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IPLA2β-mediated lipid detoxification controls p53-driven ferroptosis independent of GPX4.

ABSTRACT: Here, we identify iPLA2β as a critical regulator for p53-driven ferroptosis upon reactive oxygen species (ROS)-induced stress. The calcium-independent phospholipase iPLA2β is known to cleave acyl tails from the glycerol backbone of lipids and release oxidized fatty acids from phospholipids. We found that iPLA2β-mediated detoxification of peroxidized lipids is sufficient to suppress p53-driven ferroptosis upon ROS-induced stress, even in GPX4-null cells. Moreover, iPLA2β is overexpressed in human cancers; inhibition of endogenous iPLA2β sensitizes tumor cells to p53-driven ferroptosis and promotes p53-dependent tumor suppression in xenograft mouse models. These results demonstrate that iPLA2β acts as a major ferroptosis repressor in a GPX4-independent manner. Notably, unlike GPX4, loss of iPLA2β has no obvious effect on normal development or cell viability in normal tissues but iPLA2β plays an essential role in regulating ferroptosis upon ROS-induced stress. Thus, our study suggests that iPLA2β is a promising therapeutic target for activating ferroptosis-mediated tumor suppression without serious toxicity concerns.

SUBMITTER: Chen D

PROVIDER: S-EPMC8206155 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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iPLA2β-mediated lipid detoxification controls p53-driven ferroptosis independent of GPX4.

Chen Delin D Chu Bo B Yang Xin X Liu Zhaoqi Z Jin Ying Y Kon Ning N Rabadan Raul R Jiang Xuejun X Stockwell Brent R BR Gu Wei W

Nature communications 20210615 1

Here, we identify iPLA2β as a critical regulator for p53-driven ferroptosis upon reactive oxygen species (ROS)-induced stress. The calcium-independent phospholipase iPLA2β is known to cleave acyl tails from the glycerol backbone of lipids and release oxidized fatty acids from phospholipids. We found that iPLA2β-mediated detoxification of peroxidized lipids is sufficient to suppress p53-driven ferroptosis upon ROS-induced stress, even in GPX4-null cells. Moreover, iPLA2β is overexpressed in human ...[more]

PMID: 34131139

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Project description:BackgroundGastric cancer is one of the most prevalent malignant tumors within the digestive system, and ferroptosis playing a crucial role in its progression. Glutathione peroxidase 4 (GPX4), a key negative regulator of ferroptosis, is highly expressed in gastric cancer and contributes to tumor growth. Targeting the regulation of GPX4 has emerged as a promising approach to induce ferroptosis and develop effective therapy for gastric cancer.MethodsTo confirm that OTUD5 is a deubiquitinase of GPX4 and regulates ferroptosis, we performed Western blotting, Co-IP, immunofluorescence, quantitative real-time PCR, Ub assay and flow cytometry experiments. To explore the physiological function of OUTD5, we knocked out the Otud5 gene in the mouse gastric cancer cell line (MFC) using CRISPR-Cas9 and eatablished the subcutaneous tumour model. Immunohistochemistry (IHC) analysis was used to inveatigate the pathological correlation in human gastric cancer.ResultsWe report that ovarian tumor domain-containing 5 (OTUD5) interacts with, deubiquitylates and stabilizes GPX4. OTUD5 depletion destabilizes GPX4, promotes lipid peroxidation and sensitizes gastric cancer cells to ferroptosis. Moreover, the p53 activator nutlin-3a suppresses OTUD5 transcription, leading to GPX4 degradation and ferroptosis of gastric cancer cells. Notably, only wild-type p53 has the capacity to inhibit OTUD5 transcription, while p53 mutations or deficiencies correlate with increased OTUD5 expression, promoting gastric cancer progression. Additionally, OTUD5 silencing and nutlin-3a-induced GPX4 degradation enhances the sensitivity of gastric cancer cells to ferroptosis in vivo. Subsequently, the p53/OTUD5/GPX4 axis is confirmed in clinical gastric cancer samples.ConclusionCollectively, these findings elucidate a mechanism whereby p53 inactivation upregulates OTUD5 transcription to deubiquitylate and stablize GPX4, resulting in ferroptosis inhibition and gastric cancer progression. This discovery highlights the potential therapeutic value of targeting OTUD5 to promote ferroptosis in p53-inactivated gastric cancer.Key pointsOTUD5 mediates GPX4 deubiquitination to regulate its stability. Deletion of OTUD5 promotes ferroptosis and inhibits tumor growth. Wild type p53 inhibits OTUD5 transcription, thereby promoting GPX4 degradation and inhibiting the development of gastric cancer. OTUD5, GPX4 expression and p53 activity are highly correlated and correlates with clinical progression in STAD.

