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. Author manuscript; available in PMC: 2026 Jun 25.
Published in final edited form as: Biochimie. 2025 Jun 25;236:127–137. doi: 10.1016/j.biochi.2025.06.013

Regulation of Ferroptosis by Transcription Factor E2F1

Nishanth Kuganesan 1, Samkeliso Dlamini 2, Safiyyah Hasan 1, LM Viranga Tillekeratne 2,*, William R Taylor 1,*
PMCID: PMC12333391  NIHMSID: NIHMS2098118  PMID: 40578750

Abstract

The E2F family of transcription factors plays multiple roles in cell cycle regulation. E2F can be inhibited by binding to RB proteins, an interaction that is regulated by CDK phosphorylation of RB. We previously observed that CDKs, RB, and E2F regulate ferroptosis, a type of programmed cell death characterized by catastrophic peroxidation of membrane lipids. Here we investigate the impact of E2F on ferroptosis. E2F1 regulates both pro and anti-ferroptotic proteins including ALOX5, MYC SLC7A11, ATF4, and GPX4 and finally renders a net inhibitory role in ferroptosis. Interestingly, we also obtained evidence for a cell type dependent compensatory effect of E2F3 upon E2F1 depletion. Specifically, downregulation of ferroptotic genes upon E2F1 knockdown fails to occur in an osteosarcoma cell line which upregulates E2F3 under these conditions. Taken together, our study identifies a number of E2F targets with the potential to affect ferroptotic sensitivity.

Keywords: ferroptosis, SLC7A11, E2F, GPX4, MYC

Graphical Abstract:

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Introduction

The E2F family is a critical regulator of cell cycle progression. Tumor suppressor proteins including p53, p21 and p16INK in turn regulate E2F in part by affecting the state of RB phosphorylation. Many cancers develop as a result of bypassing this checkpoint system through mutation or suppression of p53 activity or elevated cyclin dependent kinase (CDK) activity leading to aberrant E2F transactivation [1] [2]. p53 and several of its downstream cell cycle targets can modulate sensitivity to ferroptosis, a non-apoptotic form of cell death caused by iron-dependent lipid peroxidation [3, 4]. Ferroptosis has gained attention due to rapid killing and more selective targeting of mesenchymal cancer stem cells and drug resistant persister cells [5, 6].

Our group discovered a class of small molecules called CETZOLEs that rapidly kill cells with many of the hallmarks of ferroptosis [4, 6–9]. For example, CETZOLE-induced death is blocked by iron chelators (deferoxamine, hydroxyurea, ciclopiroxolamine), anti-oxidants (butylated hydroxyanisole (BHA), trolox), and a lipophilic radical trap (liproxstatin) [6, 7]. CETZOLEs elevate cellular ROS and induce lipid peroxidation [6, 7]. CETZOLE-treated cells have less glutathione (GSH) and show reduced import of cystine-FITC [6]. The active moiety of CETZOLEs is a thiazole-alkyne group that can participate in a Michael reaction to covalently bind to thiols in vitro [9]. In cells, CETZOLEs directly bind to GPX4 and other proteins [9]. GPX4 is an enzyme that reduces toxic lipid peroxides to non-toxic alcohols. Our current working model is that CETZOLEs induce ferroptosis mainly by acting as small covalent GPX4 inhibitors.

E2F1 transcription factors are major RB targets that control cell cycle dependent and independent cellular functions. We previously observed that E2F1 inhibits ferroptosis partially through p21 [4]. We now characterize several other effects of E2F1 that are relevant to ferroptotic sensitivity. For example, SLC7A11 was upregulated in response to E2F1 overexpression. SLC7A11 encodes an essential subunit of the system xc− transporter that imports cystine needed for synthesis of antioxidant GSH [10]. Two E2F targets, namely ATF4 and c-MYC both appear to contribute to the upregulation of SLC7A11. Further analysis indicates that E2F1-dependent regulation of SLC7A11 is cell-type dependent and possibly related to regulation of other E2F-family members by E2F1. Cells overexpressing SLC7A11 are protected from multiple ferroptotic stimuli, which helps to explain the ability of E2F1 to suppress ferroptosis.

Materials and Methods

Cell Lines and Culture Conditions.

Cell lines were cultured in a humidified atmosphere containing 10% CO2 in Dulbecco’s Modified Eagle’s Medium (Mediatech, Inc.) supplemented with 10% fetal bovine serum (Phoenix Scientific). Cell types used; HT1080-LXSN (human fibrosarcoma cells expressing empty vector LXSN), NARF2 (osteosarcoma). Chemicals were obtained from Cayman Chemicals unless otherwise noted. CETZOLE 1 was synthesized as we have described in [7].

