Description of transcriptome in non-small cell lung cancer cell lines without KEAP1/NRF2 mutations in response to an electrophilic reagent dimethylmaleate (DEM)
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE118842 | GEO | 2020/09/23
REPOSITORIES: GEO
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Project description:Description of transcriptome in non-small cell lung cancer cell lines without KEAP1/NRF2 mutations in response to an electrophilic reagent dimethylmaleate (DEM)
| PRJNA486962 | ENA
Project description:We report NRF2-dependent changes in the transcriptome of A549, H460 and H2023 cell lines.
2020-09-23 | GSE118841 | GEO
Project description:Nrf2 is the major transcription factor that regulates many of the cytoprotective enzymes involved in the adaptive stress response. Modulation of Nrf2 could be therapeutically useful in a number of disease states. Activation can occur through either an electrophilic or non-electrophilic mechanism. To date, most of the research has focused on electrophilic Nrf2 activators, but there is increasing interest in non-electrophilic modulators of Nrf2. This Digest examines the current selection of small molecules that modulate Nrf2 through non-electrophilic mechanisms, and it highlights new opportunities for this important therapeutic target.
| S-EPMC4643947 | biostudies-literature
Project description:The Keap1-Nrf2 system is the major regulatory pathway of cytoprotective gene expression against oxidative and/or electrophilic stresses. Keap1 acts as a stress sensor protein in this system. While Keap1 constitutively suppresses Nrf2 activity under unstressed conditions, oxidants or electrophiles provoke the repression of Keap1 activity, inducing the Nrf2 activation. However, the precise molecular mechanisms behind the liberation of Nrf2 from Keap1 repression in the presence of stress remain to be elucidated. We hypothesized that oxidative and electrophilic stresses induce the nuclear accumulation of Nrf2 by affecting the Keap1-mediated rapid turnover of Nrf2, since such accumulation was diminished by the protein synthesis inhibitor cycloheximide. While both the Cys273 and Cys288 residues of Keap1 are required for suppressing Nrf2 nuclear accumulation, treatment of cells with electrophiles or mutation of these cysteine residues to alanine did not affect the association of Keap1 with Nrf2 either in vivo or in vitro. Rather, these treatments impaired the Keap1-mediated proteasomal degradation of Nrf2. These results support the contention that Nrf2 protein synthesized de novo after exposure to stress accumulates in the nucleus by bypassing the Keap1 gate and that the sensory mechanism of oxidative and electrophilic stresses is closely linked to the degradation mechanism of Nrf2.
| S-EPMC1317630 | biostudies-literature
Project description:Electrophilic neurite outgrowth-promoting prostaglandin (NEPP) compounds protect neurons from oxidative insults. At least part of the neuroprotective action of NEPPs lies in induction of hemeoxygenase-1 (HO-1), which, along with other phase II enzymes, serve as a defense system against oxidative stress. Here, we found that, by using fluorescent tags and immunoprecipitation assays, NEPPs are taken up preferentially into neurons and bind in a thiol-dependent manner to Keap1, a negative regulator of the transcription factor Nrf2. By binding to Keap1, NEPPs prevent Keap1-mediated inactivation of Nrf2 and, thus, enhance Nrf2 translocation into the nucleus of cultured neuronal cells. In turn, Nrf2 binds to antioxidant/electrophile-responsive elements of the HO-1 promoter to induce HO-1 expression. Consistent with this notion, NEPP induction of an HO-1 reporter construct is prevented if the antioxidant-responsive elements are mutated. We show that NEPPs are neuroprotective both in vitro from glutamate-related excitotoxicity and in vivo in a model of cerebral ischemia/reperfusion injury (stroke). Our results suggest that NEPPs prevent excitotoxicity by activating the Keap1/Nrf2/HO-1 pathway. Because NEPPs accumulate preferentially in neurons, they may provide a category of neuroprotective compounds, distinct from other electrophilic compounds such as tert-butylhydroquinone, which activates the antioxidant-responsive element in astrocytes. NEPPs thus represent a therapeutic approach for stroke and neurodegenerative disorders.
