Mutation of TP53 Confers Ferroptosis Resistance in Lung Cancer Through the FOXM1/MEF2C Axis
2023; Elsevier BV; Volume: 193; Issue: 10 Linguagem: Inglês
10.1016/j.ajpath.2023.05.003
ISSN1525-2191
AutoresMuyun Peng, Qikang Hu, Zeyu Wu, Bin Wang, Cheng Wang, Fenglei Yu,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoFerroptosis is a highly regulated tumor suppressor process. Loss or mutation of TP53 can cause changes in sensitivity to ferroptosis. Mutations in TP53 may be associated with the malignant or indolent progression of ground glass nodules in early lung cancer, but whether ferroptosis may also be involved in determining this biological process has not yet been determined. Using in vivo and in vitro gain- and loss-of-function approaches, this study used clinical tissue for mutation analysis and pathological research to show that wild-type TP53 inhibited the expression of forkhead box M1 (FOXM1) by binding to peroxisome proliferator-activated receptor-γ coactivator 1α, maintaining the mitochondrial function and thus affecting the sensitivity to ferroptosis. This function was absent in mutant cells, resulting in overexpression of FOXM1 and ferroptosis resistance. Mechanistically, FOXM1 activated the transcription level of myocyte-specific enhancer factor 2C in the mitogen-activated protein kinase signaling pathway, leading to stress protection when exposed to ferroptosis inducers. This study provides new insights into the mechanism of association between TP53 mutation and ferroptosis tolerance, which can aid a deeper understanding of the role of TP53 in the malignant progression of lung cancer. Ferroptosis is a highly regulated tumor suppressor process. Loss or mutation of TP53 can cause changes in sensitivity to ferroptosis. Mutations in TP53 may be associated with the malignant or indolent progression of ground glass nodules in early lung cancer, but whether ferroptosis may also be involved in determining this biological process has not yet been determined. Using in vivo and in vitro gain- and loss-of-function approaches, this study used clinical tissue for mutation analysis and pathological research to show that wild-type TP53 inhibited the expression of forkhead box M1 (FOXM1) by binding to peroxisome proliferator-activated receptor-γ coactivator 1α, maintaining the mitochondrial function and thus affecting the sensitivity to ferroptosis. This function was absent in mutant cells, resulting in overexpression of FOXM1 and ferroptosis resistance. Mechanistically, FOXM1 activated the transcription level of myocyte-specific enhancer factor 2C in the mitogen-activated protein kinase signaling pathway, leading to stress protection when exposed to ferroptosis inducers. This study provides new insights into the mechanism of association between TP53 mutation and ferroptosis tolerance, which can aid a deeper understanding of the role of TP53 in the malignant progression of lung cancer. Lung cancer is one of the most common cancers globally, with approximately 2 million new cases each year. As one of the deadliest cancers, the prognosis of lung cancer is poor, with 5-year overall survival of 10% to 20% and an estimated 1.76 million deaths a year.1Neal R.D. Sun F. Emery J.D. Callister M.E. Lung cancer.BMJ. 2019; 365: l1725Crossref PubMed Scopus (54) Google Scholar,2Thai A.A. Solomon B.J. Sequist L.V. Gainor J.F. Heist R.S. Lung cancer.Lancet. 2021; 398: 535-554Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar Despite great advances made in lung cancer therapies, including surgery, chemotherapy, radiation, and targeted therapy, the outcomes are still unsatisfactory for patients with advanced lung cancer.3Lemjabbar-Alaoui H. Hassan O.U. Yang Y.W. Buchanan P. 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Regulation of the p53 transcriptional activity.J Cell Biochem. 