Susceptibility To Ferroptosis Research Articles

Intermittent hypoxia exacerbates metabolic dysfunction-associated fatty liver disease by aggravating hepatic copper deficiency-induced ferroptosis.

Intermittent hypoxia (IH) is an independent risk factor for metabolic dysfunction-associated fatty liver disease (MAFLD). Copper deficiency can disrupt redox homeostasis, iron, and lipid metabolism. Here, we investigated whether hepatic copper deficiency plays a role in IH-associated MAFLD and explored the underlying mechanism(s). Male C57BL/6 mice were fed a western-type diet with adequate copper (CuA) or marginally deficient copper (CuD) and were exposed separately to room air (RA) or IH. Hepatic histology, plasma biomarkers, copper-iron status, and oxidative stress were assessed. An in vitro HepG2 cell lipotoxicity model and proteomic analysis were used to elucidate the specific targets involved. We observed that there were no differences in hepatic phenotypes between CuA-fed and CuD-fed mice under RA. However, in IH exposure, CuD-fed mice showed more pronounced hepatic steatosis, liver injury, and oxidative stress than CuA-fed mice. IH induced copper accumulation in the brain and heart and exacerbated hepatic copper deficiency and secondary iron deposition. In vitro, CuD-treated cells with IH exposure showed elevated levels of lipid accumulation, oxidative stress, and ferroptosis susceptibility. Proteomic analysis identified 360 upregulated and 359 downregulated differentially expressed proteins between CuA and CuD groups under IH; these proteins were mainly enriched in citrate cycle, oxidative phosphorylation, fatty acid metabolism, the peroxisome proliferator-activated receptor (PPAR)α pathway, and ferroptosis. In IH exposure, CuD significantly upregulated the ferroptosis-promoting factor arachidonyl-CoA synthetase long chain family member (ACSL)4. ACSL4 knockdown markedly eliminated CuD-induced ferroptosis and lipid accumulation in IH exposure. In conculsion, IH can lead to reduced hepatic copper reserves and secondary iron deposition, thereby inducing ferroptosis and subsequent MAFLD progression. Insufficient dietary copper may worsen IH-associated MAFLD.

Zeb1 mediates EMT/plasticity-associated ferroptosis sensitivity in cancer cells by regulating lipogenic enzyme expression and phospholipid composition.

Therapy resistance and metastasis, the most fatal steps in cancer, are often triggered by a (partial) activation of the epithelial-mesenchymal transition (EMT) programme. A mesenchymal phenotype predisposes to ferroptosis, a cell death pathway exerted by an iron and oxygen-radical-mediated peroxidation of phospholipids containing polyunsaturated fatty acids. We here show that various forms of EMT activation, including TGFβ stimulation and acquired therapy resistance, increase ferroptosis susceptibility in cancer cells, which depends on the EMT transcription factor Zeb1. We demonstrate that Zeb1 increases the ratio of phospholipids containing pro-ferroptotic polyunsaturated fatty acids over cyto-protective monounsaturated fatty acids by modulating the differential expression of the underlying crucial enzymes stearoyl-Co-A desaturase 1 (SCD), fatty acid synthase (FASN), fatty acid desaturase 2 (FADS2), elongation of very long-chain fatty acid 5 (ELOVL5) and long-chain acyl-CoA synthetase 4 (ACSL4). Pharmacological inhibition of selected lipogenic enzymes (SCD and FADS2) allows the manipulation of ferroptosis sensitivity preferentially in high-Zeb1-expressing cancer cells. Our data are of potential translational relevance and suggest a combination of ferroptosis activators and SCD inhibitors for the treatment of aggressive cancers expressing high Zeb1.

Reactive oxygen species regulation by NCF1 governs ferroptosis susceptibility of Kupffer cells to MASH

Reactive oxygen species regulation by NCF1 governs ferroptosis susceptibility of Kupffer cells to MASH

A lipid atlas of human and mouse immune cells provides insights into ferroptosis susceptibility.