| S-EPMC1334635 | biostudies-literature
Project description:Background and purposeTargeted therapy and immunotherapy have led to dramatic change in the treatment of lung cancer, however, the overall 5-year survival rate of lung cancer patients is still suboptimal. It is important to exploit new potential of molecularly targeted therapies. High-frequency somatic mutations in KEAP1/NRF2 (27.9%) have been identified in lung squamous cell carcinoma. In this research, we explored the role of KEAP1 somatic mutations in the development of LSCC and whether a nuclear factor erythroid 2-related factor 2(NRF2) inhibitor be potential to target lung cancer carrying KEAP1/NRF2 mutations.MethodsLung cancer cell lines A549 and H460 with loss-of-function mutations in KEAP1 stably transfected with wild-type (WT) KEAP1 or somatic mutations in KEAP1 were used to investigate the functions of somatic mutations in KEAP1. Flow cytometry, plate clone formation experiments, and scratch tests were used to examine reactive oxygen species, proliferation, and migration of these cell lines.ResultsThe expression of NRF2 and its target genes increased, and tumor cell proliferation, migration, and tumor growth were accelerated in A549 and H460 cells stably transfected with KEAP1 mutants compared to control cells with a loss-of-function KEAP1 mutation and stably transfected with WT KEAP1 in both in vitro and in vivo studies. The proliferation of A549 cell line trasfected with the R320Q KEAP1 mutant was inhibited more apparent than that of the A549 cell line trasfected with WT KEAP1 after treatment with NRF2 inhibitor ML385.ConclusionSomatic mutations of KEAP1 identified from patients with LSCC likely promote tumorigenesis mediated by activation of the KEAP1/NRF2 antioxidant stress response pathway. NRF2 inhibition with ML385 could inhibit the proliferation of tumor cells with KEAP1 mutation. Video abstract.
| S-EPMC7310414 | biostudies-literature
Project description:Cancer-derived loss-of-function mutations in the KEAP1 tumor suppressor gene stabilize the NRF2 transcription factor, resulting in a prosurvival gene expression program that alters cellular metabolism and neutralizes oxidative stress. In a recent genotype-phenotype study, we classified 40% of KEAP1 mutations as ANCHOR mutants. By immunoprecipitation, these mutants bind more NRF2 than wild-type KEAP1 and ubiquitylate NRF2, but they are incapable of promoting NRF2 degradation. BioID-based protein interaction studies confirmed increased abundance of NRF2 within the KEAP1 ANCHOR mutant complexes, with no other statistically significant changes to the complexes. Discrete molecular dynamic simulation modeling and limited proteolysis suggest that the ANCHOR mutations stabilize residues in KEAP1 that contact NRF2. The modeling supports an intramolecular salt bridge between the R470C ANCHOR mutation and E493; mutation of the E493 residue confirmed the model, resulting in the ANCHOR phenotype. In live cells, the KEAP1 R320Q and R470C ANCHOR mutants colocalize with NRF2, p62/SQSTM1, and polyubiquitin in structured spherical droplets that rapidly fuse and dissolve. Transmission electron microscopy coupled with confocal fluorescent imaging revealed membraneless phase-separated biomolecular condensates. We present a model wherein ANCHOR mutations form p62-dependent biomolecular condensates that may represent a transitional state between impaired proteasomal degradation and autophagy.
