A genome-wide CRISPR screen identifies mediators of ferroptosis sensitivity in polyunsaturated fatty acid-treated cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE130982 | GEO | 2020/01/01
REPOSITORIES: GEO
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Project description:Ferroptosis is a regulated form of necrotic cell death caused by iron-dependent phospholipid peroxidation. It can be induced by inhibiting glutathione peroxidase 4 (GPX4), the key enzyme for efficiently reducing peroxides within phospholipid bilayers. Recent data suggest that cancer cells undergoing EMT (dedifferentiation) and those resistant to standard therapy expose a high vulnerability toward ferroptosis. Although recent studies have begun to identify and characterize the metabolic and genetic determinants underlying ferroptosis, many mechanisms that dictate ferroptosis sensitivity remain unknown. Here, we show that low cell density sensitizes primary mammary epithelial and breast cancer cells to ferroptosis induced by GPX4 inhibition, whereas high cell density confers resistance. These effects occur irrespective of oncogenic signaling, cellular phenotype and expression of the fatty acid ligase acyl-CoA synthetase long chain family member 4 (ACSL4). By contrast, we show that a massive accumulation of neutral triacylglycerides (TAG) enriched with polyunsaturated fatty acids (PUFA) is induced at low cell density. In addition, de novo lipogenesis and desaturation pathways were found to be reduced at low cell density, indicative of increased fatty acid uptake. Our study suggests that PUFA-mediated toxicity is limited by the enrichment in TAGs that in turn might pose a vulnerability towards ferroptosis. Conclusively, cell density regulates lipid metabolism of breast epithelial and cancer cells, which results in a ferroptosis-sensitive cell state with the potential to be exploited therapeutically during metastatic dissemination.
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Project description:Clear-cell carcinomas (CCCs) are a histological group of highly aggressive malignancies commonly originating in the kidney and ovary. CCC tumors are distinguished by aberrant lipid and glycogen accumulation and are inherently refractory to a broad range of anti-cancer therapies. In the study associated with this dataset, we identified an intrinsic vulnerability to ferroptosis associated with the unique metabolic state in CCC cells. However, the mediators of this sensitivity to ferroptosis are unknown. Here we perform a genome-wide CRISPR screen to identify genes that mediate the sensitivity to ML210, a selective inhibitor of glutathione peroxidase 4 (GPX4) and a potent inducer of ferroptotic cell death in 786-O, a clear-cell renal cell carcinoma cell line.
2019-03-04 | GSE126696 | GEO
Project description:Ferroptotic cell death is dependent on unrestricted lipid peroxidation rather than activation of apoptotic effector caspases. A large number of ferroptosis activators have been reported recently. We used gene sequencing to analyze the effects of four ferroptosis activators (RSL3, N6F11, Fin56 and Erastin) on global gene expression in human PDAC1 cell line.
2023-10-18 | E-MTAB-12241 | biostudies-arrayexpress
Project description:Ferroptosis is a form of regulated cell death driven by the iron-dependent accumulation of oxidized polyunsaturated-fatty-acid-containing phospholipids (PUFA-PLs). Three key molecular features of ferroptosis are peroxidation of PUFA-PLs, accumulation of redox-active ferrous iron, and defective lipid peroxide repair (Dixon and Stockwell, 2019). However, there is currently no reliable way to selectively stain ferroptotic cells in tissue sections to characterize the extent of ferroptosis in animal models of disease and in patient tissue samples. We sought to address this gap by immunizing mice with membranes from lymphoma cells treated with the ferroptosis inducer piperazine erastin (PE), and screening ~4,750 of the resulting monoclonal antibodies generated for their ability to selectively detect cells undergoing ferroptosis. We found that one antibody, termed 3F3 Ferroptotic Membrane Antibody (3F3-FMA), was effective as a selective ferroptosis-staining reagent using immunofluorescence (IF). The antigen of 3F3-FMA was identified by immunoprecipitation and mass spectrometry as the human transferrin receptor 1 protein (TfR1), which imports iron from the extracellular environment into cells. We validated this finding with several additional anti-TfR1 antibodies, and compared them to other potential ferroptosis-detecting reagents via immunofluorescence staining, using fluorescence microscopy and flow cytometry. We found that anti-TfR1 and anti-malondialdehyde (MDA) adduct antibodies were effective at reliably staining ferroptotic tumor cells in numerous cell culture and tissue contexts. In summary, these findings suggest that anti-TfR1 antibodies can be used to selectively label cells undergoing ferroptosis.
