Metabolism-based Molecular Sub-phenotyping And Its Indication For Cancer Metabolic Moduation Therapy In Colon Carcinoma

Objective: Current studies have confirmed that the sensitivity of ketogenic diet(KD)for tumors is dependent on the low expression of ketolysis enzymes.However,due to the heterogeneity of tumor metabolism,ketolysis enzymes in some tumor cells can be reactivated under KD intervention,resulting in failure of treatment.This study aims to construct colon cancer metabolism related molecular subtyping.Exploring the metabolic heterogeneity of different colon cancer cells and the mechanisms of mitochondrial metabolic reprogramming.Thus,providing theoretical basis for ketogenic therapy and combined intervention measures based on metabolic molecular phenotyping.Methods: 1.The transcriptome data of 275 colon cancer patients in the TCGA database were analyzed by GEPIA to screen out target moleculars involved in glucose and ketone body metabolism.Based on the information of 807 colon cancer patients obtained from GEO database,the above differentially expressed moleculars were analyzed by survival and cluster analysis.Furthermore,to construct prognosis related metabolic phenotypes by verifing at the tissue level of tissue chips at 180 points.2.Establishing low-glucose hunger model through culturing different colon cancer cells in low glucose condition.Invesgating distortion of metabolism by glycolysis pressure curve and metabolic,observing mitochondrial morphological changes by confocal microscopy.To verify the metabolic heterogeneity of colon cancer cells with different P53 mutation states,and to further investigate the mechanism of metabolic reprogramming in cells with nutritional deficiency.3.By detecting the mitochondrial membrane potential level,intracellular ATP production and cell proliferation rate of different colon cancer cells in the ketogenic culture state.To verify the ketobody utilization differences caused by the heterogeneity of ketogenic enzyme levels.The effect of the core catabolic enzyme in ketogenic process was explored through specifically interfering the gene expression with lentivirus.4.By detecting the effect of P53 allosteric activator on the changes of cellular energy metabolism phenotype,mitochondrial membrane potential,intracellular ATP production,cell proliferation,content of intracellular Acetyl-Co A,expression of metabolic enzymes,and mitochondria morphology in P53 mutant cells,To verify the P53 activator could reverse mutant P53 into wild-type P53 and change the processes of mitochondria reprogramming.5.Establishing the HT29 colon cancer-BALB/C nude mice subcutaneously tumor-burdened model,the mice were divided into 4 groups: the standard diet group,the ketogenic diet group,P53 activation agent group,the combination of ketogenic diet and P53 activation agent group.The nude mouse tumors' size,nutritional status,blood glucose,ketone,and tissue metabolism enzyme expression level changes were measured to prove the efficacy and safety of graduate methadone treatment and P53 activation agent combination therapy on the inhibiting of HT29 colon cancer transplantation tumor growth.Results: 1.The target moleculars,GLUT1,PFKFB3,PKM,LDHA,PGC1 a,BDH1,OXCT1,and ACAT1 which related to glucose and ketone body metabolism were screened out from transcriptional database of colon cancer.Based on the characteristics of ketogenic diet metabolism regulation treatment and the results of survival analysis,the colon cancer patients were divided into three types with significant difference in prognosis: Glycolysis+/ Ketolysis-: GLUT1 high or PFKFB3 high,OXCT1low or ACAT1 low,and the median survival(m OS)was 22 months.Intermediate type: Glycolysis/Ketolysis metabolism(Glycolysis+/ Ketolysis+: GLUT1 high or PFKFB3 high,OXCT1high and ACAT1high),m OS 47.5 months;Glycolysis-/Ketolysis-: GLUT1 low and PFKFB3 low,OXCT1low or ACAT1 low,m OS for 30 months.The best prognosis was Glycolysis-/Ketolysis+: GLUT1 low and PFKFB3 low,OXCT1high and ACAT1 high,and less than 50% of the patients passed away at the observation cut-off point which mean the m OS needs to be further extended for follow-up.2.There was significant heterogeneity in the expression levels of glycolysis and ketolysis related metabolic enzymes among colon cancer cells.Under the condition of low glucose culture,the wild-type P53 cells showed enhanced glycolysis metablism,while the mutant P53 R273 cells could be induced into oxidative phosphorylation 48 h after culturing which was observed as increased mitochondrial fusion,increased expression of respiratory chain complex I,and induced activation of ketosomal metabolic enzymes.3.In the ketogenic culture state,there were significant differences in the utilization ability of ketobdy in different colon cancer cells.Cancer cells with high expression of ketolysis metabolic enzymes BDH1,OXCT1 and ACAT1 could use ketobody as an energy source to generate ATP and promote cell proliferation.Specific interference with the core catabolic enzyme OXCT1 of ketolysis signal pathway could significantly reduce the ketobody utilization capacity.4.P53 activator could reverse mutant P53 into wild type.Afterwards,promoting mitochondrial fission and inhibiting the expression of mitochondrial respiratory chain complex I.It reversed the metabolic reprogramming of cells in the ketogenic state,inhibited the level of oxidative phosphorylation,reduced the content of intracellular acetyl-coenzyme A,and thereby inhibited the expression of ketolysis enzyme OXCT1.5.Animal experiments verified the sensitivity of Glucolysis+/ketolysis-subtype transplanted tumor to ketogenic therapy.As results showed,KD alone and KD combined P53 agonist group showed significant inhibitory effects on tumor growth.Molecular detection of the tumors showed that ketolysis enzymes of P53 mutant tumors could be significantly induced after ketogenic therapy,and the combined use of P53 activator could reverse the expression and activation,thus exerting synergistic tumor inhibition.Conclusion: The metabolic typing of multi-target combination based on P53 mutation status can predict the sensitivity and effectiveness of ketogenic diet metabolism regulation therapy.It proved to be a foundation for individualized molecular stratified metabolism regulation therapy and provided a theoretical basis for the clinical trial of ketogenic therapy for patients with specific metabolism molecular subtypes of colon cancer with poor prognosis.