2006; 97: 448-458Crossref PubMed Scopus (77) Google Scholar,13Fridman J.S. Lowe S.W. Control of apoptosis by p53.Oncogene. 2003; 22: 9030-9040Crossref PubMed Scopus (1207) Google Scholar However, TP53 is often mutated or depleted in cancers, limiting its antitumor activity and allowing cancer to develop. Ferroptosis, a novel iron-dependent type of cell death, has opposing roles in tumor suppression and promotion.14Chen X. Kang R. Kroemer G. Tang D. Broadening horizons: the role of ferroptosis in cancer.Nat Rev Clin Oncol. 2021; 18: 280-296Crossref PubMed Scopus (932) Google Scholar Particularly, inducing ferroptosis may suppress the tumorigenesis of lung cancer, implying that ferroptosis may be a promising therapeutic target for lung cancer.15Xiong R. He R. Liu B. Jiang W. Wang B. Li N. Geng Q. Ferroptosis: a new promising target for lung cancer therapy.Oxid Med Cell Longev. 2021; 20218457521Crossref Scopus (17) Google Scholar TP53 is widely known as a key regulator in ferroptosis, and depletion of TP53 confers cell insensitivity to ferroptosis.16Tarangelo A. Magtanong L. Bieging-Rolett K.T. Li Y. Ye J. Attardi L.D. Dixon S.J. p53 Suppresses metabolic stress-induced ferroptosis in cancer cells.Cell Rep. 2018; 22: 569-575Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar Mechanically, p53 modulates the ferroptosis response induced by ferroptosis inducers, such as glutathione peroxidase 4 inhibitors, or high levels of reactive oxygen species (ROS) through its metabolic targets.17Liu Y. Gu W. p53 in ferroptosis regulation: the new weapon for the old guardian.Cell Death Differ. 2022; 29: 895-910Crossref PubMed Scopus (146) Google Scholar,18Kuganesan N. Dlamini S. Tillekeratne L.M.V. Taylor W.R. Tumor suppressor p53 promotes ferroptosis in oxidative stress conditions independent of modulation of ferroptosis by p21, CDKs, RB, and E2F.J Biol Chem. 2021; 297101365Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar However, TP53 is often mutated in tumors, and there are 6 exemplary hotspot mutations in the DNA-binding domain of the TP53 gene (R273H, R248Q, R282W, R175H, G245S, and R249S).19Thompson L.R. Oliveira T.G. Hermann E.R. Chowanadisai W. Clarke S.L. Montgomery M.R. Distinct TP53 mutation types exhibit increased sensitivity to ferroptosis independently of changes in iron regulatory protein activity.Int J Mol Sci. 2020; 21Crossref Scopus (19) Google Scholar Previous findings suggest that the high frequency of TP53 mutants (R175H, R248W, and R273H/L) in GGO tissues may determine the malignant or indolent progression of GGO.20Lin X. Peng M. Chen Q. Yuan M. Chen R. Deng H. Deng J. Liu O. Weng Y. Chen M. Zhou C. Identification of the unique clinical and genetic features of chinese lung cancer patients with EGFR germline mutations in a large-scale retrospective study.Front Oncol. 2021; 11774156Crossref Scopus (4) Google Scholar Considering the critical role of TP53 in ferroptosis, the effect of these mutants on ferroptosis and the molecular mechanisms were investigated. Forkhead box M1 (FOXM1), a transcriptional factor that regulates cell proliferation, tumor initiation, and progression in multiple cancer types, including lung cancer,21Liao G.B. Li X.Z. Zeng S. Liu C. Yang S.M. Yang L. Hu C.J. Bai J.Y. Regulation of the master regulator FOXM1 in cancer.Cell Commun Signal. 2018; 16: 57Crossref PubMed Scopus (211) Google Scholar,22Liang S.K. Hsu C.C. Song H.L. Huang Y.C. Kuo C.W. Yao X. Li C.C. Yang H.C. Hung Y.L. Chao S.Y. Wu S.C. Tsai F.R. Chen J.K. Liao W.N. Cheng S.C. Tsou T.C. Wang I.C. FOXM1 is required for small cell lung cancer tumorigenesis and associated with poor clinical prognosis.Oncogene. 