NCOA4-mediated Ferritinophagy Research Articles

Genome-wide DNA methylation sequencing reveals the involvement of ferroptosis in hepatotoxicity induced by dietary exposure to food-grade titanium dioxide

BackgroundFollowing the announcement by the European Food Safety Authority that the food additive titanium dioxide (E 171) is unsafe for human consumption, and the subsequent ban by the European Commission, concerns have intensified over the potential risks E 171 poses to human vital organs. The liver is the main organ for food-grade nanoparticle metabolism. It is increasingly being found that epigenetic changes may play an important role in nanomaterial-induced hepatotoxicity. However, the profound effects of E 171 on the liver, especially at the epigenetic level, remain largely unknown.MethodsMice were exposed orally to human-relevant doses of two types of E 171 mixed in diet for 28 and/or 84 days. Conventional toxicology and global DNA methylation analyses were performed to assess E 171-induced hepatotoxicity and epigenetic changes. Whole genome bisulfite sequencing and further ferroptosis protein detection were used to reveal E 171-induced changes in liver methylation profiles and toxic mechanisms.ResultsExposed to E 171 for 28 and/or 84 days resulted in reduced global DNA methylation and hydroxymethylation in the liver of mice. E 171 exposure for 84 days elicited inflammation and damage in the mouse liver, whereas 28-day exposure did not. Whole-genome DNA methylation sequencing disclosed substantial methylation alterations at the CG and non-CG sites of the liver DNA in mice exposed to E 171 for 84 days. Mechanistic analysis of the DNA methylation alterations indicated that ferroptosis contributed to the liver toxicity induced by E 171. E 171-induced DNA methylation changes triggered NCOA4-mediated ferritinophagy, attenuated the protein levels of GPX4, FTH1, and FTL in the liver, and thereby caused ferroptosis.ConclusionsLong-term oral exposure to E 171 triggers hepatotoxicity and induces methylation changes in both CG and non-CG sites of liver DNA. These epigenetic alterations activate ferroptosis in the liver through NCOA4-mediated ferritinophagy, highlighting the role of DNA methylation and ferroptosis in the potential toxicity caused by E 171 in vivo.

Abstract IA-01: Targeting autophagy in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most vexing problems in cancer with 5-year overall survival rates of 13%, thus there is a need for new therapeutic targets. PDAC have a distinct dependence on autophagy, a conserved eukaryotic catabolic pathway that serves to degrade proteins, other macromolecules, and organelles via the lysosome as part of a recycling process to maintain cellular homeostasis during basal and stress conditions. The unique importance of autophagy in PDAC suggests that there may be a subset of proteins that are specifically targeted by autophagy for degradation, which would promote proliferation and survival, and that these may represent new targets for therapy. Using a quantitative PDAC cellular autophagosomal proteomics approach, we identified NCOA4 as the autophagy receptor for ferritin, the cellular iron storage complex, and demonstrated that ferritin autophagy (ferritinophagy) is a highly regulated process and plays a central role in the larger context of cellular and organismal iron homeostasis. Given the reliance of PDAC, on both high levels of autophagy as well as iron, we studied the role of NCOA4 and ferritinophagy in PDAC thereby identifying NCOA4-mediated ferritinophagy and more broadly lysosomal iron metabolism as a PDAC dependency and therapeutic target. We recently determined the cryo-EM structure of the NCOA4-Ferritin complex for future drug design. To identify the role of autophagy and the lysosome in PDAC metastatic progression, we co-developed an in vivo capable lysosomal enrichment that enables capture and proteomic profiling of lysosomes at different stages of tumor evolution, including early stage, late stage, and metastatic disease. Employing this strategy, we successfully captured lysosomal proteins from primary tumors (pancreas), as well as lung and liver metastases. Our preliminary analysis revealed an enrichment of resident lysosomal membrane and luminal proteins in the datasets, confirming successful isolation of intact lysosomes from these complex tissues. Additionally, we identified non-resident lysosomal proteins (termed cargo) that are enriched and co-evolve with advancing tumor stages. Notably, our results reveal distinct differences in the lysosomal proteome of tumor cells across different tissues. These findings emphasize the critical role of lysosomal function in tumor progression and suggest that targeting lysosomal components could be a promising therapeutic strategy for treating metastatic PDAC. Finally, given the challenging pharmacokinetic and pharmacodynamic properties and limited clinical efficacy of hydroxychloroquine as an autophagy/lysosomal inhibition strategy, we are now exploring novel autophagy inhibition strategies in PDAC to effectively target this pathway for clinical benefit. Citation Format: Joseph D. Mancias. Targeting autophagy in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr IA-01.

FOXO1-NCOA4 Axis Contributes to Cisplatin-Induced Cochlea Spiral Ganglion Neuron Ferroptosis via Ferritinophagy.

