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RNAi-mediated ablation of Nrf2 in PyMT-1099 LT, Py2T LT and MT∆Ecad mesenchymal breast cancer cells

ABSTRACT: Background: Epithelial-mesenchymal transition (EMT) has been implicated in metastasis, drug resistance, survival under stress and also conferring stem cell-like traits to cancer cells. We have aimed at exploring the crosstalk between ROS and an EMT induced by TGFb in breast cancer cells. Methods: We observed that cells exposed to TGFb displayed a decrease of ROS levels. Furthermore, we identified an activation of the Nrf2 transcription factor, master regulator of the antioxidant response in TGFb-induced mesenchymal cells. In order to know the effect of Nrf2 ablation in epithelial and mesenchymal cells, RNAi-mediated ablation for Nrf2 was performed. Results: RNAi-mediated ablation of Nrf2 led to mesenchymal cancer cell death due to ferroptosis, an iron- and oxidative stress-dependent cell death. Moreover, analysis revealed that several glutathione pathway genes were specifically regulated by Nrf2, suggesting a role of glutathione-related pathways in mesenchymal cell survival. Conclusion: In this study we report the criticial role of the Nrf2-mediated glutathione metabolism in the protection of metastatic breast cancer cells from ferroptosis. Therapeutic targeting of antioxidant pathways may thus be beneficial for patients with metastatic disease.

ORGANISM(S): Mus musculus

PROVIDER: GSE187478 | GEO | 2022/10/15

REPOSITORIES: GEO

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MTflEcad I80 and MTflEcad DI150 cells, long-term treated murine derived breast cancer cell lines with H2O2

Project description:Background: Epithelial-mesenchymal transition (EMT) has been implicated in metastasis, drug resistance, survival under stress and also conferring stem cell-like traits to cancer cells. We have aimed at exploring the crosstalk between ROS and an EMT induced by TGFb in breast cancer cells. Methods: We observed that cells exposed to TGFb displayed a decrease of ROS levels. To identify the antioxidant pathways activated during EMT, we investigated the effect of long-term oxidative stress, for example by H2O2 (80 µM and 150 µM), on MTflECad epithelial cells derived from the MMTV-Neu mouse model. Results: We show that long-term treated MTflEcad epithelical cells with H2O2 display both higher migratory capability and tumorigenicity. Furthermore, we identified an activation of the Nrf2 transcription factor, master regulator of the antioxidant response, in H2O2-resistant cells, as well as in TGFb-induced mesenchymal cells. Conclusion: In this study we report the criticial role of the Nrf2-mediated glutathione metabolism in the protection of metastatic breast cancer cells from ferroptosis. Therapeutic targeting of antioxidant pathways may thus be beneficial for patients with metastatic disease.

2022-10-15 | GSE187476 | GEO

Py2T C40 and Py2T I60 cells, long-term treated murine derived breast cancer cell lines with H2O2

Project description:Background: Epithelial-mesenchymal transition (EMT) has been implicated in metastasis, drug resistance, survival under stress and also conferring stem cell-like traits to cancer cells. We have aimed at exploring the crosstalk between ROS and an EMT induced by TGFb in breast cancer cells. Methods: We observed that cells exposed to TGFb displayed a decrease of ROS levels. To identify the antioxidant pathways activated during EMT, we investigated the effect of long-term oxidative stress, for example by H2O2 (40 µM and 60 µM), on Py2T epithelial cells derived from the MMTV-PyMT mouse model. Results: We show that long-term treated Py2T epithelical cells with H2O2 display both higher migratory capability and tumorigenicity. Furthermore, we identified an activation of the Nrf2 transcription factor, master regulator of the antioxidant response, in H2O2-resistant cells, as well as in TGFb-induced mesenchymal cells. Conclusion: In this study we report the criticial role of the Nrf2-mediated glutathione metabolism in the protection of metastatic breast cancer cells from ferroptosis. Therapeutic targeting of antioxidant pathways may thus be beneficial for patients with metastatic disease.

2022-10-15 | GSE187364 | GEO

Pancreatic acinar cell fate relies on system xc- to prevent ferroptosis during stress

Project description:Acinar cell dedifferentiation is one of the most notable features of acute and chronic pancreatitis. It can also be the initial step that facilitates pancreatic cancer development. In the present study, we further decipher the precise mechanism and regulation using murine experimental models. Our RNAseq analysis indicates that, early acinar cell dedifferentiation is accompanied by multiple pathways related to cell survival that are highly enriched, and where SLC7A11 (xCT) is transiently upregulated. xCT is the specific subunit of the cystine/glutamate antiporter system xC-. Acinar cells with depleted or reduced xCT function show an increase in ferroptosis relating to lipid peroxidation. Lower glutathione levels and more lipid ROS accumulation could be rescued by the antioxidant N-acetylcysteine or the ferroptosis inhibitor Ferrostatin-1. In caerulein-induced acute pancreatitis in mice, xCT also prevents lipid peroxidation in acinar cells. In conclusion, during stress, acinar cell fate seems to be poised for avoiding several forms of cell death. xCT specifically prevents acinar cell ferroptosis by fueling the glutathione pool and maintaining ROS balance. The data suggest that xCT offers a druggable tipping point to steer the acinar cell fate in stress conditions.