Measurement of Cell Viability.

The effect of ferroptotic compounds was determined using one of two methods as indicated in appropriate figure legends. Cells were stained with either methylene blue or MTT with similar results. In previous work, we observed both assays perform similarly when cells are undergoing ferroptosis [8]. While MTT measures ongoing cellular reductive capacity methylene blue stains nucleic acids of cells remaining on the culture plate after drug treatment. For methylene blue staining, plates were stained with a saturated solution of the dye dissolved in 50% ethanol. Plates were then rinsed with tap water, retained dye solubulized, and measured by spectrophotometry (absorbance at 650nm). For MTT staining, a 12 mM MTT (Goldbio) stock solution (10 X) was prepared in 1X PBS. The stock was diluted to 1X in DMEM plus FBS and added to plates (100 μl for 96 well plates and 300 μl for 24-well plates). Cells were incubated for 2–4 h at 37°C. Dye crystals were solubilized with an equal volume of 0.01N HCl in 10% SDS. Absorbance at 540 nm was determined.

Adenoviral experiments.

Adenoviruses were amplified in HEK293 as follows: HEK293 cells were plated in 10 cm plate and next day infected with recombinant adenoviruses. Virions were collected 2–3 days later from both the supernatant and cells by freeze-thawing process three times (frozen in dry ice and thawed at 37 °C). To determine viral titers, HEK293 cells were seeded in 96 well plates and the next day, the virus suspension was diluted at 1:100. Later, 10μl virus supernatant was added to cells in 90μl culture media. One week later, cytopathic effect (CPE) was assessed by phase contrast microscopy. Alternatively, infected cells were fixed in 100% methanol at −20°C for 20 min. Cells were blocked by 1% PBSP. One hour later, cells were probed with FITC conjugated Hexon antibody at 1:5000 in blocking buffer for 1 h. Cells were washed 3X with PBSP and infected cells visualized by fluorescence microscopy using an EVOS microscope. Titers determined using CPE or hexon immunofluorescence were similar. For adenoviral infections 2000–4000 NARF2 or 2000–3000 HT1080 cells were plated into each well of a 24 well plate. Next day, cells were infected with viruses and one day later, fresh media was replaced. Drugs were added 48–72h post adenoviral infection and viability determined after 1–3 days as indicated.

SLC7A11-GFP overexpression in HT1080 cells.

Phoenix retroviral packaging cells were plated to be 90% confluency on the day of transduction into a 6 well plate. The cells were transfected with SLC7A11-GFP plasmid, a gift from Alec Kimmelman [11] or control Rv-GFP plasmid (2.5 μg) using lipofectamine 3000 (Life technologies) according to manufacturer’s instructions. After 16hrs, media was replaced. Generated retroviruses were collected 48hrs post transfection. HT1080 cells from 24 well plates were infected with 100 μl of 48hr-viruses separately along with polybrene (4μg/ml). Next day, cells were expanded into 10 cm plates separately. SLC7A11-GFP cells were grown in culture media containing neomycin (400μg/ml). Since control GFP cells had no antibiotic-resistant genes, individual colonies were selected by GFP expression using a fluorescence microscope. Moreover, collected Rv-GFP clone was sub-cultured to isolate individual clones again. After 10–14 days, GFP or GFP- SLC7A11expressing colonies were selected, expanded, grown and confirmed by western blot analysis.

Western Blotting.

Cells were harvested by scraping and lysed in a buffer solution containing: 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% NP-40, 1 mg/ml aprotinin, 2 mg/ml leupeptin, 1 mg/ml pepstatin A, 1 mM DTT, 0.1M phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium fluoride and 1 mM sodium vanadate for 20 min on ice. Insoluble debris was removed by centrifugation at 16,000 g for 20 min at 4°C. Equal amounts of protein for each sample (determined using BCA protein assay kit - Pierce) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Gels were transferred to polyvinylidene difluoride membranes (Millipore), blocked in a solution containing 5% (w/v) non-fat dry milk dissolved in PBST [PBS containing 0.05% (v/v) Tween 20], and probed with antibodies as indicated. For phospho antibodies, membranes were blocked, or primary antibodies were diluted in 5% (w/v) bovine serum albumin in tween containing tris buffered saline.

