PSAT1 Regulates Non-small Cell Lung Cancer Progression Through The De Novo Serine Synthesis Pathway

Background:Cancer is one of the important public issues worldwide.Lung cancer is one of the most common cancers,and accounts for the highest mortality rate among all cancers.Non-small cell lung cancer(NSCLC)is the main pathological type of lung cancer,accounting for about 80% of all lung cancers.Due to the lack of effective early diagnostic methods,limited effectiveness of chemotherapy,and susceptibility to tumor metastasis,the five-year survival rate of patients with NSCLC is still relatively low.Therefore,it is necessary to find new treatment methods and targets for NSCLC.Metabolic reprogramming is a hallmark of malignant tumors.It refers to the process by which cancer cells autonomously change metabolic flux through various metabolic pathways to meet increased energy and biosynthetic needs and maintain the oxidative stress required for cancer cell proliferation and survival.The “Warburg effect” is currently one of the most widely studied modes of cell metabolism reprogramming.The “Warburg effect” is a “selfish” metabolic reprogramming of tumor cells.Glucose is no longer used to provide energy,but is transferred into other metabolic pathways through various glycolysis intermediates to promote the synthesis of nucleotides,non-essential amino acids and other molecules.Among them,serine,a non-essential amino acid,plays an important role in the rapid proliferation of malignant tumors.There are two main sources of serine: one is the intake of serine from the microenvironment,and the other is the synthesis of serine through the de novo serine synthesis pathway(SSP).In SSP,3-phosphoglycerate(3-PG),an intermediate product of glycolytic metabolism,is catalyzed by 3-phosphoglycerate dehydrogenase(PHGDH),phosphoserine aminotransferase 1(PSAT1),and phosphoserine phosphatase(PSPH)to produce serine.Through this process,glucose in tumor cells completes the transition from energy production to biosynthesis,playing a crucial role in the rapid proliferation of cancer cells.As the second key enzyme of SSP,PSAT1 is closely related to the occurrence and development of various tumors.The preliminary bioinformatics analysis results of this project and previous literature research results all showed that compared with the control group,the expression of multiple genes in NSCLC tissues was changed,among which the expression of PSAT1 was significantly increased.Further analysis suggested that the patients with high PSAT1 expression had significantly shorter overall survival(OS).However,the regulatory function of PSAT1 on NSCLC remains to be further clarified,and its regulatory mechanism also requires more in-depth study.In conclusion,the study of PSAT1 not only helps to understand the tumor metabolic process but also helps to find new metabolic treatment options,which have certain value in improving the prognosis of NSCLC patients.Objective:This study aims to clarify the expression level of PSAT1 in NSCLC tissues and cells,further investigate the regulatory function of PSAT1 on rapid proliferation and apoptosis of NSCLC and the impact of PSAT1 on the therapeutic efficacy of NSCLC drugs.The experiment focuses on the combination of knockdown of psat1 and restriction of serine intake.This study delves into the mechanism of action of PSAT1 in NSCLC cells,especially the mechanism of insufficient serine synthesis and abnormal cellular redox levels caused by the decrease of PSAT1,activating the mitochondrial apoptosis pathway,and provides direction for metabolic therapy of NSCLC.Methods:1、The expression of PSAT1 in NSCLC(1)Conduct bioinformatics analysis using the GEPIA online website(http://gepia.cancer-pku.cn/)to investigate the expression of PSAT1 in NSCLC and using the Kaplan-Meier Plotter database(http://kmplot.com/analysis/)to study the impact of PSAT1 expression levels on OS in NSCLC patients.(2)Tissues of 15 cases of NSCLC and para-cancer tissues were collected,and tissue chips containing 75 pairs of NSCLC and para-cancer tissues were used for immunohistochemical staining to analyze the expression level of PSAT1 in NSCLC and para-cancer tissues and the relationship between PSAT1 expression and pathological features of patients.