Mammalian cochlea spiral ganglion neurons (SGNs) are crucial for sound transmission, they can be damaged by chemotherapy drug cisplatin and lead to irreversible sensorineural hearing loss (SNHL), while such damage can also render cochlear implants ineffective. However, the mechanisms underlying cisplatin-induced SGNs damage and subsequent SNHL are still under debate and there is no currently effective clinical treatment. Here, this study demonstrates that ferroptosis is triggered in SGNs following exposure to cisplatin. Inhibiting ferroptosis protects against cisplatin-induced SGNs damage and hearing loss, while inducing ferroptosis intensifies these effects. Furthermore, cisplatin prompts nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy in SGNs, while knocking down NCOA4 mitigates cisplatin-induced ferroptosis and hearing loss. Notably, the upstream regulator of NCOA4 is identified and transcription factor forkhead box O1 (FOXO1) is shown to directly suppress NCOA4 expression in SGNs. The knocking down of FOXO1 amplifies NCOA4-mediated ferritinophagy, increases ferroptosis and lipid peroxidation, while disrupting the interaction between FOXO1 and NCOA4 in NCOA4 knock out mice prevents the cisplatin-induced SGN ferroptosis and hearing loss. Collectively, this study highlights the critical role of the FOXO1-NCOA4 axis in regulating ferritinophagy and ferroptosis in cisplatin-induced SGNs damage, offering promising therapeutic targets for SNHL mitigation.

COPZ1 regulates ferroptosis through NCOA4-mediated ferritinophagy in lung adenocarcinoma

BackgroundFerroptosis, a type of autophagy-dependent cell death, has been implicated in the pathogenesis of lung adenocarcinoma (LUAD). This study aimed to investigate the involvement of coatomer protein complex I subunit zeta 1 (COPZ1) in ferroptosis and ferritinophagy in LUAD. MethodsPublicly available human LUAD sample data were obtained from the TCGA database to analyze the association of COPZ1 expression with LUAD grade and patient survival. Clinical samples of LUAD and para-carcinoma tissues were collected. COPZ1-deficient LUAD cell model and xenograft model were established. These models were analyzed to evaluate tumor growth, lipid peroxidation levels, mitochondrial structure, autophagy activation, and iron metabolism. ResultsHigh expression of COPZ1 was indicative of malignancy and poor overall survival. Clinical LUAD tissues showed increased COPZ1 expression and decreased nuclear receptor coactivator 4 (NCOA4) expression. COPZ1 knockdown inhibited xenograft tumor growth and induced apoptosis. COPZ1 knockdown elevated the levels of ROS, Fe2+ and lipid peroxidation. COPZ1 knockdown also caused mitochondrial shrinkage. Liproxstatin-1, deferoxamine, and z-VAD-FMK reversed the effects of COPZ1 knockdown on LUAD cell proliferation and ferroptosis. Furthermore, COPZ1 was directly bound to NCOA4. COPZ1 knockdown restricted FTH1 expression and promoted NCOA4 and LC3 expression. NCOA4 knockdown reversed the regulation of iron metabolism, lipid peroxidation, and mitochondrial structure induced by COPZ1 knockdown. COPZ1 knockdown induced the translocation of ferritin to lysosomes for degradation, whereas NCOA4 knockdown disrupted this process. ConclusionThis study provides novel evidence that COPZ1 regulates NCOA4-mediated ferritinophagy and ferroptosis. These findings provide new insights into the pathogenesis and potential treatment of LUAD.

Selenium nanoparticles alleviate renal ischemia/reperfusion injury by inhibiting ferritinophagy via the XBP1/NCOA4 pathway

Acute kidney injury (AKI) is closely related to lysosomal dysfunction and ferroptosis in renal tubular epithelial cells (TECs), for which effective treatments are urgently needed. Although selenium nanoparticles (SeNPs) have emerged as promising candidates for AKI therapy, their underlying mechanisms have not been fully elucidated. Here, we investigated the effect of SeNPs on hypoxia/reoxygenation (H/R)-induced ferroptosis and lysosomal dysfunction in TECs in vitro and evaluated their efficacy in a murine model of ischemia/reperfusion (I/R)-AKI. We observed that H/R-induced ferroptosis was accompanied by lysosomal Fe2+ accumulation and dysfunction in TECs, which was ameliorated by SeNPs administration. Furthermore, SeNPs protected C57BL/6 mice against I/R-induced inflammation and ferroptosis. Mechanistically, we found that lysosomal Fe2+ accumulation and ferroptosis were associated with the excessive activation of NCOA4-mediated ferritinophagy, a process mitigated by SeNPs through the upregulation of X-box binding protein 1 (XBP1). Downregulation of XBP1 promoted ferritinophagy and partially counteracted the protective effects of SeNPs on ferroptosis inhibition in TECs. Overall, our findings revealed a novel role for SeNPs in modulating ferritinophagy, thereby improving lysosomal function and attenuating ferroptosis of TECs in I/R-AKI. These results provide evidence for the potential application of SeNPs as therapeutic agents for the prevention and treatment of AKI.

Targeting ferritinophagy impairs quiescent cancer stem cells in acute myeloid leukemia in vitro and in vivo models.