The cellular lipidome comprises thousands of unique lipid species. Here, using mass spectrometry-based targeted lipidomics, we characterize the lipid landscape of human and mouse immune cells ( www.cellularlipidatlas.com ). Using this resource, we show that immune cells have unique lipidomic signatures and that processes such as activation, maturation and development impact immune cell lipid composition. To demonstrate the potential of this resource to provide insights into immune cell biology, we determine how a cell-specific lipid trait-differences in the abundance of polyunsaturated fatty acid-containing glycerophospholipids (PUFA-PLs)-influences immune cell biology. First, we show that differences in PUFA-PL content underpin the differential susceptibility of immune cells to ferroptosis. Second, we show that low PUFA-PL content promotes resistance to ferroptosis in activated neutrophils. In summary, we show that the lipid landscape is a defining feature of immune cell identity and that cell-specific lipid phenotypes underpin aspects of immune cell physiology.

Abstract 5837: Aryl hydrocarbon receptor is critical for enzalutamide-resistant prostate cancer growth

Abstract Therapy resistance is a ubiquitous and major challenge for curing cancer including prostate cancer. As the most prevalent non-skin cancer for men, prostate cancer is responsible for more than 30,000 deaths annually in the U.S. Despite a favorable prognosis at early stages, prostate cancer progressively develop into an incurable castration-resistant stage (CRPC) where prostate cancer grows in a low-androgen environment. Enzalutamide (ENZ) is the first drug approved by FDA for treating CRPC by inhibiting the residual androgen signaling. While ENZ significantly improves overall survival and quality of life, resistance inevitably develops and greatly limits its benefit. To understand ENZ resistance and identify novel therapeutic targets with the hope of overcoming resistance, we performed RNA-seq to analyze mRNA expression profiles from three ENZ-resistant human CRPC cell lines (C4-2BENZR, CWR-R1ENZR and VCaPENZR) and identified the aryl hydrocarbon receptor (AHR) among the 114 commonly upregulated genes. AHR is a ligand-dependent transcription factor that has been shown to drive disease progression in multiple types of cancer. Stable shRNA-mediated knockdown of AHR substantially decreased growth of ENZ-resistant CRPC cells. Our pilot study also showed that AHR knockdown resulted in fewer and smaller CWR-R1ENZR tumors in a xenograft mouse model. To elucidate the underlying mechanism of AHR-mediated cancer growth, gene set enrichment analysis (GSEA) revealed a declining trend of enrichment of the 114 commonly ENZ-upregulated genes among differentially altered genes upon AHR knockdown in ENZ-resistant cells, suggesting that AHR may act as a master regulator of ENZ resistance. Moreover, we found that the Hallmark androgen response gene set is downregulated in AHR-silenced ENZ-resistant cells, implying a role of AHR in sustaining androgen response for ENZ-resistance. Consistently, we performed AHR ChIP-seq and confirmed that AHR bound to promoter regions of several androgen-regulated genes in CWR-R1ENZR cells. In addition, three peroxisome-related Hallmark gene sets are also negatively enriched in AHR-knockdown cells, which provides a rationale for further exploring a potential AHR-dependent connection of peroxisomal function to ferroptosis as a newly emerging mechanism of action for ENZ. In summary, our study suggests that AHR plays a critical role in maintaining ENZ-resistant CRPC growth with the likely involvement of modulating androgen signaling and ferroptosis susceptibility. Citation Format: Chia-Hui Chen, Boyang Jason Wu. Aryl hydrocarbon receptor is critical for enzalutamide-resistant prostate cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5837.

Coculture with macrophages alters ferroptosis susceptibility of triple-negative cancer cells

Various treatment options, such as molecular targeted drugs and immune checkpoint blockades, are available for patients with cancer. However, some cancer types are refractory to molecular targeted therapies or acquire drug resistance after long-term treatment. Thus, ferroptosis, a newly defined type of programmed cell death caused by the iron-dependent accumulation of lipid peroxidation, has gained attention as a novel cancer treatment strategy. Understanding cell–cell interactions in the tumor microenvironment is important for the clinical application of ferroptosis inducers. However, the effects of cell–cell interactions on ferroptosis sensitivity remain unclear. Thus, we aimed to evaluate the effects of macrophage–cancer cell interactions on ferroptosis induction. Coculture experiments showed that conditioned medium prepared from macrophages did not alter the ferroptosis sensitivity of cancer cells. By contrast, coculture via transwell, which enables cell–cell interactions through secretion, increased the sensitivity of cancer cells to ferroptosis inducers. Additionally, direct coculture increased the susceptibility of cancer cells to RSL3-induced ferroptosis. Mechanistically, coculture with macrophages upregulated the levels of intracellular ferrous ions and lipid peroxidation in cancer cells. These findings provide novel insights into the mechanisms by which cell–cell interactions influence ferroptosis induction and application of ferroptosis inducers as a cancer treatment option.