2021; 40: 4847-4858Crossref PubMed Scopus (17) Google Scholar affects ferroptosis.23Tang B. Yan R. Zhu J. Cheng S. Kong C. Chen W. Fang S. Wang Y. Yang Y. Qiu R. Lu C. Ji J. Integrative analysis of the molecular mechanisms, immunological features and immunotherapy response of ferroptosis regulators across 33 cancer types.Int J Biol Sci. 2022; 18: 180-198Crossref PubMed Scopus (18) Google Scholar Previous studies have shown that FOXM1 and forkhead box O3 (FOXO3), a reported downstream target of TP53,24Renault V.M. Thekkat P.U. Hoang K.L. White J.L. Brady C.A. Kenzelmann Broz D. Venturelli O.S. Johnson T.M. Oskoui P.R. Xuan Z. Santo E.E. Zhang M.Q. Vogel H. Attardi L.D. Brunet A. The pro-longevity gene FoxO3 is a direct target of the p53 tumor suppressor.Oncogene. 2011; 30: 3207-3221Crossref PubMed Scopus (100) Google Scholar form a competitive balance mechanism to determine tumorigenesis and drug resistance.25Yao S. Fan L.Y. Lam E.W. The FOXO3-FOXM1 axis: a key cancer drug target and a modulator of cancer drug resistance.Semin Cancer Biol. 2018; 50: 77-89Crossref PubMed Scopus (125) Google Scholar Nevertheless, the effect of mutant TP53 on these events remains unclear, and whether ferroptosis is involved needs to be investigated. The current study first demonstrated that mutant TP53 (carrying the common mutation site: R248W) conferred lung cancer cell resistance to ferroptosis. Second, the effect of wild-type TP53 and mutant TP53 on FOXM1 expression levels was investigated and a novel mechanism was identified by which mutant TP53 impaired peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α)/FOXM1–dependent mitochondrial function, thereby affecting ferroptosis. Third, pathways through which up-regulation of FOXM1 affects ferroptosis and is related to the activation of mitogen-activated protein kinase (MAPK) signaling were elucidated, which confer greater resistance to ferroptosis inducers. Tumor and paracancerous normal tissues were collected from patients diagnosed with lung cancer at the Second Xiangya Hospital of Central South University from 2020 to 2021. All patients provided written informed consent. Our study was approved by the Ethics Committee of the Second Xiangya Hospital of Central South University. Human lung cancer cell lines CALU-1 and NCI-H358 (ATCC, Manassas, VA) were maintained in McCoy’s 5A medium/10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA). For inducing ferroptosis, cells were treated with RSL3 (Selleck, Shanghai, China) or erastin (Selleck) for 24 hours. For stable overexpression, coding regions of wild-type TP53 and mutant TP53 were cloned into pLVX-Puro lentiviral vector (Clontech, Mountain View, CA), respectively. Lentiviral particles were packaged in 293T cells with Lenti-X HTX Packaging System (Clontech) and harvested for infecting CALU-1 and NCI-H358 cells. shRNAs against myocyte-specific enhancer factor 2C (MEF2C) and scrambled controls were obtained from GenePharma (Shanghai, China). Coding regions of FOXM1 were inserted into pcDNA3.1 vector (Thermo Fisher Scientific). Cells were transfected with Lipofectamine 3000 (Thermo Fisher Scientific). Cells were seeded in 96-well plates and treated with RSL3 (concentration range: 0–20 μmol/L for CALU-1 cells; 0–30 μmol/L for NCI-H358 cells) or erastin (concentration range: 0-30 μmol/L for CALU-1 and NCI-H358 cells) for 24 hours. [The half maximal inhibitory concentration was selected as the subsequent experimental drug concentration treatment (see Figure 1).] Subsequently, medium was removed, and 100 μL of fresh medium and 10 μL of cell counting kit 8 (CCK-8) (Beyotime, Shanghai, China) were added to each well. Cells were incubated for 4 hours, and the absorbance (450 nm) was recorded. A total of 10 μmol/L boron dipyrromethene C-11 probe (catalog number MX5211, Millipore, Burlington, MA) was added and incubated with cells in a 5% carbon dioxide incubator at 37°C for 1 hour. Then fluorescence images were taken under an flow cytometer (catalog number FACSCanto II, BD Biosciences, Franklin Lakes, NJ) to analyze intracellular lipid ROS. Cell samples were lysed or tissues ground into tissue homogenates and then centrifuged (10,000 × g for 10 