Research Paper Volume 11, Issue 24 pp 12600—12623

Genome-wide global identification of NRF2 binding sites in A549 non-small cell lung cancer cells by ChIP-Seq reveals NRF2 regulation of genes involved in focal adhesion pathways

Akhileshwar Namani 1, *, , Kaihua Liu 2, *, , Shengcun Wang 1, *, , Xihang Zhou 1, , Yijiao Liao 1, , Hongyan Wang 1, , Xiu Jun Wang 2, , Xiuwen Tang 1, ,

  • 1 Department of Biochemistry and Department of Thoracic Surgery of the First Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang University, Hangzhou 310003, PR China
  • 2 Department of Pharmacology and Cancer Institute of the Second Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang University, Hangzhou 310058, PR China
* Equal contribution

received: September 20, 2019 ; accepted: November 26, 2019 ; published: December 28, 2019 ;

https://doi.org/10.18632/aging.102590
How to Cite

Copyright © 2019 Namani et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Nuclear factor erythroid-derived-2-like 2(NRF2) regulates its downstream genes through binding with antioxidant responsive elements in their promoter regions. Hyperactivation of NRF2 results in oncogenesis and drug resistance in various cancers including non-small cell lung cancer (NSCLC). However, identification of the genes and pathways regulated by NRF2 in NSCLC warrants further investigation. We investigated the global NRF2 genomic binding sites using the high-throughput ChIP-Seq technique in KEAP1 (Kelch-like ECH-associated protein 1)-mutated A549 (NSCLC) cells. We next carried out an integrated analysis of the ChIP-Seq data with transcriptomic data from A549 cells with NRF2-knockdown and RNA-Seq data from TCGA patients with altered KEAP1 to identify downstream and clinically-correlated genes respectively. Furthermore, we applied transcription factor enrichment analysis, generated a protein-protein interaction network, and used kinase enrichment analysis. Moreover, functional annotation of NRF2 binding sites using DAVID v7 identified the genes involved in focal adhesion. Putative focal adhesion genes regulated by NRF2 were validated using qRT-PCR. Further, we selected one novel conserved focal adhesion gene regulated by NRF2–LAMC1 (laminin subunit gamma 1) and validated it using a reporter assay. Overall, the identification of NRF2 target genes paves the way for identifying the molecular mechanism of NRF2 signaling in NSCLC development and therapy. Moreover, our data highlight the complexity of the pathways regulated by NRF2 in lung tumorigenesis.

Abbreviations

3′-UTR: 3′-untranslated regions; ARE: Antioxidant responsive element; ChIP-Seq: Chromatin Immunoprecipitation Sequencing; CUL3: Cullin-dependent E3 ligase; DAVID: Database for annotation visualization and integrated discovery; EGFR: Epidermal growth factor receptor; FDR: False discovery rate; HOMER: Hypergeometric Optimization of Motif EnRichment; KEA: Kinase Enrichment Analysis; KEAP1: Kelch-like ECH-associated protein 1; KEGG: Kyoto Encyclopedia of Genes and Genomes; LUAD: Lung adenocarcinoma; NRF2/ NFE2L2: Nuclear factor erythroid 2-related factor; NSCLC: Non small cell lung cancer; PPI: Protein-protein interaction; PPP: Pentose phosphate pathway; qRT-PCR: quantitative real-time PCR; sMAF: Small musculoaponeurotic fibrosarcoma; TCA: Tricarboxylic acid; TCGA: The Cancer Genome Atlas; TFBS: transcription factor binding sites; TFEA: Transcription Factor Enrichment Analysis; TRAP: Transcription factor Affinity Prediction; X2K: eXpression2Kinases.