Cell density impacts the susceptibility to ferroptosis by modulating IRP1-mediated iron homeostasis.

Ferroptosis has been implicated in several neurological disorders and may be therapeutically targeted. However, the susceptibility to ferroptosis varies in different cells, and inconsistent results have been reported even using the same cell line. Understanding the effects of key variables of invitro studies on ferroptosis susceptibility is of critical importance to facilitate drug discoveries targeting ferroptosis. Here, we showed that increased cell seeding density leads to enhanced resistance to ferroptosis by reducing intracellular iron levels. We further identified iron-responsive protein 1 (IRP1) as the key protein affected by cell density, which affects the expression of ferroportin or transferrin receptor and results in altered iron levels. Such observations were consistent across different cell lines, indicating that cell density should be tightly controlled in studies of ferroptosis. Since cell densities vary in different brain regions, these results may also shed light on selective regional vulnerability observed in neurological disorders.

Closed‐loop of Catalyzed Lactate Oxidization Synergistically Sensitizes Apoptosis and Ferroptosis for Cancer Therapy

AbstractLactate accumulated in Tumor is a key oncometabolite and associated with various oncogenic processes, including proliferation, invasion, angiogenesis, metastasis, immunosuppression and therapeutic resistance. Particularly, lactate contributes to the apoptosis and ferroptosis resistance in cancer cells. Consequently, blockade of lactate provides a promising opportunity for cancer therapy. However, due to abnormal tumor microenvironment (TME) and intracellular glutathione (GSH) mediated ferroptosis resistance, the current approaches for regulating lactate to sensitize cells to apoptosis and ferroptosis are still challenging. Herein, we developed a catalytic lactate oxidizing liposomes (Lip‐LM) incorporated with lactate oxidase (LOX)‐loaded MnO2 nanoparticles. O2 regenerated from H2O2 constitute closed‐loop of lactate oxidation catalyzed by LOX, enhancing the lactate‐consuming efficacy in TME. Besides, glutathione is consumed by MnO2 to amplify the ferroptosis susceptibility of cancer cells. The results showed that Lip‐LM achieved synergistical apoptosis and ferroptosis to enhance the anti‐tumor efficacy of amplified oxidative stress in cancer cells through the strategy of self‐sufficient O2 supplying and GSH consumption. The results of transcriptomics and proteomics analyses also support the synergistical anti‐tumor mechanisms of Lip‐LM. Hence, it is a promising catalytic nanoplatform combined lactate regulation and oxidative stress amplification and have great potential to further enhance therapeutic outcomes based on synergistical apoptosis and ferroptosis.

Induction of stearoyl-CoA desaturase confers cell density-dependent ferroptosis resistance in melanoma.

Ferroptosis is a form of regulated cell death that is induced by inhibiting glutathione peroxidase 4 (GPX4), which eliminates lipid peroxidation. Ferroptosis induction is influenced by the cell environment. However, the cellular states altering ferroptosis susceptibility remain largely unknown. We found that melanoma cell lines became resistant to ferroptosis as cell density increased. Comparative transcriptome and metabolome analyses revealed that cell density-dependent ferroptosis resistance was coupled with a shift toward a lipogenic phenotype accompanied by strong induction of stearoyl-CoA desaturase (SCD). Database analysis of gene dependency across hundreds of cancer cell lines uncovered a negative correlation between GPX4 and SCD dependency. Importantly, SCD inhibition, either pharmacologically or through genetic knockout, sensitized melanoma cells to GPX4 inhibition, thereby attenuating ferroptosis resistance in cells at high density. Our findings indicate that transition to an SCD-inducing, lipogenic cell state produces density-dependent resistance to ferroptosis, which may provide a therapeutic strategy against melanoma.

