IFNy enhances ferroptosis by increasing JAK-STAT pathway activation to suppress SLCA711 expression in adrenocortical carcinoma
XINBO YU1*, DANDAN ZHU1*, BIXIAN LUO2*, WEI KOU1, YULING CHENG1 and YU ZHU1
Departments of Urology and 2General Surgery, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, P.R. China
Received November 10, 2021; Accepted March 2, 2022
DOI: 10.3892/or.2022.8308
Abstract. Adrenocortical carcinoma (ACC) is a rare type of tumor with a poor prognosis. Ferroptosis is a relatively novel form of programmed cell death driven by iron-dependent lipid peroxidation accumulation. Recent evidence suggests that IFNy facilitates erastin-induced ferroptosis, which contributed to anticancer therapy in various types of cancer. However, it has remained elusive whether the regulation of IFNy on ferrop- tosis has a positive role in the treatment of ACC. Thus, the aim of the present study was to explore the effects of IFNy on erastin-induced ferroptosis in the ACC cell line NCI-H295R and investigate the underlying mechanisms. Cell viability was assessed using a Cell Counting Kit-8 assay, an ethynyl- dioxyuridine proliferation assay and Live/Dead staining. The levels of iron, reactive oxygen species, lipid peroxidation and mitochondrial damage were also assessed. Western blot and reverse transcription-quantitative PCR analyses were used to determine the underlying molecular mechanisms involved in the erastin-induced ferroptosis of NCI-H295R cells. The results suggested that IFNy promoted erastin-induced ferrop- totic cell death. Furthermore, IFNy enhanced erastin-induced ferroptosis, as evidenced by the accumulation of iron, as well as the increase in lipid peroxidation and promotion of mitochondrial damage. Further analysis suggested that IFNy enhanced ferroptosis by suppressing the expression of solute carrier family 7 member 11, an important negative regulator of ferroptosis, and this was achieved via activation of the JAK/STAT pathway in NCI-H295R cells. The present study provided experimental evidence on the activity and mechanism
Correspondence to: Dr Yu Zhu, Department of Urology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, 97 2nd Ruijin Road, Shanghai 200025, P.R. China E-mail: zy10478@rjh.com.cn
*Contributed equally
Key words: adrenocortical carcinoma, IFNy, SLC7A11, ferroptosis, JAK/STAT pathway
of ferroptosis enhanced by IFNy in ACC and may give critical insight into the immunotherapeutic management of ACC.
Introduction
Adrenocortical carcinoma (ACC) is rare endocrine neoplasia with an annual incidence rate of 0.7-2 cases/million individ- uals (1). It is an aggressive cancer type and lacks efficacious therapies. Early diagnosis, complete surgical resection and adjuvant therapy with mitotane are recommended as the cura- tive treatment for ACC (2). Immunotherapy is also feasible and rational in the management of ACC; however, an increasing body of evidence suggests that the clinical efficacy of immu- notherapy is modest in patients with ACC (3-5). Productive CD8+ T-cell infiltrates induce antitumor immune responses to promote tumor clearance. TP53 germline mutations and Wnt/B-catenin activation have important roles in ACC tumorigenesis, which result in impaired CD8+ function and immunoresistance (6). Therefore, novel therapeutic strategies are required to overcome immunoresistance in ACC.
IFNy is an essential cytokine for antitumor immunity and may inhibit tumor growth by inducing cell cycle arrest and apoptosis (7). CD8+ T cells exhibit cytolytic activities involving perforin or the Fas mechanism to achieve tumor clearance (8). CD8+ T cells are the major source of IFNy production (9,10). Previous studies have indicated that the IFNy produced by CD8+ T cells appears to improve antitumor responses by enhancing tumor sensitivity to ferroptosis (11,12).
Ferroptosis is an iron-dependent cell death that differs from traditional apoptosis, necrosis and autophagy with regard to both the cellular morphological changes and the and gene expression profiles (13). This relatively recently discovered form of regulated cell death is characterized by lipid peroxida- tion and accumulation of reactive oxygen species (ROS) (14). Acyl-CoA synthase long chain family member 4 (ACSL4) participates in the synthesis of oxidized cellular membrane phospholipids capable of controlling the sensitivity of cells to ferroptosis (15).