Antibodies were generally diluted in blocking solution at 1:1000. Primary antibodies recognizing SLC7A11 (cell signaling technologies, CST#12691S), ATF4 (Abclonal #A0201), c-Myc (CST #9402S), p21 (Santa Cruz #C-19/HRP), p53 (Santa Cruz #D0–1/HRP), E2F1 (Santa Cruz #56661), Nrf2 (Abclonal #A0674), p-Nrf2 (S40)(Abclonal #AP1133), GPX4 (Abclonal #A11243), ALOX5 (Abclonal#A2877), α-Tubulin (Sigma #T5168), GAPDH (Abclonal #AC002) and β-Actin (Abcam #3280) were generally incubated at 4° C overnight. Signals were detected using horse-radish peroxidase conjugated secondary antibodies (Biorad) and enhanced chemiluminescence (Biorad). Western blot images were mostly taken by a chemi doc and the digital images were analyzed using ImageJ software. Uncropped images and higher exposures are available in supplementary figures.

siRNA transfection.

NARF2 and HT1080 cells were seeded in 6 well plates at a density of 2 × 105 and 3 × 105 respectively. Next day, the cells were transfected by 50 pmol of siRNAs against E2F1 or 50 pmol non-specific (scrambled/ siNeg) or Myc (10–50pmol) using Lipofectamine RNAi Max reagent according to the manufacturer’s instructions (Invitrogen life technologies). One day later, cells from multiple wells were expanded into 96 well plate for ferroptosis sensitivity assay or 6cm/ 9cm plates for western blot/ RNA extraction. siRNAs were obtained from Life technologies siE2F1 (cat#4427038), siMyc (cat#4427037) and siNeg (cat#4390843)

Transient c-Myc overexpression.

NARF2 (2 × 105) or HT1080 (4 × 105) cells were plated into 6 well plates. The next day, cells were transfected with indicated amount of pSVHmycMoneo plasmid (0.5–2.5μg) using Lipofectamine LTX Plus reagent (Life technologies). pSVHmycMoneo expresses human c-Myc under the control of an SV40 promoter [12]. One day post-transfection, cells were expanded into 6cm or 7cm plate according to the experiment. 48h later post transfection, cells were harvested and analyzed by western blotting. As a transfection control, cells were transfected with a GFP reporter plasmid. We observed >90% transfection efficiency by GFP positive cells.

Real time PCR (qRT-PCR).

Cellular RNA was extracted using TRIzol (Invitrogen Life technologies) method according to the manufacturer’s protocol. 1μg RNA was used to synthesize cDNA by reverse transcription reaction using oligo-dT primer according to NEB protocol (MuLV RT kit #BO2535). qPCR reactions were performed using iTaq Universal SYBR Green Supermix (Bio-Rad #1725121) and run it on Biorad CFX96 machine. Relative mRNA expression level was normalized to GAPDH and quantified using comparative CT method (ΔΔCT). n=6, biological triplicates and technical duplicates. Primer sequences for the genes are shown in Supplemental Table 1.

Statistical analysis.

Statistical significance was determined (* p < .05) by Student’s t test. Measurements of cell viability involved the preparation of triplicate samples, used to calculate averages and standard deviations. With one excpetion, cell viability studies were repeated a total of three times (total n = 9). Cell viability of SLC7A11-overexpressing cells in Figure 10 were repeated once in triplicate (total n=6). IC50 curves of SLC7A11-GFP clones were plotted using GraphPad Prism 9.0. Gene expression (mRNA) level was determined using comparative CT method (ΔΔCT). n=6, biological triplicates and technical duplicates. Technical duplicates were first averaged before statistical analysis of triplicate values.

Results

Ferroptosis antagonism by E2F1

The E2F1 transcription factor originally implicated in cell cycle regulation also modules apoptosis, senescence, and ferroptosis . To investigate the influence of E2F1 on ferroptosis, we modulated its expression by overexpression or RNAi knockdown in two ferroptosis-sensitive cell lines. HT1080 cells are a fibrosarcoma cell line previously used to study ferroptosis [3]. In the current study we used a subclone infected with an empty retrovirus “HT1080-LXSN”. We also analyzed NARF2 cells which are a derivative of osteosarcoma cell line U2OS. NARF2 were previously engineered to contain an IPTG-inducible p14ARF gene that allows for induction of p53 [13]. We used this feature in a previous study to investigate the effect of p53 on ferroptosis [4]. Here we analyze NARF2 cells without IPTG addition, to serve as a second ferroptotic-sensitive cell line that we can compare to our previous observations.

