Deregulation Of AIF Renders Cell Metabolism Shifts To Regulate Non Small Cell Lung Cancer Cell Survival By ROS Induced HIF1-α Stabilizition

Objective:Non small cell lung cancer(NSCLC)is the one of leading cause of cancer death.Growing evidences showed that person who diagnosed with NSCLC own poor outcomes and high recurrence rate.Although,more and more strategies of lung cancer therapy were developed,it is not efficiently to improve the outcomes of the patients.Based on the pathological type,lung cancer were classified into two major categories as non small cell lung cancer(NSCLC)and small cell lung cancer,and almost80-90% lung cancer patients were non small cell lung cancer patients.According to the majority of NSCLC patients,it is eager to develop more efficient and novel drug to improve the motility and outcomes of NSCLC patients.Until now,great improvement in NSCLC treatment were obtained,such as monoclonal antibodies against EGFR,PD1 and PD-L1,as well as promising immune therapy,but it is also urgently to discover novel target and prognostic maker due to the individual difference and chemo-resistance.Apoptosis-inducing factor(AIF)is a mitochondrial flavoprotein primarily named based on its pro-apoptotic activity by its nucleus translocation in various model systems.AIF can induce isolated nuclei to acquire features of apoptosis such as chromatin condensation and DNA fragmentation,Upon mitochondrial outer membrane permeabilization(MOMP)apoptotic pathways,AIF is released from mitochondria and subsequently translocated to the cytosol and then to the nucleus,where it fulfills its lethal function by inducing chromatin condensation and DNA degradation.Recently,evidences reported that AIF has a vital role inmitochondria: AIF is indispensable for the optimal function of the mitochondrial respiratory chain and is involved in redox metabolism.In addition,AIF was proposed as a tumor suppressor based on the crucial role in apoptosis.Though the role of AIF in modulating tumor cell growth and death was well elucidated in prostate cancer,colon cancer,and other cancer types via executing diversely functions,the potential role of AIF in NSCLC carcinogenesis remains largely unknown.This study focuses on investigating the potential role of AIF in regulating NSCLC cancer cell growth,whether it could be considered as an essential biomaker and target for NSCLC diagnosis and therapy.Methods1: Western blot and q RT-PCR were used to analyze the protein and m RNA level of indicated molecules;2: CCK-8 cell proliferation detection kit was performed to determine relative cell proliferation rate of sh Cont and AIF knockdown A549 and H1299 cells in vitro;xenograft tumor assay was applied to evaluate cancer growth of sh Cont and AIF knockdown of H1299 cells in vivo;3: The alteration of cellular overall OCR and aerobic glycolytic rate of sh Cont and AIF knockdown cells were detected by seahorse XF96 bioenergetics analyzer;4: Cell total ROS level was confirmed by flow cytometry analysis;5: Glucose uptake detection kit and extracellular lactate level were used to evaluate the level of glucose and lactate level due to AIF depletion,respectively;6: The survival curve of NSCLC patients with different AIF and HIF1-α expression were carried out by Kaplan-Meier survival analysis,χ2 test was applied to investigate the association between AIF or HIF1-α level and various clinical and pathological parameters of NSCLC patients.Results1: Dramatically overexpressed AIF(both protein and m RNA level)in tumor tissues compared to that in adjacent non-cancerous tissues of NSCLC patients;2: AIF knockdown led to NSCLC cancer cell growth in vitro and in vivo;3: Depletion of AIF caused significant reduction of certain subunits of mitochondrial respiration chain complexes,but not m RNA level and mt DNA contents;4: Upon normoxia,reduced OXPHOS activity was found in AIF downregulated cells,but not aerobic glycolysis;5: Under hypoxia condition,downregulation of AIF destabilized HIF1-α and subsequently suppressed downstream target gene transcription,while repressed glucose uptake and lactate generation;6: AIF knockdown inhibited cancer cell metabolic reprogramming under hypoxia;7: AIF depletion reduced ROS level which further destabilized HIF1-α stability as well as suppressed cell metabolism;8: Due to Kaplan-Meier analysis,overexpression of AIF and HIF1-α indicated poor outcomes of NSCLC patients,χ2 test showed that closely correlation between AIF/HIF1-α and clinical and pathological indexes.Conclusion Here,we found overexpressed AIF in NSCLC tissues and AIF deletion led to cancer cell growth delay.And our data indicated that AIF play a crucial role in mitochondrial respiration homeostasis.Additionally,AIF functioned as a potential factor in regulating cancer cell metabolic reprogramming through ROS mediated HIF1-αstability.Based on the survival analysis,we found AIF and HIF1-α overexpression indicated much poor outcomes of NSCLC patients.Moreover,our data suggested that AIF overexpression closely related to clinical stage and metastasis,while aberrant upregulation of HIF1-α associated with tumor size,clinical stage and metastasis.In conclusion,our data suggested that AIF may play a fundamental role in NSCLC carcinogenesis beyond its apoptosis inducible activity.These findings may provide a novel mechanism of NSCLC tumorigenesis and an essential biomaker for early diagnosis and therapeutic target for NSCLC.