Study On The Role And Mechanism Of L-carnitine/CPT1A Pathway In Regulating Ferroptosis Resistance And Immune Escape Of Lung Cancer Stem Cell

Cancer stem cells（CSCs）and their microenvironment-driven immune escape is the main reason for the failure of immune checkpoint blockade therapy,while tumor cell ferroptosis mediated by cytotoxic T lymphocytes is key to tumor immune clearance.However,the regulatory mechanism of CSCs ferroptosis in non-small cell lung cancer is not clear.Tumor-associated macrophages（TAMs）,as the most abundant immune cells in the tumor microenvironment,are closely related to the maintenance of CSCs stemness and the formation of tumor immune tolerance.However,there are few reports on the regulation of CSCs ferroptosis by TAMs.On the other hand,numerous studies have found that the occurrence of ferroptosis is closely related to cell fatty acid metabolism.Carnitine palmitoyl transferase 1A（CPT1A）is an important limiting enzyme in the process of fatty acid oxidation（FAO）.With the help of the fatty acid carrier L-carnitine,CPT1A converts long-chain fatty acids into acylcarnitine and transports them to the mitochondria for βoxidation of fatty acids.Although studies have shown that L-carnitine and CPT1A are closely related to tumor progression,the role and mechanism of L-carnitine and CPT1A in CSCs stemness maintenance and ferroptosis regulation are not clear.In this study,we first found that metabolic products secreted by TAMs can inhibit ferroptosis of lung cancer cells.Using metabolomics technology and cell experiments,we confirmed that L-carnitine secreted by TAMs can inhibit ferroptosis of lung cancer cells,maintain the stemness phenotype of lung cancer cells,and ultimately promote the development of mouse lung cancer.Further using stable knockdown of the CPT1A gene in lung cancer cell lines and the spontaneous lung cancer(Cpt1a^{Flox Flox}-Sftpc-creER^T2;Kras^LSL-G12D/-)mouse model with lung epithelial cell-specific knockout of the Cpt1a gene,we found that L-carnitine drives CSCs stemness maintenance and ferroptosis resistance,and the formation of a tumor immune suppressive microenvironment through its downstream metabolic enzyme CPT1A.Analysis of lung cancer patient tissue chips found that CPT1A is highly expressed in human lung cancer tissue and increases with disease progression.Bioinformatics analysis found that the CPT1A transcript is significantly negatively correlated with the clinical prognosis of lung cancer patients.In the mechanism study,we found that CPT1A increases the stability of c-Myc protein by inhibiting its ubiquitination and degradation.At the same time,c-Myc can directly transcribe and activate the expression of CPT1A,thus forming a CPT1A/c-Myc positive feedback regulatory loop.This molecular loop further inhibits the expression of ACSL4 to reduce the synthesis of polyunsaturated fatty acids,and activates the NRF2/GPX4 pathway to enhance the antioxidant activity of lung cancer cells,thereby inhibiting ferroptosis of LCSCs.We confirmed the correlation between the activity of the CPT1A/c-Myc loop and the expression of ferroptosis-related genes ACSL4 and GPX4 using lung cancer clinical samples,transgenic mouse models,and bioinformatics analysis.Finally,using a mouse subcutaneous tumor model and cell co-culture experiments,we confirmed that inhibiting CPT1A can restore the infiltration and activation of CD8+T cells.Further using knockdown or small molecule inhibitor Etomoxir targeting CPT1A combined with ferroptosis inducer Erastin or immune checkpoint inhibitor PD-1 antibody can promote anti-tumor immunity and tumor cell ferroptosis,thus more effectively inhibiting tumor progression.In summary,our study first revealed the important role of TAM-derived metabolite Lcarnitine in promoting CSCs ferroptosis resistance and immune escape through the regulation of the CPT1A/c-Myc positive feedback loop,which activates the cell antioxidant system（NRF2/GPX4）and inhibits the synthesis of polyunsaturated fatty acids（ACSL4）.We revealed the core mechanism of the CPT1A/c-Myc positive feedback loop in regulating CSCs stemness phenotype,ferroptosis resistance,and immune escape,and confirmed that CPT1A may be a potential target for improving the sensitivity of CSCs ferroptosis and improving immune therapy for lung cancer.Our findings provide new theoretical basis for the development of combination immune therapy strategies for lung cancer.