Acute myeloid leukemia (AML) remains a challenging hematological malignancy with poor prognosis and limited treatment options. Leukemic stem cells (LSCs) contribute to therapeutic failure, relapse, and adverse outcome. This study investigates the role of quiescence and related molecular mechanisms in AML pathogenesis and LSC functions to identify potential therapeutic targets. Transcriptomic analysis revealed that the LSC-enriched quiescent cell population has a distinct gene signature with prognostic relevance in patients with AML. Mechanistically, quiescent blasts exhibit increased autophagic activity, which contributes to their sustained viability. Proteomic profiling uncovered differential requirements for iron metabolism between quiescent and cycling cells, revealing a unique dependence of quiescent cells on ferritinophagy, a selective form of autophagy mediated by nuclear receptor coactivator 4 (NCOA4), which regulates iron bioavailability. We evaluated the therapeutic potential of inhibiting NCOA4-mediated ferritinophagy using genetic knockdown and chemical inhibition approaches. In vitro assays showed that suppression of NCOA4 was toxic to leukemic blasts, particularly the CD34+CD38- LSC-enriched population, without affecting normal CD34+ hematopoietic progenitors. In vivo studies using murine patient-derived xenograft (PDX) models of AML confirmed that NCOA4 inhibition reduced tumor burden and impaired LSC viability and self-renewal, indicating a specific vulnerability of these cells to ferritinophagy disruption. Our findings underscore the role of NCOA4-mediated ferritinophagy in maintaining LSC quiescence and function and suggest that targeting this pathway may be an effective therapeutic strategy for AML. This study highlights the potential of NCOA4 inhibition to improve AML outcomes and paves the way for future research and clinical development.

LACTB suppresses liver cancer progression through regulation of ferroptosis

Ferroptosis, driven by iron-dependent phospholipid peroxidation, is emerging as an intrinsic cancer defense mechanism. However, the regulatory networks involved in ferroptosis remain largely unknown. Here, we found that serine beta-lactamase-like protein (LACTB) inhibits liver cancer progression by regulating ferroptosis. LACTB is downregulated in liver cancer, and the ectopic expression of LACTB markedly inhibits cell viability, colony formation, and tumour growth. LACTB knockout exerts the opposite effects. Further investigation revealed that LACTB blocks HSPA8 transcription in a p53-dependent manner, resulting in the elevation of NCOA4-mediated ferritinophagy and inhibition of SLC7A11/GSH/GPX4 signalling, thereby triggering ferroptosis and suppressing liver cancer progression. Liver cancer cells with an endogenous mutation of p53 binding site in the HSPA8 promoter exhibited increased resistance to ferroptosis inducers, and the ferroptosis-promoting effect of LACTB was significantly weakened in these mutant cells. Importantly, LACTB is identified as a downstream target of lenvatinib, and adeno-associated virus-mediated overexpression and knockdown of LACTB notably enhance and attenuate the anti-tumour efficacy of lenvatinib in vivo, respectively. Taken together, our study reveals a novel action of LACTB and provides potential therapeutic strategies for enhancing the efficacy of lenvatinib in liver cancer.

Tumor suppressor Par-4 activates autophagy-dependent ferroptosis

Ferroptosis is a unique iron-dependent form of non-apoptotic cell death characterized by devastating lipid peroxidation. Whilst growing evidence suggests that ferroptosis is a type of autophagy-dependent cell death, the underlying molecular mechanisms regulating ferroptosis are largely unknown. In this study, through an unbiased RNA-sequencing screening, we demonstrate the activation of a multi-faceted tumor-suppressor protein Par-4/PAWR during ferroptosis. Functional studies reveal that genetic depletion of Par-4 effectively blocks ferroptosis, whereas Par-4 overexpression sensitizes cells to undergo ferroptosis. More importantly, we have determined that Par-4-triggered ferroptosis is mechanistically driven by the autophagic machinery. Upregulation of Par-4 promotes activation of ferritinophagy (autophagic degradation of ferritin) via the nuclear receptor co-activator 4 (NCOA4), resulting in excessive release of free labile iron and, hence, enhanced lipid peroxidation and ferroptosis. Inhibition of Par-4 dramatically suppresses the NCOA4-mediated ferritinophagy signaling axis. Our results also establish that Par-4 activation positively correlates with reactive oxygen species (ROS) production, which is critical for ferritinophagy-mediated ferroptosis. Furthermore, Par-4 knockdown effectively blocked ferroptosis-mediated tumor suppression in the mouse xenograft models. Collectively, these findings reveal that Par-4 has a crucial role in ferroptosis, which could be further exploited for cancer therapy.