Helicobacter pylori CagA-mediated ether lipid biosynthesis promotes ferroptosis susceptibility in gastric cancer

Helicobacter pylori, particularly cytotoxin-associated gene A (CagA)-positive strains, plays a key role in the progression of gastric cancer (GC). Ferroptosis, associated with lethal lipid peroxidation, has emerged to play an important role in malignant and infectious diseases, but the role of CagA in ferroptosis in cancer cells has not been determined. Here, we report that CagA confers GC cells sensitivity to ferroptosis both in vitro and in vivo. Mechanistically, CagA promotes the synthesis of polyunsaturated ether phospholipids (PUFA-ePLs), which is mediated by increased expression of alkylglycerone phosphate synthase (AGPS) and 1-acylglycerol-3-phosphate O-acyltransferase 3 (AGPAT3), leading to susceptibility to ferroptosis. This susceptibility is mediated by activation of the MEK/ERK/SRF pathway. SRF is a crucial transcription factor that increases AGPS transcription by binding to the AGPS promoter region. Moreover, the results demonstrated that CagA-positive cells are more sensitive to apatinib than are CagA-negative cells, suggesting that detecting the H. pylori CagA status may aid patient stratification for treatment with apatinib.

Heterogeneous ferroptosis susceptibility of macrophages caused by focal iron overload exacerbates rheumatoid arthritis

Focal iron overload is frequently observed in patients with rheumatoid arthritis (RA), yet its functional significance remains elusive. Herein, we report that iron deposition in lesion aggravates arthritis by inducing macrophage ferroptosis. We show that excessive iron in synovial fluid positively correlates with RA disease severity as does lipid hyperoxidation of focal monocyte/macrophages. Further study reveals high susceptibility to iron induced ferroptosis of the anti-inflammatory macrophages M2, while pro-inflammatory M1 are less affected. Distinct glutathione peroxidase 4 (GPX4) degradation depending on p62/SQSTM1 in the two cell types make great contribution mechanically. Of note, ferroptosis inhibitor liproxstatin-1 (LPX-1) can alleviate the progression of K/BxN serum-transfer induced arthritis (STIA) mice accompanied with increasing M2 macrophages proportion. We thus propose that the heterogeneous ferroptosis susceptibility of macrophage subtypes as well as consequent inflammation and immune disorders are potential biomarkers and therapeutic targets in RA.

The BRD4-SRPK2-SRSF2 signal modulates the splicing efficiency of ACSL3 pre-mRNA and influences erastin-induced ferroptosis in osteosarcoma cells

Lipid metabolism is the key to ferroptosis susceptibility. However, little is known about the underlying mechanisms in osteosarcoma cells. Functional restriction of bromodomain-containing protein 4 (BRD4) reduced the susceptibility to erastin-induced ferroptosis of osteosarcoma cells both in vitro and in vivo. Mechanically, BRD4 controls the splicing efficiency of the RNA precursor (pre-mACSL3) of ACSL3 (ACSL3) by recruiting serinerich/threonine protein kinase 2 (SRPK2) to assemble the splicing catalytic platform. Moreover, the AMP-binding domain of ACSL3 significantly influences arachidonic acid synthesis and thus determines the susceptibility to erastin-induced ferroptosis. Overall, we found a BRD4-mediated pre-mACSL3 splicing influences erastin-induced ferroptosis by affecting arachidonic acid synthesis in osteosarcoma cells. Data in this study fills some of the gap in understanding the post-transcriptional regulatory mechanisms of ACSL3 and provides new insights into the mechanisms of lipid metabolism regulation and its effect on susceptibility to ferroptosis in osteosarcoma cells.

Inhibition of PKA/CREB1 pathway confers sensitivity to ferroptosis in non-small cell lung cancer

