Identification Of NRF2 Regulated Pathways And Biomarkers In LUAD And HNSCC

BackgroundTrancriptomic and genomic profiling of cancers such as microarray,RNA-Seq and ChIP-Seq are widely using effective approaches for the identification of dysregulated genes and pathways in tumor biology.Specifically,Chromatin immunoprecipitation followed by next generation sequencing(ChIP-Seq)is an indispensable tool for identifying novel transcription factor binding sites and their regulated pathways in different cancers.Recent studies of The Cancer Genome Atlas(TCGA)have provided the wealth of information about next generation high-throughput platforms and technologies in wide variety of cancers.This highly valuable data provided by the TCGA network allows the researchers to identify the somatic nutations,copy number alterations of their genes of interest across thousands of patient's samples.NRF2 is the key transcription factor which regulates oxidative stress in normal cells through binding with the cis-acting regulatory sequence present in the promoter regions of its downstream gens which is termed as antioxidant response element(ARE).The well known NRF2 encoding genes include phase ?/? drug metabolism enzymes,cytoprotective proteins.NRF2 negatively regulated by KEAP1 via CUL3 mediated ubiquitination and proteosomal degradation.Aberrant expression of NRF2 pathway due to genetic alterations of KEAP1-NRF2-CUL3 axis leads to tumorigenesis and drug resistance in many cancers.Thus,NRF2 became an important therapeutic target in human cancers and inhibition of NRF2 overexpression could pave way to inhibit tumor growth and drug resistance.Stable KEAP1 overexpression and NRF2 siRNA knock down of Human Non Small Cell Lung Cancer(NSCLC)A549 cells were generated in our lab.To identify novel NRF2 regulated genes in A549 cells,microarray analysis of both cell lines was performed.By utilizing RNA-Seq data of TCGA network and with other bioinformatics tools,multi-omics approach was applied to discover the NRF2-dependent genes,pathways and potential biomarkers in two human cancers such as lung adeno carcinoma(LUAD)and Head and Neck Squamous Cell Cancer(HNSCC).In addition,ChIP-Seq analysis of NSCLC-A549 cells that contain dysfunctional KEAP1 mutation that shows NRF2 overexpression was also carried out to identify known and novel NRF2 regulated binding sites and pathways.ObjectiveThis research focused on the identification of known and novel NRF2 regulated genes,pathways,and potential biomarkers in human cancers such as LUAD and HNSCC by using high-throughput omics data.Method1.Establishment of stable KEAP1 overexpression A549 cell linesa.Cultures of the A549 lung adeno carcinoma cell line were transfected with empty pEGFP plasmid and mKeap1-pEGFP plasmid using the LipofectamineTM 2000 reagent.b.mRNA and protein expression levels of NRP2 and its regulatory genes such as HO-1,NQO1 in KEAP1 overexpression A549 cell lines were examined by using RT-PCR and western blot.c.Total RNA extraction was carried out with TRizol(Invitrogen)reagent,following fragmentation and hybridization on Affymetrix Human Gene Expression Array-Prime View.2.Identification NRF2 regulated metabolic gene signature(NRMGS)as a predictive biomarkers in Non small cell lung cancer(NSCLC).a.Using Agilent GeneSpring GX software(Version 11.0),individual and combinatorial microarray analysis was carried out for KEAP1 overexpression and previously developed NRF2 siRNA knock down A549 NSCLC cells.b.Using DAVID bioinformatics tool and STRING database,functional annotation and protein-protein interaction network analyses were carried out for upregulated genes in both microarrays.c.RSAT tool was used to identify ARE sequences in the promoter regions of known and putative NRF2 regulated genes.d.Correlation analysis of NRF2 regulated metabolic genes expression with KEAP1 altered TCGA-LUAD RNA-Seq gene expression data was carried out for the identification of NRF2 regulated metabolic gene signature(NRMGS).e.Overall survival analysis and Kaplan Meier analysis of NRMGS was performed by using an online multi cancer biomarker validation tool "SurvExpress."3.Identification KEAP1-NRF2-CUL3 axis regulated gene expression signature in TCGA-Head and Neck Squamous Cell Cancer(HNSCC).a.RNA-Seq V2 level 3 data of 279 tumor samples along with 37 adjacent normal samples that are enrolled in TCGA-HNSCC study was used to identify the upregulated genes in two methods(KEAP1-NRF2-CUL3 altered versus normal and KEAP1-NRF2-CUL3 altered versus wild type).b.Combinatorial analysis of upregulated genes in two methods was performed to identify KEAP1-NRF2-CUL3 axis regulated gene signature in HNSCC.c.Subsequently this gene signature was tested in 4 independent HNSCC datasets to validate its prognostic value.d.In addition,functional annotation and PPI analysis of this gene signature was performed by using DAVID v6.8 and STRING v10 databases respectively.4.Genome-wide global identification of NRF2 binding sites in A549 NSCLC by ChIP-Seq includes four steps.a.Chromatin Immunoprecipitation followed by sequencingb.Peak calling and de novo motif discovery analysis by HOMER softwarec.Functional pathway analysis by DAVIDd.Validation of ChIP-Seq data by Real-time RT-PCRResults1.Successfully established the stably transfected A549 NSCLC cells contain mKeap1-pEGFP plasmid encodes KEAP1 overexpression along with cell line contains empty pEGFP plasmid for control.NRF2 and its regulatory genes HO-1,NQO1 mRNA and protein expression levels were significantly reduced in clones labeled A549-mKeapl-5p as compared with the control A549-pEGFP clone.Thus,the lower NRF2 and HO-1,NQO1 genes expression was indicating the sucessful negative regulation ofNRF2 in A549 cells.2.By focusing on the intersection of genes with significant downregulation in KEAP1 overexpression and NRF2 siRNA A549 knockdown cells with a fold change cutoff>1.5,214 overlapping common NRF2 regulated genes were identified in both A549 cells microarray data.A cluster of 34 genes involves in 'small molecule metabolic process'was identified by using GO analysis.Further study on identification of AREs in the promoters of these genes deciphered evidence that NRF2 may directly regulates 27 genes through its ARE.12 key genes among 27 genes were significantly upregulated in KEAP1 altered samples of TCGA-LUAD RNA-Seq gene expression data as compared with wild type samples.Because of the aggressive expression in KEAP1 altered samples,these 12 genes were named as NRF2 regulated metabolic genes signature(NRMGS).Subsequent overall survival analysis in 8 independent LUAD cohorts revealed that,genes present in NRMGS are the prognostic biomarkers in LUAD.3.A subset of seventeen KEAP1-NRF2-CUL3 axis regulated genes signature was identified by overlapping both upregulated genes of altered versus normal(251 genes)and altered versus wild type(25 genes)datasets in TCGA-HNSCC.An increased expression of this gene expression was significantly associated with poor survival in 4 independent HNSCC datasets including TCGA-HNSCC.Furthermore,GO,KEGG and PPI analysis revealed that majority of the genes present in this signature are associated with drug metabolism and glutathione metabolism pathways.4.ChIP-Seq peak calling analysis by HOMER software identified 2395 strongly enriched genome-wide Nrf2-binding sites with 150 bp peak size using tags on both strands in A549 cells.KEGG pathway analysis NRF2-binding sites by DAVID identified novel NRP2 regulated pathway known as 'focal adhesion' in lung cancer cells.Presence of ARE sequences and qRT PCR results suggests that,a direct association of NRF2 with the set of genes involved in focal adhesion pathway.ConclusionsAltogether,our research emphasized on the discovery of NRF2 regulated metabolic genes signature(NRMGS),KEAP1-NRF2-CUL3 axis regulated gene signature in LUAD and TCGA-HNSCC respectively.These signatures are highly implicated in tumorigenesis and drug resistance of respective cancers and could be used as potential biomarkers and therapeutic targets for NRF2 pathway activated tumor cases.In addition,the ChIP-Seq results discovered a novel NRF2 regulated focal adhesion pathway that plays a major role in invasion,migration,metastasis of tumor cells.