Excessive autophagy of myocardial cells promotes ferroptosis and exacerbates heart failure in the state of myocardial infarction

IntroductionThis study investigates the molecular mechanisms by which excessive autophagy exacerbates post-myocardial infarction heart failure(post-MI HF) through nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy and ferroptosis.Material and methodsWe developed a post-MI heart failure model in Sprague-Dawley rats via coronary artery ligation, alongside an in vitro heart failure model using hypoxia/reoxygenation-stimulated H9C2 cells. Intervention with rapamycin (autophagy activator), 3-methyladenine (autophagy inhibitor), desferrioxamine, and ferredoxin-1(ferroptosis inhibitors). Various techniques, including echocardiography, immunofluorescence colocalization, C11 BODIPY 581/591 staining, flow cytometry, transmission electron microscopy, western blotting, and RT-qPCR, were employed.ResultsIn vivo analyses revealed that NCOA4-mediated ferritinophagy and ferroptosis are significant in post-MI HF. Manipulating autophagy through rapamycin and 3-methyladenine influenced the expression of NCOA4 and glutathione peroxidase 4 (GPX4), subsequently affecting ferroptosis and modulating heart failure severity. Our in vitro experiments corroborated these findings, demonstrating that heightened autophagy amplifies NCOA4 expression, which in turn fosters ferroptosis and exacerbates myocardial injury. Interestingly, silencing of NCOA4 partially mitigated autophagy-induced iron deficiency, indicating a crucial intersection between autophagy and iron metabolism. Moreover, the cardioprotective effects observed following NCOA4 silencing were negated by concurrent GPX4 silencing.ConclusionsOur findings elucidate that autophagy precedes NCOA4 in its regulatory pathway and directly influences ferritinophagy. Enhanced autophagy augments intracellular free iron and unstable iron pools, triggering lipid peroxidation through ferritinophagy, which promotes ferroptosis and impairs cardiac function. These insights offer a novel scientific basis for developing therapeutic strategies for heart failure post-MI HF.

Nuciferine Protects Cochlear Hair Cells from Ferroptosis through Inhibiting NCOA4-Mediated Ferritinophagy.

Cisplatin is a widely used antineoplastic drug for treating various types of cancers. However, it can cause severe side effects, such as bilateral and irreversible hearing loss, which significantly impacts quality of life. Ferroptosis, an iron-dependent form of programmed cell death, has been implicated in the pathogenesis of cisplatin-induced ototoxicity. Here, we investigated the effects of nuciferine, a natural active ingredient isolated from lotus species, on the ferroptosis of cochlear hair cells. Firstly, our results demonstrated that nuciferine can protect hair cells against RSL3-induced and cisplatin-induced damage. Secondly, nuciferine treatment reduced ferrous iron (Fe2+) overload in cochlear hair cells via inhibiting NCOA4-mediated ferritinophagy. Inhibition of ferritinophagy by knocking down Ncoa4 alleviated cisplatin-induced ototoxicity. Importantly, nuciferine treatment mitigated cochlear hair cell loss and damage to ribbon synapse, and improved mouse hearing function in an acute cisplatin-induced hearing loss model. Our findings highlight the role of NCOA4-mediated ferritinophagy in the pathogenesis of cisplatin-induced ototoxicity and provide evidence for nuciferine as a promising protective agent for treating cisplatin-induced hearing loss.

Targeting ferroptosis using Chinese herbal compounds to treat respiratory diseases

BackgroundRespiratory diseases pose a grave threat to human life. Therefore, understanding their pathogenesis and therapeutic strategy is important. Ferroptosis is a novel type of iron-dependent programmed cell death, distinct from apoptosis, necroptosis, and autophagy, characterised by iron, reactive oxygen species, and lipid peroxide accumulation, as well as glutathione (GSH) depletion and GSH peroxidase 4 (GPX4) inactivation. A close association between ferroptosis and the onset and progression of respiratory diseases, including chronic obstructive pulmonary disease, acute lung injury, bronchial asthma, pulmonary fibrosis, and lung cancer, has been reported. Recent studies have shown that traditional Chinese medicine (TCM) compounds exhibit unique advantages in the treatment of respiratory diseases owing to their natural properties and potential efficacy. These compounds can effectively regulate ferroptosis by modulating several key signalling pathways such as system Xc− -GSH-GPX4, NCOA4-mediated ferritinophagy, Nrf2-GPX4, and Nrf2/HO-1, thus playing a positive role in improving respiratory diseases. PurposeThis comprehensive review systematically outlines the regulatory role of ferroptosis in the onset and progression of respiratory diseases and provides evidence for treating respiratory diseases by targeting ferroptosis with TCM compounds. These insights aim to offer potential remedies for the clinical prevention and treatment of respiratory diseases. Study design and methodsWe searched scientific databases PubMed, Web of Science, Scopus, and CNKI using keywords such as “ferroptosis”,“respiratory diseases”,“chronic obstructive pulmonary disease”,“bronchial asthma”,“acute lung injury”,“pulmonary fibrosis”,“lung cancer”,“traditional Chinese medicine”,“traditional Chinese medicine compound”,“monomer”, and “natural product” to retrieve studies on the therapeutic potential of TCM compounds in ameliorating respiratory diseases by targeting ferroptosis. The retrieved data followed PRISMA criteria (preferred reporting items for systematic review). ResultsTCM compounds possess unique advantages in treating respiratory diseases, stemming from their natural origins and proven clinical effectiveness. TCM compounds can exert therapeutic effects on respiratory diseases by regulating ferroptosis, which mainly involves modulation of pathways such as system Xc− -GSH-GPX4,NCOA4-mediated ferritinophagy, Nrf2-GPX4, and Nrf2/HO-1. ConclusionTCM compounds have demonstrated promising potential in improving respiratory diseases through the regulation of ferroptosis. The identification of specific TCM-related inducers and inhibitors of ferroptosis holds great significance in developing more effective strategies. However, current research remains confined to animal and cellular studies, emphasizing the imperative for further verifications through high-quality clinical data.

