Binding partners of NRF2: Functions and regulatory mechanisms.

NRF2 is a redox-sensitive transcription factor that plays an important role in protecting organisms against diverse types of electrophiles or oxidants. The level of NRF2 is maintained low in normal cells, but highly elevated in cancer provoking chemoresistance or radioresistance. It is now recognized that NRF2 does not merely maintain the redox balance, but also plays significant roles in autophagy, apoptosis, cell cycle progression, and stem cell differentiation, all of which could be possibly attributable to the existence of multiple binding proteins. In the present manuscript, we summarize direct binding partners of NRF2 and illustrate how they bind to NRF2 and regulate its stability or activity.

Homoharringtonine stabilizes secondary structure of guanine-rich sequence existing in the 5'-untranslated region of Nrf2.

Homoharringtonine, known as omacetaxine mepesuccinate, is a pharmaceutical drug substance approved for treatment of chronic myeloid leukemia. Here, we report that homoharringtonine (HHT) is a novel chemical inhibitor of NRF2. HHT significantly suppressed NRF2 and ARE-dependent gene expression in human lung carcinoma A549 cells. HHT stabilized secondary structure of guanine-rich sequence existing in the 5'-untranslated region (5'-UTR) of Nrf2 and sensitized A549 cells to etoposide-induced apoptosis. To the best of our knowledge, HHT is the first type of transcriptional inhibitor of Nrf2 that stabilizes guanine-rich sequence existing in the 5'-UTR. Our study also provides a novel mechanism of action underlying how HHT exerts anti-carcinogenic effects in cancer cells.

Structure, Inhibitors, and Biological Function in Nervous System and Cancer of Ubiquitin-Specific Protease 46.

Deubiquitinating enzymes (DUBs) prevent ubiquitination by eliminating ubiquitin from their substrates. Deubiquitinating enzymes have important roles in a number of cell biology subfields that are highly relevant to diseases like neurodegeneration, cancer, autoimmune disorders, and long-term inflammation. Deubiquitinating enzymes feature a well-defined active site and, for the most part, catalytic cysteine, which makes them appealing targets for small-molecule drug development. Ubiquitin-specific protease 46 (USP46) is a member of the ubiquitin-specific protease family, the largest subfamily of DUBs. Over the past 10 years, some studies have steadily demonstrated the significance of USP46 in several biological processes, although it was identified later and early research progress was modest. Specifically, in the last few years, the carcinogenic properties of USP46 have become more apparent. In the current review, we provide a comprehensive overview of the current knowledge about USP46 including its characteristics, structure, inhibitors, function in diseases, especially in the nervous system, and the correlation of USP46 with cancers.

BAP1 Downregulates NRF2 Target Genes and Exerts Anti-Tumorigenic Effects by Deubiquitinating KEAP1 in Lung Adenocarcinoma.

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.

Triptolide Downregulates the Expression of NRF2 Target Genes by Increasing Cytoplasmic Localization of NRF2 in A549 Cells.

We have identified triptolide as a novel NRF2 inhibitor, which significantly attenuates ARE-luciferase activity at nanomolar concentrations. Triptolide did not affect the level of NRF2, but significantly inhibited the expression of NRF2 target genes in A549 cells. We found that NRF2 possesses a previously unrecognized NES in the Neh2 domain, and that triptolide promotes an interaction between NRF2 and CRM1. Triptolide also decreased nuclear accumulation of NRF2, suggesting that it promotes nuclear export of NRF2. In addition, we show that triptolide decreased the expression of NRF2 target genes and increased intracellular oxidative stress, suppressing invasion and promoting cisplatin-induced apoptosis in A549 cells. Finally, oral administration of triptolide suppressed the growth of A549 xenografts in athymic mice by decreasing the expression of NRF2 target genes and promoting oxidative damages via the nuclear export of NRF2 and CRM1 in vivo. To the best of our knowledge, triptolide is the first type of compound to inhibit NRF2 by increasing cytoplasmic localization of NRF2.

Regulation of NRF2 by Na+/K+-ATPase: implication of tyrosine phosphorylation of Src.

Tumors adapt well to the imbalanced redox status created by rapid growth and limited nutrient availability because they highly express high levels of NRF2 to counteract oxidative stress. Therefore, inhibition of NRF2 is currently considered a feasible strategy for development of chemotherapeutic agents. In the present study, we identified that Na+/K+-ATPase regulates NRF2 in A549 cells. Suppression of Na+/K+-ATPase by convallatoxin or siRNAs downregulates NRF2 in A549 cells, and this event is mediated by Ca2+-dependent induction of CSK1 and subsequent phosphorylation of SRC at Tyr 527. Consistent with this finding, knocking down the α1 or ß1 subunit of Na+/K+-ATPase promotes the generation of intracellular ROS by cisplatin and potentiates cisplatin-induced apoptosis and autophagy in A549 cells. Our study reveals that the signaling axis composed of Na+/K+-ATPase, CSK1, and tyrosine phosphorylation of Src could be a useful target for development of NRF2 inhibitors.

Molecular mechanisms and systemic targeting of NRF2 dysregulation in cancer.

NF-E2-related factor 2 (NRF2) is a master regulator of redox homeostasis and provides cellular protection against oxidants and electrophiles by inducing the expression of a wide array of phase II cytoprotective genes. Until now, a number of NRF2 activators have been developed for treatment of chronic diseases and some are under evaluation in the clinical studies. On the other hand, accumulating evidence indicates that NRF2 confers chemoresistance and radioresistance, and its expression is correlated with poor prognosis in cancer patients. Studies in the last decade demonstrate that diverse mechanisms such as somatic mutations, accumulation of KEAP1 binding proteins, transcriptional dysregulation, oncogene activation, and accumulation of reactive metabolites contribute to NRF2 activation in cancer. In the present review, we illustrate the molecular mechanisms governing the function of NRF2 and explain how they are hijacked in cancer. We also provide some examples of NRF2 inhibitors together with a brief explanation of their mechanisms of action.

Suppression of NRF2/ARE by convallatoxin sensitises A549 cells to 5-FU-mediated apoptosis.

NF-E2-related factor 2 (NRF2) regulates transcription of phase II cytoprotective enzymes to protect normal cells against oxidative stress. However, a high level of NRF2 offers a growth advantage, chemoresistance, and radioresistance in cancer. In the present study, we have identified convallatoxin as a novel inhibitor of NRF2/ARE. Suppression of NRF2 by convallatoxin was not transcriptionally mediated, but regulated at the level of proteolysis. Convallatoxin activated GSK-3ß and suppression of NRF2 by convallatoxin required the Neh6 domain. Convallatoxin sensitised A549 cells to 5-fluorouracil-mediated cell death by promoting apoptosis. Together, our results provide evidence that convallatoxin might be useful as a chemotherapeutic adjuvant due to its ability to suppress NRF2/ARE.