2023-08-07 | GSE240106 | GEO

Deletion of thioredoxin reductase disrupts redox homeostasis and impairs β-cell function

Project description:Reactive oxygen species (ROS) have been implicated as mediators of pancreatic β-cell damage. While β-cells are thought to be vulnerable to oxidative damage, we have shown, using inhibitors and acute depletions, that thioredoxin reductase, thioredoxin, and peroxiredoxins are the primary mediators of antioxidant defense in β-cells. However, the role of this antioxidant cycle in maintaining redox homeostasis and β-cell survival in vivo remains unclear. Here, we generated mice with a β-cell specific knockout of thioredoxin reductase 1 (Txnrd1.fl/fl; Ins1.Cre/+, βKO). Despite blunted glucose-stimulated insulin secretion, knockout mice maintain normal whole body glucose homeostasis. Unlike pancreatic islets with acute Txnrd1 inhibition, βKO islets do not demonstrate increased sensitivity to continuous ROS. RNA-sequencing analysis revealed that Txnrd1-deficient β-cells have increased expression of Nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated genes, and altered expression of genes involved in heme and glutathione metabolism, suggesting an adaptive response. Txnrd1-deficient β-cells also have decreased expression of factors controlling β-cell function and identity which may explain the mild functional impairment. Together, these results suggest that Txnrd1-knockout β-cells compensate for loss of this essential antioxidant pathway by increasing expression of Nrf2-regulated antioxidant genes, allowing for protection from excess ROS at the expense of normal β-cell function and identity.

2022-07-05 | GSE200965 | GEO

Nrf2-mediated enhancer activation via phase separation ameliorates oxidative stress and cystogenesis in autosomal dominant polycystic kidney disease

Project description:Mitochondrial dysfunction is emerging as a crucial contributor to the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD), but the molecular mechanisms underlying the disturbed mitochondrial homeostasis in cystic cells remain elusive. In the present study, we identify impaired activity of NRF2 antioxidant pathway as a driver mechanism for mitochondrial dysfunction and ADPKD progression. Using a quantitative proteomic approach, we find that NRF2 antioxidant pathway is suppressed in ADPKD kidneys. In a cohort of ADPKD patients, reactive oxygen species (ROS) levels are frequently elevated, and the increased ROS levels inversely correlates with decreased NRF2 expression and positively correlates with disease severity. Genetic deletion of NRF2 increases ROS generation and promotes cyst growth in an orthologous ADPKD mouse model, while pharmacological induction of NRF2 reduces ROS levels and retards cystogenesis and disease progression. Mechanistically, NRF2 activates its antioxidant target genes mainly through remodeling enhancer landscapes. The activation domain of NRF2 forms phase-separated condensates with MED16, a Mediator complex subunit in vitro, and NRF2 is required for optimal Mediator recruitment to target genomic sites in vivo. Taken together, these findings indicate that NRF2 remodels enhancer landscapes and activates its target genes through a phase-separation mechanism, and that activation of NRF2 represents a promising strategy for restoring mitochondrial homeostasis and combatting ADPKD.

2021-12-12 | PXD016312 | Pride

D2.0R mouse breast cancer cells cultured on stiff or soft ECM hydrogels

Project description:Metastatic breast cancer cells disseminate to organs with a soft microenvironment. Whether and how tissue mechanical properties influence their response to treatment remains unclear. Here we found that a soft ECM empowers redox homeostasis. Cells cultured on a soft ECM display increased peri-mitochondrial F-actin promoted by Spire1C and Arp2/3 nucleation factors, and increased DRP1- and MIEF1/2-dependent mitochondrial fission. Changes in mitochondrial dynamics lead to increased mtROS production and activate the NRF2 antioxidant transcriptional response, including increased cystine uptake and glutathione metabolism. This retrograde response endows cells with resistance to oxidative stress and ROS-dependent chemotherapy drugs. This is relevant in a mouse model of metastatic breast cancer cells dormant in the lung soft tissue, where inhibition of DRP1 and NRF2 restored cisplatin sensitivity and prevented disseminated cancer cell awakening. We propose that targeting this mitochondrial dynamics- and redox-based mechanotransduction pathway could open new avenues to prevent metastatic relapse.