Melatonin alleviates septic ARDS by inhibiting NCOA4-mediated ferritinophagy in alveolar macrophages

Ferroptosis is a novel form of programmed cell death which can exacerbate lung injury in septic acute respiratory distress syndrome (ARDS). Alveolar macrophages, crucial innate immune cells, play a pivotal role in the pathogenesis of ARDS. Ferritinophagy is a process of ferritin degradation mediated by nuclear receptor coactivator 4 (NCOA4) which releases large amounts of iron ions thus promoting ferroptosis. Recent evidence revealed that inhibiting macrophage ferroptosis can effectively attenuate pulmonary inflammatory injury. Melatonin (MT), an endogenous neurohormone, has antioxidant and anti-inflammatory effects and can reduce septic ARDS. However, it is not clear whether MT’s pulmonary protective effect is related to the inhibition of macrophage ferritinophagy. Our in vitro experiments demonstrated that MT decreased intracellular malondialdehyde (MDA), Fe2+, and lipid peroxidation levels, increased glutathione (GSH) levels and cell proliferation, and upregulated glutathione peroxidase 4 (GPX4) and ferritin heavy chain 1 (FTH1) protein levels in LPS-treated macrophages. Mechanistically, the antiferroptotic effect of MT on LPS-treated macrophages was significantly compromised by the overexpression of NCOA4. Our in vivo experiments revealed that MT alleviated the protein expression of NCOA4 and FTH1 in the alveolar macrophages of septic mice. Furthermore, MT improved lipid peroxidation and mitigated damage in alveolar macrophages and lung tissue, ultimately increasing the survival rates of septic mice. These findings indicate that MT can inhibit ferroptosis in an NCOA4-mediated ferritinophagy manner, thereby ameliorating septic ARDS.

#289 Lipocalin-2 promotes cardiovascular calcification in chronic kidney disease by upregulating STAT3/NCOA4-mediated ferritinophagy and ferroptosis

Abstract Background and Aims Cardiovascular calcification (CVC) is a common complication of chronic kidney disease (CKD) and contributes to cardiovascular morbidity and mortality without any effective therapies. To date, the underlying mechanism of CKD-CVC has not been elucidated. Recent studies suggest that ferroptosis is a novel programmed cell death that may play a role in the process of CKD-CVC, but the mechanism is largely unclear. Ferritinophagy is an important inducer of ferroptosis that is regulated by nuclear receptor coactivator 4 (NCOA4). However, the relationship between ferritinophagy and CKD-CVC has not been explored. Lipocalin-2 (LCN2) is an iron-trafficking protein that has been associated with both osteogenesis and ferroptosis, but the exact role and molecular mechanism of LCN2 in CKD-CVC needs to be explored. Method A cross-sectional clinical study was utilized to examine the association between LCN2 expression and CKD-CVC in serum and calcified radial arteries of CKD patients. LCN2 knockout mice, their wild type littermates and mice infected with VSMCs-targeted adeno-associated virus encoding LCN2 gene or negative control gene were all fed by the high adenine and phosphate diet to induce CKD-CVC.VSMCs transfected with LCN2-overexpressing lentivirus, LCN2-shRNA lentivirus or LCN2 siRNA and recombinant LCN2 stimulated VSMCs were treated under the condition of calcifying medium to investigate the role of LCN2 in CKD-CVC in vitro. Cardiovascular calcification was evaluated by Von-kossa staining and tissue calcium content assay. VSMCs calcification were checked by the Alizarin Red Staining and cellular calcium content assay. Cellular reactive oxygen species (ROS) were measured by the DCFDA assay. Lipid peroxidation was measured by the fluorescence of the fatty acid analog C11-BODIPY581/591 probe. Intracellular labile ferrous iron levels were measured by fluorescence of Phen Green SK probe and ferro-orange probe. Transmission electron microscopy was also used to examine mitochondrial change of ferroptosis. Results The cross-sectional study identified that LCN2 levels in serum and radial arteries were significantly increased as the severity of CKD-CVC increased. High adenine and phosphate diet intake provokes renal fibrosis and cardiovascular calcification in vivo. In vitro, five-days high phosphate sodium stimulation induced calcium deposition and osteoblastic trans differentiation of VSMCs. Meanwhile, high phosphate also increased the levels of ferroptosis in VSMCs as evidenced by decreased cell viability, iron and ROS accumulation, upregulated PTGS2 and MDA levels, reduced glutathione peroxidase 4 (GPX4) levels and significantly mitochondrial damage in VSMCs. Intriguingly, calcium deposition in VSMCs could be reversed by the ferroptosis inhibitor ferrostatin-1. LCN2 expression was significantly upregulated both in the aorta of mice and calcified VSMCs. Compared with WT mice, deletion of LCN2 inhibited cardiovascular calcification whereas VSMC-targeted LCN2 overexpression aggravated CKD-CVC. Consistently, LCN2 knockdown also alleviated calcium deposition of VSMCs. Meanwhile, inhibition of ferroptosis were observed after LCN2 silencing as evidenced by decreased ferrous and lipid oxidation while increased levels of GPX4. Mechanically, we demonstrated high phosphate increased the protein level of NCOA4 and downregulated FTH1, which was reversed by depletion of LCN2. Knockdown of NCOA4 partly alleviated the ferritin reduction, ferroptosis, and calcification in VSMCs, indicating that NCOA4-mediated ferritinophagy was required for VSMCs calcification. Furthermore, we found that the recombinant LCN2 treatment significantly upregulated phosphorylated STAT3. STAT3 signaling inhibitor abolished the recombinant LCN2 induced NCOA4 upregulation and calcification in VSMCs. Conclusion Our findings indicate that the pro-calcific effect of high phosphate is due to VSMCs ferroptosis mediated by LCN2 upregulation. LCN2 silencing alleviates CKD-CVC by suppressing the STAT3/ NCOA4/FTH1 signaling mediated ferritinophagy. LCN2 could serve as a candidate therapeutic strategy for CKD-CVC.