Overexpression of E2F1 protected from CETZOLE-1 induced ferroptotic cell death in part through upregulating p21 [4]. Overexpression of E2F1 in HT1080 cells also inhibited cell death induced by ferroptosis inducers Erastin, RSL3 and ML162 (Figure 1A–D). In these experiments, E2F1 was overexpressed for 48 hours before drugs were added. Cell death was rescued by lipid ROS scavenger, liproxstatin-1 when co-treated with the highest concentration of the above drugs. This confirms that cells are undergoing ferroptosis.

Figure 1. E2F1 protects from ferroptotic cell death.

Figure 1.

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HT1080 cells were transduced with E2F1 adenoviruses for 2 days and then ferroptosis inducers CETZOLE-1 (A), Erastin (B), RSL3 (C) or ML162 (D) were added at the indicated concentrations. Highest dose of each inducer was co-treated with Liproxsatin-1 to confirm ferroptotic cell death. One day later, cell viability was measured using methylene blue staining. (* p < 0.05, from three biological replicates)

E2F1 upregulates SLC7A11 mRNA.

To understand how E2F regulates ferroptosis we performed an mRNA screen of ferroptosis related genes by real time PCR (qPCR). mRNA encoding the xc− subunit SLC7A11 was upregulated in HT1080 cells upon E2F1 overexpression (Figure 2A). SLC7A11 expression was not significantly altered by E2F1 in NARF2 cells (Figure 2B). Thus, the effect of E2F1 appears to be cell type dependent. Publicly available chromatin immunoprecipitation (ChIP) data were downloaded from ChIP-Atlas [14, 15] and viewed using the Integrative Genomics Viewer (IGV) [16, 17]. These traces suggest that E2F1, along with other factors, may interact with the SLC7A11 promoter (Figure 3), potentially explaining the regulation that we have observed (Figure 2).

Figure 2. Expression of ferroptotic genes upon E2F1 overexpression.

Figure 2.

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Adenovirus (muliplicity of infection (MOI) of 100) expressing E2F1 or CMV control viruses were transduced for two days into (A) HT1080 or (B) NARF2 cells. Then, qPCR was performed to measure the mRNA levels of SLC7A11, NFE2L2, GPX4, ATF4, and CCNE1 as a reference of E2F1 target using SYBR green. (n=3 and * p < 0.05)

Figure 3.

Figure 3.

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Transcription factors binding to the SLC7A11 promoter. Public databases of ChIP-seq data (ChIP-Atlas) [14, 15] were aligned using Integrative Genomics Viewer (IGV) [16, 17]. Tracks of particular interest are indicated by arrows: MYC, E2F1, and ATF4. For this display, the threshold for statistical significance values calculated by peak-caller MACS2 (−10*Log10[MACS2 Q-value]) was set at 50 (only peaks with Q value < 1E−05 are shown). Colors shown in IGV indicate the statistical significance values as follows: blue (50), cyan (250), green (500), yellow (750), and red (> 1,000).

CCNE1 (cyclin E) was used as a positive control for E2F1 targets (Figure 2). Nuclear erythroid factor 2 (Nrf2)) was reported to transactivate several antioxidant genes including SLC7A11 [18] and GPX4 [19]. E2F1 had no effect on the amount of NFE2L2 (encoding Nrf2) mRNA in either NARF2 or HT1080 cells (Figure 2A, B). Similarly, E2F1 had no significant effect on GPX4 mRNA in either HT1080 or NARF2 cells (Figure 2A, B).

SLC7A11 is modulated by the activation transcription factor (ATF/CREB) family. ATF has 6 family members. ATF4 upregulates SLC7A11 [20, 21] while ATF3 was reported to repress SLC7A11 [22]. Since SLC7A11 was upregulated, and E2F1 is mostly an activating transcription factor, we measured mRNA levels of ATF4 upon E2F1 overexpression. ATF4 was upregulated upon E2F1 overexpression in both HT1080 and NARF2 (Figure 2A, B). These results indicate that E2F1 may partly upregulate SLC7A11 through ATF4. ATF1–4 showed the potential to interact with the SLC7A11 promoter (Figure 3).

Effect of E2F1 on expression of ferroptotic proteins.

Next, we used western blot analysis to measure several proteins implicated in ferroptosis. We overexpressed E2F1 in NARF2 and HT1080 cells by means of recombinant adenovirus (Figure 4). Two days post infection, E2F1 induced only a marginal induction of SLC7A11 expression in NARF2 cells (Figure 4A). When we repeated this same experiment, SLC7A11 showed a biphasic induction, an effect that was not consistently observed (Supplemental Figure 1). Overall, the induction of SLC7A11 protein in NARF2 by E2F1 at two days was marginal. However, SLC7A11 was induced 3 and 4 days after E2F1 overexpression in this cell line (Figure 4B). p21 was induced by E2F1 as previously observed (Figure 4A)[4]. No change in p53 expression was observed upon E2F1 overexpression (Figure 4B).SLC7A11 was also induced by E2F1 overexpression in HT1080 cells (Figure 4C).