Solute carrier family 7 member 11 (SLC7A11) is an impor- tant negative regulator of ferroptosis and encodes the light chain subunit of system Xc -. System Xc- is an amino acid antiporter located in the plasma membrane, which allows for
cystine uptake and glutamate release (16). Once cystine enters cells, it is reduced to cysteine, which in turn is used in the biosynthesis of glutathione (GSH). GSH is the major intracel- lular antioxidant and cofactor of GSH peroxidase 4 (GPX4), which prevents cells from experiencing oxidative stress and therefore undergoing ferroptosis (17). Erastin is able to inhibit system Xc-activity and subsequently cause GSH depletion, resulting in increased ferroptosis. In addition to killing tumor cells, several studies have demonstrated that erastin enhances the sensitivity of cancers to chemotherapy and radio- therapy (18-20). Previous studies have also indicated that IFNy facilitates erastin-induced ferroptosis, which contributes to anticancer therapy in various types of cancer (11,12). However, it remains elusive whether the regulatory effect of IFNy on ferroptosis has a positive role in the therapy of ACC.
In the present study, it was demonstrated that IFNy promoted erastin-induced ferroptotic cell death. In addition, IFNy enhanced erastin-induced ferroptosis, as evidenced by the accumulation of iron, elevation of lipid peroxidation and increase in mitochondrial damage. Further analysis indicated that IFNy enhanced ferroptosis by suppressing the expression of SLC7A11, which was achieved through the activation of the JAK/STAT pathway in NCI-H295R cells. Taken together, these results suggest that targeting SLC7A11 predisposes ACC cells to ferroptosis and this may provide a novel strategy to improve the efficacy of immunotherapy in ACC.
Materials and methods
Identification of prognostic ferroptosis-related differentially expressed genes (DEGs). The RNA sequencing data of 128 normal samples and 79 ACC samples were downloaded from the UCSC Xena platform (https://xena.ucsc.edu/). In addi- tion, the clinical information of 79 patients with ACC was obtained from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/). A total of 60 ferroptosis-related genes (FRGs) were acquired from previous research (21). All DEGs were analyzed using R (version 4.10) (22). For DEGs, a false discovery rate of <0.05 was used as a cutoff criterion and was processed using the limma R package (23). Univariate Cox analysis was performed using the survival R package, with the aim of evaluating the interaction between FRGs and overall survival (OS). All genes with P<0.001 were considered prognostic genes. The Venn R package was used to draw Venn diagrams and identify the intersection genes between the DEGs and prognostic genes. Next, GEPIA2 (http://gepia2. cancer-pku.cn/#index) was used to perform survival analysis of the intersection genes (24). Overall survival was estimated by Kaplan-Meier analysis, and the P-values were determined by log-rank test. P<0.05 was considered to indicate a statisti- cally significant difference.
Cell culture and drugs. The human ACC cell line NCI-H295R was purchased from Procell Life Science & Technology Co., Ltd. Cells were incubated in DMEM/F12 media with added 6.25 µg/ml insulin, 6.25 µg/ml transferrin, 6.25 ng/ml selenium, 5.35 µg/ml linoleic acid, 10% FBS and 1% penicillin/streptomycin (all from Procell Life Science & Technology Co., Ltd.). Cells were maintained in a humidi- fied incubator supplied with 5% CO2 at 37℃. Recombinant
human IFNy (cat. no. HY-P7025A), erastin (cat. no. HY-15763) and fludarabine (cat. no. HY-B0069) were purchased from MedChemExpress. Erastin and fludarabine were dissolved in DMSO and then diluted in complete culture medium to the desired concentration, with a final DMSO concentration of <0.1%.
Cell viability assay. NCI-H295R cells were seeded into a 96-well plate at a density of 1x104 cells/well in 100 ul culture medium. After incubation for 24 h, cells were pretreated with or without IFNy (10 ng/ml) for 24 h, followed by treatment with erastin (10 or 20 uM) for 24, 48 or 72 h. Cell viability was detected using a Cell Counting Kit-8 (CCK-8) assay (Epizyme Biotech). A total of 10 ul CCK-8 solution was added to each well, followed. By further incubation for 4 h at 37°℃. The optical density value was measured at 450 nm using a plate reader (Tecan infinite F200PRO; Tecan Group, Ltd.) and the percentage of viable cells compared with the control cells was calculated.