NCOA4-mediated ferritinophagy participates in cadmium-triggered ferroptosis in spermatogonia

Cadmium (Cd) is a common pollutant with reproductive toxicity. Our previous study revealed that Cd triggered spermatogonia ferroptosis. However, the underlying mechanisms remain unclear. Nuclear receptor coactivator 4 (NCOA4) mediates ferritinophagy and specific degradation of ferritin through lysosomes, resulting in the release of ferrous ions. Excessive autophagy can lead to ferroptosis. This study investigated the role of autophagy in Cd-triggered ferroptosis using GC-1 spermatogonial (spg) cells which exposed to CdCl2 (5 μM, 10 μM, or 20 μM) for 24 without/with CQ. The cells which transfected with Ncoa4-siRNA were used to explore the role of NCOA4-mediated ferritinophagy in Cd-triggered ferroptosis. The results revealed that Cd caused mitochondrial swelling, rupture of cristae, and vacuolar-like changes. The Cd-treated cells exhibited more autophagosomes. Simultaneously, Cd increased intracellular iron, reactive oxygen species, and malondialdehyde concentrations while decreasing glutathione content and Superoxide Dismutase-2 activity. Moreover, Cd upregulated mRNA levels of ferritinophagy-associated genes (Ncoa4, Lc3b and Fth1), as well as enhanced protein expression of NCOA4, LC3B, and FTH1. While Cd decreased the mRNA and protein expression of p62/SQSTM1. These results showed that Cd caused ferritinophagy and ferroptosis. The use of chloroquine to inhibit autophagy ameliorated Cd-induced iron overload and ferroptosis. Moreover, Ncoa4 knockdown in spermatogonia significantly reduced intracellular iron concentration and alleviated Cd-triggered ferroptosis. In conclusion, our findings demonstrate that Cd activates the ferritinophagy pathway mediated by NCOA4, resulting in iron accumulation through ferritin degradation. This causes oxidative stress, ultimately initiating ferroptosis in spermatogonia. Our results may provide new perspectives and potential strategies for preventing and treating Cd-induced reproductive toxicity.

Ganoderic acid A mitigates dopaminergic neuron ferroptosis via inhibiting NCOA4-mediated ferritinophagy in Parkinson’s disease mice

Ethnopharmacological relevanceGanoderma lucidum, a renowned tonic traditional Chinese medicine, is widely recognized for the exceptional activity in soothing nerves and nourishing the brain. It has been extensively employed to alleviate various neurological disorders, notably Parkinson's disease (PD). Aim of the studyTo appraise the antiparkinsonian effect of GAA, the main bioactive constituent of G. lucidum, and clarify the molecular mechanism through the perspective of ferritinophagy-mediated dopaminergic neuron ferroptosis. Materials and methodsPD mouse and cell models were established using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP+), respectively. Cell viability, behavioral tests and immunofluorescence analysis were performed to evaluate the neurotoxicity, motor dysfunction and dopaminergic loss, respectively. Biochemical assay kits were used to determine the levels of iron, lipid reactive oxygen species (ROS), malondialdehyde (MDA), total ROS and glutathione (GSH). Western blot and immunofluorescence were applied to detect the expressions of nuclear receptor co-activator 4 (NCOA4), ferritin heavy chain 1 (FTH1), p62 and LC3B. Additionally, NCOA4-overexpressing plasmid vector was constructed to verify the inhibitory effect of GAA on the neurotoxicity and ferroptosis-related parameters in PD models. ResultsGAA significantly mitigated MPP+/MPTP-induced neurotoxicity, motor dysfunction and dopaminergic neuron loss (p＜0.01 or p＜0.05). In contrast to MPP+/MPTP treatment, GAA treatment decreased the levels of iron, MDA, lipid and total ROS, while increasing the GSH level. GAA also reduced the levels of NCOA4 and LC3B, and enhanced the expressions of FTH1 and p62 in PD models (p＜0.01 or p＜0.05). However, the protective effect of GAA against the neurotoxicity, NCOA4-mediated ferritinophagy and ferroptosis in PD model was abolished by the overexpression of NCOA4 (p＜0.01). ConclusionGAA exerted a protective effect on PD, and this effect was achieved by suppressing dopaminergic neuron ferroptosis through the inhibition of NCOA4-mediated ferritinophagy.