Project description:Rationale: As a key endogenous negative regulator of ferroptosis, glutathione peroxidase 4 (GPX4) can regulate its antioxidant function through multiple post-translational modification pathways. However, the effects of the phosphorylation/dephosphorylation status of GPX4 on the regulation of inducible ferroptosis in hepatocellular carcinoma (HCC) remain unclear. Methods: To investigate the effects and molecular mechanism of GPX4 phosphorylation/dephosphorylation modification on ferroptosis in HCC cells. Sorafenib (Sora) was used to establish the ferroptosis model in HCC cells in vitro. Using the site-directed mutagenesis method, we generated the mimic GPX4 phosphorylation or dephosphorylation HCC cell lines at specific serine sites of GPX4. The effects of GPX4 phosphorylation/dephosphorylation modification on ferroptosis in HCC cells were examined. The interrelationships among GPX4, p53, and protein phosphatase 2A-B55β subunit (PP2A-B55β) were also explored. To explore the synergistic anti-tumor effects of PP2A activation on Sora-administered HCC, we established PP2A-B55β overexpression xenograft tumors in a nude mice model in vivo. Results: In the Sora-induced ferroptosis model of HCC in vitro, decreased levels of cytoplasmic and mitochondrial GPX4, mitochondrial dysfunction, and enhanced p53 retrograde signaling occurred under Sora treatment. Further, we found that mitochondrial p53 retrograded remarkably into the nucleus and aggravated Sora-induced ferroptosis. The phosphorylation status of GPX4 at the serine 2 site (GPX4Ser2) revealed that mitochondrial p-GPX4Ser2 dephosphorylation was positively associated with ferroptosis, and the mechanism might be related to mitochondrial p53 retrograding into the nucleus. In HCC cells overexpressing PP2A-B55β, it was found that PP2A-B55β directly interacted with mitochondrial GPX4 and promoted Sora-induced ferroptosis in HCC. Further, PP2A-B55β reduced the interaction between mitochondrial GPX4 and p53, leading to mitochondrial p53 retrograding into the nucleus. Moreover, it was confirmed that PP2A-B55β enhanced the ferroptosis-mediated tumor growth inhibition and mitochondrial p53 retrograde signaling in the Sora-treated HCC xenograft tumors. Conclusion: Our data uncovered that the PP2A-B55β/p-GPX4Ser2/p53 axis was a novel regulatory pathway of Sora-induced ferroptosis. Mitochondrial p-GPX4Ser2 dephosphorylation triggered ferroptosis via inducing mitochondrial p53 retrograding into the nucleus, and PP2A-B55β was an upstream signal modulator responsible for mitochondrial p-GPX4Ser2 dephosphorylation. Our findings might serve as a potential theranostic strategy to enhance the efficacy of Sora in HCC treatment through the targeted intervention of p-GPX4 dephosphorylation via PP2A-B55β activation.

