Ferritinophagy is a selective form of autophagy in which ferritin, the primary intracellular iron storage protein complex, is targeted by NCOA4 (Nuclear receptor coactivator 4) to the lysosome for degradation. NCOA4-mediated ferritinophagy plays a crucial role in cellular iron metabolism, influencing iron homeostasis, heme synthesis, mitochondrial respiratory function, and ferroptosis, an iron-dependent form of cell death. Targeting ferritinophagy has emerged as a potential anticancer therapeutic strategy. In this context, we provide a flowchart of the procedures and accompanying protocols for monitoring ferritinophagic flux.

BACKGROUND: Pancreatic cancer (PC) is characterized by a poor prognosis and limited treatment options. Ferroptosis plays an important role in cancer, SET and MYND domain-containing protein 2 (SMYD2) is widely expressed in various cancers. However, the role of SMYD2 in regulating ferroptosis in PC remains unexplored. This study aimed to investigate the role of SMYD2 in mediating ferroptosis and its mechanistic implications in PC progression.
METHODS: The levels of SMYD2, c-Myc, and NCOA4 were assessed in PC tissues, and peritumoral tissues. SMYD2 expression was further analyzed in human PC cell lines. In BxPC3 cells, the expression of c-Myc, NCOA4, autophagy-related proteins, and mitochondrial morphology, was evaluated following transfection with si-SMYD2 and treatment with autophagy inhibitors and ferroptosis inhibitors. Ferroptosis levels were quantified using flow cytometry and ELISA assays. RNA immunoprecipitation was conducted to elucidate the interaction between c-Myc and NCOA4 mRNA. A xenograft mouse model was constructed to validate the impact of SMYD2 knockdown on PC growth.
RESULTS: SMYD2 and c-Myc were found to be highly expressed in PC tissues, while NCOA4 showed reduced expression. Among the PC cell lines studied, BxPC3 cells exhibited the highest SMYD2 expression. SMYD2 knockdown led to decreased c-Myc levels, increased NCOA4 expression, reduced autophagy-related protein expression, mitochondrial shrinkage, and heightened ferroptosis levels. Additionally, an interaction between c-Myc and NCOA4 was identified. In vivo, SMYD2 knockdown inhibited tumor growth.
CONCLUSIONS: Targeting SMYD2 inhibits PC progression by promoting ferritinophagy-dependent ferroptosis through the c-Myc/NCOA4 axis. These findings provide insights into potential diagnostic and therapeutic strategies for PC.

Hidrocystoma is thought to be a benign retention cyst of sweat ductal units. The lesion is usually located in the periorbital skin; however, lesions with similar histopathological features are rarely observed in extra-facial sites. Herein, we present four cases of hidrocystoma-like tumours in extra-facial skin sites that harboured a RET or ALK rearrangement. This study features a 67-year-old female with a 10 mm-sized digital tumour (Case 1), a 62-year-old male with an 8 mm-sized clavicular tumour (Case 2), a 61-year-old male with a 19 mm-sized digital tumour (Case 3), and an 11-year-old female with a 10 mm-size lower leg tumour (Case 4) as well as five control cases (Cases 5-9) of classical periorbital hidrocystoma. In Cases 1-4, multicystic tumours comprising a two-cell layer of inner cuboidal ductoglandular (p63- and SOX10+/-) and outer flat myoepithelial (p63+ and SOX10+) cells were observed. The inner ductoglandular tumour cells exhibited micropapillary projections and Roman bridging structures. No apparent atypical cells were observed. NCOA4::RET in Cases 1 and 3, CCDC6::RET in Case 2, and SLC12A2::ALK in Case 4 were revealed by next-generation sequencing or Sanger sequencing. In contrast, control cases of classical hidrocystoma (Cases 5-9) did not show intracystic proliferation, abundant cytoplasm, ALK immunoreactivity, or NCOA4::RET detection in the tumour cells. RET/ALK-rearranged hidrocystoma-like tumours are tumour entities that can be distinguished from classical hidrocystoma. This RET/ALK-rearranged neoplasm is benign and is frequently observed in the digits. Future studies will establish the concept, detailed clinicopathological characteristics, and genetic variations of hidrocystoma-like tumours.

