
==== Front
Redox Biol
Redox Biol
Redox Biology
2213-2317
Elsevier

S2213-2317(24)00283-0
10.1016/j.redox.2024.103305
103305
Research Paper
A potential therapeutic strategy based on acute oxidative stress induction for wild-type NRF2/KEAP1 lung squamous cell carcinoma
Sánchez-Ortega M. a1
Garrido A. antonio.garrido@universidadeuropea.es
ab⁎⁎1
Cirauqui C. c
Sanz-Gonzalez L. a
Hernández M.C. a
González-García A. a
Obregon K. a
Ferrer I. c
Paz-Ares L. c
Carrera A.C. acarrera@cnb.csic.es
a⁎
a Department of Immunology and Oncology, National Centre for Biotechnology (CNB), Spanish Research Council (CSIC), Autonomous University of Madrid, Cantoblanco, Madrid, E-28049, Spain
b Department of Biosciences, School of Biomedical and Health Sciences, European University of Madrid, Villaviciosa de Odón, Madrid, E-28670, Spain
c H12O-CNIO Lung Cancer Clinical Research Unit, Health Research Institute Hospital 12 de Octubre, Spanish National Cancer Research Center (CNIO), Madrid, E28029, Spain
⁎ Corresponding author. acarrera@cnb.csic.es
⁎⁎ Corresponding author. antonio.garrido@universidadeuropea.es
1 These authors contributed equally to this work.

08 8 2024
9 2024
08 8 2024
75 1033051 7 2024
31 7 2024
6 8 2024
© 2024 The Authors. Published by Elsevier B.V.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Extensive efforts have been conducted in the search for new targetable drivers of lung squamous cell carcinoma (LUSC); to date, however, candidates remain mostly unsuccessful. One of the oncogenic pathways frequently found to be active in LUSC is NFE2L2 (NRF2 transcription factor), the levels of which are regulated by KEAP1. Mutations in NFE2L2 or KEAP1 trigger NRF2 activation, an essential protector against reactive oxygen species (ROS). We hypothesized that the frequency of NRF2 activation in LUSC (∼35 %) may reflect a sensitivity of LUSC to ROS. Results from this study reveal that whereas tumors containing active forms of NRF2 were protected, ROS induction in wild-type NFE2L2/KEAP1 LUSC cells triggered ferroptosis. The mechanism of ROS action in normal-NRF2 LUSC cells involved transient NRF2 activation, miR-126-3p/miR-126-5p upregulation, and reduction of p85β and SETD5 levels. SETD5 levels reduction triggered pentose pathway gene levels increase to toxic values. Simultaneous depletion of p85βPI3K and SETD5 triggered LUSC cell death, while p85βPI3K and SETD5 overexpression rescued survival of ROS-treated normal-NRF2 LUSC cells. This shows that the cascade involving NRF2 > miR-126-3p, miR-126-5p > p85βPI3K and SETD5 is responsible for ROS-induced cell death in normal-NRF2 LUSC. Transient ROS-induced cell death is shown in 3D spheroids, patient-derived organoids, and in xenografts of wild-type NFE2L2/KEAP1 LUSC cells, supporting the potential of acute local ROS induction as a therapeutic strategy for LUSC patients with normal-NRF2.

Highlights

• ROS triggers cell death in lung squamous cancer cells with wild type NRF2/KEAP1.

• ROS-cell death was confirmed in xenografts, spheroids and patient-derived organoids.

• p85βPI3K and SETD5 downregulation are key events in ROS-induced LUSC cell death.

• SETD5 restricts PPP genes, SETD5 attenuation increases G6PD PPP gene to toxic levels.

• ROS generation is a novel therapeutic strategy for wild-type NRF2/KEAP1 LUSC cancer.

Keywords

LUSC treatment
Transient ROS inducers
SETD5
MicroRNA126
PI3K
p85βPI3K
==== Body
pmc1 Introduction

Lung cancer, which has a 5-year overall survival rate of ∼15 %, is a common cause of cancer-related death worldwide [1,2]. Approximately 85 % of lung tumors belong to a group known as non-small cell lung cancer (NSCLC), as opposed to small-cell lung cancer (15 %). Within NSCLC, lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) are the most common subtypes [1,2]. Targeted therapies have improved the treatment landscape for NSCLC, but these advances have mainly benefited patients with oncogene-driven LUAD, while most attempts to develop targeted treatment for LUSC remain poorly successful [3].

The Cancer Genome Atlas project, which identified the most frequent genetic alterations in LUSC, found that the mutational profile of this tumor type is distinct from other forms of NSCLC. LUSC mutations include alterations in genes such as TP53, CDKN2A, PTEN, PIK3CA, KEAP1, KMT2D, and NFE2L2. The existence of these mutations, points to the cell response to oxidative stress (KEAP1 and NFE2L2), the PI3K (phosphoinositide 3-kinase) pathway (PTEN, PIK3CA), the squamous differentiation, and the cell proliferation pathways, as drivers of LUSC tumor progression [4,5]. To date, however, actionable driver mutations have proven elusive, and attempts to develop targeted treatment for LUSC patients exhibit low success rates [[6], [7], [8], [9]].

One frequently altered pathway in LUSC is activation of the NRF2 (NFE2L2) transcription factor, which provides essential protection for cells against reactive oxygen species (ROS). NRF2 stability is dependent on KEAP1 (Kelch Like ECH Associated Protein 1). KEAP1 is a substrate-specific adapter of a BCR (BTB-CUL3-RBX1) E3 ubiquitin ligase that interacts with NRF2 in non-stress conditions and promotes its ubiquitination and degradation [[10], [11], [12]]. ROS triggers the modification of several cysteine residues in KEAP1, leading to inactivation of the E3 ligase, release of NRF2, and its translocation to the nucleus. The alterations that NFE2L2/KEAP1 genes present in LUSC include activating mutations of NFE2L2, loss of function of KEAP1, and altered copy numbers [4]. The high proportion of LUSC samples exhibiting active-NRF2 (∼35 %) [9] lends support to the hypothesis that LUSC tumors with wild-type forms of NFE2L2/KEAP1 (normal-NRF2) might be highly sensitive to ROS.

The urgent need to identify new targetable drivers of LUSC prompted us to study the potential sensitivity of LUSC to ROS inducers in samples with either wild-type- or active-NFR2. Treatment with ROS inducers caused cell death selectively in normal-NRF2 LUSC cells. The mechanism of action of ROS involved transient activation of NRF2, upregulation of miR-126-3p and miR-126-5p, and subsequent downregulation of p85βPI3K and of SETD5 (SET Domain Containing 5), respectively. p85βPI3K (PIK3R2) is a regulatory subunit of Class IA PI3K with oncogenic capacities and overexpressed in LUSC [14], whereas SETD5 is an epigenetic modifier regulating gene expression [15]. Simultaneous depletion of p85β and of SETD5 triggered LUSC cell death, while co-overexpression of p85βPI3K and SETD5 rescued survival after intracellular ROS (iROS) induction in normal-NRF2 LUSC cells. These observations indicate that the cascade NRF2 activation > miR-126-3p and -5p increase > reduction of p85βPI3K and of SETD5 levels triggers cell death in LUSC cells with normal-NRF2. ROS-induced LUSC cell death was detected in 3D spheroids, patient-derived organoids, and xenografts, raising expectations for a new therapeutic strategy for normal-NRF2 LUSC.