Project description:Human cytomegalovirus (HCMV) modulates numerous cellular pathways to facilitate infection, including key components in cellular iron homeostasis. Iron is essential to many cellular processes but, if present in excess, drives cell death through ferroptosis. Ferroptosis is a process that is dependent upon the accumulation of oxidatively damaged phospholipids (lipid peroxides); when these lipid peroxides accumulate in membranes, this culminates in plasma membrane rupture and eventual cell lysis. Here, we demonstrate that HCMV infection downregulates the expression of a key modulator of lipid peroxidation, glutathione peroxidase 4 (GPX4). HCMV infection also markedly increased levels of lipid peroxides within infected cells. Despite the marked downregulation of GPX4 by HCMV, further inhibition of GPX4 impaired virus replication. Interestingly, overexpression of GPX4 did not reduce the production of lipid peroxides within infected cells. In contrast, lipid peroxide levels were reduced by treatment with ferrostatin-1, a ferrous iron-dependent scavenger of alkoxyl radicals, indicating a role for iron in the production of lipid peroxides. HCMV-infected cells became less sensitive to GPX4 inhibition as infection progressed, requiring substantially higher levels of GPX4 inhibitors to induce ferroptosis compared to uninfected cells. This observed difference in sensitivity to ferroptosis upon infection correlated with a large increase in lipid production by infected cells. Therefore, the marked stimulation of lipid peroxidation by HCMV likely proceeds through a pathway that is independent of GPX4 regulation, but the ability of lipid peroxides to stimulate ferroptosis by modulating plasma membrane rupture is likely blunted by the massive increase in lipid production during HCMV infection.ImportanceHuman cytomegalovirus (HCMV) infection is intimately linked with countless host cell pathways that are modulated in a coordinated fashion to facilitate infection. Here, we describe HCMV-induced regulation of lipid peroxidation, a precursor of the iron-regulated cell death pathway known as ferroptosis, during human cytomegalovirus infection. These studies reveal hitherto unidentified changes in metabolism mediated by HCMV that decrease sensitivity to ferroptosis, despite increases in lipid peroxidation and transient increases in intracellular iron levels in infected cells.

Project description:Gankyrin is found in high levels in triple-negative breast cancer (TNBC) and has been established to form a complex with the E3 ubiquitin ligase MDM2 and p53, resulting in the degradation of p53 in hepatocarcinoma cells. Therefore, this study sought to determine whether gankyrin could inhibit ferroptosis through this mechanism in TNBC cells. The expression of gankyrin was investigated in relation to the prognosis of TNBC using bioinformatics. Co-immunoprecipitation and GST pull-down assays were then conducted to determine the presence of a gankyrin and MDM2 complex. RT-qPCR and immunoblotting were used to examine molecules related to ferroptosis, such as gankyrin, p53, MDM2, SLC7A11, and GPX4. Additionally, cell death was evaluated using flow cytometry detection of 7-AAD and a lactate dehydrogenase release assay, as well as lipid peroxide C11-BODIPY. Results showed that the expression of gankyrin is significantly higher in TNBC tissues and cell lines, and is associated with a poor prognosis for patients. Subsequent studies revealed that inhibiting gankyrin activity triggered ferroptosis in TNBC cells. Additionally, silencing gankyrin caused an increase in the expression of the p53 protein, without altering its mRNA expression. Co-immunoprecipitation and GST pull-down experiments indicated that gankyrin and MDM2 form a complex. In mouse embryonic fibroblasts lacking both MDM2 and p53, this gankyrin/MDM2 complex was observed to ubiquitinate p53, thus raising the expression of molecules inhibited by ferroptosis, such as SLC7A11 and GPX4. Furthermore, silencing gankyrin in TNBC cells disrupted the formation of the gankyrin/MDM2 complex, hindered the degradation of p53, increased SLC7A11 expression, impeded cysteine uptake, and decreased GPX4 production. Our findings suggest that TNBC cells are able to prevent cell ferroptosis through the gankyrin/p53/SLC7A11/GPX4 signaling pathway, indicating that gankyrin may be a useful biomarker for predicting TNBC prognosis or a potential therapeutic target.

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IPLA2β-mediated lipid detoxification controls p53-driven ferroptosis independent of GPX4.

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iPLA2β-mediated lipid detoxification controls p53-driven ferroptosis independent of GPX4.

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