Figure 4. Ferroptotic protein levels upon E2F1 overexpression.

Figure 4.

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(A, B) NARF2 or (C) HT1080 cells were transduced with E2F1or CMV adenoviruses at increasing MOIs. (A, C) 2 days or (B) indicated times later cells were collected and immunoblotted for given proteins. All western blots shown are representative of at least two independent experiments.

We detected no change of Nrf2 proteins level after E2F1 overexpression in HT1080 cells similar to the mRNA level. Interestingly, E2F1 overexpression led to increased expression of GPX4 protein in both HT1080 and NARF2 cells (Figure 4A, C). This indicates that E2F1 may indirectly stabilize GPX4 protein since we observed no difference at the mRNA level. Further study is needed to confirm the mechanism of E2F regulation of these targets.

E2F1 knockdown and downstream targets.

To better understand the effect of E2F1 on ferroptosis we used siRNA to deplete the protein (Figure 5). Western blot shows that more than 90% knock down of the protein was achieved (Figure 5D, Supplemental Figure 2). Consistent with our previous results, knockdown of E2F1 in HT1080 cells increased cell death in response to various ferroptotic inducers (Figure 5A, B, C). Similarly results were obtained with NARF2 cells (Supplemental Figure 2). Cell death in HT1080 cells was rescued by co-treatment liproxstatin-1 and not with pan caspase inhibitor Z-vad (FMK) (Figure 6) suggesting E2F1 knockdown increases ferroptosis.

Figure 5. E2F1 knock-down sensitizes sarcoma cells to ferroptosis.

Figure 5.

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HT1080 cells were transfected with E2F1 siRNA or Negative control siRNA (siNeg/scramble) or no transfection (parental). Next day, cells were expanded into a 96 well plate and 3 days post transfection, cells were treated with RSL3 (A), CETZOLE-1 (B), or ML162 (C) at the concentratiosn indicated. Cell viability was measured using MTT assay one day later. Western blot analysis was performed to visualize the protein level (D). (* p < 0.05). All viability assays are averages of three independent samples. Western blots are representative examples of two independent experiments.

Figure 6. Enhanced killing after E2F knockdown is due to ferroptosis.

Figure 6.

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E2F1 was depleted by siRNA in HT1080 cells. Three days post transfection, the cells were treated with indicated ferroptosis inducers along with liproxstatin-1 (LIP- 0.25μM) or Zvad-FMK(10μM). After 24h, cell viability was determined using methylene blue. (* p < 0.05)

Next, we measured the protein levels of SLC7A11, ATF4 and ALOX5 after E2F1 knock down (Figure 7A, B). ALOX5 is a key pro-ferroptotic enzyme that catalyzes the oxidation polyunsaturated fatty acids (PUFAs). In HT1080 cells, SLC7A11 was repressed >90% (n=3) in 3 days. Downregulation of ATF4 (~40%) and ALOX5 (~55%) was also observed (Figure 7B). Surprisingly, these protein levels were not altered in NARF2 cells. Next, we analyzed mRNA expression of several genes in both HT1080 and NARF2 cells. SLC7A11, ATF4, Myc and ALOX5 were downregulated in HT1080 cells after E2F1 knockdown (Figure 7C). In this context, we measured Myc levels since it is reported to be an E2F target [23]. Modest, but significant downregulation of ATF4 transcripts was observed in HT1080 cells. Neither SLC7A11, ATF4, Myc, nor ALOX5 were altered in NARF2 cells after E2F1 knockdown, similar to the lack of effect at the protein level (Figure 7D).

Figure 7. Cell type dependent regulation of SLC7A11, ALOX5, and ATF4 by E2F1.

Figure 7.