AMPK activation eliminates senescent cells in diabetic wound by inducing NCOA4 mediated ferritinophagy.

Diabetic wounds are one of the long-term complications of diabetes, with a disordered microenvironment, diabetic wounds can easily develop into chronic non-healing wounds, which can impose a significant burden on healthcare. In diabetic condition, senescent cells accumulate in the wound area and suppress the wound healing process. AMPK, as a molecule related to metabolism, has a close relationship with aging and diabetes. The purpose of this study was to investigate the effects of AMPK activation on wound healing and explore the underlying mechanisms. AMPK activator A769662 was topically applied in wound models of diabetic mice. Alterations in the wound site were observed and analyzed by immunohistochemistry. The markers related to autophagy and ferritinophagy were analyzed by western blotting and immunofluorescence staining. The role of AMPK activation and ferritinophagy were also analyzed by western blotting. Our results show that AMPK activation improved diabetic wound healing and reduced the accumulation of senescent cells. Intriguingly, we found that AMPK activation-induced ferroptosis is autophagy-dependent. We detected that the level of ferritin had deceased and NCOA4 was markedly increased after AMPK activation treatment. We further investigated that NCOA4-mediated ferritinophagy was involved in ferroptosis triggered by AMPK activation. Most importantly, AMPK activation can reverse the ferroptosis-insensitive of senescent fibroblast cells in diabetic mice wound area and promote wound healing. These results suggest that activating AMPK can promote diabetic wound healing by reversing the ferroptosis-insensitive of senescent fibroblast cells. AMPK may serve as a regulatory factor in senescent cells in the diabetic wound area, therefore AMPK activation can become a promising therapeutic method for diabetic non-healing wounds.

Ferritinophagy mediates adaptive resistance to EGFR tyrosine kinase inhibitors in non-small cell lung cancer

Osimertinib (Osi) is a widely used epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI). However, the emergence of resistance is inevitable, partly due to the gradual evolution of adaptive resistant cells during initial treatment. Here, we find that Osi treatment rapidly triggers adaptive resistance in tumor cells. Metabolomics analysis reveals a significant enhancement of oxidative phosphorylation (OXPHOS) in Osi adaptive-resistant cells. Mechanically, Osi treatment induces an elevation of NCOA4, a key protein of ferritinophagy, which maintains the synthesis of iron-sulfur cluster (ISC) proteins of electron transport chain and OXPHOS. Additionally, active ISC protein synthesis in adaptive-resistant cells significantly increases the sensitivity to copper ions. Combining Osi with elesclomol, a copper ion ionophore, significantly increases the efficacy of Osi, with no additional toxicity. Altogether, this study reveals the mechanisms of NCOA4-mediated ferritinophagy in Osi adaptive resistance and introduces a promising new therapy of combining copper ionophores to improve its initial efficacy.

JWA binding to NCOA4 alleviates degeneration in dopaminergic neurons through suppression of ferritinophagy in Parkinson's disease

Parkinson's disease (PD) poses a significant challenge in neurodegenerative disorders, characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc). The intricate mechanisms orchestrating DA neurodegeneration in PD are not fully understood, necessitating the exploration of innovative therapeutic approaches. Recent studies have implicated ferroptosis as a major contributor to the loss of DA neurons, revealing a complex interplay between iron accumulation and neurodegeneration. However, the sophisticated nature of this process challenges the conventional belief that mere iron removal could effectively prevent DA neuronal ferroptosis. Here, we report JWA, alternatively referred to as ARL6IP5, as a negative regulator of ferroptosis, capable of ameliorating DA neuronal loss in the context of PD. In this study, synchronized expression patterns of JWA and tyrosine hydroxylase (TH) in PD patients and mice were observed, underscoring the importance of JWA for DA neuronal survival. Screening of ferroptosis-related genes unraveled the engagement of iron metabolism in the JWA-dependent inhibition of DA neuronal ferroptosis. Genetic manipulation of JWA provided compelling evidence linking its neuroprotective effects to the attenuation of NCOA4-mediated ferritinophagy. Molecular docking, co-immunoprecipitation, and immunofluorescence studies confirmed that JWA mitigated DA neuronal ferroptosis by occupying the ferritin binding site of NCOA4. Moreover, the JWA-activating compound, JAC4, demonstrated promising neuroprotective effects in cellular and animal PD models by elevating JWA expression, offering a potential avenue for neuroprotection in PD. Collectively, our work establishes JWA as a novel regulator of ferritinophagy, presenting a promising therapeutic target for addressing DA neuronal ferroptosis in PD.