2 Materials and methods

2.1 Antibodies, reagents, and siRNA transfections

Antibodies used: anti-phospho (p)-AKT T308, -total AKT and -active Caspase 3 (Cell Signaling Technology), -α-tubulin (Calbiochem) LC3B (NOVUS) and -β-actin (Sigma). Anti-SETD5 antibody was a gift from Dr. Mazur (Stanford University, USA) [16]. The ROS inducers used were dipyridone, oxidized PAPC (oxPAPC), and anisomycin. The dipyridone sophocarpidine (1R,2R,9S,17S)-7,13 diazatetracyclo [7.7.1.02, 7.013,17] heptadecan-6-one (IUPAC), will be referred to as dipyridone (Sigma-Aldrich). Dipyridone solutions were prepared in culture media with the exception of ROS quantitation analysis (in ROS buffer). 1-palmitoyl-2-archidonoyl-sn-glycero-3-phosphocholine (PAPC, 10 mg/ml) was from Avanti. PAPC was oxidized to oxPAPC by exposure to air (40 h) in a cell culture cabinet, then dissolved in chloroform and stored at −80 °C. Chloroform was evaporated with nitrogen and lipids re-suspended in media and sonicated. Anisomycin (Sigma) was dissolved in ethanol. Other reagents were: ON-TARGETplus PIK3R2, SETD5, KEAP1 and NFE2L2 siRNA pools were from Dharmacon, Lipofectamine RNAiMax (Invitrogen), and OptiMem (Life Technologies); anti-miR-126 (MIMAT0000445) and anti-miR™ miRNA inhibitor negative control #1 (Thermofisher). AG1 was from MedChemExpress; Vitamin E, Necrostatin and N-acetylcysteine (NAC) from Sigma and ZVAD and Ferropstatin1 from Abcam.

2.2 Cell lines, cell culture, generation of SETD5 and PIK3R2 stable cell lines

The following LUSC cell lines were purchased from the American Type Culture Collection [SK-MES-1 (HTB-58), H520 (HTB182), and H226 (CRL-5826)] and one from the DSMZ collection [HCC-15 (ACC-496)]. The normal airway epithelial cells, HSAEC1/KT, were from ATCC and were used as controls. HEK-293T cells (CRL-3216) were used for virus production. These cells were maintained in DMEM or RPMI (Gibco, Thermofisher Scientific) containing 10 % fetal bovine serum, 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C, 5 % CO2, and 95 % humidity. HSAEK1/KT cells were cultured in SABM™ small airway epithelial cell growth basal medium plus with SAGM™ Medium SingleQuots™ (Lonza). Cells were used in passages 3 to 12.

H226 cells overexpressing PIK3R2 and/or SETD5 genes were generated using lentiviral particles containing the appropriate plasmids. pWPI-IRES-GFP-PIK3R2 was produced from pWPI-IRES-GFP (green fluorescence protein) plasmid (Addgene 12254), while pCMV1 (w117.1)-Hygro-SETD5 was a gift from Dr. Mazur (Stanford University, USA). For infection, 293T cells were transfected using JetPei (DNA transfection reagent; Polyplus transfection) with either the vector of PIK3R2, SETD5, or the empty vector, plus pMD2.G (Addgene 12259) and psPax2 plasmids (Addgene 12260). At 48 h, supernatants containing viral particles were filtered (0.45 μm; Millipore), supplemented with polybrene (8 μg/ml, Sigma), and added to the target cells. The cells plus viruses were centrifuged at 1500 rpm for 90 min and cultured. After repeating infection on day 2, the cells were grown (∼5 d). Clones were selected by cell sorting of GFP + cells (for PIK3R2 overexpression) or in a medium with hygromycin (200 μg/ml/7 days) (Sigma) for SETD5 overexpressing cells. Cells with PIK3R2 and SETD5 overexpression were first selected by sorting and then with hygromycin. The efficiency of infections was screened by Western blot (WB).

2.3 Real-time quantitative PCR (RT-qPCR)

The RNeasy kit (Qiagen) was used to extract total cellular RNA. For microRNA extractions, mirVana™ microRNA (miR) isolation kit (Thermofisher Scientific) was used. Regarding tumor xenograft samples, total tissue RNA was extracted with Trizol™ reagent (Invitrogen). All extraction kits were used according to the respective manufacturer's protocol. cDNA was synthesized with a High-Capacity cDNA Reverse Transcription (RT) kit (Invitrogen) with 1 μg of RNA. RT products were stored at −20 °C. mRNA levels were quantified by RT-qPCR in an ABI PRISM 7900HT System (Applied Biosystems) using a HOT FIREPolR EvaGreenR RT-qPCR Mix Plus (Solis Biodyne). Specific primers are listed in Supplementary Table 1. GAPDH (and TBP) were used for normalization. Gene expression [RQ ± standard deviation (SD)] was calculated using the 2-ΔΔCt method [17].

2.4 Cell death, apoptosis, ferroptosis ROS analysis, lipid peroxidation, and Western blot

Cell viability was analyzed by flow cytometry with propidium iodide (PI). After the cells were washed twice with PBS, they were resuspended in 350 μl of PBS +0.5 % BSA, and 10 μl of PI (30 min). Cell death was analyzed in an FC500 Flow Cytometer (Beckman Coulter; Life Sciences) and the percentage of cell death (PI-positive cells) was calculated based on 50.000 events using Kaluza software (Beckman Coulter; Life Sciences). For cell death, alternatively, flow cytometry can be fixed for 90 s each sample and the number of dead or alive cells examined. For apoptosis, the cells were stained with Anexin V (Thermo Fisher) and propidium iodide (PI), then examined by flow cytrometry [18]. To measure ferroptosis, the cells were collected, stained with the lipid peroxidation sensor BODIPY™ 581/591 C11 (Thermofisher) and then analyzed according to manufacturers.

Intracellular ROS (iROS) was measured using an DCFDA/H2DCFDA assay kit (Abcam). Cells were incubated with 5 μM of DCFDA (30 min at 37 °C in darkness) and then were exposed to dipyridone (0.5nmg/ml) for different lengths of time. iROS was then detected in fluorescence microscopy and quantified in parallel by flow cytometry. Similarly, lipid peroxidation was analyzed by Bodipy™ 581/591 C11 probe (Thermofisher). Cells were exposed to dipyridone (0.5 mg/ml), and the probe was added to growth media (45 min at 37 °C in darkness). Cells were washed with PBS, and lipid peroxidation analyzed by flow cytometry. WB was performed as described elsewhere [19]. iROS was also measured using as lipid peroxidation test the malondialdehyde (MDA) production (nmols MDA/gr) performed according to manufacturers (MDA assay kit, Sigma).

2.5 Spheroid generation

Ten thousand cells/well were seeded in 96-well Round (U)-bottom ultralow-adhesion plates (Thermofisher). The plates were then centrifuged at 1200 rpm for 5 min and incubated for 96 h. Then, 100 μl of medium was replaced with 100 μl of fresh media containing dipyridone (0.75 mg/ml or 1 mg/ml) (24 h), the DRAQ7 probe (Thermofisher) was added to each well (1:100) (30 min). A complete image of each spheroid was taken after 24, 48, and 72 h of dipyridone treatment using a confocal microscope (Stellaris, Leica Microsystems). Three spheroids were generated per condition and experiment. Results are presented as fold change (dead cells) relative to the controls.

2.6 LUSC PDX-derived organoid culture

Organoid cultures were performed at the Spanish National Cancer Center (CNIO). Two cultures derived from PDX (patient derived xenograft) models (TP13 and TP34) were selected from a collection of PDX models established at the Institute of Biomedicine in Seville (IBIS) and the CNIO. Early-stage lung tumors resected from patients (Hospital Universitario Virgen del Rocío in Seville and Hospital 12 de Octubre in Madrid, Spain) and were cultured as follows: PDX tumors were minced into 1–2 mm3 fragments, then digested with 5 ml of digestion solution (1.2 mg/ml collagenase IA in DMEM/F12 with 10 mM HEPES, 1X GlutaMax, 2.5 % FBS, and 0.25 % Primocin) (1–2 h at 37 °C); red blood cell lysis buffer (eBiosicences) was used to remove erythrocytes. For organoid culture, 40,000 of the recovered PDX cells were embedded in 40 μl of Matrigel and seeded into 8 well-chamber slides (Nunc Ref. 154534). Once Matrigel was solidified, 300 μl of complete human feeding media was added (DMEM/F12 plus 10 mM HEPES, GlutaMax, 0.25 % Primocin, 2 % FBS, 5 μg/ml insulin, 3 ng/ml hEGF, 1 μg/ml hydrocortisone, 1X B-27 supplement, and 10.5 μM Rho Kinase inhibitor). When 3D structures were formed, the organoids were treated with different concentrations of dipyridone (0.25 mg/ml to 1.5 mg/ml) (24 h). Viability was measured with DRAQ7™ Dye.

