Aberrant NALP3 Regulates Cellular Bioenergetics To Facilitate Non Small Cell Lung Cancer Cell Growth

Purpose:Lung cancer is the leading cause of cancer death and the non-small cell lung carcinorma?NSCLC?which represents 80%of all cases.Although the targeted therapeutics for lung cancer harboring some benefits,the chemoresistence and survival post operation of patients should be deeply concerned.Previous work has reported the closely correlation between inflammation and carcinogenesis,while the role of NALP3,the key component of inflammasome activation in NSCLC remains elusive.The objective of this study was to unravel the mechanism of NALP3 execution on NSCLC cancer cell growth and the prognostic value of NALP3.Experimental methods:1:Western blot was used to analysis the protein level of indicated molecules,real-time PCR was performed to determine the alteration of mRNA transcripts;2::In vitro cell growth curve was graphed by cell number counting by trypan blue staining;3:In vivo cancer cell growth was evaluated by xenograft tumor assay;4:XF96 seahorse cellular bioenergetics analyzer was used to detect the alteration of cellular oxygen consumption rate and extracellular acidification rate in control and NALP3downregulated cells;5:Glucose uptake assay kit and lactate production detection kit were performed to determine the glucose uptake rate and relative lactate production in control and NALP3depleted A549 and H1299 cells;6:Cell total ROS level was detected with DCFH-DA cell ROS kit by flow cytometry;7:The activity of mitochondrial respiration activity was detected by spectrophotometry;8:Co-Immunoprecipitation assasy was performed to confirm the interaction between NALP3 and DMAP1;9:Collect the tumor and adjacent non-cancerous tissues of NSCLC patients and construct the Tissue Micro Array?TMA?,immunohistochemistry assay was performed to visualize the expression level of NALP3 in normal and NSCLC tissues;10:?² test was applied to evaluate the association between NALP3 and various clinical and pathological parameters,and Cox`proportional hazards model was used assess the hazard ratio of these parameters;11:The survival time of NSCLC was analyzed by Kaplan-Meier curve.Results1:Our results showed dramatically increased NALP3,NF-B,STAT3 and IL-1which were key components of inflammation activation in tumor tissues of NSCLC patients compared to that in adjacent non-cancerous tissues;2:In A549 and H1299 cells,NALP3 depletion caused delayed cell growth and decreased colony formation activity in vitro as well suppressed tumor growth in vivo;3:Our data showed that NALP3 knockdown caused enhanced cellular oxygen consumption rate while decreased extracellular acidification rate in A549 and H1299 cell lines.4:Depletion of NALP3 led to excessive ROS production,and promoted mtDNA replication;5:NALP3 knockdown decreased cell glucose uptake and lactate production,as well as the expression of key enzymes which participate in aerobic glycolysis;downregulation of NALP3 significantly enhanced mitochondrial biogenesis and activity of mitochondrial respiration;6:NALP3 directly interacted with DMAP1,and NALP3 knockdown decreased DNMT1 protein level,but not mRNA transcript;7:Downregulation of DNMT1 reversed aerobic glycolysis to oxidative phosphorylation;8:Dramatically overexpressed DNMT1 was found in NSCLC tumor tissues,and knockdown DNMT1 significantly suppressed A549 and H1299 tumor cell growth;9:IHC data indicated that NALP3 was significantly overexoressed in tumor sections of NSCLC patients;and Kaplan-Meier survival curve analysis results suggested much poor outcomes post-operation of NSCLC patients owning relative higher NALP3 staining;10:?² test results validated that NALP3 level was closely associated TNM stage,tumor grade and lymph node metastases,but not age,gender,smoking history,alcohol history and T stage.Conclusions:Here,we demonstrated that RNAi mediated NALP3 depletion delayed cancer cell growth in vitro and in vivo which suggests the potential role of NALP3 in NSCLC carcinogenesis.In addition,downregulation of NALP3 promotes metabolic reprogramming from OXPHOS to aerobic glycolysis.NALP3 knockdown leads to metabolic switch via reversing Warburg effect.Furthermore,we found that NALP3 could directly interact with DMAP1 and regulate DNMT1,which finally promotes metabolicreprogramming.And we also demonstrated that high NALP3 was closely correlated with TNM stage,tumor grade and lymph node metastases,and indicating poor outcomes of NSCLC patients.Based on these data,we suggest that NALP3 may play a crucial role in regulating NSCLC cancer cell growth through promotes cellular metabolic reprogramming,and could be considered as a novel diagnostic biomarker and therapeutic target for NSCLC patients.