(3)Compare the expression levels of PSAT1 between NSCLC cell lines(A549,H1299)and bronchial epithelial cell line(BEAS-2B)using real-time fluorescence quantitative PCR(RT-q PCR)and Western Blot experiments.2、The regulatory function of PSAT1 on the malignant behavior of NSCLC(1)Construct stable knockdown PSAT1 H1299 and A549 cell lines by transfecting lentivirus plasmids,observe the transfection status of the cells under fluorescence microscopy,and verify the knockdown efficiency of the plasmids through RTq PCR and Western Blot experiments.Using a customized serine-free culture medium to simulate conditions of insufficient exogenous serine intake,investigate the synergistic effect of serine deficiency and knockdown of PSAT1.(2)The effects of knocking down PSAT1 on the proliferation function of NSCLC cells were studied through CCK-8 proliferation experiments and plate cloning experiments.Flow cytometry was used to detect the effect of knocking down PSAT1 on the cell cycle of NSCLC cells.(3)Flow cytometry was used to detect the effect of knocking down PSAT1 on the apoptosis of NSCLC cells.3、The effect of PSAT1 on the drug treatment of NSCLC(1)Construct stable knockdown PSAT1 H1299 and A549 cell lines by transfecting lentivirus plasmids and verify the knockdown efficiency of the plasmids through RT-q PCR and Western Blot experiments.(2)Analyze the effect of knocking down PSAT1 on the half inhibitory concentration(IC50)of chemotherapy drugs such as gefitinib through the CCK-8 proliferation experiment.(3)Investigate the effect of knocking down PSAT1 on the survival of NSCLC cells at the same drug concentration through the Calcein/PI cell viability/cytotoxicity assay.4、The mechanism of PSAT1 in NSCLC(1)Investigate the effect of knocking down PSAT1 on the synthesis of serine in NSCLC through the isotope tracing experiment.(2)Explore the effect of knocking down PSAT1 on reactive oxygen species(ROS)levels in NSCLC cells using the Reactive Oxygen Species Assay Kit.Explore the effect of knocking down PSAT1 on the redox homeostasis of NSCLC cells by detecting the ratio of reduced glutathione(GSH)to oxidized glutathione disulfide(GSSG),as well as the ratio of oxidized nicotinamide adenine dinucleotide phosphate(NADP+)to reduced nicotinamide adenine dinucleotide phosphate(NADPH).(3)Study the effect of PSAT1 knockdown on NF-κB signaling pathway and mitochondrial apoptosis pathway through Western Blot experiments.5、The function and mechanism of PSAT1 in in vivo experiments(1)Construct stably PSAT1-knockdown H1299 cell line for xenograft experiments in nude mice,and use serine-free diet to simulate the absence of exogenous serine intake.(2)Measure the change in mouse body weight and tumor size at regular intervals during the experiment,and euthanize the mouse when the tumor grows to about1-1.5 cm.(3)Tumor tissue samples were stained by immunohistochemistry,Ki67,TUNEL and CASP3 to detect the effect of PSAT1 knockdown on the growth and apoptosis of tumor cells.Results:1、 PSAT1 is significantly overexpressed in NSCLC and is related to the OS of patients with NSCLC.(1)Using the GEPIA online database for bioinformatics analysis,the results indicate that compared to normal lung tissue,PSAT1 expression is significantly increased in LUAD and LUSC(P<0.05).Survival analysis using the Kaplan-Meier Plotter online database shows that patients with high PSAT1 expression have lower overall survival(logrank P<0.05)and worse prognosis.(2)Immunohistochemistry staining results suggest that PSAT1 is highly expressed in NSCLC tissues(P<0.05).Further analysis reveals a correlation between PSAT1 expression and NSCLC histological types,with significant differences observed in LUAD and LUSC(P<0.05),while no association was found with patient age,gender,tumor size,TNM staging,etc.(3)Western blot experiments demonstrate that PSAT1 expression is significantly higher in NSCLC cell lines(H1299 and A549)compared to bronchial epithelial cell line BEAS-2B(P<0.05).These findings are consistent with the bioinformatics analysis and immunohistochemistry results.2、 Knockdown of PSAT1 inhibits the proliferation of NSCLC cells and increases apoptosis,which has a synergistic effect with insufficient exogenous serine intake.