minutes) to collect supernatant. Malondialdehyde (MDA) content in all samples were then tested and calculated according to kit operating instructions (catalog number S0131S, Beyotime). Cells were seeded in six-well plates at a density of 300 cells per dish, and 2 mL of medium was added to each dish and evenly dispersed by turning gently. Then the cells were cultured in a cell incubator at 37°C and 5% carbon dioxide for 3 weeks. After the visible clones were grown, they were fixed with 4% paraformaldehyde, stained with crystal violet (catalog number C0121, Beyotime), and then photographed under white light to calculate the clone formation rate. Total RNA was extracted from cells and clinical specimens using TRIzol reagent (Thermo Fisher Scientific) following the manual. Subsequently, RNA was reversely transcribed into cDNA with a QuantiTect Reverse Transcription Kit from Qiagen (Germantown, MD). The expression of FOXM1 and MEF2C was analyzed by quantitative real-time RT-PCR (RT-qPCR), which was normalized to glyceraldehyde-3-phosphate dehydrogenase. The 2−ΔΔCT formula was applied, and primers were given in Table 1.Table 1Quantitative Real-Time RT-PCR PrimersMarkerPrimer sequencesFOXM1Forward: 5′-TTCTGGACCATTCACCCCAGTG-3′Reverse: 5′-GAGCTCTGGATTCGGTCGTT-3′MEF2CForward: 5′-GCATTCGTTCCTGCACATACATCC-3′Reverse: 5′-TGCTGACGGGTACAACT-3′GAPDHForward: 5′-CCAGGTGGTCTCCTCTGA-3′Reverse: 5′-GCTGTAGCCAAATCGTTGT-3′FOXM1, forkhead box M1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MEF2C, myocyte-specific enhancer factor 2C. Open table in a new tab FOXM1, forkhead box M1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MEF2C, myocyte-specific enhancer factor 2C. Cells were lysed in radioimmunoprecipitation assay lysis buffer for 30 minutes on ice, and cell lysates were collected. Protein was quantified using a bicinchoninic acid kit (Bio-Rad, Hercules, CA). Protein (30 μg) was electrophoresed and transferred to polyvinylidene fluoride membranes, which were blocked for 1 hour. Membranes were incubated with anti-p53 (ab26, 1:1000, Abcam, Cambridge, UK), antimutant p53 (ab32049, 1:2000, Abcam), anti–PGC-1α (ab191838, 1:1000, Abcam), anti-FOXM1 (ab207298, 1:1000, Abcam), anti-MEF2C (ab211493, 1:500, Abcam), or anti–β-actin (1:5000) overnight at 4°C. The next day membranes were washed and incubated with horseradish peroxidase–conjugated secondary antibodies for 1 hour. Bands were visualized with ECL substrate (Beyotime), and the intensity was analyzed using ImageJ software version 1.49 (NIH, Bethesda, MD; http://imagej.nih.gov/ij). First, the expression vector pcDNA3.1 TP53 (wild-type or mutated) was constructed and transfected into lung cancer cell lines with TP53 loss. Second, immunoprecipitation grade antibodies of the labeled protein p53 (5 μg, Abcam) was used for the immunoprecipitation process. Third, the contents of the targeted protein (PGC-1α) in the precipitated products were detected by Western blotting (1:1000, Abcam). Cells were crosslinked in formaldehyde solution, detached, and lysed in lysis buffer. Cell lysates were harvested and sonicated for DNA-protein fragments. DNA-protein fragments were incubated with rabbit primary antibodies against FOXM1 (5 μg, Abcam) at 4°C overnight. Normal rabbit IgG was used as an isotype control. The next day protein A/G Magnetic Beads (Thermo Fisher Scientific) were added, mixed, and incubated with gentle rotation. Subsequently, DNA was recovered for RT-qPCR and electrophoresis analysis. Promoter regions of FOXM1 and MEF2C were cloned into pGL3 luciferase vector (Promega, Madison, WI) as FOXM1, and MEF2C luciferase reporters. wild-type TP53, mutant TP53, or FOXM1-overexpressing lung cancer cell lines were transfected with FOXM1 or MEF2C luciferase reporter. After 48 hours, luciferase activity was measured with the Dual-Glo Luciferase System (Promega). The bait recombinant plasmid, pAbAi- MEF2C, and