2020-03-10 | PXD017425 | Pride
Project description:Ferroptosis is a recently identified program of regulated cell death, defined by excessive accumulation of hydroperoxy-arachidonoyl (C20:4)- or adrenoyl (C22:4)- phosphatidyl-ethanolamine (OOH-PE). The selenium-dependent glutathione peroxidase 4 (GPX4) inhibits ferroptosis, converting unstable ferroptotic lipid hydroperoxides to non-toxic lipid alcohols. The effect of GPX4 ablation is divergent among cells and tissues, suggesting that additional regulators may guard trophoblasts against ferroptosis. While placental oxidative stress and lipotoxicity are hallmarks of placental dysfunction, the possible role of ferroptosis in placental function has not been hitherto investigated. Here we found that placental dysfunction is associated with ferroptosis, and that inhibition of GPX4 causes trophoblast injury and ferroptosis in vitro and in vivo during mouse pregnancy. Importantly, we uncover a role for the phospholipase PLA2G6 (PNPLA9, iPLA2beta), known to metabolizes OOH-PE to lyso-PE and oxidized fatty acid, in mitigating ferroptosis induced by GPX4 inhibition in vitro or by hypoxia-reoxygenation injury in vivo. Together, we identified ferroptosis in the human and mouse placenta, and established a novel role for PLA2G6 in attenuating trophoblastic ferroptosis. Our data provide mechanistic insights to the ill-defined entity of placental lipotoxicity, and may inspire new therapeutic strategies that target PLA2G6 as a means to modulate ferroptosis in placental and non-placental tissue.
2020-09-27 | GSE147625 | GEO
Project description:Overexpression of MYC family members is linked to poor clinical outcome in many human cancers. These oncoproteins drive proliferation, alter metabolism, and mediate an antioxidant response to maintain tumor cell redox balance. However, to date, there are no effective inhibitors that specifically target MYC-amplified tumors. We demonstrate that MYCN-amplified, aggressive childhood neuroblastoma cells undergo ferroptotic cell death in vivo in the absence of intracellular cysteine, thus implicating MYCN as a predictive biomarker for ferroptosis sensitivity in neuroblastoma. Although cysteine is provided by both uptake from the microenvironment and MYCN-induced transsulfuration of methionine, glutathione levels remain low in these highly proliferative cancer cells due to concomitant cysteine utilization for protein and nucleotide synthesis. Consequently, MYCN-amplified neuroblastoma cells are highly susceptible to lipid peroxidation and ferroptosis, which must be counteracted by GPX4 activity. Pharmacological inhibition of both cystine uptake and transsulfuration combined with GPX4 inactivation resulted in tumor remission in an orthotopic MYCN-amplified neuroblastoma model. Our data show that MYCN-amplified neuroblastoma is sensitized to ferroptosis, which can be exploited therapeutically, by depleting the intracellular cysteine pool with concomitant GPX4 inactivation. These findings may help to develop novel clinical strategies to target MYCN-amplified tumors by inducing ferroptotic cell death.
2022-01-18 | GSE189174 | GEO
Project description:Ferroptosis, a non-apoptotic form of programmed cell death, is triggered by oxidative stress in cancer, heat stress in plants and hemorrhagic stroke. A homeostatic transcriptional response to ferroptotic stimuli is unknown. We show that neurons respond to ferroptotic stress by induction of a transcriptional adaptive response. Pharmacological selenium (Se) augments mitochondrial and nuclear GPX4 and other genes in this transcriptional program, the selenome, via coordinated activation of the transcription factors TFAP2c and Sp1, to protect neurons. This RNA seq data uses primary cortical neurons in cultures treated with either homocysteic acid (HCA), which induces ferroptosis and/or Se, which augments the adaptation to ferroptosis. Both protective (1mM Se) and non-protective (0.1mM Se) of selenium are used. Cells are collected after 6 hours when the transcriptional response is observed.
