Abstract
KELCH-ECH-associated protein 1 (KEAP1) is an adaptor protein of Cullin 3 (CUL3) E3 ubiquitin ligase that targets a redox sensitive transcription factor, NF-E2-related factor 2 (NRF2). BRCA1-associated protein 1 (BAP1) is a tumor suppressor and deubiquitinase whose mutations increase the risk of several types of familial cancers. In the present study, we have identified that BAP1 deubiquitinates KEAP1 by binding to the BTB domain. Lentiviral transduction of BAP1 decreased the expression of NRF2 target genes, suppressed the migration and invasion, and sensitized cisplatin-induced apoptosis in human lung adenocarcinoma (LUAD) A549 cells. Examination of the lung tissues in KrasG12D/+ mice demonstrated that the level of Bap1 and Keap1 mRNAs progressively decreases during lung tumor progression, and it is correlated with NRF2 activation and the inhibition of oxidative stress. Supporting this observation, lentiviral transduction of BAP1 decreased the growth of A549 xenografts in athymic nude mice. Transcriptome analysis of human lung tissues showed that the levels of Bap1 mRNA are significantly higher in normal samples than LUAD samples. Moreover, the expression of Bap1 mRNA is associated with a better survival of LUAD patients. Together, our study demonstrates that KEAP1 deubiquitination by BAP1 is novel tumor suppressive mechanism of LUAD.
Highlights
NF-E2-related factor 2 (NRF2) is a transcription factor that plays an important role in the detoxification of reactive oxygen species (ROS) by transcriptional activation of phaseII cytoprotective enzymes [1]
In order to identify the DUBs of KELCH-ECH-associated protein 1 (KEAP1), pcDNA3-FLAG-DUB plasmids were transfected in 293T cells and the level of endogenous KEAP1 was measured by Western blot analysis
Among DUBs, we observed that overexpression of FLAG-BRCA1-associated protein 1 (BAP1) or FLAG-Ubiquitin C-terminal hydrolase L3 (UCHL3) induced KEAP1 (Figure 1A)
Summary
NF-E2-related factor 2 (NRF2) is a transcription factor that plays an important role in the detoxification of reactive oxygen species (ROS) by transcriptional activation of phaseII cytoprotective enzymes [1]. NRF2 is located in the cytosol and constantly targeted for polyubiquitination by KELCH-like ECH-associated protein. Exposure of oxidative insults halts polyubiquitination of NRF2, which causes NRF2 to translocate into the nucleus and initiates transcriptional activation of phase II cytoprotective enzymes by binding to the antioxidant response element (ARE), a cis-acting motif sequence existing in the promoter of NRF2 target genes [3]. Antioxidants 2022, 11, 114 in the last decade have demonstrated that NRF2 is constitutively activated in cancer due to somatic mutations in the KEAP1/NRF2 pathway, conferring chemoresistance and radioresistance [5]. Comprehensive genomic analyses have identified that somatic Keap mutations in cancer frequently occur together with the alterations in other tumor suppressor genes or oncogenes, such as Tp53, Cdkn2a, Pten, and Pik3ca [7]. A homodimer of KEAP1 binds to a single NRF2 at the DLG and ETGE motifs existing in the Neh domain [8]
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