| S-EPMC6206457 | biostudies-literature
Project description:The transcription factor Nrf2 and its repressor protein Keap1 play key roles in the regulation of antioxidant stress responses and both Keap1-Nrf2 signalling and oxidative stress have been implicated in the pathogenesis of the ALS-FTLD spectrum of neurodegenerative disorders. The Keap1-binding partner and autophagy receptor SQSTM1/p62 has also recently been linked genetically to ALS-FTLD, with some missense mutations identified in patients mapping within or close to its Keap1-interacting region (KIR, residues 347-352). Here we report the effects on protein function of four different disease associated mutations of SQSTM1/p62 which affect the KIR region. Only mutations mapping precisely to the KIR (P348L and G351A) were associated with a loss of Keap1 binding in co-immunoprecipitations comparable to wild-type SQSTM1/p62. These selective effects on Keap1 recognition were entirely rational based on protein structural models. Consistent with impaired Keap1 binding, the P348L and G351A KIR mutants showed reduced ability to activate Nrf2 signalling compared to wild-type SQSTM1/p62 in antioxidant response element (ARE)-luciferase reporter assays. The results suggest that SQSTM1 mutations within the KIR of SQSTM1/p62 contribute to aetiology of some cases of ALS-FTLD through a mechanism involving aberrant expression or regulation of oxidative response genes.
| S-EPMC5062946 | biostudies-literature
Project description:NRF2 is a transcription factor that mediates stress responses. Oncogenic mutations in NRF2 localize to one of its two binding interfaces with KEAP1, an E3 ubiquitin ligase that promotes proteasome-dependent degradation of NRF2. Somatic mutations in KEAP1 occur commonly in human cancer, where KEAP1 may function as a tumor suppressor. These mutations distribute throughout the KEAP1 protein but little is known about their functional impact. In this study, we characterized 18 KEAP1 mutations defined in a lung squamous cell carcinoma tumor set. Four mutations behaved as wild-type KEAP1, thus are likely passenger events. R554Q, W544C, N469fs, P318fs, and G333C mutations attenuated binding and suppression of NRF2 activity. The remaining mutations exhibited hypomorphic suppression of NRF2, binding both NRF2 and CUL3. Proteomic analysis revealed that the R320Q, R470C, G423V, D422N, G186R, S243C, and V155F mutations augmented the binding of KEAP1 and NRF2. Intriguingly, these "super-binder" mutants exhibited reduced degradation of NRF2. Cell-based and in vitro biochemical analyses demonstrated that despite its inability to suppress NRF2 activity, the R320Q "superbinder" mutant maintained the ability to ubiquitinate NRF2. These data strengthen the genetic interactions between KEAP1 and NRF2 in cancer and provide new insight into KEAP1 mechanics.
| S-EPMC3932503 | biostudies-literature
Project description:Lung squamous cell carcinoma (LSCC) pathogenesis remains incompletely understood, and biomarkers predicting treatment response remain lacking. Here, we describe novel murine LSCC models driven by loss of Trp53 and Keap1, both of which are frequently mutated in human LSCCs. Homozygous inactivation of Keap1 or Trp53 promoted airway basal stem cell (ABSC) self-renewal, suggesting that mutations in these genes lead to expansion of mutant stem cell clones. Deletion of Trp53 and Keap1 in ABSCs, but not more differentiated tracheal cells, produced tumors recapitulating histologic and molecular features of human LSCCs, indicating that they represent the likely cell of origin in this model. Deletion of Keap1 promoted tumor aggressiveness, metastasis, and resistance to oxidative stress and radiotherapy (RT). KEAP1/NRF2 mutation status predicted risk of local recurrence after RT in patients with non-small lung cancer (NSCLC) and could be noninvasively identified in circulating tumor DNA. Thus, KEAP1/NRF2 mutations could serve as predictive biomarkers for personalization of therapeutic strategies for NSCLCs. SIGNIFICANCE:We developed an LSCC mouse model involving Trp53 and Keap1, which are frequently mutated in human LSCCs. In this model, ABSCs are the cell of origin of these tumors. KEAP1/NRF2 mutations increase radioresistance and predict local tumor recurrence in radiotherapy patients. Our findings are of potential clinical relevance and could lead to personalized treatment strategies for tumors with KEAP1/NRF2 mutations. Cancer Discov; 7(1); 86-101. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1.
| S-EPMC5222718 | biostudies-literature