2019-02-20 | GSE126787 | GEO
Project description:Ferroptosis is associated with lipid hydroperoxides generated by oxidation of polyunsaturated acyl chains. Lipid hydroperoxides are reduced by glutathione peroxidase 4 (GPX4) and GPX4 inhibitors induce ferroptosis. However, the therapeutic potential of triggering ferroptosis in cancer cells with polyunsaturated fatty acids is unknown. We identified conjugated linoleates including α-eleostearic acid (αESA) as novel ferroptosis inducers. αESA did not alter GPX4 activity but was incorporated into cellular lipids and promoted lipid peroxidation and cell death in diverse cancer cell types. αESA-triggered death was mediated by acyl-CoA synthetase long-chain isoform 1, which promoted αESA incorporation into neutral lipids including triacylglycerols. Interfering with triacylglycerol biosynthesis suppressed ferroptosis triggered by αESA but not by GPX4 inhibition. Orally administered tung oil, naturally rich in αESA, limited tumor growth and metastasis with transcriptional changes consistent with ferroptosis. Overall, these findings illuminate a novel approach to ferroptosis, complementary to GPX4 inhibition, with therapeutic potential.
2021-02-08 | GSE162069 | GEO
Project description:Ferroptosis is an iron-dependent programmed cell death associated with severe kidney diseases, linked to decreased glutathione peroxidase 4 (GPX4). However, the spatial distribution of renal GPX4-mediated ferroptosis and the molecular events causing GPX4 reduction during ischemia-reperfusion (I/R) remain largely unknown. Using spatial transcriptomics, we identify that GPX4 is situated at the interface of the inner cortex and outer medulla, a hyperactive ferroptosis site post-I/R injury. We show that OTU deubiquitinase 5 (OTUD5) is a GPX4-binding protein that confers ferroptosis resistance by stabilizing GPX4. During I/R, ferroptosis is induced by mTORC1-mediated autophagy, causing OTUD5 degradation and subsequent GPX4 decay. Functionally, OTUD5 deletion intensifies renal tubular cell ferroptosis and exacerbates acute kidney injury, while AAV-mediated OTUD5 delivery mitigates ferroptosis and promotes renal function recovery from I/R injury. In this work, our study highlights a new autophagy-dependent ferroptosis module: hypoxia/ischemia-induced OTUD5 autophagy triggers GPX4 degradation, offering a potential therapeutic avenue for I/R-related kidney diseases.
2023-11-29 | GSE244330 | GEO
Project description:Ferroptosis is a form of regulated cell death driven by lipid peroxidation of polyunsaturated fatty acids (PUFAs). Endogenous PUFAs are nonconjugated PUFAs and their peroxidation proceeds via the hydrogen-atom transfer (HAT) mechanism. We previously reported that lipids with conjugated double bonds undergo lipid peroxidation mostly via a different mechanism, peroxyl radical addition (PRA), and were much more readily oxidizable than nonconjugated ones. In this study, we aim to elucidate the effects of various unsaturated lipids in sensitizing ferroptosis. We found that while some peroxidation-reactive lipids, such as 7-dehydrocholesterol, vitamins D3 and A, and coenzyme Q10, suppress ferroptosis, both nonconjugated and conjugated PUFAs enhanced cell death induced by RSL3, a ferroptosis inducer. Importantly, we showed that conjugated linolenic acid (CLA 18:3) could act as a ferroptosis inducer by itself and conjugated linoleic acid (CLA 18:2) was more potent in sensitizing cells to RSL3-induced cell death than any nonconjugated PUFAs. We next sought to elucidate the mechanism underlying the different ferroptosis-inducing potency of conjugated and nonconjugated PUFAs. Lipidomics revealed that conjugated and nonconjugated PUFAs are incorporated into distinct cellular lipid species. Furthermore, the different peroxidation mechanisms predict the formation of higher levels of reactive electrophilic aldehydes from conjugated PUFAs than nonconjugated PUFAs, which was confirmed by aldehyde-trapping and mass spectrometry. RNA sequencing revealed that protein processing in the endoplasmic reticulum and proteasome are among the most significantly upregulated pathways in cells treated with CLA 18:3, suggesting increased ER stress and activation of unfolded protein response. Significantly, using click chemistry, we observed increased protein adduction by oxidized lipids in cells treated with an alkynylated CLA 18:2 probe. These results suggest that protein damage by lipid electrophiles is a key step in ferroptosis.
2023-04-17 | GSE224249 | GEO