Regulating NCOA4-Mediated Ferritinophagy for Therapeutic Intervention in Cerebral Ischemia-Reperfusion Injury.

Ischemic stroke presents a global health challenge, necessitating an in-depth comprehension of its pathophysiology and therapeutic strategies. While reperfusion therapy salvages brain tissue, it also triggers detrimental cerebral ischemia-reperfusion injury (CIRI). In our investigation, we observed the activation of nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy in an oxygen-glucose deprivation/reoxygenation (OGD/R) model using HT22 cells (P < 0.05). This activation contributed to oxidative stress (P < 0.05), enhanced autophagy (P < 0.05) and cell death (P < 0.05) during CIRI. Silencing NCOA4 effectively mitigated OGD/R-induced damage (P < 0.05). These findings suggested that targeting NCOA4-mediated ferritinophagy held promise for preventing and treating CIRI. Subsequently, we substantiated the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway effectively regulated the NCOA4-mediated ferritinophagy, by applying the cGAS inhibitor RU.521 and performing NCOA4 overexpression (P < 0.05). Suppressing the cGAS-STING pathway efficiently curtailed ferritinophagy (P < 0.05), oxidative stress (P < 0.05), and cell damage (P < 0.05) of CIRI, while NCOA4 overexpression could alleviate this effect (P < 0.05). Finally, we elucidated the specific molecular mechanism underlying the protective effect of the iron chelator deferoxamine (DFO) on CIRI. Our findings revealed that DFO alleviated hypoxia-reoxygenation injury in HT22 cells through inhibiting NCOA4-mediated ferritinophagy and reducing ferrous ion levels (P < 0.05). However, the protective effects of DFO were counteracted by cGAS overexpression (P < 0.05). In summary, our results indicated that the activation of the cGAS-STING pathway intensified cerebral damage during CIRI by inducing NCOA4-mediated ferritinophagy. Administering the iron chelator DFO effectively attenuated NCOA4-induced ferritinophagy, thereby alleviating CIRI. Nevertheless, the role of the cGAS-STING pathway in CIRI regulation likely involves intricate mechanisms, necessitating further validation in subsequent investigations.

Emodin induces ferroptosis in colorectal cancer through NCOA4-mediated ferritinophagy and NF-κb pathway inactivation.

Ferroptosis is a new programmed cell death characterized by iron-dependent lipid peroxidation. Targeting ferroptosis is considered a promising strategy for anti-cancer therapy. Recently, natural compound has gained increased attention for their advantage in cancer treatment, and the exploration of natural compounds as ferroptosis inducers offers a hopeful avenue for advancing cancer treatment modalities. Emodin is a natural anthraquinone derivative in many widely used Chinese medicinal herbs. In our previous study, we predicted that the anti-cancer effect of Emodin might related to ferroptosis by using RNA-seq in colorectal cancer (CRC). Thus, in this study, we aim to investigate the molecular mechanism underlying Emodin-mediated ferroptosis in CRC. Cell-based assays including CCK-8, colony formation, EdU, and Annexin V/PI staining were employed to assess Emodin's impact on cell proliferation and apoptosis. Furthermore, various techniques such as FerroOrange staining, C11-BODIPY 581/591 staining, iron, MDA, GSH detection assay and transmission electron microscopy were performed to examine the role of Emodin in ferroptosis. Additionally, specific NCOA4 knockdown cell lines were generated to elucidate the involvement of NCOA4 in Emodin-induced ferroptosis. Moreover, the effects of Emodin on ferroptosis were further confirmed through the application of inhibitors, including Ferrostatin-1, 3-MA, DFO, and PMA. As a results, Emodin inhibited proliferation and induced apoptosis in CRC cells. Emodin could decrease GSH content, xCT and GPX4 expression, meanwhile increasing ROS generation, MDA, and lipid peroxidation, and these effects could reverse by ferroptosis inhibitor, Ferostatin-1, iron chelator DFO, autophagy inhibitor 3-MA and NCOA4 silencing. Moreover, Emodin could inactivate NF-κb pathway, and PMA, an activator of NF-κb pathway could alleviate Emodin-induced ferroptosis in CRC cells. Xenograft mouse model also showed that Emodin suppressed tumor growth and induced ferroptosis in vivo. In conclusion, these results suggested that Emodin induced ferroptosis through NCOA4-mediated ferritinophagy by inactivating NF-κb pathway in CRC cells. These findings not only identified a novel role for Emodin in ferroptosis but also indicated that Emodin may be a valuable candidate for the development of an anti-cancer agent.