The Prognostic And Therapeutic Value Of Hypoxia And Oxidative Stress In Non-Small-Cell Lung Cancer

Malignant tumor is a major threat to human health and is one of the leading causes of death in China.According to the latest statistical data in China,lung cancer is the most common malignant tumor now.The five-year survival rate of advanced lung cancer is less than 10%.According to pathological types,lung cancer can be divided into Non-small-cell lung cancer(NSCLC)and Small-cell lung cancer(SCLC).NSCLC is the main component of lung cancer,accounting for nearly 80-85%of all lung cancer.Due to the lack of typical clinical manifestations,NSCLC is often already advanced at their first diagnosis.In recent years,although NSCLC treatment has made some progress in surgery,radiotherapy and chemotherapy,the five-year survival rate is still low.Hypoxia is one of the important biological characteristics of malignant solid tumors.Clinical research in the past two decades has clearly demonstrated that a certain range of hypoxic regions exists in most malignant solid tumors and is a characteristic pathophysiological feature of locally advanced solid tumors.Causes of tumor hypoxia are diversified,but their root causes are inadequate oxygen supply and increased oxygen consumption.In normal tissues,the supply of oxygen and the need for tissue metabolism are in equilibrium,but in locally advanced tumors,tumor hypoxia is eventually generated due to the lack of perfect blood supply and the high metabolic aerobic state of the tumor.At the same time,due to the tumor itself and chemotherapy-related anemia,the oxygen content in the blood decreases and the oxygen supply to the tumor is insufficient.Hypoxia can have a huge impact on tumor cells,such as tumor genome instability,tumor angiogenesis,tumor metastasis,tolerance to radiotherapy and chemotherapy,and poor prognosis.With respect to hypoxia,oxidative stress is also common.But there is no contradiction between hypoxia and oxidative stess.Oxygen is required for mitochondria to produce adenosine triphosphate(ATP)by oxidative phosphorylation.At the same time,due to the chemical activity of oxygen molecules,reactive oxygen species(ROS)are easily formed.ROS is a normal product in cell metabolism,including superoxide,peroxides,and hydroxyl radicals.The electron transport chain in mitochondrial metabolism is the main source of ROS.ROS plays an important role in the developmentof tumors.In vitro studies have shown that ROS production in tumor cells is much faster than normal cells.A certain level of ROS increases mutation frequency,activates proliferation-related signaling pathways,and promotes tumor progression.Although ROS promotes the development of tumors to a certain extent,when ROS levels are too high,oxidative stress can cause fatal damage to tumor cells.Under normoxic conditions,intracellular ROS levels are controlled in equilibrium by redox mechanisms.The balance of redox levels depends on the production rate of ROS and the scavenging capacity of the scavenging system.Superoxide dismutase,catalase,glutathione(GSH),thioredoxin,glutaredoxin,and other ROS scavengers play an important role in redox homeostasis.LKB1 is a key molecule for maintaining energy and redox homeostasis in cells.LKB1 mutation alone is considered to be unable to induce lung cancer,but KRAS-LKB1 co-mutation(KL tumor)induces lung adenocarcinoma,squamous cell carcinoma,or large cell carcinoma.And KL tumors grow faster than KRAS mutant tumors(K tumors)and are more likely to metastasize.KEAP1 is an important molecule in cellular oxidative stress reaction and is an adaptable protein of E3 ubiquitin ligase.Under normal circumstances,KEAP1 binds to and induces ubiquitination of NRF2 and degrades NRF2 through the proteasome pathway.This in turn affects the transcription of antioxidant-related genes.Deletion of LKB1 leads to an increase in intracellular ROS levels.Interestingly,LKB1 is usually co-mutated with KEAP1.Our study found that LKB1 mutant tumors have enhanced anti-oxidative stress abiltiy,which may be closely related to the upregulation of NRF2 caused by LKB1 mutations and increased glutamine metabolism.The metabolism of glutamine is critical in the progression of tumors.Its importance is mainly reflected in the fact that,like glucose,it can provide a large amount of ATP for the growth of tumor cells,and it is a precursor amino acid that generates GSH,a major reduction product in cells.Both normal cells and tumor cells need to use glucose and glutamine,but the demand is very different.The demand for glutamine in tumor cells is quite large,and normal cells mainly provide energy through the aerobic oxidation of sugar,so the demand for glutamine is much lower than tumor cells.Glutamine glutamate(GLS)catalyzed the formation of glutamate is the first step in the metabolism of glutamine.Currently,GLS inhibitors have entered the clinic,but are not effective for all patients.Finding suitable biomarkers to guide the application of GLS inhibitors is critical for the clinical application of this drug.Part I Effect of Erythrocyte Related Indexes on Prognosis of Patients with Non-small Cell Lung CancerObjectives:1.Analyze the influence of preoperative erythrocyte-related index on prognosis of patients with NSCLC using hypoxic predisposition;2.To find the independent prognostic factors of RBC-related index in NSCLC through multi-factor analysis;3.Explore the relationship between this indicator and the clinical characteristics of patients with NSCLC.Methods:1.Patients enrolled in the study:Patients who underwent radical lung cancersurgery at the Department of Thoracic Surgery of the Provincial Hospital Affiliated toShandong University from January 2005 to December 2009 were selected for screening.2.Data collection:We collected patient’s