NRF2/KEAP1 Pathway Feedback And Its Impact On Sensitivity Of Chemotherapy And Radiotherapy In Non-small Cell Lung Cancer Promoter

Part I Cisplatin treatment activates NRF2 signal in lung cancer cell line,and the degree of activation depends on KEAP1 mutant statusPurpose: NRF2 is a pivotal b ZIP transcription factor involved in maintaining redox homeostasis and cellular metabolism.However,activation of NRF2 during chemotherapy in lung cancer cell line hasn’t been widely studied.In this part,we focused on variety of NRF2 signal during cisplatin treatment in lung cancer cell line,and also on what influence would interventing towards this pathway will bring to cisplatin’s cytotoxity.Experimental Design: 1.Specify KEAP1 mutant status in A549,H460,H292 and SKMES-1 with Sanger sequencing,depict baseline protein expression of NRF2 signal in above lung cancer cell line with westernblot.2.Observe variation of NRF2 signal by q-PCR,westernbot and immunofluorescence during cisplatin treatment.3.Construction of NRF2 downstream gene’s promoter into reporter plasmid and detect their activity during cisplatin treatment.4.Mutagenesis of KEAP1 ORF and observe the influence of the mutation on cisplatin cytotoxity.5.Interfere NRF2 activity by t BHQ or si RNA,observe variance of nuclear damage marker γ-H2 AX and after the intervention detect cells’ sensitivity to cisplatin.Results: 1.A549 and H460 cell carry KEAP1 mutation while H292 and SKMES-1 don’t.Mutant cell lines tend to express higher nuclear NRF2 and its downstream transcripts.2.At m RNA and protein level,during cisplatin treatment,NRF2 signal in KEAP1 mutant cell lines elevates violently while in KEAP1 wildtype cell lines doesn’t.3.Promoter activity of NRF2 transcripts is induced significantly during cisplatin treatment in A549 cell line while in H292 it is not.4.Modificaiton of inheret KEAP1 mutant status of lung cancer cell line changes NRF2 signal pattern and also the sensitivity to cisplatin.5.Intervention NRF2 activity confers to altered cisplatin toxity putatively by changing nuclear damage.Conclusions: NRF2 signal reaction after cisplatin depends on KEAP1 mutant status in lung cancer cell line.Baseline NRF2 activity and KEAP1 mutant status affects cisplatin toxity in lung cancer cell line.Part II Radiation activates NRF2 signal in lung cancer cell line,and the degree of activation depends on KEAP1 mutant statusPurpose: Reactive Oxygen Species and electrophile groups caused by indirect biological effect of ionizing radiation are appropriate trigger for NRF2 signal activation.Within this part,we will explore alteration of NRF2 signal after irradiation in lung cancer cell line,as well as NRF2 signal influence on lung cancer cells’ radiation sensitivity.Experimental Design: 1.Observe variation of NRF2 signal by reporter assay and westernbot during irradiation in A549 and H292.2.Verify activation of NRF2 signal in GSE10547,which detect polysome m RNA alteration during radiation.3.Interfere NRF2 activity in A549 and H292 cell line and observe apoptosis fraction caused by irradiation.Detect nuclear damage within this process by westernblot and evaluate capacity of mentioned cell line to form colony by clone formation assay.Results: 1.For promoter activity and protein expression,upon radiation,NRF2 signal in A549 elevates violently while in H292 doesn’t.2.Polysome m RNA expression profile for A549 and HOP62 demonstrate same trend for NRF2 signal during radiation.3.Depletion of NRF2 confers to radiosensitizing in A549,and enhancement of NRF2 confers to radioresistant in H292.Conclusions: NRF2 signal reaction towards radiation depends on KEAP1 mutant status in lung cancer cell line.Baseline NRF2 activity and KEAP1 mutant status affects radiation sensitivity in lung cancer cell line.Part III NRF2 transactivates KEAP1 by binding to antioxidant responsive element located in KEAP1 promoterPurpose: To identify the phenomenon of NRF2 transactivating KEAP1 and explore the molecular mechanisms by which NRF2 regulate KEAP1 expression.Experimental Design: 1.Detect KEAP1 protein abundance under t BHQ treatment in Bease2 B,H292 and SKMES-1 cell line,or under si NRF2 transfection in A549 cell line.2.Evaluate significance of putative ARE in KEAP1 promoter by mutagenic reporter assay.3.Re-analysis Ch IP-seq data produced by GSE37589.Call peak between SFN and Control group to see KEAP1 promoter enrichment by NRF2 activation.4.Genomic edit ARE in HEK293 T and H292 cell line,then quatify baseline KEAP1 m RNA expression.Results: 1.NRF2 activation by t BHQ robust increases KEAP1 protein expression in a transient manner,while NRF2 knockdown by si RNA diminishes KEAP1 protein level.2.In H292 and HEK293 T cell line,ΔARE Mutagenic KEAP1 promoter reporter showed significant altered activity after NRF2 activation or overexpression,while ΔSP1 mutation doesn’t.In A549 cell line,ΔARE mutant only confers to baseline activity inhibition.3.KEAP1 promoter was robust enriched under SFN treatment(p=1E-59,FDR q=1E-52).The enriched region includes our interest region in reporter assay.4.CRISPR-Cas9 directed ΔARE in genomic region significantly reduced KEAP1 m RNA expression in H292 and HEK293 T cells.Conclusions: NRF2 regulates KEAP1 expression in lung epithelia and cancer cell line by binding to ARE in KEAP1 promoter.Part IV Evidence in clinical specimen for NRF2 regulation upon KEAP1Purpose: Seek for evidence for putative feedback regulation of NRF2 on KEAP1 in lung cancer genetic and genomic profile.Experimental Design: 1.Retrieve gene-normalized RSEM,methylation 450 K array and DNA copy number variation data for TCGA lung adenocarcinoma and squamous cell lung cancer respectively,analyze genetic factor that influence KEAP1 expression in both pathology subgroup by multiple linear regression.2.Static model NRF2 downstream gene and make correlation between module eigenvalue with KEAP1.3.Compare baseline m RNA expression between squamous cell lung cancer and adenocarcinoma by meta-analysis.Results: 1.KEAP1 m RNA expression highly correlates with NRF2 expression in squamous cell lung cancer.2.In multiple dataset,KEAP1 m RNA expression highly correlates with module eigenvalue produced from NRF2 downstream gene expression.And the direction was same with NRF2 expression.3.For both KEAP1 and NRF2 gene,m RNA expression in squamous cell lung cancer is significant higher than in lung adenocarcinoma.KEAP1 mutant/NRF2 pathway mutant tumor express higher KEAP1 m RNA.Conclusions: NRF2 correlated with KEAP1 expression in lung squamous cell lung cancer specimen.NRF2 and KEAP1 expression are higher in squamous cell lung cancer than in adenocarcinoma.Mutant tumors expresses higher KEAP1 m RNA than wildtype ones.