceride kits to detect total cholesterol and triglyceride content.5.Western blot was performed to detect ACADVL expression;immunofluorescence assay was performed to detect mitochondrial localization and ACADVL expression level;UCSC Xena database was used to analyze the correlation between PKM2 and ACADVL expression in breast cancer patients at m RNA level.6.After further knockdown of ACADVL expression in PKM2 knockout MDA-MB-231 cells,changes in proliferation and migration ability were detected by CCK8,Ed U and wound healing assays.7.Western blot was performed to detect the expression of transcription factor KLF4(Krüppel-like factor 4,KLF4)and energy-sensing protein AMPK in ACADVL;Western blot was performed to detect the expression of autophagy-related proteins LC3A/B and Beclin1;the introduction of AMPK activator(AICAR)and inhibitor(Compound C)was introduced to detect the expression of KLF4 and ACADVL.Results:1.High PKM2 expression positively correlates with breast cancer progression.PKM2 was upregulated in breast cancer;Kaplan-Meier analysis showed that high PKM2 expression was associated with poorer overall patient survival;PKM2expression levels were elevated in all subtypes of breast cancer and at all stages of disease progression.2.Western blot assay confirmed the successful knockout of PKM2 in MDA-MB-231 cells,and the knockout of PKM2 downregulated pyruvate kinase activity by about 90%,inhibited glucose uptake by about 50%,decreased lactate production by about 40%,and decreased the level of ATP produced by aerobic glycolysis by about 50%.The knockout of PKM2 to some extent diminished the proliferative capacity,and lateral and longitudinal migration ability of MDA-MB-231 cells.3.The results of metabolomic analysis showed that glucose metabolism,lipid metabolism and amino acid metabolism were significantly changed after PKM2 knockout,and the significantly different metabolites were enriched in lipid catabolic signaling pathway,pyruvate metabolism and glycolytic metabolism signaling pathway,alanine,aspartate and glutamate metabolism pathway and AMPK signaling pathway.The RNA-seq results suggested that a total of 3235 genes were up-regulated and3419 genes were down-regulated after PKM2 knockout(≧2-fold,adjusted P<0.05),among which the up-regulated expression of very long chain fatty acid oxidative dehydrogenase(ACADVL)was more obvious.KEGG analysis of differentially expressed significant genes showed that cholesterol homeostasis and fatty acid synthesis were the most down-regulated pathways,and fatty acid oxidation was the more up-regulated pathway;KEGG enrichment analysis of lipid metabolism-related genes in RNA-seq showed that knockout of PKM2 caused MDA-MB-231 cells to have "fatty acid degradation ","fatty acid metabolism" and "metabolic pathways" were disrupted in MDA-MB-231 cells.4.Knockout of PKM2 in MDA-MB-231 cells resulted in a decrease in lipid droplet abundance and neutral lipid content;an increase in fatty acid oxidation levels;and a decrease in total cholesterol content.5.Western blot assay showed that the expression level of ACADVL increased after PKM2 knockout in MDA-MB-231 cells;immunofluorescence assay showed that the expression of ACADVL localized in mitochondria increased;and the m RNA levels of PKM2 and ACADVL were negatively correlated in breast cancer by UALCAN database analysis.6.After further knockdown of ACADVL expression in PKM2 knockout MDA-MB-231 cells,the proliferation and migration ability of the cells were further impaired.7.Knockout of PKM2 expression in MDA-MB-231 cells induced autophagy and activated the energy receptor AMPK,leading to inhibition of downstream ACC activity and enhanced expression of the ACADVL transcription factor KLF4;this effect was enhanced or diminished by AMPK activators or inhibitors.Conclusions:1.Knockout of PKM2 in MDA-MB-231 cells blocking the Warburg effect resulted in significant changes in cellular glucose metabolism,lipid metabolism and amino acid metabolism,with oxidative catabolism of fatty acids being particularly important.2.Knockout of PKM2 in MDA-MB-231 cells after blocking the Warburg effect through activation of the AMPK-KLF4-ACADVL pathway to increase fatty acid oxidative energy supply.