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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.

Alterations in KEAP1/ NF-E2 p45-related factor 2 (NFE2L2/Nrf2) signaling pathway have been reported in 23% lung adenocarcinoma patients, suggesting that deregulation of the pathway is a major cancer driver. Here we report that mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1) drives tumor growth and drug resistance by up regulating transcription factor Nrf2. In non-small cell lung cancer (NSCLC) cells and xenografts, MKP-1 knockdown triggered the down-regulation of the metabolic enzymes and cytoprotective proteins, which are the target genes of Nrf2. Consequently, the cell growth was markedly inhibited with decrease of tumor metabolisms and GSH contents. Moreover, MKP-1 silencing sensitized NSCLC cells to cisplatin treatment. Mechanistically, MKP-1 inhibited the ubiquitylation of Nrf2 via a direct interaction with the transcription factor. Nrf2 was hence stabilized and its transcriptional program was activated. Notably, Nrf2 elevated MKP-1 expression at transcriptional level. In human lung adenoma tumor samples, high levels of expression of MKP-1, Nrf2, and its target gene heme oxygenase 1 were closely correlated. Thus, MKP-1 and Nrf2 form a forward feedback loop in lung cancer cells, which stabilizing and activating Nrf2 to promote anabolic metabolism and GSH biosynthesis. This study uncovers a novel role of MKP-1 in the malignant evolution of cancers.