OBJECTIVE: Bietti crystalline dystrophy (BCD) is an inherited retinal degeneration disease caused by mutations in the CYP4V2 gene. Currently, there is no clinical therapy approach available for BCD patients. Previous research has suggested that polyunsaturated fatty acids (PUFAs) may play a significant role in the development of BCD, implicating the involvement of ferroptosis in disease pathogenesis. In this work, we aimed to investigate the interplay between ferroptosis and BCD and to detect potential therapeutic strategies for the disease.
METHODS: Genetic-edited RPE cell line was first established in this study by CRISPR-Cas9 technology. Cyp4v3 (the homologous gene of human CYP4V2) knock out (KO) mice have also been used. Lipid profiling and transcriptome analysis of retinal pigment epithelium (RPE) cells from Cyp4v3 KO mice have been conducted. Ferroptosis phenotypes have been first investigated in BCD models in vitro and in vivo, including lipid peroxidation, mitochondrial changes, elevated levels of reactive oxygen species (ROS), and altered gene expression. Additionally, an iron chelator, deferiprone (DFP), has been tested in vitro and in vivo to determine its efficacy in suppressing ferroptosis and restoring the BCD phenotype.
RESULTS: Cyp4v3 KO mice exhibited progressive retinal degeneration and lipid accumulation, similar to the BCD phenotype, which was exacerbated by a high-fat diet (HFD). Increased levels of PUFAs, such as EPA (C22:5) and AA (C20:4), were observed in the RPE of Cyp4v3 KO mice. Transcriptome analysis of RPE in Cyp4v3 KO mice revealed changes in genes involved in iron homeostasis, particularly an upregulation of NCOA4, which was confirmed by immunofluorescence. Ferroptosis-related characteristics, including mitochondrial defects, lipid peroxidation, ROS accumulation, and upregulation of related genes, were detected in the RPE both in vitro and in vivo. Abnormal accumulation of ferrous iron was also detected. DFP, an iron chelator administration suppressed ferroptosis phenotype in CYP4V2 mutated RPE. Oral administration of DFP also restored the retinal function and morphology in Cyp4v3 KO mice.
CONCLUSIONS: This study represented the first evidence of the substantial role of ferroptosis in the development of BCD. PUFAs resulting from CYP4V2 mutation may serve as substrates for ferroptosis, potentially working in conjunction with NCOA4-regulated iron accumulation, ultimately leading to RPE degeneration. DFP administration, which chelates iron, has demonstrated its ability to reverse BCD phenotype both in vitro and in vivo, suggesting a promising therapeutic approach in the future.

Iron homeostasis is vital for the host\'s defense against pathogenic invasion and the ferritinophagy is a crucial mechanism in maintaining intracellular iron homeostasis by facilitating the degradation and recycling of stored iron. The nuclear receptor coactivator 4 (NCOA4) serves as a ferritinophagy receptor, facilitating the binding and delivery of ferritin to the autophagosome and lysosome. However, NCOA4 of the sea cucumber Apostichopus japonicus (AjNCOA4) has not been reported until now. In this study, we identified and characterized AjNCOA4 in A. japonicus. This gene encodes a polypeptide containing 597 amino acids with an open reading frame of 1794 bp. The inferred amino acid sequence of AjNCOA4 comprises an ARA70 domain. Furthermore, a multiple sequence alignment demonstrated varying degrees of sequence homology between AjNCOA4 from A. japonicus and other NCOA4 orthologs. The phylogenetic tree of NCOA4 correlates with the established timeline of metazoan evolution. Expression analysis revealed that AjNCOA4 is expressed in all tested tissues, including the body wall, muscle, intestine, respiratory tree, and coelomocytes. Following challenge with Vibrio splendidus, the coelomocytes exhibited a significant increase in AjNCOA4 mRNA levels, peaking at 24 h. We successfully obtained recombinant AjNCOA4 protein through prokaryotic expression and prepared a specific polyclonal antibody. Immunofluorescence and co-immunoprecipitation experiments demonstrated an interaction between AjNCOA4 and AjFerritin in coelomocytes. RNA interference-mediated knockdown of AjNCOA4 expression resulted in elevated iron ion levels in coelomocytes. Bacterial stimulation enhanced ferritinophagy in coelomocytes, while knockdown of AjNCOA4 reduced the occurrence of ferritinophagy. These findings suggest that AjNCOA4 modulates ferritinophagy induced by V. splendidus in coelomocytes of A. japonicus.

We show that redox active iron can induce a regulated form of non-apoptotic cell death and tissue damage called ferroptosis that can contribute to secondary damage and functional loss in the acute and chronic periods after spinal cord injury (SCI) in young, adult, female mice. Phagocytosis of red blood cells at sites of hemorrhage is the main source of iron derived from hemoglobin after SCI. Expression of hemeoxygenase-1 that induces release of iron from heme, is increased in spinal cord macrophages 7 days after injury. While iron is stored safely in ferritin in the injured spinal cord, it can, however, be released by NCOA4-mediated shuttling of ferritin to autophagosomes for degradation (ferritinophagy). This leads to the release of redox active iron that can cause free radical damage. Expression of NCOA4 is increased after SCI, mainly in macrophages. Increase in the ratio of redox active ferrous (Fe2+) to ferric iron (Fe3+) is also detected after SCI by capillary electrophoresis inductively coupled mass spectrometry. These changes are accompanied by other hallmarks of ferroptosis, i.e., deficiency in various elements of the antioxidant glutathione (GSH) pathway. We also detect increases in enzymes that repair membrane lipids (ACSL4 and LPCAT3) and thus promote on-going ferroptosis. These changes are associated with increased levels of 4-hydroxynonenal (4-HNE), a toxic lipid peroxidation product. Mice with mild SCI (30 kdyne force) treated with the ferroptosis inhibitor (UAMC-3203-HCL) either early or delayed times after injury showed improvement in locomotor recovery and secondary damage. Cerebrospinal fluid and serum samples from human SCI cases show evidence of increased iron storage (ferritin), and other iron related molecules, and reduction in GSH. Collectively, these data suggest that ferroptosis contributes to secondary damage after SCI and highlights the possible use of ferroptosis inhibitors to treat SCI.