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(A) Western blot analysis of the effect of E2F1 knock-down in HT1080 and NARF2 cells. Three days after transfection, protein levels of E2F1, SLC7A11, ALOX5, and ATF4 were determined using β-actin as a loading control. (n=3) (B) Bar graph shows the fold of protein levels of HT1080 cells in siE2F1 vs control siNeg. Averages of three biological replicates run on two gels are shown after correcting for exposure differences between the gels and then normalizing to actin loading. (* p < 0.05). Transcripts level of the indicated genes were measured in both HT1080 (C) or NARF2 (D) cells after E2F1 or scrambled (siNeg) knock down for 3 days. (* p < 0.05)

We speculated that cell-type differences in the regulation of the genes we measured may be due to other members of the E2F family. E2F regulates its own family members [23]. In both HT1080 and NARF2 cells, E2F1 knockdown led to reduced nRNA expression of E2F2, E2F4, and E2F8 and increased expression of E2F7 as determined by real time PCR analysis (Figure 8A, B). No change was observed in E2F5 and E2F6 mRNA. Interestingly, E2F1 knockdown resulted in upregulation of E2F3 mRNA in NARF2 cells but not HT1080 cells (Figure 8A, B). E2F1, E2F2 and E2F3 primarily activate transcription of their target genes [24]. Therefore, cell-type dependent compensatory effect of E2F3 may distinguish NARF2 cells from HT1080 cells. This may possibly explain the differential expression of ferroptotic genes in NARF2 and HT1080 cells.

Figure 8. E2F1 knockdown affects its own family members.

Figure 8.

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Transcripts levels of E2F family members were measured after E2F1 knockdown (3 days) in both (A) HT1080 and (B) NARF2 cells. (* p < 0.05)

C-Myc regulates SLC7A11

Public ChIP-seq data suggests that Myc has the potential to bind to the SLC7A11 promoter Figure 3). Consistent with this, overexpression of c-Myc in HT1080 and NARF2 cells upregulated SLC7A11 protein levels (Figure 9A, B). Western blotting indicated that transfection led to a clear elevation of Myc protein in NARF2. This was less obvious in HT1080 cells, even though SLC7A11 was upregulated in both cell types (Figure 9A, B). It is possible that a threshold effect in HT1080 cells result in a large change in SLC7A11 in response to a more marginal upregulation of Myc expression. To confirm the effects of Myc, we used siRNA to knockdown the protein in HT1080 cells (Figure 9C). c-Myc knock down led to downregulation of SLC7A11 protein within 3 days (Figure 9C). At 2 days post-transfection, a modest suppression of SLC7A11 was observed. These results suggest that Myc may transcriptionally upregulate SLC7A11. This effect may contribute to the upregulation of SLC7A11 in response to E2F1 overexpression in HT1080 cells. This regulation appears to be cell-type dependent since neither Myc nor SLC7A11 mRNAs were induced in NARF2 cells upon E2F1 overexpression. Depending on the cellular context, Myc family members have been found to either enhance or suppress ferroptosis [25–31]. While our work suggests that SLC7A11 is one potential Myc target important for ferroptosis, many other Myc targets likely contribute to ferroptotic sensitivity.

Figure 9. Transcription factor c-Myc upregulates SLC7A11.

Figure 9.

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(A) HT1080 or (B) cells were transiently reconstituted with c-Myc and harvested 48h later. (C) HT1080 cells were transfected with siRNA against Myc (10–50 pmol) or non-targeted sequence (50 pmol, siNeg/ control) and the cells collected after 48h or 72h. (A-C) Harvested cells were analyzed by western blotting and probed for Myc, SLC7A11, and β-actin as a loading control. Different blots are shown together in right and left panel of A and B.

SLC7A11 overexpression protects from multiple ferroptotic signals

SLC7A11-GFP fusion protein was stably expressed in HT1080 cells to study its effect on ferroptosis. GFP expressing HT1080 cells served as a control. Clone #3 and #4 were chosen for further analysis since they represented intermediate and high level of transgene expression (Figure 8A).. SLC7A11 clones #3 and #4 were relatively resistant to both SLC7A11 inhibitors (Erastin) and GPX4 inhibitors (RSL3, ML162) compared to either control HT1080 Rv-GFP or parental cells (HT1080) (Figure 10B–E).

Figure 10. Overexpression of SLC7A11 protects from various ferroptotic inducers.

Figure 10.

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SLC7A11-GFP fusion protein was stably expressed in HT1080 cells by retroviral transduction and individual cell lines were generated. Control GFP expressing cell line was generated using lentivirus (Rv-GFP#1). (A) Western blot analysis demonstrates the level of endogenous and ectopic SLC7A11-GFP. SLC7A11-GFP clones #3 and #4 were tested for ferroptosis sensitivity using ferroptosis inducers: (B) CETZOLE-1, (C) Erastin, (D) RSL3, (E) ML162 and (F, G) cystine deprivation. Parental and Rv-GFP cells were used as control cells. Cells were deprived of cystine for 16 or 24h. All other inducers were treated for 3 days and cell viability was measured by methylene blue staining. (* p < 0.05). (H, I) Effect of ferroptotic inducers on SLC7A11 expression. NARF2 cells were treated with CETZOLE-1 (C1), Erastin, or RSL3 at the indicated concentration for 24h. Western blot was performed, and membranes were probed with antibodies against SLC7A11 and α-tubulin as a loading control.