2.7 Subcutaneous tumor xenografts

The Ethics Committee of the National Centre for Biotechnology (CNB/CSIC) approved all procedures using mice in accordance with EU/Spanish legislation (RD53/2013). For xenografts, ∼107 cells were mixed with Matrigel (BD) (at 50 %, v:v) in 0.2 ml and then inoculated subcutaneously (s.c.) into both flanks of 8-week-old female immunodeficient athymic nude mice (Envigo) under isoflurane anesthesia. Tumors formed for 3–7 days until the volume ranged from 75 to 100 mm3. At this point, dipyridone treatment (100 mg/kg dissolved in sterile PBS) or vehicle was injected intraperitoneally (three times weekly for three weeks). All animals were weighed, the volume of the tumors measured with calipers three times a week. Tumor volume was calculated as V = (smaller side length2 × larger)/2. We established (i) loss of 20 % of initial weight or (ii) tumor size greater than 1500 mm3 as endpoints for the experiment.

2.8 Statistical analysis

Statistical analysis was performed using GraphPad Prism software (GraphPad Software, Inc.). Data were expressed as mean ± SD for ≥ three independent experiments. Student's t-test (*p < 0.05; **p < 0.01; and ***p < 0.001), Chi-Square (and Fisher's exact test) [**] P < 0.01; [***] P < 0.001 or one-way analysis of variance (ANOVA) with multiple comparisons used to analyze differences between more than two groups. For xenograft studies, two-way ANOVA was used (*) p < 0.05; (**) p < 0.01; and (***) p < 0.001.

3 Results

3.1 Human LUSC cell lines are sensitive to intracellular ROS induction

To test the hypothesis that LUSC cells with wild type (WT) KEAP1 and NFE2L2 genes are sensitive to ROS, we used dipyridone, which contains an electron-donating group (Fig. S1A) and does not cause severe side effects in vivo [20]. The capacity of dipyridone to increase iROS was examined in normal-NRF2 H226 cells, by DCFDA staining and was detectable from 5 min to 1h (Fig. S1B). This was confirmed by measurement of lipid peroxidation, a sign of iROS [21], using Bodipy C11 (Fig. S1C). In contrast, in active-NRF2 HCC15 cells only a small and transient increase in iROS was seen at 10 min of dipyridone treatment (Fig. S1B). iROS greater formation in normal-NRF2 cells versus active-NRF2 cells was confirmed by testing malondialdehyde (MDA) formation, one of the final products of polyunsaturated fatty acids peroxidation [22]. Both dipyridone, oxPAPC and anisomycin (see below) increased MDA levels in normal-NRF2 H226 cells, and had a very low effect in active-NRF2 HCC15 cells (Fig. S1D).

The sensitivity of LUSC cells to dipyridone was assessed in: HCC15 cells expressing active-NRF2 (KEAP1 p.304G > C mutation); H226 cells containing WT forms of KEAP1/NFE2L2 (normal-NRF2); SK-MES-1 and H520 cells with NFE2L2/KEAP1 increased copy number ratio; and as a control, normal lung airway epithelial cells (HSAEC1/KT) (Fig. 1A–D; Figs. S1E and F).Fig. 1 High sensitivity to iROS of LUSC cell lines with wild type NFE2L2/KEAP1. (A–D) Cell death in LUSC cell lines treated with different doses of dipyridone (indicated). Cells examined included: HCC15 (exhibiting activation of the NRF2/KEAP1 pathway) (A), H226 (with normal NRF2) (B), SK-MES-1 (with altered copy number; NFE2L2>KEAP1) (C), and normal epithelial airway cells (HSAEC1/KT cells) (D). All cell lines were seeded in parallel, treated for different times with dipyridone, and maintained in exponential growth. The figure shows representative images ( × 10) of the different cells at 24h of the treatment. After 24, 48, and 72h of treatment, cells were collected and incubated with propidium iodide (PI). The percentage of PI-positive cells (dead cells) was analyzed by flow cytometry. Flow cytometry square plots show the number of PI- (alive cells) and PI+(death cells, in red) at 0.5 mg/ml of dipyridone (24h) vs untreated cells. Bar graphs show the percentage of PI+ cells per condition subtracting the background (PI+ cells in untreated cells). Percentage of cell death is expressed in relation to total cell number (100 %) (Mean ± SD, n = 3). The dashed line at 30 % cell death (maximum death in ROS treated-normal lung cells) is drawn for comparison. ***P > 0.001 unpaired t-test; n.s. non-statistically significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 1

HCC15 cells showed resistance to dipyridone treatment, as detected in PI incorporation plots, and cell death quantitation (Fig. 1A). In normal-NRF2 H226 cells, dipyridone decreased cell numbers (representative images) (Fig. 1B); staining for these cells was positive for PI, and the cells exhibited a dose- and time-proportional increase of cell death. In SK-MES-1 and H520 cells (with NFE2L2/KEAP1 copy-number increase), dipyridone treatment induced cell death to a lesser extent than in H226 cells but more than in HCC15 cells (Fig. 1C, Fig. S1F), showing that NFE2L2/KEAP1 copy-number altered cells exhibit an intermediate phenotype. Lung normal epithelial cells (HSAEC1/KT) showed the lowest proportion of cell death (not significant at 0.25–0.50 mg/ml of dipyridone) (Fig. 1). Therefore, all LUSC cell lines with WT NRF2/KEAP1 genes were more sensitive to dipyridone than physiological epithelial lung cells or active-NRF2 cells.

3.2 Induction of iROS induces transient NRF2 activation

iROS trigger activation of NRF2, the master transcription factor induced to cope with ROS cellular stress [9]. H226 and HCC15 cells were treated with dipyridone and the mRNA levels of NFE2L2 or its targets EGFL7, NQO1, and AKR1C3, were analyzed by RT-qPCR. The mRNA expression of NFE2L2, NQO1, and AKR1C3 was higher in dipyridone-treated normal-NRF2 cells than in untreated cells or in HCC15 cells (Fig. 2A, Fig. S2A). In the active-NRF2 HCC15 cells, NRF2 effectors were constitutively high, as supported by the low RT-qPCR cycle number in which NQO1 or AKR1C3 were found, but were unaffected by dipyridone (Fig. 2A, Fig. S2A); EGFL7 was at lower levels in HCC15 cells than H226 cells; and iROS tended to increase EGFL7 in H226 cells. Protein levels analysis upon dipyridone treatment confirmed an iROS-dependent NRF2 and NQO1 protein increase in H226 cells (Fig. 2B), not seen in HCC15. Both HCC15 and H226 decreased KEAP1 levels following iROS (Fig. S2B).Fig. 2 Different iROS inducers trigger LUSC cell death.(A) mRNA expression of NFE2L2 and its target gene NQO1 determined in H226 and HCC15 cells treated with dipyridone at different concentrations and times (indicated). The initial cycle (indicated as C) at which the genes are detected in untreated cells is indicated. Graph bars represents the Mean±SD of the RQ levels referred to gene expression in control cells, n = 3. (B) NRF2 and NQO1 protein expression levels in cells treated as in (A) were tested in WB. Graph bars (at the bottom) represent NRF2 and NQO1 signals (mean ± SD, n = 2) corrected by the loading control (α tubulin) and referred to the levels at 24h of dipyridone treatment (100 %). (C, D) Representative images of H226 and HCC15 cells ( × 10) treated with vehicle, oxPAPC, or dipyridone (positive control) for 24h (C); or of H226 and HCC15 cells treated with anisomycin for 24h (D). Graphs on the right show the percentage of PI+ cells in untreated vs treated cells (indicated) as determined by flow cytometry. Graph bars represent the Mean ± SD, n = 3(C, D). Statistical significance was determined by one-way ANOVA with Dunett test as a post hoc analysis; (*) P < 0.05, (**) P < 0.01, (***) P < 0.001 or using Unpaired t-test: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2

Thus, the constitutive activation of NRF2 in HCC15 cells drives sustained NRF2 effectors gene expression, explaining HCC15 resistance to iROS (Fig. 1A). In normal-NRF2 H226 cells, iROS induced transient NRF2 effector's gene expression (Fig. 2A and B). Compared to control airway epithelial cells, H226, SK-MES1, and H520 cells were sensitive to ROS-induced cell death (Fig. 1). Therefore, the WT status of NFE2L2/KEAP1 predicts the susceptibility of a LUSC sample to iROS; NFE2L2/KEAP1 normal copy number cells shows a higher iROS sensitivity than LUSC cell lines with an increased NFE2L2/KEAP1 copy number ratio.

