Research On Mechanism Of CPT1A-Mediated Fatty Acid Oxidation Promotes The Proliferation And Survival Of Nasopharyngeal Carcinoma Cells

Tumor cells maintain their growth energy requirements by efficiently using energy substances such as glucose,glutamine,fatty acids,and acetic acid.It also provides the metabolic basis for resistance to stressful conditions and therapeutics.These demonstrate the metabolic plasticity of tumor cells.Lipid metabolism is essential to tumor metabolic reprogramming,regulating tumor cell proliferation,survival,and determining cell fate.It is critical to maintain cellular energy homeostasis.Fatty acid oxidation(FAO)occurs dominantly as β-oxidation.Mitochondria is the main site for fatty acid β-oxidation.Fatty acids undergo a series of steps to generate acetyl CoA,which enters the tricarboxylic acid cycle coupled with oxidative phosphorylation to produce ATP and NADPH,providing energy and reduced power for cells.Carnitine palmitoyl transferase 1(CPT1)is located in the outer mitochondrial membrane to catalyze the esterification of long-chain acyl groups with carnitine.This is the key rate-limiting step in FAO and mediates activation of FAO.Recently,fatty acid metabolism has been recognized as a crucial component in tumor metabolic reprogramming,and a series of progress has been made in this field.As an essential regulator in FAO,the role of CPT1A in tumor biology and treatment needs to be further explored.This paper focus on CPT1A-mediated FAO,exploring its expression,metabolic function,and biological effects in nasopharyngeal carcinoma(NPC).In the first part,the role of CPT1A in NPC was analyzed using the public database,clinical specimens,and NPC cell lines.We investigated the expression characteristics of CPT1A in NPC and explored the mechanism.High expression of CPT1A in head and neck squamous carcinoma(HNSCC)and NPC tissues was associated with poor overall survival of patients.Elevation of CPT1A in the serum of NPC patients by ELISA.Amplification is a predominant alteration of the CPT1A gene in the HNSCC database and NPC cells,and occurred in approximately 15%of patients to cause its enhanced expression.The RNA-seq dataset enriched pathway suggested that CPT1A expression might be associated with the NF-κB signaling pathway.Transcription factor P65 binds to the CPT1A promoter region and was enhanced in NPC cells by ChIP assay.Targeting NFκB signaling pathway with small molecule inhibitor Bay-11 or P65 siRNA decreased CPT1A protein levels,lowered biological enzyme activity,diminished mitochondrial localization,and altered intracellular free fatty acid distribution.Taken together,these results indicate that the NF-κB signaling pathway mediated cellular FAO through transcriptional regulation of CPT1 A.Cellular metabolic remodeling and cell cycle progression synergistically regulate carcinogenesis.In the second part,we clarified the metabolic phenotype with active FAO in NPC cells,and determined how CPT1A-mediated FAO connects the cell cycle progression and cell proliferation.Metabolomic analysis revealed that both fatty acid transport and enzymatic bioactivities of CPT1 A were enhanced.CPT1A downregulated cellular neutral lipid content and promoted FAO and ATP levels.Using the GEO database,we analyzed the gene expression profiles of NPC patients and found that CPT1A may be involved in DNA replication,and the pyrimidine metabolic pathway.Nucleoside metabolic pathways are also activated in EBV-positive NPC by metabolomics analysis.The metabolic flux assay discovered that FAO provides a carbon source for nucleoside metabolic intermediates,and the knockdown of CPT1 A significantly reduced the proportion of 13 C-palmitate-derived carbon incorporated into pyrimidine synthesis.Knockdown of CPT1A or blocking its enzymatic activity by etomoxir(Eto)decreased the proliferation of NPC cells.The cell cycle was arrested in the G1 phase,reduced the protein levels of Cyclin D1 and CDK4,and decreased phosphorylation of RB(Thr821).Autophagy is a key regulatory manner to maintain lipid homeostasis during metabolic stress,and FAO is an essential pathway to promote survival by autophagy.In the third part,we explored the role of autophagy on lipid metabolism and cellular energy homeostasis.The results indicated that CPT1Amediated FAO activation promotes survival under nutrient-deficient conditions in NPC cells.We further elucidated the mechanism of activated FAO and tumor survival from the perspective of cellular autophagy.By laser confocal and transmission electron microscopy,we found that autophagosome formation increased and selective lipophagy occurred in NPC cells under energy stress conditions.CPT1A-mediated activation of FAO in NPC cells facilitated increased free fatty acids uptake and enhanced expression of autophagy-related genes in NPC.Autophagy and extracellular lipids promote cell survival through FAO.Inhibition of autophagy increases sensitivity to Eto,and combing chloroquine and Eto synergistically inhibits tumor cell growth.In summary,this paper elucidated the metabolic profile of FAO in NPC and identified CPT1A as a critical molecule in the aberrant activation of FAO.Our findings demonstrated that CPT1 A links FAO to cell cycle progression and promotes cell proliferation,survival,and metabolic adaptation.We discovered the molecular mechanisms based on the link between energy production and tumor cell growth.This work provides an experimental basis on FAO-targeted tumor therapy.