2021-12-21 | GSE189803 | GEO

Nrf2-mediated enhancer activation ameliorates oxidative stress and cystogenesis in autosomal dominant polycystic kidney disease [RNA-seq]

Project description:Oxidative stress is emerging as a crucial contributor to the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD), but the molecular mechanisms underlying the disturbed redox homeostasis in cystic cells remain elusive. In the present study, we identify impaired activity of Nrf2 antioxidant pathway as a driver mechanism for oxidative damage and ADPKD progression. Using a quantitative proteomic approach, together with biochemistry analyses, we find that Nrf2 antioxidant pathway is suppressed due to increased degradation of Nrf2 protein in ADPKD kidneys. In a cohort of ADPKD patients, reactive oxygen species (ROS) levels are frequently elevated, and the ROS levels inversely correlates with Nrf2 abundance and positively correlates with disease severity. Genetic deletion of Nrf2 further increases ROS generation and promotes cyst growth in an orthologous ADPKD mouse model, while pharmacological induction of Nrf2 reduces ROS levels and retards cystogenesis and disease progression. Mechanistically, pharmacological induction of Nrf2 remodels enhancer landscapes and activates Nrf2-bound enhancer-associated genes in ADPKD cells. The activation domain of Nrf2 forms phase-separated condensates with Mediator subunit MED16 in vitro, and Nrf2 is required for optimal Mediator recruitment to target genomic loci in vivo. Taken together, these findings indicate that Nrf2 remodels enhancer landscapes and activates its target genes through a phase-separation mechanism, and that activation of Nrf2 represents a promising strategy for restoring redox homeostasis and combatting ADPKD.

2021-01-26 | GSE141740 | GEO

Nrf2-mediated enhancer activation ameliorates oxidative stress and cystogenesis in autosomal dominant polycystic kidney disease [ChIP-seq]

2021-01-26 | GSE141739 | GEO

Enhanced glutathione levels confer resistance to apoptotic and ferroptotic programmed cell death in NEIL DNA glycosylase deficient HAP1 cells

Project description:The NTHL1 and NEIL1-3 DNA glycosylases are major enzymes in the removal of oxidative DNA base lesions, via the base excision repair (BER) pathway. To investigate their interplay in human cells, we engineered single, double, triple, and quadruple DNA glycosylase deficient HAP1 cells using the Crisp-cas 9 technology. We found that these DNA glycosylase deficient cells are more resistant to oxidative stress caused by genotoxic agents than wild type cells, and a further augment in resistance was observed upon inhibition of glutathione synthesis. Gene expression analysis revealed that GSH depletion activates the NRF2/KEAP1 pathway and ER stress induced apoptosis. Furthermore, glutathione depleted NEIL triple KO cells are also more resistant to cell death induced via ferroptosis than wild type cells. Finally, we observed higher basal level of glutathione and differential expression of NRF2-regulated genes associated with glutathione homeostasis in these cells. We propose that NEIL deficiency leads to aberrant transcription and subsequent errors in protein synthesis, which cause ER- and proteotoxic stress. This triggers the unfolded protein response, which consequently upregulates intracellular antioxidant defence mechanisms. Altogether, our data suggest that NEIL deficient cells developed an oxidative stress defence mechanism termed hormesis mediated by elevated GSH levels in response to the increased intracellular stress inflicted by loss of NEIL123 activities.

2023-09-08 | GSE242290 | GEO

RNA helicase DDX5 mediates adaptive response to multi-kinase inhibitors in liver cancer

Project description:Earlier studies linked sorafenib effectiveness to induction of ferroptosis. Herein we demonstrate sorafenib and mTKIs downregulate DDX5 in vitro and in vivo. To understand the effect of DDX5 downregulation, we compared TCGA-derived HCCs expressing low vs. high DDX5 focusing on ferroptosis-related genes. Glutathione Peroxidase 4 (GPX4), a key ferroptosis regulator, was significantly overexpressed in DDX5LOW HCCs. Importantly, DDX5-knockdown (DDX5KD) HCC cell lines lacked lipid peroxidation by GPX4 inhibition, indicating DDX5 downregulation suppresses ferroptosis. RNAseq analyses comparing wild type vs. DDX5KD cells in the presence or absence of sorafenib, identified a unique set of genes repressed by DDX5 and upregulated by sorafenib. This set significantly overlaps genes from Wnt/β-catenin and non-canonical NF-κB pathways, including NF-κB inducing Kinase required for non-canonical NF-κB activation. Pharmacologic inhibition of these pathways in combination with sorafenib reduced DDX5KD cell viability. Mechanistically, sorafenib-mediated NF-κB activation induced NRF2 transcription, while DDX5KD extended NRF2 half/life by stabilizing p62/SQSTM1, leading to enhanced expression of GPX4 and ferroptosis escape. Conclusion: Sorafenib/mTKI-mediated DDX5 downregulation results in adaptive mTKI resistance by enhancing NRF2 expression, leading to ferroptosis escape. We propose inhibition of the pathways leading to NRF2 expression will enhance the therapeutic effectiveness of sorafenib/mTKIs.

2023-12-01 | GSE199092 | GEO

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