3.3 LUSC cell lines are sensitive to different iROS inducers

Cell death induction by iROS was confirmed using different iROS inducers. Whereas iROS oxidizes phospholipids, it can also be employed to trigger iROS [23]. Oxidized forms of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho-choline (oxPAPC) were prepared and assessed to determine their capacity to trigger cell death. H226 and HCC15 cells were treated with dipyridone (0.5 mg/ml, as positive control) or oxPAPC (30 μg or 100 μg/ml; 48 h). Cell death rate was analyzed by flow cytometry. OxPAPC triggered cell death in H226 cells but not in HCC15 cells (Fig. 2C). Similarly, anisomycin, an alternative iROS inducer [24], triggered stronger cell death (25 or 50 μM, 48 h) in H226 than in HCC15 cells (Fig. 2D).

Treatment with oxPAPC selectively induced a dose-proportional expression of NFE2L2 and its effector AKR1C3 mRNAs as well as an increase in AKR1C3 protein levels in H226 cells and not in HCC15 cells (with constitutively high AKR1C3 levels) (Figs. S2C and D). Anisomycin also increased NQO1 mRNA and protein expression in H226 cells but not in HCC15 cells, where it was expressed at high levels (Figs. S2E and F). In the presence of anisomycin, both H226 and HCC15 cells showed a reduction in NRF2 and KEAP1 protein levels (Fig. S2F), which might be related to the reduction of translation efficiency caused by anisomycin [25]. Wild type NFE2L2/KEAP1 LUSC cells are therefore sensitive to different iROS inducers.

3.4 Cell death induced by iROS is reduced by antioxidants and requires NRF2 expression

Pretreatment of H226 with the antioxidants Vitamin E and NAC prior to addition of the iROS inducers dipyridone, anisomycin or oxPAPC reduced AKR1C3 mRNA levels and reduced iROS-induced cell death selectively in H226 cells (Fig. 3A). No significant effects were seen in HCC15 cells, except for a small proportion of cell death with anisomycin, which was not reverted by antioxidants (Fig. 3A).Fig. 3 Cell death triggered by exogenous ROS inducers is reduced with antioxidants and by NRF2 depletion. (A) H226 or HCC15 cells were seeded and after 12h, NAC (2 mM) was added to some of the samples (12h). At 24h after seeding, other cells were pretreated with Vitamin E (250 μM) for 3h. Then the samples were treated with dipyridone (0,75 mg/ml), anisomycin (30 μM) or oxPAPC (100 mg/ml) (48h). The mRNA levels for the NRF2 effector AKR1C3 and the percentage of cell death were examined. Graphs show the Mean ± SD (n = 3). (B–D) H226 or HCC15 cells were seeded (24h), transfected with control siRNA or with siRNA specific for KEAP1 or NFE2L2 (72h) and then incubated with medium or dipyridone (0,75 mg/ml) (48h). NRF2 and KEAP1 protein expression was examined in WB (B). The percentage of cell death was examined by flow cytometry (C). KEAP1, NFE2L2, AKR1C3 and NQO1 mRNA levels were examined by RT-qPCR. (E) H226 or HCC15 cells were seeded (24h) pretreated with necrostatin (NEC, 20 μM) or ZVAD (50 μM) (2h) and then treated with dipyridone, anisomycin or oxPAPC (as in A) (48h). Apoptosis (using annexin V-FITC) was examined as described in methods. (F) H226 or HCC15 cells were pretreated with ferrostatin (Ferr.) (10 μM, 2h), then treated with RSL3 (4.5 μM) or Dipyridone (0.75 mg/ml) for 48h. The graph shows the percent of ferroptotic cells as examined using Bodipy C11 (mean ± SD). (C, D-F) Graphs as in (A). *P < 0.05; **P < 0.01; ***P < 0.001, Unpaired Student t-test (A, C, F). (***) P < 0.001 (A) one-way ANOVA test (D); [***] P < 0.001, Chi-Square (and Fisher's exact test) percent alive vs death cells (E).

Fig. 3

To support NRF2′s involvement in iROS-induced cell death, the consequences of depleting NRF2 or KEAP1 were examined. Western blotting confirmed KEAP1 and NRF2 efficient depletion (Fig. 3B, Fig. S3A). Dipyridone increased cell death in NRF2-control H226 cells to greater extent than in active-NRF2 HCC15 cells (Fig. 3C).

KEAP1 depletion increased H226 basal cell death but reduced dipyridone-induced cell death (Fig. 3C). iROS modifies the Cysteines in KEAP1 to activate NRF2 [10,11,13], if KEAP1 is depleted, the lower activation of NRF2 by iROS explains the lower cell induction of death. In NFR2-active HCC15 cells, KEAP1 depletion reduced basal cell death (Fig. 3C and D), this is likely caused by the lower NRF2 activation when KEAP1 (bearing an activating mutation) is at low levels. Dipyridone treatment of HCC15 cells, however, triggered a similar (low) cell death than in control (Fig. 3C); the NRF2 remaining in KEAP1 depleted cells would be more stable and nuclear, permitting a partial induction of NRF2 effectors expression and cell death. As for NRF2 depletion, in H226 there was a higher basal cell death (due to lack of protection against metabolic ROS), but iROS failed to trigger cell death (Fig. 3C and D). Also, in HCC15 cells, NRF2 depletion reduced cell death (Fig. 3C and D). These results show that NRF2 is needed for iROS-induced cell death.

To better characterize the cell death induced by iROS, it was considered that ROS induced lipid peroxidation affects apoptosis, autophagy and ferroptosis [26]. To evaluate apoptosis, staurosporine was used as positive control, which efficiently induced apoptosis (sensitive to ZVAD), in both H226 and HCC15 cells (Figs. S3B–C). In contrast, dypiridone induced a very small proportion of apoptosis in H226 cells and a marked increase in necrosis (Fig. 3E), which was low in HCC15 cells. This necrosis is unrelated to apoptosis as was not reduced by ZVAD and is probably not necroptosis [27], as it was only slightly reduced by necrostatin (Fig. 3E). In control-NRF2 cells, oxPAPC and more clearly anisomycin induced a low proportion of apoptosis (sensitive to ZVAD) and, to greater extent, necrosis (Fig. S3C). This necrosis was reduced by ZVAD, suggesting that part of it represent late apoptosis (Fig. S3C). The contribution of autophagy was examined by LC3BII detection in immunoblot [26]; both dipyridone and oxPAPC induced LC3BII formation in H226 cells, dipyridone also increased LC3BII in HCC15 cells (Fig. S3D). Immunoblot confirmed the presence of active-Caspase 3 (formed during apoptosis) in staurosporin- and anisomycin-treated H226 cells; staurosporin also induced active-Caspase 3 in HCC15 apoptotic cells (Fig. S3D).

Ferroptosis was examined using RLS3 (an inhibitor of GPX4 [26]) as positive control. RLS3-triggered ferroptosis (sensitive to the ferroptosis inhibitor ferrostatin-1) in H226 and HCC15 cells (Fig. S3E). Dipyridone, oxPAPC and anisomycin also induced a marked increase in ferroptosis (sensitive to ferrostatin) selectively in H226 cells (Fig. 3F, Fig. S3F). Therefore, iROS triggers ferroptosis, which induces plasma membrane permeability and explains the high degree of necrosis (cells that take PI) found in i-ROS-treated normal-NRF2 cells. iROS inducers also triggered a low-level of autophagy and apoptosis in normal-NRF2 cells.