the prey recombinant plasmid, pGADT7-FOXM1, were constructed and transferred into engineering yeast. When the fusion expression vector of the transcription factor (FOXM1) was transferred into the yeast, it activated the pmin promoter. However, the reporter gene expression was promoted after binding with the target-acting element (MEF2C promoter fragment). Yeast growing on the SD/–Leu/+AbA (250 ng/mL) plate means that pAbAi-MEF2C and pGADT7-FOXM1 could interact and activate the reporter gene expression of host bacteria. Animal studies were performed in accordance with Central South University and approved by the Second Xiangya Hospital of Central South University Animal and Life Sciences Research Ethics Committee. CALU-1 cells (4 × 106) were subcutaneously injected into the left flanks of BALB/c nude mice (8- to 10-week–old, male mice). For RSL3 and erastin administration, mice were intraperitoneally injected with RSL3 at 5 mg/kg per day and erastin at 20 mg/kg per day for 5 days. Mice were divided into four groups: wild-type TP53 plus erastin, mutant TP53 plus erastin, wild-type TP53 plus RSL3, and mutant TP53 plus RSL3. Mice were weighted every day. Tumor volume was monitored and calculated with the formula length × width2/2. After 20 days, the mice were anesthetized via intraperitoneal injection of 2% pentobarbital sodium (3 mg/100 g) and then sacrificed by cervical dislocation. Tumors were excised from mice for subsequent immunohistochemistry staining. Subcutaneous tumors from mice and clinical specimens were fixed and embedded in paraffin. Subsequently, samples were sliced into 5-μm sections. Sections were deparaffinized, rehydrated, and retrieved in pH 8.0 antigen retrieval solution (Thermo Fisher Scientific). After blocking, sections were incubated with a rabbit anti-MEF2C (ab211493, 1:200, Abcam) and anti-FOXM1 (ab207298, 1:200, Abcam) overnight. The next day sections were rinsed and incubated with an horseradish peroxidase–conjugated secondary antibody. Signals were visualized with diaminobenzidine (Beyotime). Sections were stained with hematoxylin and imaged under the BX51 microscope (Olympus, Tokyo, Japan). Targeted fatty acid detection and analysis of cells were performed by Sci-tech Innovation (Shan Dong, China). Extraction of lipid samples from cells, saponification and methyl esterification, and gas chromatography were used to the quantify the unsaturated fatty acids. Finally, the data analysis was performed by the data acquisition instrument system (catalog number 7890A; Agilent, Santa Clara, CA). Data from three independent assays are represented as means ± SD. The t-test for two independent groups and one-way analysis of variance for multiple groups were applied for variance analysis. P < 0.05 was statistically significant. In a previous study, gene mutation analysis in 213 patients with early-stage non–small cell lung cancer (stage I and II) showed a high frequency of mutants (R175H, R248W, and R273H/L) present in these early lung cancers (Figure 1A) which were closely related to the malignant progression of lung cancer during GGO.20Lin X. Peng M. Chen Q. Yuan M. Chen R. Deng H. Deng J. Liu O. Weng Y. Chen M. Zhou C. Identification of the unique clinical and genetic features of chinese lung cancer patients with EGFR germline mutations in a large-scale retrospective study.Front Oncol. 