(1)RT-q PCR and Western Blot results demonstrated that the constructed lentiviral plasmid effectively suppressed the expression of PSAT1(P<0.05),and the absence of exogenous serine intake did not interfere with the knockdown of PSAT1 expression by sh RNA.(2)The results from CCK-8 proliferation experiment and plate cloning experiment revealed that both PSAT1 knockdown and serine intake significantly inhibited the proliferation of NSCLC cells(P<0.05),exhibiting a synergistic effect.(3)Flow cytometry analysis indicated that both PSAT1 knockdown and serine intake induced cell cycle arrest,characterized by a decrease in G1 phase cells and an increase in S phase cells(P<0.05).Furthermore,these two factors exhibited a synergistic effect.(4)Flow cytometry analysis demonstrated that both PSAT1 knockdown and serine intake promoted apoptosis in NSCLC cells(P<0.05),displaying a synergistic effect.3、 Knockdown of PSAT1 improved the killing effect of gefitinib on NSCLC,which had a synergistic effect with insufficient exogenous serine intake.(1)Following the addition of appropriate concentration gradients of chemotherapy drugs to NSCLC cells,the results of the CCK-8 experiment demonstrated a significant enhancement in gefitinib’s killing effect on NSCLC after PSAT1 knockdown at multiple concentration gradients(P<0.05).Further calculations revealed that PSAT1 knockdown significantly reduced the IC50 of gefitinib(P<0.05).(2)The live cell/dead cell staining experiment indicated that both PSAT1 knockdown and serine deficiency could promote gefitinib’s killing effect on NSCLC cells,with synergistic effects observed between them.4、 Knockdown of PSAT1 inhibits serine synthesis,increases the level of reactive oxygen species in NSCLC,activates NF-κB signaling pathway and mitochondrial apoptosis pathway,and has a synergistic effect with insufficient exogenous serine intake.(1)The results of GSH/GSSG ratio,ROS level,and NADPH/NADP+ ratio detection suggest that PSAT1 knockdown and serine deficiency can decrease the GSH/GSSG ratio of NSCLC cells(P<0.05),increase ROS levels,and decrease the NADPH/NADP+ ratio(P<0.05),indicating an abnormal redox level in NSCLC cells.PSAT1 knockdown and serine deficiency exhibit synergistic effects.(2)Western Blot experiments show that PSAT1 knockdown and serine deficiency increase the protein expressions of NF-κB and p-NF-κB(P<0.05),decrease the protein expression of Bcl-2,increase the protein expressions of Bax,Cytochrome C,and Caspase 3(P<0.05),indicating activation of the NF-κB signaling pathway and mitochondrial apoptosis pathway.PSAT1 knockdown and serine deficiency exhibit synergistic effects.(3)Isotope tracing experiments show that PSAT1 knockdown has no significant effect on pentose phosphate pathway or tricarboxylic acid cycle but directly leads to insufficient endogenous serine synthesis,suggesting that PSAT1 functions through SSP pathway.5、 In vivo experiments verified the function of PSAT1 to maintain the proliferation of NSCLC and inhibit its apoptosis.(1)Through the establishment of a nude mice xenograft model,it was observed that knockdown of PSAT1 and serine deficiency resulted in a significant reduction in both volume and weight of NSCLC xenografts.(2)When subjected to a serine-free diet,mice exhibited initial weight loss followed by gradual recovery;however,their body weight remained lower compared to those fed with a normal diet under identical conditions.(3)The findings from staining experiments conducted on xenograft samples indicated that PSAT1 knockdown and serine deficiency effectively suppressed the proliferation of NSCLC while promoting apoptosis.Conclusions:1、 PSAT1 is significantly overexpressed in NSCLC and indicates a poor prognosis for patients2、 Knockdown of PSAT1 inhibits SSP,reduces DNA synthesis in NSCLC cells,arrests the cell cycle,and weakens cell proliferation.3、 Knockdown of PSAT1 inhibits serine synthesis,reduces glutathione synthesis,breaks the redox balance of NSCLC cells,increases the level of reactive oxygen species,causes apoptosis,and can enhance the killing effect of gefitinib on NSCLC.4、 Knockdown of PSAT1 activates the NF-κB signaling pathway and mitochondrial apoptosis pathway,causing apoptosis in NSCLC cells.5、 Insufficient exogenous serine intake and knockdown of PSAT1 have a synergistic effect in function and mechanism.