Ferroptosis is a type of regulated cell death characterized by iron accumulation and lipid peroxidation. The molecular mechanisms underlying ferroptosis regulation in non-small cell lung cancer (NSCLC) are poorly understood. In this study, we found that protein kinase A (PKA) inhibition enhanced ferroptosis susceptibility in NSCLC cells, as evidenced by reduced cell viability and increased lipid peroxidation. We further identified cAMP-responsive element protein 1 (CREB1), a transcription factor and a substrate of PKA, as a key regulator of ferroptosis. Knockdown of CREB1 sensitized NSCLC cells to ferroptosis inducers (FINs) and abolished the effects of PKA inhibitor and agonist, revealing the pivotal role of CREB1 in ferroptosis regulation. Using a high-throughput screening approach and subsequent validation by chromatin immunoprecipitation (ChIP) and dual-luciferase assays, we discovered that CREB1 transcriptionally activated stearoyl-CoA desaturase (SCD), an enzyme that catalyzes the conversion of saturated fatty acids to monounsaturated fatty acids. SCD conferred ferroptosis resistance by decreasing the availability of polyunsaturated fatty acids for lipid peroxidation, and its overexpression rescued the effect of CREB1 knockdown on ferroptosis in vitro. Besides, CREB1 knockdown suppressed xenograft tumor growth in the presence of Imidazole Ketone Erastin (IKE), a potent FIN, and this effect was reversed by SCD. Finally, we showed that high expression of CREB1 was associated with poor prognosis in NSCLC patients from public datasets and our institution. Collectively, this study illustrates the effect of PKA/CREB1/SCD axis in regulating ferroptosis of NSCLC, targeting this pathway may provide new strategies for treating NSCLC patients.

The Novel Link between Gene Expression Profiles of Adult T-Cell Leukemia/Lymphoma Patients' Peripheral Blood Lymphocytes and Ferroptosis Susceptibility.

Ferroptosis, a regulated cell death dependent on iron, has garnered attention as a potential broad-spectrum anticancer approach in leukemia research. However, there has been limited ferroptosis research on ATL, an aggressive T-cell malignancy caused by HTLV-1 infection. Our study employs bioinformatic analysis, utilizing dataset GSE33615, to identify 46 ferroptosis-related DEGs and 26 autophagy-related DEGs in ATL cells. These DEGs are associated with various cellular responses, chemical stress, and iron-related pathways. Autophagy-related DEGs are linked to autophagy, apoptosis, NOD-like receptor signaling, TNF signaling, and the insulin resistance pathway. PPI network analysis revealed 10 hub genes and related biomolecules. Moreover, we predicted crucial miRNAs, transcription factors, and potential pharmacological compounds. We also screened the top 20 medications based on upregulated DEGs. In summary, our study establishes an innovative link between ATL treatment and ferroptosis, offering promising avenues for novel therapeutic strategies in ATL.

The interplay of miRNAs and ferroptosis in diseases related to iron overload.

Ferroptosis has been conceptualized as a novel cell death modality distinct from apoptosis, necroptosis, pyroptosis and autophagic cell death. The sensitivity of cellular ferroptosis is regulated at multiple layers, including polyunsaturated fatty acid metabolism, glutathione-GPX4 axis, iron homeostasis, mitochondria and other parallel pathways. In addition, microRNAs (miRNAs) have been implicated in modulating ferroptosis susceptibility through targeting different players involved in the execution or avoidance of ferroptosis. A growing body of evidence pinpoints the deregulation of miRNA-regulated ferroptosis as a critical factor in the development and progression of various pathophysiological conditions related to iron overload. The revelation of mechanisms of miRNA-dependent ferroptosis provides novel insights into the etiology of diseases and offers opportunities for therapeutic intervention. In this review, we discuss the interplay of emerging miRNA regulators and ferroptosis players under different pathological conditions, such as cancers, ischemia/reperfusion, neurodegenerative diseases, acute kidney injury and cardiomyopathy. We emphasize on the relevance of miRNA-regulated ferroptosis to disease progression and the targetability for therapeutic interventions.

Emerging Role of Ferroptosis in Breast Cancer: Characteristics, Therapy, and Translational Implications for the Present and Future.