3.5 Increased microRNA-126 levels trigger ROS-induced LUSC cell death

LUSC cell lines with high p85βPI3K levels are sensitive to p85βPI3K-depletion [28]. p85βPI3K protein levels are restricted by miR-126 [19], an NRF2 effector encoded in EGFL7 intron 7 [29]. The parallel sensitivity of the LUSC lines to iROS (here) and p85βPI3K [28], led us to propose that p85βPI3K might be involved in iROS-induced WT NFE2L2/KEAP1 LUSC cell's death. Whether iROS increased miR-126 levels, and these in turn reduce p85βPI3K levels was examined. Dipyridone, oxPAPC, and anisomycin increased miR-126(3p) expression in normal-NRF2 H226 cells while low variations were found in active-NRF2 cells (Fig. 4A). The first cycle at which miR-126 is found is indicated and shows its lower content in HCC15 cells. As for p85βPI3K levels, the different iROS-inducers triggered a reduction in p85βPI3K levels in H226 cells (Fig. 4B), which was not detected in HCC15 cells (Fig. 4C).Fig. 4 Transient iROS reduces p85βPI3Klevels through an increase of miR-126. (A)miR-126 expression levels in H226 or HCC15 cells treated with dipyridone (24, 48 and 72h) [left panel], with oxPAPC (48h) [medium], or with anisomycin (24h) [right panel]. The initial cycle at which this miR is detected is indicated on the left-most column. Graphs represent the Mean±SD of RQ levels referred to miR-126 expression in control H226 cells, n = 3. (B) Cell lysates (50 μg) from H226 cells treated as in (A) with iROS inducers doses and times indicated were examined in blots specific for p85βPI3K. Graphs represent the mean p85βPI3K signal ± SD, n ≥ 3, compared to that in untreated cells (100 %). (C) HCC15 cells treated with dipyridone (0.5 mg/ml) or oxPAPC (indicated) for 24 or 48h were lysed and extracts tested in WB. Graphs as in (B). (D) H226 and HCC15 cells transfected with anti-miR control or anti-miR-126 (α-miR) (48h) were treated with dipyridone (0.5 mg/ml) for 24h. Lysates (50 μg) were examined in WB. The graph shows p85βPI3K signal in the different conditions compared to p85βPI3K signal in controls (100 %). (E) H226 and HCC15 cells were treated with dipyridone as in (D), representative images of the different conditions ( × 10). The graph represents the percentage of dead cells (PI+ cells) analyzed by flow cytometry. α-miR-126 decreases cell death induced by iROS. (F) Representative images of control H226 or HCC15 cells or of cells infected with an empty vector or a vector encoding the PIK3R2 gene (72h) and treated with vehicle (medium) or dipyridone (0.5 mg/ml, 24h). The graphs represent the difference between percentage of PI+ cells in the three groups (control, empty vector-infected, and PIK3R2 overexpression) prior to and upon dipyridone treatment. p85βPI3K overexpression reduced H226 cell death ∼30 %. (***) P < 0.001, one-way ANOVA, Dunett test as a post hoc analysis. *P < 0.05; **P < 0.01; ***P < 0.001, Unpaired Student t-test.

Fig. 4

To determine whether miR-126 contributes to ROS-induced cell death an anti-miR-126 (α-miR-126) molecule was transfected into H226 and HCC15 cells (48 h) and the cells were treated with dipyridone (0.5 mg/ml, 24h). Transfection of α-miR-126 restored p85βPI3K expression levels in normal-NRF2 cells but did not change p85βPI3K levels in HCC15 (Fig. 4D). Accordingly, α-miR-126 reduced the death of H226 cells, but not of HCC15 cells (Fig. 4E). α-miR-126-reduction of miR-126 levels was confirmed by RT-qPCR; α-miR-126 did not affect NRF2 effector's mRNA levels, as miR-126 upregulation is posterior to NRF2 activation (Fig. S4A). In H226 cells, EGFL7 levels increased with dipyridone and α-miR-126 (Fig. S4A), as expected from the negative feedback regulation between miR-126 and EGFL7 [29]. Thus, iROS activates NRF2 and upregulates miR-126, which reduces p85βPI3K levels and decreases cell survival in LUSC cells with normal NFE2L2/KEAP1 genes.

3.6 Overexpression of p85βPI3K partially rescued iROS-induced LUSC cell death

As p85βPI3K levels play a role in iROS-inducted cell death, overexpression of p85βPI3K could rescue cell survival. Indeed, p85βPI3K expression reduced iROS-induced cell death by ∼ one-third in H226 cells, without affecting HCC15 cells (Fig. 4F). p85βPI3K overexpression did not alter mir-126 miR levels, or mRNA levels for NQO1, AKR1C3, EGFL7 or NFE2L2 in dipyridone-treated H226 or HCC15 cells (Fig. S4B).

WB analysis of protein levels certified p85βPI3K overexpression (Fig. S4C). Considering that p85βPI3K is a PI3K subunit, it was tested whether it activated AKT (estimated as phospho(p)Thr308 AKT levels). p85βPI3K overexpression in H226 cells (but not in HCC15 cells) increased basal and dipyridone-induced pAKT levels (Fig. S4C). p85βPI3K expression partially rescues dipyridone-induced H226 cell death (Fig. 4F); this action is likely related to PI3K/AKT activation. Together, in human LUSC lines with WT NFE2L2/KEAP1, miR-126 contributes to iROS-induced cell death by reducing p85βPI3K and pAKT levels; accordingly, p85βPI3K overexpression partially rescues cell survival.

3.7 SETD5 and p85βPI3K cooperate to regulate cell death in human LUSC cells

miR-126 depletion by α-miR-126 reduced iROS-induced H226 cell death by ∼50 % (Fig. 4E), while p85βPI3K overexpression rescued cell death by only ∼30 % (Fig. 4F). This suggests that additional mediators contribute to iROS-induced cell death. Other genes regulated by miR-126 such as IRS-1 affect cell survival [30], but its involvement was not supported by the preliminary assays. miR-126-5p is synthetized from the same transcript than miR-126 (3p) and they coregulate [31]. miR126-5p represses SETD5 levels in other cell types (leukocytes) [32]; it was tested if miR-126-5p could regulate SETD5 levels in LUSC.

SETD5 mRNA and protein levels were analyzed in control and dipyridone-treated cells. SETD5 mRNA levels were reduced by dipyridone treatment in H226, but not in HCC15 cells (Fig. 5A). WB analysis confirmed that SETD5 protein levels were reduced by dipyridone treatment selectively in H226 cells (Fig. 5B); HCC15 cells express lower SETD5 levels that were unaffected by dipyridone. To link SETD5 protein levels to miR-126-5p, its levels were examined upon treatment with dipyridone or oxPAPC in H226 cells. miR-126-5p increased in i-ROS induced H226 cells but not in HCC15 cells (Fig. 5C). The selective increase in miR-126-5p levels in iROS-treated H226 cells concurs with the reduction in SETD5 levels. SETD5 might then be a critical effector for iROS-induced death in normal-NRF2 LUSC cells.Fig. 5 SETD5 regulates iROS-induced cell death. (A) SETD5 mRNA levels in control and dipyridone-treated H226 and HCC15 cells for 24 or 48h. Graph bars represent mean ± SD (n = 3) of the RQ value (vs GAPDH) normalized to the control sample of H226 cells, considered 1. (B) H226 or HCC15 cells were treated with medium or dipyridone for 6, 24, or 48 h. Cell extracts (50 μg) were used to examine SETD5 levels by WB. Graphs show mean ± SD (n = 3) of SETD5 signal referred to SETD5 signal in H226 untreated cells at 24h (100 %). MW, molecular weight. (C) miR-126-5p levels in control and dipyridone- or oxPAPC-treated H226 or HCC15 cells (indicated). Graphs as in (A), miR levels (RQ) were referred to untreated control H226 cells (considered 1). (D) H226 cells were infected with lentivirus carrying an empty vector, the PIK3R2 gene, SETD5, or both. Cell lysates (50 μg) were examined in WB using the indicated antibodies. The graphs [at the right] show the SETD5 and p85βPI3K signals in the different conditions, normalized to their loading controls, and referred to the maximal SETD5 or p85βPI3K signal in cells overexpressing both (100 %). (E) Representative images of H226 cells ( × 10) infected as in (D) (72 h) and then treated with medium or dipyridone (0.5 mg/ml; 24 h) (indicated). The graph shows the percentage (%) of PI+ cells in empty, PIK3R2-, SETD5-, or PIK3R2-and SETD5-overexpressing H226 cells after dipyridone treatment (0.5 mg/ml; 24 h) (right) subtracting the percentage of PI+ cells in untreated conditions; total cell number 100 %. (F) H226 cells were transfected with siRNA control (Ctr), for PIK3R2, SETD5, or both (72h). Cell extracts (50 μg) were tested in WB (indicated). Graphs as in D, the signal was referred to that of non-transfected cells (100 %). A fraction of these cells was collected for RT-qPCR. Graphs (as in A). (G) Representative images of H226 cells transfected with siRNA as in (F). Graphs show the percentage of cell death, or cell count of PI− cells in control and siRNA transfected cells. All graphs represent the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. Student's t-test.