We also tested the effect of SLC7A11-GFP expression on the cellular response to cystine deprivation. Clone #3 was significantly protected from cell death at 16h and 24h after removal of cystine from culture media (Figure 10F, G). Cell death was rescued by Liproxstatin-1 and N-acetyl cysteine (NAC). NAC is a stable form of cysteine transported inside the cells independent of system xc−. These observations provide evidence that death is due to ferroptosis (Figure 10F, G). At first glance, this observation seems at odds with the known role of SLC7A11. For example, under conditions where there is no cystine to import, it is not immediately clear why a cell line expressing higher levels of a subunit of a cystine importer should be protected from ferroptosis. We speculate that Clone #3, even before cystine is removed from culture medium, exhibits increased system xc− activity. This may result in a higher starting pool of intracellular cysteine resulting in delayed death upon cystine deprivation. Clone #4 was not protected from cystine deprivation possibly due to lower levels of expression of SLC7A11-GFP compared to clone #3 (Figure 10). Taken together, SLC7A11 overexpression protected cells from both SLC7A11 and GPX4 inhibitors. The ability of a system xc− subunit to protect from GPX4 inhibitors may be a result of higher intracellular cysteine leading to increased GSH levels needed to detoxify lipid peroxides.

SLC7A11 upregulation occurs in response to both GPX4 and system xc− inhibition

System xc− is comprised of two subunits, SLC7A11 and SLC3A2 (4F2hc). In 2004, Sato et al reported that cystine uptake via system xc− and SLC7A11 transcriptional activity were upregulated upon deprivation of several amino acids including cysteine and arginine in NIH3T3 fibroblasts [10]. Both SLC7A11 and SLC3A2 proteins were upregulated under these conditions [10]. They also showed that ATF4 was involved in SLC7A11 expression [10]. In 2008, Lo et al observed that both SLC7A11 and SLC3A2 subunits were elevated at mRNA and protein levels upon cystine deprivation in pancreatic cancer cell lines [32]. Several studies have implied that the upregulation of system xc−/ SLC7A11 is a characteristic features of system xc− inhibitors [33][34]. Therefore, we performed western blot analysis in NARF2 cells after treatment of CETZOLE-1, Erastin and RSL3 with (Figure 10H) and without Liproxstatin-1 (Figure 10I). SLC7A11 upregulation was observed with both system xc− inhibitors (Erastin and CETZOLE-1) and GPX4 inhibitors (RSL3 and CETZOLE-1). SLC7A11 was slightly reduced when ferroptotic inducers were combined with Liproxstatin-1. This suggests that the presence of lipid peroxides may be one of the signals that trigger upregulation of SLC7A11. ROS accumulation can lead to Nrf2 signaling and Nrf2 can upregulate SLC7A11 which may explain some of our results [34]. Overall, our results and previous findings [35] suggest that SLC7A11 is upregulated is response to ROS accumulation during ferroptosis but is not limited to system xc− inhibition.

Discussion

We previously observed that E2F1 protects from CETZOLE-1 induced ferroptosis in NARF2 and HT1080 cells [4]. Our present study indicates that the suppressing effect of E2F1 in ferroptosis is not limited to CETZOLE-1. Rather, E2F1 protects from multiple ferroptosis inducers including Erastin, RSL3 and ML162. We previously determined that E2F1 partially protects from ferroptotic cell death through CDK inhibitor, p21 [4]. In order to identify other targets of E2F1 responsible for ferroptosis inhibition, we analyzed a number of ferroptosis candidate genes. Interestingly, we found that SLC7A11 mRNA and protein expression were upregulated in HT1080 cells after E2F1 overexpression. Transcription factors ATF4 and Nrf2 can upregulate SLC7A11 and block ferroptosis [36, 37]. Thus, we measured the mRNA levels of these genes and found NFE2L2 levels were unchanged but ATF4 was upregulated by E2F1. Interestingly, the level of GPX4 protein was increased upon E2F1 overexpression. However, GPX4 mRNA levels were not altered, suggesting that E2F1 regulates GPX4 post-transcriptionally. Moreover, we identified a pro ferroptotic gene ALOX5 as another target of E2F1.