Ferroptosis is a nonapoptotic, iron-dependent form of cell death that can be actuated in disease cells by expected improvements and manufactured specialists. Different studies have recently resurrected the role of this newly discovered cell death pathway and demonstrated its efficacy in treating breast cancer. Breast cancer is the most well-known type of cancer among women worldwide. Despite many years of research focusing on cell death in breast cancer, counting apoptosis, clinical treatment leftovers are difficult due to the high likelihood of recurrence. Ferroptosis is defined by a lack of lipid peroxide repair capacity by phospholipid hydroperoxides GPX4, accessibility of redox-active iron, and followed oxidation of polyunsaturated fatty acids acid-containing phospholipids signalling, amino acid and iron metabolism, ferritinophagy, epithelial-to-mesenchymal transition, cell adhesion, and mevalonate and phospholipid biosynthesis can all be factors that influence ferroptosis susceptibility. Ferroptosis, an iron-dependent controlled cell death caused by excessive lipid peroxidation, has been entwined in breast cancer development and therapeutic response for the past decade. Advances in enhancing clinical drugs targeting ferroptosis are developing silver linings to treat breast cancer. Ferroptosis is influenced by metabolism and the expression of certain genes, making it a prospective therapeutic target for monitoring malignant growth and an appealing target for precision cancer medication disclosure. In the coming years, research into biomarkers to follow ferroptosis in patients with breast cancer and the course of events and the subsequent use of novel ferroptosis-based treatments will be captious. We present a fundamental analysis of the actual understanding of molecular mechanisms along with regulatory networks associated with ferroptosis, expected physiological functions in growth concealment, ferroptosis-associated differentially expressed genes, treatment targeting potential, and recent advances in the development of therapeutic strategies in this review.

Patient-derived zebrafish xenografts of uveal melanoma reveal ferroptosis as a drug target

Uveal melanoma (UM) has a high risk to progress to metastatic disease with a median survival of 3.9 months after metastases detection, as metastatic UM responds poorly to conventional and targeted chemotherapy and is largely refractory to immunotherapy. Here, we present a patient-derived zebrafish UM xenograft model mimicking metastatic UM. Cells isolated from Xmm66 spheroids derived from metastatic UM patient material were injected into 2 days-old zebrafish larvae resulting in micro-metastases in the liver and caudal hematopoietic tissue. Metastasis formation could be reduced by navitoclax and more efficiently by the combinations navitoclax/everolimus and flavopiridol/quisinostat. We obtained spheroid cultures from 14 metastatic and 10 primary UM tissues, which were used for xenografts with a success rate of 100%. Importantly, the ferroptosis-related genes GPX4 and SLC7A11 are negatively correlated with the survival of UM patients (TCGA: n = 80; Leiden University Medical Centre cohort: n = 64), ferroptosis susceptibility is correlated with loss of BAP1, one of the key prognosticators for metastatic UM, and ferroptosis induction greatly reduced metastasis formation in the UM xenograft model. Collectively, we have established a patient-derived animal model for metastatic UM and identified ferroptosis induction as a possible therapeutic strategy for the treatment of UM patients.

Clostridium butyricum and its metabolite butyrate promote ferroptosis susceptibility in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with limited therapeutic options. The diversity and composition of the intratumoral microbiota are associated with PDAC outcomes, and modulating the tumor microbiota has the potential to influence tumor growth and the host immune response. Here, we explore whether intervention with butyrate-producing probiotics can limit PDAC progression. Based on the TCGA (PAAD) database, we analyzed the differential communities of intratumoral microbiota in PDAC patients with long survival and short survival and explored the relevant mechanisms of Clostridium butyricum and its metabolite butyrate in the treatment of PDAC. Treatment with Clostridium butyricum or butyrate in combination with the ferroptosis inducer RSL3 in a PDAC mouse model has an inhibitory effect on PDAC progression. The potential molecular mechanisms were verified by flow cytometry, RNA-seq, Western blotting, qRT‒PCR and immunofluorescence. We found that the tumoral butyrate-producing microbiota was linked to a better prognosis and less aggressive features of PDAC. Intervention with Clostridium butyricum or its metabolite butyrate triggered superoxidative stress and intracellular lipid accumulation, which enhanced ferroptosis susceptibility in PDAC. Our study reveals a novel antitumor mechanism of butyrate and suggests the therapeutic potential of butyrate-producing probiotics in PDAC.