2021; 11774156Crossref Scopus (4) Google Scholar Lung cancer cell line A549 was confirmed to harbor TP53, whereas CALU-1 and NCI-H358 cells lacked TP53 gene through sequencing of PCR fragments (Figure 1B). Next, overexpression plasmids with wild-type TP53 and mutant TP53 (hereinafter referred to as R248W mutant unless otherwise specified) were constructed and transfected into CALU-1 and NCI-H358 cells to observe their susceptibility to ferroptosis (Figure 1C). Erastin or RSL3 treatment reduced cell viability in a dose-dependent manner (Figure 1D). Compared with mutant TP53, wild-type TP53–overexpressing cells showed relatively poor cell viability when exposed to ferroptosis inducers (Figure 1E). These observations suggest that TP53 sensitizes lung cancer cells to ferroptosis, whereas mutant TP53 does the opposite. Global gene expression analyses revealed that cell cycle regulatory genes and transcription factors E2F1, MYBL2, and FOXM1 were disproportionately up-regulated in many TP53 mutant cancer types.26Parikh N. Hilsenbeck S. Creighton C.J. Dayaram T. Shuck R. Shinbrot E. Xi L. Gibbs R.A. Wheeler D.A. Donehower L.A. Effects of TP53 mutational status on gene expression patterns across 10 human cancer types.J Pathol. 2014; 232: 522-533Crossref PubMed Scopus (56) Google Scholar Whether wild-type or mutant TP53 played a role in ferroptosis by affecting FOXM1 expression was investigated. The Cancer Genome Atlas database analysis showed that FOXM1 expression level was significantly higher in lung adenocarcinoma and lung squamous carcinoma than that in normal lung tissues (Figure 2A). The high expression of FOXM1 was negatively correlated with survival time of patients with lung cancer (Figure 2B), demonstrating that FOXM1 may act as a carcinogen in lung cancer. In addition, the expression of FOXM1 in lung cancer tissues with TP53 mutation was higher than that in wild-type TP53 (Figure 2C). Overexpression of wild-type TP53 could significantly bring down the mRNA and protein expression of FOXM1, whereas overexpression of the mutant had little effect (Figure 2, D and E), suggesting that the increased expression of FOXM1 in lung cancer may be a consequence of TP53 mutation or deletion. Next, FOXM1 was overexpressed in wild-type and mutant TP53 cells to examine the effect on ferroptosis. Up-regulation of FOXM1 protected cells from ferroptosis, which was more significant in TP53 mutant cells than in the wild-type TP53 cells, as reflected by relatively higher cell viabilities and clonogenesis and less lipid peroxides accumulation when exposed to the same dose of ferroptosis inducers (Figure 3, A–C ). These data suggest that ferroptosis resistance caused by FOXM1 overexpression can be attributed to TP53 mutation.Figure 3TP53 affects ferroptosis by regulating the transcription level of of forkhead box M1 (FOXM1). A–C: The cell viability (A), colony-forming abilities (B), and lipid reactive oxygen species (ROS) (C) of each group tested by the same method as shown in Figure 1. n = 3. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 (one-way analysis of variance). Mut, mutation; NC, negative control; OE, overexpression; WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The peroxisome proliferator-activated receptor-γ coactivator 1 α (PGC-1α) transcriptional coactivator is a master regulator of mitochondrial biogenesis.27Hayes J.D. Dinkova-Kostova A.T. Tew K.D. Oxidative stress in cancer.Cancer Cell. 2020; 38: 167-197Abstract Full Text Full Text PDF PubMed Scopus (956) Google Scholar In cancer, activation of PGC-1α can exert both positive and negative effects in that it supports survival and metabolic flexibility of tumor cells.28Gravel S.P. Deciphering the dichotomous effects of PGC-1alpha on tumorigenesis and metastasis.Front Oncol. 2018; 8: 75Crossref PubMed Scopus (27) Google Scholar Among the TP53-binding proteins, PGC-1α was selected because it may alter cellular metabolic characteristics by regulating mitochondria (Figure 4A), which is critical for sensitivity to ferroptosis. The results of co-immunoprecipitation showed that the binding of TP53 and PGC-1α occurred in the wild-type cells but was weak in the mutants (Figure 4B). Overexpression of PGC-1α in wild-type cells resulted in a significant down-regulation of FOXM1 transcript and protein levels than in mutant cells (Figure 4, C–D). Furthermore, PGC-1α was overexpressed in cells (wild-type or mutant TP53) transfected