Fig. 5

To test whether the reduction in SETD5 and/or p85βPI3Klevels cooperate to induce cell death, the consequences of overexpressing SETD5 (alone or in combination with PIK3R2) in cell survival rescue upon iROS treatment were examined. SETD5-plus PIK3R2 (p85βPI3K)-expressing viruses increased SETD5 and p85βPI3K protein levels in the iROS sensitive H226 LUSC cells (Fig. 5D). Cells were infected with SETD5-or PIK3R2 (p85βPI3K) viruses (72 h) and were treated with dipyridone (24h). As with p85βPI3K, SETD5 overexpression alone partially rescued cell death (Fig. 5E). Nonetheless, combined overexpression of SETD5 and p85βPI3K rescued ∼50 % of the i-ROS-induced cell death (Fig. 5E). Therefore, SETD5 and p85βPI3Kcooperate in mediating cell survival in iROS-treated normal-NRF2 LUSC cells.

To confirm the synergy of reduced SETD5 and p85βPI3K levels to mediate iROS- cell death, the consequences of simultaneous depletion of SETD5 and p85βPI3K levels using siRNAs (72h) was performed (Fig. 5F). Depletion of p85βPI3K or of SETD5 alone, and particularly depletion of both, increased cell death and markedly reduced cell number (Fig. 5G). Cell number is a better indicator of cell loss, as fragmented dying cells are not considered in the PI analysis. Altogether, the reduction of SETD5 and PIK3R2 levels triggered by iROS in normal-NFE2L2/KEAP1 LUSC cell lines is at least one of the mechanisms for iROS-induced cell death, as SETD5 and PIK3R2 double depletion triggers cell death by itself (without ROS). These results support that iROS, by transiently activating NRF2, increases miR-126 levels (3P and 5P), and in turn reduces SETD5 and p85βPI3K levels, which trigger cell death.

3.8 SETD5-controlled G6PD expression induces cell death

Whereas p85βPI3K levels modulated pAKT, the action of SETD5 in cell death is unknown. SETD5 belongs to the SET family of histone methyltransferases that lacks methyl-transferase activity but associates histone deacetylase 3 and G9a methyl transferase thereby regulating H3K9me3 levels [16]. One of the genes repressed by H3K9me3 is G6PD (glucose-6-phosphate dehydrogenase), the first enzyme of the pentose phosphate pathway (PPP) [33]. The mechanism of H3K9me3 modulation in G6PD promoter is unknown. Since SETD5 complex induces H3K9 methylation, we tested whether SETD5 could restrict G6PD expression. First, it was confirmed that iROS triggers PPP gene expression in normal-NRF2 cells. G6PD basal levels in HCC15 cells were higher than in H226 (Fig. 6A). Nonetheless, both G6PD and TKT mRNA levels were selectively increased in dipyridone-treated H226 cells (Fig. 6A); TALDO was not induced by i-ROS. Immunoblotting confirmed selective iROS-increased G6PD protein levels in H226 cells (Fig. 6B).Fig. 6 SETD5 silencing induces G6PD de-repression and triggers cell death in normal-NRF2 LUSC cells. (A) Comparison of G6PD, TKT, and TALDO levels (RQ values vs GAPDH and TBP) in control and dipyridone-treated (0.5 mg/ml; 24 or 48 h) H226 or HCC15 cells. Graphs show the mRNA levels of the genes (as RQ values) for the different conditions. The cycle at which G6PD, TKT, and TALDO appear in untreated cells is indicated and shows higher levels that do not change with dipyridone in active-NFR2 cells. (B) H226 or HCC15 cells were activated with dipyridone (0.5 mg/ml; 24 and 48h). Lysates (50 μg) were examined in WB (indicated). Graph represent G6PD WB signal at 48h, corrected with the loading control and referred to maximal (100 %). (C) 4×105 H226 or HCC15 cells were seeded in p60 plates 24h prior to transfection with control or SETD5 specific siRNA (72 h). Cells were collected for RT-qPCR. Graphs as in (A). (D) 105 H226 or HCC15 cells were seeded in 12-well plates 24 h prior to addition of vehicle, dipyridone (0.75 mg/ml), or AG1 (indicated). Representative flow cytometry images of the cells after 75 μM AG1 treatment (24 h); on the right, percentage of death. *P < 0.05; **P < 0.01; ***P < 0.001 Student's t-test; (*) P < 0.05, (**) P < 0.01, (***) P < 0.001; two-way ANOVA with Bonferroni post-test.

Fig. 6

SETD5 depletion triggered cell death in normal-NRF2 cells (Fig. 5G) and increased G6PD and TKT levels (Fig. 6C), confirming that SETD5 represses G6PD and TKT genes in normal-NRF2 cells. This suggesting that G6PD upregulation might trigger cell death in normal-NRF2 cells. To test whether normal-NRF2 LUSC cells were sensitive to G6PD upregulation, the cells were treated with a selective activator of G6PD, AG1 [33]. Whereas AG1 treatment (24 h) triggered H226 cell death, only a low percentage of cell death was detected in active-NRF2 HCC15 cells at the maximum AG1 dose (Fig. 6D). Thus, the reduction of SETD5 by iROS in normal-NRF2 cells contributes to cell death, at least partially, by mediating an excessive activation of G6PD.

3.9 iROS induces cell death in 3D spheroids and in LUSC PDX-derived organoids

To evaluate the potential interest of acute iROS inducers for LUSC therapy, the consequences of treating normal-NRF2 or active-NRF2 cells was tested in 3D spheroid cultures. Cell lines were cultured in ultra-low adhesion plates (96 h) and dipyridone was added to the culture (0.75 and 1 mg/ml; 24 h). DRAQ7 probe was used to determine cell death after the treatment. While H226 spheroids were sensitive to dipyridone (Fig. 7A); no significant cell death increase was observed in HCC15 cells. Thus, transient iROS-triggers cell death of normal-NRF2 LUSC spheroids.Fig. 7 Transient iROS slows WT NFE2L2/KEAP1 LUSC organoid growth. (A) 104 H226 or HCC15 cells were seeded per well in 96-well ultralow attachment plates. After 96 h, medium was renovated or replaced with media plus dipyridone (0.75 or 1 mg/ml). 24h later, DRAQ7 probe was added (30 min). Representative images ( × 10) of confocal microscopy. Graphs (mean ± SD) represent the cell-death fold change (n ≥ 4) compared to untreated spheroids at different times. (B) mRNA levels of PIK3R2 and SETD5 in a battery of LUSC PDX (patient derived xenografts). Graphs show the RQ values (versus GAPDH) relative to gene expression levels in HCC15 control. Squared samples represent high RQs; dashed squared those with low RQs. (C) Extracts obtained from PDX (50 μg) were examined in WB with indicated antibodies (n > 20 tumors examined). The graphs show the percent of signal referred to maximal p85β signal (TP3 signal, 100 %) or, in the case of SETD5, to SETD5 levels in tumor T34 (100 %). These blots were performed twice with similar results. (D) mRNA levels of NFE2L2, AKR1C3, and NOQ1 in a collection of LUSC PDX. Graphs as in (B). PDX expressing NRF2 effectors constitutively (squared high RQs; e.g.TP13) or normal-NRF2 organoids with low constitutive expression of NRF2 targets (dashed squared, low RQs; e.g.TP34) but high levels of SETD5 and PIK3R2 mRNA (TP34) were chosen. (E) Cells from the two selected LUSC PDX-derived organoids were seeded (40,000 cells/well) in confocal plates, after 24h, culture media were replaced with media or media containing dipyridone (0.75; 1 mg/ml) (24h). DRAQ7 probe was added (30 min). Representative images from confocal microscopy. Graphs represent the number of DRAQ7+ dead cells per field (mean ± SD) in each organoid (n ≥ 4). *P < 0.05; **P < 0.01; ***P < 0.001 Unpaired t-test.