Ethynyldioxyuridine (EdU) proliferation assay. An EdU staining assay was performed using an EdU staining kit (Beyotime Institute of Biotechnology) according to the manufacturer’s protocol. Cells were stained with 20 uM EdU solution for 4 h and then fixed with fixative solution provided in the staining kit. The cell nuclei were stained with DAPI. The images were acquired using a fluorescence microscope (Eclipse 80i; Nikon Corporation) and stained cells were analyzed using ImageJ software (version 1.53; National Institutes of Health).
Cell death assay. Cell death was detected using a Calcein-acetoxymethyl ester (AM)/propidium iodide (PI) LIVE/DEAD double staining kit (cat. no. CA1630; Beijing Solarbio Science & Technology Co., Ltd.). Cells were harvested and washed using the included Assay Buffer three times. The harvested cells were resuspended in Assay Buffer and subsequently stained with 2 uM calcein-AM (for live cells) and 4.5 uM PI (for dead cells) at 37℃ for 30 min in the dark. Stained cells were imaged using a fluorescence microscope (Eclipse 80i; Nikon Corporation) and analyzed using ImageJ (version 1.53; National Institutes of Health).
Western blot analysis. After treatment, cells were lysed with RIPA lysis buffer supplemented with a protease and phospha- tase inhibitor cocktail (Epizyme Biotech). The concentration of the protein samples was quantified using a BCA protein assay kit (Takara Bio, Inc.), total protein (20 µg/lane) was resolved by SDS-PAGE on 10% gels and transferred to PVDF membranes (MilliporeSigma). Membranes were blocked with protein free rapid blocking buffer (Epizyme Biotech) at room temperature for 15 min. Subsequently, the PVDF membrane was incubated with primary antibodies against B-actin (1:1,000 dilution; cat. no. ab8226; Abcam), SLC7A11 (1:1,000 dilution; cat.ARG57998; Arigobio), phosphorylated signal transducer and activator of transcription 1 (p-STAT1; 1:1,000 dilution; cat. no. ab109461; Abcam), STAT1 (1:10,000 dilution; cat. no. ab109320; Abcam) p-STAT3 (1:1,000 dilu- tion; cat. no. ab267373; Abcam), STAT3 (1:1,000 dilution; cat. no. ab68153; Abcam) or IFN regulatory factor 1 (IRF1;
1:1,000 dilution; cat. no. ab243895; Abcam) at 4℃ over- night, followed by horseradish peroxidase (HRP)-labeled secondary antibody (1:1,000 dilution; cat. no. LF101 or LF102; Epizyme Biotech). Protein signals were visualized using Chemiluminescent HRP substrate (MilliporeSigma) and analyzed using ImageJ (version 1.53; National Institutes of Health).
Reverse transcription-quantitative (RT-q) PCR. Total RNA was extracted from cells using Total RNA isolation reagent (Biosharp) and then reverse-transcribed into cDNA using a Takara RT kit (Takara Bio, Inc.). The mixture was incubated at 37℃ for 15 min, followed by 85℃ for 5 sec and then cooled at 4℃. qPCR was performed using TB Green® Premix Ex Taq™M (Takara Bio, Inc.) in a final volume of 20 ul in an ABI 7500 reactor (Applied Biosystems; Thermo Fisher Scientific, Inc.). The qPCR conditions consisted of an initial denaturation step for 30 sec at 95℃, followed by 40 cycles of 3 sec dena- turation at 95℃ and annealing/elongation for 30 sec at 60℃. The samples were quantified using the 2-44Cq method and B-actin was used as the internal control (25). The PCR primer sequences (Sangon Biotech, Co., Ltd.) are listed in Table I.