with a luciferase vector carrying the FOXM1 promoter fragment. FOXM1 promoter activity decreased most significantly in the presence of both wild-type TP53 and PGC-1α, whereas in mutant cells, the FOXM1 promoter activity was relatively strong (Figure 4E). These data indicated that wild-type TP53 can repress FOXM1 transcription with PGC-1α, whereas the mutant has no significant effect because of weak binding to PGC1α. The effect of PGC-1α on ferroptosis in wild-type and mutant TP53 cells was studied. Up-regulation of PGC-1α enhanced the expression levels of oxidized phosphate and electron transport chain complexes I and III (HADHα, HADHβ, NDUFB8, and CYTB) (Figure 5A), making cells sensitive to ferroptosis (Figure 5, B–D). This effect was more pronounced in wild-type cells than in mutant cell lines. Collectively, these results suggest that wild-type TP53 affects FOXM1 transcription by interacting with PGC-1α, which is important for sensitivity to ferroptosis as determined by metabolic characteristics. Subsequently, unsaturated fatty acids, such as stearic acid, palmitic acid, arachidonic acid, dihomo-γ-linolenic acid, icosapentaenoic acid, and docosapentaenoic acid, decreased to varying degrees in mutant cells compared with that in the wild type (Supplemental Figure S1), which may reduce lipid ROS production and thus susceptibility to ferroptosis. The downstream targets of the transcription factor FOXM1 in the regulation of ferroptosis were explored. Hence, FOXM1 overexpressing lung cancer cells treated with erastin were prepared for RNA sequencing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed of differently expressed genes (Figure 6A). Compared with the control group, there were more genes, including SOS1, NGFR, and MEF2C, enriched in the MAPK signaling pathway (activated in ferroptosis) in the FOXM1 overexpressed group. Because MEF2C is in ferroptosis, whether FOXM1 regulates MEF2C transcription and thus influences ferroptosis was studied next. Chromatin immunoprecipitation/PCR assay showed that FOXM1 was enriched in the MEF2C promoter region (Figure 6B). In addition, the yeast one-hybrid system (Figure 6C) and the dual-luciferase assay (Figure 6D) demonstrated that FOXM1 activated the promoter of MEF2C to initiate its transcription. Furthermore, both the mRNA and the protein expression levels of MEF2C were up-regulated after FOXM1 overexpression, and in the mutant TP53 cell types, the expression level of MEF2C was much more higher than that of the wild type (Figure 6, E–F), indicating that FOXM1 and mutant TP53 can synergistically promote MEF2C expression. In CALU-1 cells, shRNA interfered with MEF2C expression, whereas FOXM1 was overexpressed, and ferroptosis was then detected to assess whether FOXM1 affects ferroptosis through MEF2C. Cells in FOXM1-overexpressing groups had a higher survival rate, lower lipid ROS and MDA levels, and more clones of cancer cells (Figure 7, A–E ) with isodose erastin (or RSL3) compared with the empty vector group, demonstrating that cells are less sensitive to ferroptosis. However, knockdown of MEF2C negated the survival benefits of FOXM1 overexpression in the presence of ferroptosis inducers. Similar results were verified in another lung cancer cell line, NCI-H358 (Figure 8). These studies confirmed that FOXM1 affected ferroptosis by up-regulating MEF2C in lung cancer cells.Figure 8The forkhead box M1 (FOXM1)/myocyte-specific enhancer factor 2C (MEF2C) axis causes ferroptosis insensitivity in NCI-H358 cells. A–E: The cell viability (A), lipid reactive oxygen species (ROS) (B), relative MEF2C protein expression levels (C), malondialdehyde contents (D), and the colony-forming abilities (E) of each group tested by the same method as shown in Figures 1, 2, and 4. ∗P < 0.05, ∗∗P < 0.01 (one-way analysis of variance). NC, negative contr
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