gender,blood routine examination(the first test after admission),smoking history,pathological type,tumor size,number of lymph node metastases,surgical methods and other information.Patients’ clinical information and contact information were obtained through the medical record management system of Provincial Hospital Affiliated to Shandong University.3.Statistical methods:The primary endpoint was overall survival(OS),and the secondary endpoint was Recurrence free survival(RFS).Kaplan-Meier method was used to analyze the influence of single factor on the prognosis of patients and draw the survival curve.The log-rank test was used to compare the differences between the two groups.The ability of the different indicators to predict the prognosis was determined by the receiver operating characteristic curve and the best cutoff was determined.The χ2 test(with Fisher’s exact test if necessary)or the t-test was used to analyze the relationship with clinicopathological features.Multivariate analysis of prognostic factors was performed using the Cox risk regression model.Variables with a p<0.05 in univariate analysis were entered into a multivariate analysis.Statistical differences were considered significant when p<0.05(two-sided test).Results:1.Basic characteristics of patients:The study included 649 patients,456 males and 193 females.In the patient’s pathological distribution,most patients were at their early stage.Stage Ⅰ/Ⅱ patients accounted for 72.9%of the total number of patients,patients with stage Ⅲ accounted for 27.1%,no IV patients were enrolled.2.Univariate analysis:Patient’s gender,age,surgical method,tumor differentiation,N stage,T stage,tumor size,HGB,HCT,MCV,MCH,and MCHC can affect patients’survival in univariate analysis.Among the RBC-related indices,higher levels of HGB,HCT,MCHC,MCV,and MCH were associated with better OS in patients with NSCLC.At the same time,in the study of RFS,higher levels of MCH,MCV,and MCHC were also closely related to better RFS,but HGB and HCT have no statistically significant effect on RFS.3.Multivariate analysis:After correction with other defined prognostic factors,only MCHC was both an independent prognostic factor for OS(p=0.032)and an independent prognostic factor for RFS(p =0.010).MCV was an independent prognostic factor for OS only(p=0.018),but it was not an independent prognostic factor for RFS(p=0.059).Other erythrocyte-related indicators such as HCT,HGB,and MCH are only significant in the factor analysis,but they have no significance in multivariate analysis,demonstrating that these prognostic factors are not independent prognostic factors.4.Correlation analysis of MCHC and clinicopathologic factors:low MCHC levels was associated with male(p=0.02),higher smoking index(p=0.002),higher T stage(p=0.018),and larger tumor volume(p= 0.004).Conclusion:1.In the univeriate analysis,MCHC,MCH,and MCV all had effects on OS and RFS of NSCLC patients,but HGB and HCT only affected the patient’s OS.2.In patients with non-anemic NSCLC,MCHC is an independent prognostic factor predicting OS and RFS.MCV is an independent predictor for OS,but not an independent prognostic factor for RFS.3.Lower MCHC levels were closely related to male,higher smoking index,higher T-stage,and larger tumor volume.Part II Effects of Energy and Oxidative Stress on KRAS Mutant NSCLCObjectives:1.The relationship between LKB1 and KEAP1/NRF2 pathways.2.Effects of LKB1 and KEAP1/NRF2 pathways on NSCLC energy and redox homeostasis.3.Mechanisms of LKB1 and KEAP1/NRP2 on anti-oxidative stress.4.Treatment value of GLS inhibitors on NSCLC.5.The sensitivity of KRAS-LKB1-KEAP1 co-mutation to GLS inhibitors and glutamine dependence.Methods:1.Use TCGA data to perform co-mutation analysis on KRAS mutant NSCLC and use GSEA to study the possible effects of KEAP1 mutation on NSCLC.2.Use Lentivirus infection or CRISPR/cas9 to construct stable cell lines(LKB1 and KEAP1 overexpression),knock out NRF2 using siRNA.3.Cell proliferation was detected by sulforhodamine B,clone formation assay and CellTiter-Glo assay.Cell cycle was detected by BrdU and PI methods.Apoptosis was detected by Annexin V/7-AAD,and ROS level was measured by DCFDA.Western blot was used to detect protein expression.ATPlite was used to detect intracellular ATP levels.The content of various metabolites in the cells were detected by kits.2-NBDG was used to detect the cellular glucose uptake ability.Result:1.There is a significant co-mutation between KRAS and LKB1 in NSCLC,and there are obvious co-mutations between LKB1 and KEAP1.2.LKB1 plays an important role in maintaining the energy and redox homeostasis of KRAS mutant tumor cells,but LKB 1-deficient cells are sensitive to energy stress but resistant to oxidative stress.3.LKB1 can regulate NRF2 expression through KEAP1-independent pathway.4.Glutamine plays an important role in KLK tumors on proliferation and maintaining redox homeostasis.5.GLS inhibitors CB-839 and IACS-006274 have good anti-tumor activity and can inhibit tumor cell proliferation through cycle inhibition,but they can not cause tumor cell apoptosis.6.GLS inhibitors can induce tumor oxidative stress and energy stress,and its tumor inhibitory effect can be rescued by pyruvate and reducing agent NAC.7.KRAS-LKB1-KEAP1 tumors are most sensitive to GLS inhibitors,KRAS-LKB1 mutations or KRAS-KEAP1 mutations are more sensitive to GLS inhibitors than KRAS single mutation.Conclusion:1.LKB1 mutant tumors can induce upregulation of NRF2 expression through KEAP1 mutation or KEAP1 independent pathway and produce dependence on glutamine.2.Anti-tumor effects of GLS inhibitors are induced by the induction of energy and oxidative stress.3.GLS inhibitors have the best effect on KRAS-LKB1-KEAP1 co-mutation tumors,and may become biomarkers for clinical use of GLS inhibitors.