Our E2F1 knockdown experiments in HT1080 and NARF2 cells indicate that both cell types become more prone to ferroptosis upon depletion of E2F1. Furthermore, SLC7A11, ATF4, ALOX5 and c-Myc mRNA were reduced after E2F1 siRNA knockdown in HT1080 cells. Surprisingly, these proteins or mRNAs were not altered in NARF2 cells after 3 days of E2F1 knockdown. To try to understand the differential effect of E2F1 knockdown in the two cell types we analyzed other E2F family members. In both HT1080 and NARF2, E2F4, and E2F8 were downregulated and E2F7 was upregulated after E2F1 knockdown. Interestingly, E2F3 was upregulated after E2F1 knockdown, but only in NARF2 and not in HT1080 cells. These experiments show that simply knocking down one E2F family member can have complicated effects on expression of the E2F family. Furthermore, these effects can be cell-type specific.

We speculate the elevation of E2F3 may compensate for E2F1 in NARF2 cells such that E2F1 knockdown ultimately has minimal effect on the expression of SLC7A11, c-MYC, ATF4, and ALOX5. However, it remains elusive how NARF2 cells becoming more sensitive to ferroptosis upon knockdown of E2F1. This suggests that E2F1 has various targets to overcome oxidative stress depending on the cell type that we have not yet uncovered. Analysis of reduced thiols using monochlorobimane showed no effect of when p53 was induced by adding IPTG, or when p21, or E2F1 were overexpressed using recombinant adenoviruses (Supplemental Figure 3). In recently published work, we observed that overexpressing RB (wild-type WT, or a mutant, RBΔCDK, no longer phosphorylated by CDKs) could modulate ferroptosis [38]. Neither WT-RB nor RBΔCDK affected reduced thiols (Supplemental Figure 3). This suggests that these proteins do not affect ferroptosis by modulating the abundance of GSH which is the most abundant thiol in most mammalian cells [39].

Based on our qPCR results, we found ATF4 and c-Myc were upregulated with E2F1 overexpression. Myc is a known target of E2F1 [23]. Oncogene MYC regulates proliferation, glycolysis, oxidative phosphorylation, glutaminolysis, fatty acid metabolism and apoptosis [40, 41]. N-MYC regulates ferroptosis by modulating cysteine and glutamine utilization [42, 43]. Furthermore, N-Myc binds to the promoter region and transcriptionally activates SLC3A2 [44]. Meanwhile SLC7A11 was upregulated at both protein and mRNA levels by N-MYC [44]. According to publicly available global ChIP seq data, Myc may bind to the promoter of SLC7A11 [45]. Furthermore, overexpression of c-MYC elevated SLC7A11. Similarly, E2F1 was found to upregulate ATF4, a transcription factor previously shown to increase expression of SLC7A11 [20, 21]. Therefore, E2F1 may engage both c-MYC and ATF4 to upregulate SLC7A11 to suppress ferroptosis.

Conclusion

Overall, we have found that several ferroptosis markers are regulated by E2F1 including SLC7A11, ATF4, GPX4, ALOX5 and Myc. This complex role of E2F1 provides insight into how cancer cells can prevent oxidative stress while being highly proliferative.

Supplementary Material

Supplementary material

Acknowledgements

We thank Alec Kimmelman (NYU) for providing SLC7A11-GFP plasmid. We also thank Heather Conti (University of Toledo) for help with flow cytometry.

Funding

This project was supported by National Institute of Health grants: NIH-R15CA213185 to L. M. V. T. and NIH-R15GM141712 to W. R. T.

ABBREVIATIONS:

CDK

elevated cyclin dependent kinase

BHA

butylated hydroxyanisole

GSH

glutathione

CPE

cytopathic effect

PMSF

phenylmethylsulfonyl fluoride

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

PBST

PBS containing 0.05% (v/v) Tween 20

ChIP

chromatin immunoprecipitation

IGV

Integrative Genomics Viewer

CCNE1

cyclin E

Nrf2

Nuclear erythroid factor 2

ATF/CREB

activation transcription factor

NAC

N-acetyl cysteine

MOI

multiplicity of infection

Footnotes

Competing interests

L. M. V. T. and W. R. T. appear as inventors on patents covering the compound CETZOLE 1 described in this article. N. K., S. D., and S. H. declare that they have no conflicts of interest with the contents of this article.

Data availability

All data generated or analyzed during this study are included in this published article [and its supplementary information files].

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material
NIHMS2098118-supplement-Supplementary_material.pdf (1.5MB, pdf)

Data Availability Statement

All data generated or analyzed during this study are included in this published article [and its supplementary information files].

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