Epigenetically silenced lncRNA SNAI3-AS1 promotes ferroptosis in glioma via perturbing the m6A-dependent recognition of Nrf2 mRNA mediated by SND1

BackgroundFerroptosis has been linked to tumor progression and resistance to antineoplastic therapy. Long noncoding RNA (lncRNA) exerts a regulatory role in various biological processes of tumor cells, while the function and molecular mechanism of lncRNA in ferroptosis are yet to be clarified in glioma.MethodsBoth gain-of-function and loss-of-function experiments were employed to investigate the effects of SNAI3-AS1 on the tumorigenesis and ferroptosis susceptibility of glioma in vitro and in vivo. Bioinformatics analysis, Bisulfite sequencing PCR, RNA pull-down, RIP, MeRIP and dual-luciferase reporter assay were performed to explore the low expression mechanism of SNAI3-AS1 and the downstream mechanism of SNAI3-AS1 in ferroptosis susceptibility of glioma.ResultsWe found that ferroptosis inducer erastin downregulates SNAI3-AS1 expression in glioma by increasing the DNA methylation level of SNAI3-AS1 promoter. SNAI3-AS1 functions as a tumor suppressor in glioma. Importantly, SNAI3-AS1 enhances the anti-tumor activity of erastin by promoting ferroptosis both in vitro and in vivo. Mechanistically, SNAI3-AS1 competitively binds to SND1 and perturbs the m6A-dependent recognition of Nrf2 mRNA 3’UTR by SND1, thereby reducing the mRNA stability of Nrf2. Rescue experiments confirmed that SND1 overexpression and silence can rescue the gain- and loss-of-function ferroptotic phenotypes of SNAI3-AS1, respectively.ConclusionsOur findings elucidate the effect and detailed mechanism of SNAI3-AS1/SND1/Nrf2 signalling axis in ferroptosis, and provide a theoretical support for inducing ferroptosis to improve glioma treatment.

Abstract 1376: Prx4 depletion enhances ferroptosis susceptibility in colorectal cancer

Abstract Ferroptosis is a form of cell death that is characterized by the accumulation of lipid peroxides. Dysregulation of ferroptosis has been linked to the development of cancer. Several important regulators involved in ferroptosis have been discovered, However, the molecular mechanism behind this process has yet to be completely understood. Members of the peroxiredoxin (Prx) family function as leading cellular antioxidants that react with hydrogen peroxide to keep redox homeostasis as well as contribute to oxidative signaling under both physiological and pathological conditions. In this study, we investigated the role of Prx family members in the regulation of ferroptosis in colorectal cancer cells using loss-of-function and gain-of-function experiments. Firstly, immunoblotting was used to examine endogenous levels of Prx family members in different colorectal cancer cells and cells with high or low expression of Prxs were selected for study. Secondly, each of the endogenously expressed 2-Cys containing Prxs including Prx1, Prx2, Prx3 and Prx4 in representative CRC cell lines was depleted by stably expression of ShRNA targeting their coding regions or Knockout using CRISPR-Cas9. The consequences of Prx knockdown in these cells in response to ferroptosis inducers, including erastin and RSL3, were examined by cell viability and colonogenic assays. Cell death due to ferroptosis in these cells was demonstrated by the measurement of lipid peroxidation using the sensor BODIPY 581/591 C11, Levels of MDA and 4-HNE. The mode of cell death was differentiated from other cell death types by the combined application of various inhibitors. In addition, overexpression were used to validate the results observed in Prx-depleted cells. Finally, Prx4 depleted cells were submitted for RNA sequencing and the data were subjected to GSEA analysis to identify enriched pathways. In conclusion, we found that 2-Cys containing Prxs are widely expressed in CRC cell lines, and depletion of Prx1, Prx2 or Prx4 but not Prx3 sensitizes CRC cells to erastin induced cell death. However, only depletion of Prx4 but not other Prxs sensitizes CRC cells to ferroptotic cell death that is characterized by the accumulation of lipid peroxidation and can be rescued by the treatment with inhibitors of ferroptosis. Moreover, Prx4 depleted cells show higher levels of MDA and 4-HNE. In consistence, overexpression of Prx4 also leads to the resistance of CRC cell to ferroptosis. Importantly, according to gene set enrichment analysis (GSEA) of (KEGG) pathway, pathways including P53 pathway and arachidonic acid (AA) metabolism, which have a well-known significant role in ferroptosis regulation, were activated in Prx4 depleted cells. Thus, our findings reveal an essential role of Prx4 in protecting colorectal cancer against ferroptosis and provide a potential target to enhance the antitumor activity of ferroptosis-based treatment. Citation Format: Aziza Alshahrani, Hong Jiang, Pratik Thapa, Na Ding, Yanning Hao, Qiou Wei. Prx4 depletion enhances ferroptosis susceptibility in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1376.