Fig. 7

As an alternative approach, we evaluated the influence of iROS inducers on the growth of organoid cultures stablished from LUSC PDX (patient derived xenografts) [34]. Considering that each patient tumor is different than any other from the same cancer type [35], a battery of LUSC PDX samples was tested for PIK3R2 and SETD5 mRNA levels. TP1 and TP34 showed high levels of SETD5 and PIK3R2-p85βPI3K mRNA and protein (Fig. 7B and C). The levels of NFE2L2, and it's effectors AKR1C3 and NQO1 were also examined (Fig. 7D). Whereas TP34 (or TP114) showed basal levels of NRF2 gene targets, TP13 (and TP1) mimicked NRF2-active cells as they exhibited constitutive expression of NRF2 targets (Fig. 7D). TP34 PDX-derived organoids were chosen as normal-NRF2 cells (that also expressed PIK3R2 and SETD5) and TP13 derived organoids were selected as active-NRF2 (Fig. 7D) (low-to-medium levels of SETD5 and PIK3R2-p85βPI3K) (Fig. 7B and C).

The organoids derived from TP34 and TP13 PDX were seeded and maintained in normal conditions until they reached medium-size growth. Then, dipyridone was added (24 h) and cell death was examined by DRAQ7 staining. Whereas in TP34 (normal-NRF2) dipyridone treatment increased cell death, TP13 organoids (active-NRF2) did not respond to dipyridone (Fig. 7E). Together, transient iROS triggers death of normal-NRF2 LUSC cells in spheroids and organoids, opening new possibilities for the therapeutic repertoire for LUSC.

3.10 iROS halts LUSC tumor growth in vivo

H226 cells were grown as xenografts in immunodeficient mice, and the effect of treating the mice by i.p. injection of dipyridone (100 mg/kg) or PBS (three times weekly for three weeks) was evaluated as described [14,19,28,36,37]. Dipyridone treatment halted LUSC tumor expansion, resulting in smaller tumors up to final point, at day 21 (Fig. 8A and B). The RT-qPCR analysis showed that the mRNA levels of miR-126 and SETD5 were the most altered genes in dipyridone-treated tumors (Fig. 8C). As for p85βPI3K, whereas the PIK3R2 mRNA levels were unchanged (Fig. 8C), p85βPI3K protein levels were lower in dipyridone-treated tumors, as expected from miR-126 levels increase (Fig. 8D). SETD5 protein levels were also reduced in dipyridone-treated tumors (Fig. 8D). No change in NFE2L2 or its targets AKR1C3 and NQO1 was detected by RT-qPCR, confirming the transient nature of iROS/NRF2 activation (Fig. 2A). Nonetheless, AKR1C3 protein levels were still increased in ROS-treated xenografts (Fig. 8C and D) Thus, ROS treatment halts tumor growth in LUSC xenografts from normal-NRF2 expression cells.Fig. 8 Transient iROS slows WT NFE2L2/KEAP1 LUSC tumor growth in vivo. (A) H226 cells were expanded in culture and grafted subcutaneously into nude mice (107 cells in 100 μl of PBS plus 100 μl matrigel). The graph illustrates the mean tumor volume in mm3 ± SD, n = 12) measured at different times after initiation of dipyridone (100 mg/kg) treatment. (B) Representative images of control and treated mice harboring LUSC tumors and of a sample of these tumors. (C) Different gene mRNA expression levels analyzed in control and dipyridone-treated LUSC tumors. Significant decreases were found in miR-126 and SETD5 levels. (D) Cell lysates (50 μg) from untreated (control, Ctr) and treated tumors (from A) were examined in WB. The graph shows the signal intensity in the control and treated tumors (normalized to loading control) and referred to the mean p85βPI3K content in untreated tumors (100 %). MW, Molecular weight. (***) P < 0.001 (A) two-way ANOVA test followed by Tukey posttest; (C, D) *P < 0.05; **P < 0.01; ***P < 0.001 Unpaired t-test.

Fig. 8

4 Discussion

Targeted therapies are improving the treatment landscape for NSCLC. However, the effects of these advances mainly ameliorated LUAD patients [2,3]. Given that LUSC tumors frequently present activating mutations in the NFE2L2/KEAP1 pathway, it was hypothesized that this may indicate high sensitivity of LUSC tumors to ROS induction, that is, whereas tumors with active-NRF2 would be protected against ROS, LUSC tumors with an intact NRF2 pathway could be sensitive. We examined three iROS inducers and different LUSC cell lines with WT status of NFE2L2/KEAP1. ROS triggered cell death in normal-NRF2 LUSC cell lines even if they show increased NFE2L2/KEAP1 copy number ratio, in 3D LUSC spheroids, as well as in organoids stablished from LUSC patients. Ferroptosis is the main mechanism of ROS-induced cell death. The required molecular events ending in cell death included: transient NRF2 activation, increased expression of NRF2's targets miR126-3p and miR126-5p, miR126-3p -mediated p85βPI3K levels reduction and miR126-5p-induced SETD5 downregulation, respectively. miR126-p85βPI3K axis affects the activation of PI3K survival pathway [14,19], and miR126-5p driven SETD5 downregulation releases the expression of G6PD, which is toxic for normal-NRF2 LUSC cells (Fig. 6D). In agreement with a critical role of SETD5 and p85βPI3K in normal-NRF2 iROS-induced cell death, combined downregulation of SETD5 and p85βPI3K triggered cell death, while combined overexpression inhibited cell death. In conclusion, these data describe a novel function of iROS: the upregulation of miR-126-3p and -5p following NRF2 activation, as well as the subsequent downregulation of p85βPI3K and that of SETD5. Here is reported the involvement of SETD5 epigenetic modifier in repression of the PPP pathway genes. The organization as a cascade of NRF2 > miR-126-3p, miR-126-5p > p85βPI3K and SETD5 to trigger iROS-induced cell death in normal-NRF2 LUSC is novel, and suggests that wild type NFE2L2/KEAP1 LUSC might be treated with an acute local iROS induction as a new therapeutic strategy. It will be of interest to optimize and examine the efficacy of this treatment in mouse genetic LUSC models [39].

Prior attempts at developing targeted treatments for LUSC include interventions directed at FGFR1, frequently amplified in LUSC, however, early-phase trials discard clinical benefit [38]. Another candidate for targeted therapy in LUSC is PI3K [4,5]. Activation of this pathway induces cell survival, division, and cancer-associated metabolic switch [40,41]. Currently available inhibitors for this enzyme show limited efficacy as single treatment, but pose hope in combination therapies [42]. Several immunotherapy drugs have been applied in LUSC patients, but they are effective in a low proportion of cases [43,44]. The results presented provide a rationale for the use of iROS inducers in clinical trials with LUSC patients with a normal-NRF2 status.

Regulation of redox homeostasis is fundamental for the maintenance of physiological cell functions [45]. Tumors rely on moderate increases of cellular ROS to modulate proliferation, angiogenesis, metastasis, etc. [46,47]. Nonetheless, this oxidative characteristic of tumor cells may render them more vulnerable to further ROS insults. In the case of LUSC, generation of oxidative stress by iROS did not affect all LUSC types in the same way. Whereas H226, H520, and SK-MES-1 (with WT NFE2L2/KEAP1 alleles) and organoids derived from LUCS patients with normal-NRF2, were sensitive to an acute oxidant insult, HCC15 and organoids with an active-NRF2 forms were protected. iROS also reduced KEAP1 protein levels in both WT- and active-NRF2 cell lines. This is caused by a CUL3-dependent ubiquitination of KEAP1 in cells exposed to ROS [48]. The expression of NRF2 was essential for iROS-induced cell death (Fig. 3 C). Thus, only cells carrying WT alleles for NFE2L2/KEAP1 are sensitive to iROS-induced ferroptosis. Although the common action of dipyridone, oxPAPC, and anisomycin is to induce iROS (and the cell death induced by these substances is reduced by antioxidants), it cannot be excluded that additional pathways activated by these compounds contribute to cell death.