Osteosarcoma is a primary solid bone malignancy, and surgery + chemotherapy is the most commonly used treatment. However, chemotherapeutic drugs can cause a range of side effects. Casticin, a polymethoxyflavonoid, has anti-tumor therapeutic effects. This study is aim to investigate the anti-osteosarcoma activity of casticin and explore the mechanism. Crystal violet staining, MTT assay, colony formation assay, wound healing assay, transwell assay, hoechst 33,258 staining, and flow cytometry analysis were used to investigate the effects of casticin on proliferation, migration, invasion, and apoptosis of osteosarcoma cells in vitro. The intracellular Fe2+, ROS, MDA, GSH/GSSG content changes were detected using the corresponding assay kits. The mRNA sequencing + bioinformatics analysis and western blot were used to detect the possible mechanism. We found that casticin caused G2/M phase cell cycle arrest in human osteosarcoma cells, inhibited the migration and invasion, and induced cell apoptosis and ferroptosis. Mechanistic studies showed the ferroptosis pathway was enriched stronger than apoptosis. Casticin up-regulated the expression of HMOX1, LC3 and NCOA4, meanwhile it activated MAPK signaling pathways. Animal experiments proved that casticin also inhibited the growth and metastasis of osteosarcoma cell xenograft tumor in vivo. In conclusion, casticin can induce ferroptosis in osteosarcoma cells through Fe2+ overload and ROS production mediated by HMOX1 and LC3-NCOA4. This provides a new strategy for osteosarcoma treatment.

Aims: The objective was to examine the efficacy of autologous platelet-rich gel (APG) in treating diabetic wound and investigate the association between APG and ferritinophagy. Methods: A total of 32 patients with diabetic foot (DF) and Wagner grade 1 to 2 were included. Within the APG group, individuals with DF received weekly APG treatment. In the non-APG group, DF patients received daily dressing changes. Flow cytometry quantified the proportion of endothelial progenitor cells (EPCs) in peripheral blood on days 0 and 10. The diabetic rat model was induced using Streptozotocin. Two circular skin wounds were created on the backs of rats. The normal glucose group received daily dressing changes on the wound. In the diabetic group, the left wound underwent daily dressing changes, whereas the right wound was treated with APG once a week. CD34 levels were tested 7 days after the skin damage. The levels of glutathione peroxidase 4 (GPX4), Nuclear Receptor Coactivator 4 (NCOA4), Light chain 3 (LC3), and Masson staining were quantified on 14 days. The wound area and wound healing rate were separately measured at 0 and 14 days after the injury, regardless of DF patients or diabetic rats. Results: The wound healing rate was higher in the APG group than in the non-APG group, regardless of DF patients or diabetic rats. The APG group had a greater ΔEPCs% in DF patients than the non-APG group. Regarding rat experiment, the APG group exhibited lower levels of NCOA4, and LC3 expressions and a shorter wound healing time. However, the APG group showed higher levels of CD34 expression, GPX4 protein, and collagen fibers than the non-APG group. Conclusions: Autologous platelet-rich gel accelerated the wound healing rate in diabetic populations and rats. Autologous platelet-rich gel promoted EPCs counts, collagen fiber volume, and vessel numbers. Autologous platelet-rich gel decreased LC3 and NCOA4 expression, but increased GPX4 protein expression. The possible mechanism was the inhibition of ferritinophagy.

Cerebral ischemia-reperfusion injury (CIRI) is the second leading cause of death worldwide, posing a huge risk to human life and health. Therefore, investigating the pathogenesis underlying CIRI and developing effective treatments are essential. Ferroptosis is an iron-dependent mode of cell death, which is caused by disorders in iron metabolism and lipid peroxidation. Previous studies demonstrated that ferroptosis is also a form of autophagic cell death, and nuclear receptor coactivator 4(NCOA4) mediated ferritinophagy was found to regulate ferroptosis by interfering with iron metabolism. Ferritinophagy and ferroptosis are important pathogenic mechanisms in CIRI. This review mainly summarizes the link and regulation between ferritinophagy and ferroptosis and further discusses their mechanisms in CIRI. In addition, the potential treatment methods targeting ferritinophagy and ferroptosis for CIRI are presented, providing new ideas for the prevention and treatment of clinical CIRI in the future.

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.