p85βPI3K and SETD5 are identified here as the two main effectors regulating the iROS-induced cell death. miR-126 was investigated because it is a target of NRF2 that restricts p85βPI3K levels; we previously shown that p85βPI3K depletion triggered LUSC xenografts regression [14,28], pointing at p85βPI3K as an oncogenic driver in LUSC. Remarkably, the same LUSC cell lines (with WT alleles of NFE2L2/KEAP1) that were sensitive to p85βPI3K depletion, were also susceptible to iROS (Fig. 1, S1; [28]), indicating that p85βPI3K downregulation might be involved in iROS-induced cell death. In support of this idea, miR-126 levels, which restrains p85βPI3K, increased after ROS treatment in normal-NRF2 cells. Moreover, genetic modulation of p85βPI3K expression levels confirmed its relevance in ROS-cell death. p85βPI3K actions that may affect LUSC survival include regulation of PI3K/AKT activation (Fig. S4C) and facilitation of cell-cell adhesion/invasion [19,49]. p85βPI3K overexpression in normal-NRF2 LUSC cells, however, only rescued cell survival by 30 %. To search for additional effectors involved in survival, other miR-126 regulated genes were examined, i.e. IRS-1 [30], which was excluded in preliminary tests. Since miR-126(3p) and miR-126-5p appear in the same sequence and are both effectors of NRF2 [31], we examined and detected an increase in miR-126-5p levels in iROS treated normal-NRF2 LUSC cells. Of the different miR-126-5p effectors, the focus was placed on SETD5, as it's complex increases H3K9me3 levels and modulates gene expression [16].

SET (Su(var)3–9, Enhancer-of-zeste, Trithorax) family members are predominantly lysine methyl-transferases for histone and non-histone proteins [50]; SETD enzymes are critical for cell viability in the presence of cellular stress [51]. Nonetheless, SETD5 does not have intrinsic methyl transferase activity and acts as a repressor in KRAS- pancreatic cancer by association with the NCoR1 complex, HDAC3 (histone deacetylase 3), and G9a methyl transferase that increases H3K9me3 levels [16]. Although SETD5 mutations have been recently associated with cancer progression [16], its role in LUSC is unknown. A prior comparison of WT and Nfl2e2 knockout mice showed that SETD5-regulated genes include genes involved in redox response, response to growth factors, and genes regulating NADPH [52]. It is known that NADPH levels are controlled by the PPP pathway that contributes to metabolic adaptation of cancer cells [53] but PPP excessive activation competes with glycolysis for glucose-6-p and can trigger cell death [33]. G6PD (the first PPP gene), exhibits a H3K9me3 restricting signature in its promoter [33]. This led us to propose SETD5 as a candidate for G6PD levels restriction. Indeed, reduction of SETD5 levels by iROS markedly increased G6PD levels in normal-NRF2 LUSC cells (Fig. 6A and B). Additionally, direct activation of G6PD triggered cell death in normal-NRF2 cells (Fig. 6C). Thus, SETD5 downregulation by releasing G6PD expression contributes to iROS-induced normal-NRF2 LUSC cells. This points at SETD5 inhibition as an approach to induce cell death in normal-NRF2 LUSC cells.

Some authors propose that ROS stress triggers different forms of death that cooperate to kill cells [26]. The mechanism of cell death induced by dypiridone, oxPAPC or anisomycin selectively in normal-NRF2 cells was ferroptosis (sensitive to ferrostatin). Therefore, iROS triggers ferroptosis, which induces plasma membrane permeability and explains the necrosis found in i-ROS-treated normal-NRF2 cells in the apoptosis/necrosis assays. iROS inducers also triggered a low-level of autophagy and apoptosis in normal-NRF2 cells, supporting the capacity of iROS kill cells in distinct manners.

Transient activation of NRF2 using iROS inducers is sufficient to promote the death of LUSC cells with WT forms of NFE2L2/KEAP1 genes; the treatment with iROS inducers might be a feasible approach for treating LUSC patients. Candidate patients could be selected by RT-qPCR examination of NRF2 targets expression in a surgical piece (as currently done for TP53 gene). LUSC develops near the trachea and primary bronchi [2], the final objective would be to increase iROS locally and transiently, with minimal effects for normal tissue. Aerosol therapy [54], a constantly under development approach for adult and pediatric asthma could be suitable for this treatment. Potential compounds to be used in this therapy include inhibitors of endogenous antioxidants. Compounds reducing intracellular GSH, such as buthionine sulfoximine have been clinically tested in combination with melphalan. While these compounds were well tolerated and trigger ferroptosis of cancer cells, the side effects of leukopenia and thrombocytopenia limited their clinical use [55].

For translational application, a proposal would be the use of (1°) nanoparticles (NP) as well as (2°) the development of SETD5 inhibitors. The later approach would exploit the capacity of SETD5 to restrict PPP gene expression, to use SETD5 inhibitors for upregulation of G6PD to toxic levels. G6PD activators (e.g. AG1) would act similarly. The excess of G6PD likely consumes excessive glucose that is required for glycolysis, thereby causing cell death.

Nanotechnology permits to construct small drugs with ROS regulation capacity that can be administered in aerosols; NP with an Iron core trigger direct oxidative stress in the lining of the lungs [56]. For a more controlled action, NP can be covered with different coatings. Once the Fe-coated NP are internalized in human cells, they produce iROS [57]. Zinc oxide NP have also been tested in vivo via intraperitoneal instillation and intravenous (i.v.) injection in the mouse; in i.v. injection, particles accumulate in the liver, spleen, and lung, but can also be concentrated in lung when adapted for aerosol inhalation. In the case of zinc oxide NP, lung photodynamic intranasal endoscopy would mainly affect the exposed LUSC tumor area in the lining of the lungs [58]. Other NPs with potential interest are the Iron−platinum NP assembled on the surface of graphene oxide. These materials, currently under active development, can increase ROS production thereby improving the radiation sensitivity of NSCLC lung cancer cells [59]. In this regard, a high number of different particles have been developed for single ROS treatment looking for synergistic therapeutic effect of ROS and radiotherapy [60,61]; several of them are on trials (https://clinical trials.gov). Although NP field is yet under development for increasing radiation sensitivity, the preliminary results [[60], [61], [62], [63]], the fact that aerosol therapy is achievable for LUSC, and the sensitivity to ROS of LUSC tumors (WT NFE2L2/KEAP1), pose hope on the feasibility of these treatments. The search for SETD5 inhibitors should be accomplished as an alternative potential therapy.

In conclusion, the presented data points to the development of new therapeutic strategies for LUSC based on acute ROS inducers. Since lung cancer remains one of the most lethal tumor types, and LUSC lacks a successful targeted therapy, the findings reported here open new therapeutic avenues for LUSC patients with WT NFE2L2/KEAP1.

Ethics statement

Our studies (A/ES/19/77) were approved by the ethics committees.

Funding statement

This work was supported by the Ministry of Science and innovation (MCIN) (PID2019-106937RB-I00 PIA12019; PDC-2022-133912-I00 ) by the Madrid Regional Government (S2020/BMD-7321 ), by the European Community, European COST Action CA20121 (BenBedPhar), and by the Spanish Association Against Cancer (16035CARR ). M.S.-O. was supported by the MCIN.

CRediT authorship contribution statement

M. Sánchez-Ortega: Validation, Software, Methodology, Investigation, Formal analysis. A. Garrido: Writing – review & editing, Supervision, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. C. Cirauqui: Methodology, Investigation. L. Sanz-Gonzalez: Methodology. M.C. Hernández: Methodology. A. González-García: Writing – review & editing, Methodology, Investigation. K. Obregon: Methodology, Investigation. I. Ferrer: Methodology, Investigation. L. Paz-Ares: Methodology, Investigation. A.C. Carrera: Writing – review & editing, Supervision, Software, Project administration, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors have no competing interests to declare.

Appendix A Supplementary data

The following are the Supplementary data to this article.Multimedia component 1

Multimedia component 1

Multimedia component 2

Multimedia component 2

Data availability

Original data will be available at https://doi.org/10.17632/9cwkxznpp2.1.

Acknowledgements

We thank Dr. Mazur (Stanford University) for his generous donation of reagents and R. Carballar del Valle for his help.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.redox.2024.103305.
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