Immunofluorescence analysis. After treatment, the cells were washed with PBS three times, fixed in 4% formaldehyde for 24 h and blocked with 5% goat serum (cat. no. C0265; Beyotime Institute of Biotechnology) for 60 min. Next, the cells were incubated with the anti-SLC7A11 primary anti- body (1:1,000 dilution; cat. ARG57998; Arigobio) at 4℃ overnight. The following day, the cells were washed with PBS and incubated with the secondary antibody (donkey anti-rabbit IgG H&L; 1:1,000 dilution; cat. no. ab150075; Abcam) for 2 h at room temperature, and the nuclei were stained with DAPI. Finally, the fluorescently labeled cells were visualized using fluorescence microscopy (Eclipse 80i; Nikon Corporation).
Lipid peroxidation detection using BODIPY-C11. Lipid peroxidation was detected using a C11 BODIPY 581/591 probe (ABclonal Biotech Co., Ltd.). Cells were incubated with 50 uM working solution for 1 h at 37℃ and gently washed with PBS three times. Finally, cells were imaged using a fluorescence microscope (Eclipse 80i; Nikon Corporation).
Measurement of intracellular iron levels. For assaying of intracellular iron levels, Phen Green SK (PGSK), a fluorescent probe (Cayman Chemical Company), was used. Cells were incubated with 50 µM PGSK at 37℃ for 20 min and then the fluorescence images were acquired. Finally, the intensity of green fluorescence was measured using ImageJ (version 1.53; National Institutes of Health).
Transmission electron microscopy. After treatment, cells were harvested and fixed in 2.5% glutaraldehyde at 4℃ for 12 h. The samples were rinsed, dehydrated in a gradient series of alcohol solutions, embedded and sectioned (70-90 nm). The sections were stained using uranyl acetate/lead citrate for 10 min prior to observation. Morphological changes in the mitochondria were imaged using a transmission electron microscope (H-7650; Hitachi, Ltd.). The mitochondrial area
| Gene | Primer sequence (5'-3') |
|---|---|
| SLC7A11 | |
| Forward | GGTCCATTACCAGCTTTTGTACG |
| Reverse | AATGTAGCGTCCAAATGCCAG |
| GPX4 | |
| Forward | GAGGCAAGACCGAAGTAAACTAC |
| Reverse | CCGAACTGGTTACACGGGAA |
| ACSLA | |
| Forward | CATCCCTGGAGCAGATACTCT |
| Reverse | TCACTTAGGATTTCCCTGGTCC |
| -actin | |
| Forward | GACCTGTACGCCAACACAGT |
| Reverse | CTCAGGAGGAGCAATGATCT |
SLC7A11, solute carrier family 7 member 11; ACSL4, acyl-CoA synthase long chain family member 4; GPX4, glutathione peroxidase 4.
and density were quantitatively analyzed by ImageJ software (version 1.53; National Institutes of Health).
Detection of intracellular ROS. Intracellular ROS was measured using an ROS assay kit (Beyotime Institute of Biotechnology) according to the manufacturer’s protocol. In brief, after treatment, cells were incubated with 10 µM dichloro-dihydro-fluorescein diacetate probe at 37℃ for 20 min. The fluorescent cells were observed using a fluo- rescence microscope and the intracellular ROS levels were detected using a flow cytometer (CytoFlex S; Beckman Coulter, Inc.).
Detection of mitochondrial membrane potential (MMP). The JC-1 fluorescent probe (MMP assay kit with JC-1; no. C2006; Beyotime Institute of Biotechnology) was used to evaluate the MMP of cells according to the manufacturer’s protocol. When the MMP is high, JC-1 accumulates in the matrix of the mitochondria to form a polymer (J-aggregates), which fluoresces red; when the MMP is low, JC-1 does not aggregate in the mitochondrial matrix but is present as a monomer and fluoresces green. Thus, it is convenient to detect the changes in MMP based on the change in fluorescence color. The rela- tive ratio of red to green fluorescence may be used to measure mitochondrial depolarization. NCI-H295R cells were seeded into 6-well plates at a density of 2x105 cells/well. Cells were pretreated with or without IFN-y (10 ng/ml) for 24 h, followed by treatment with erastin (20 µM) for 48 h. Cells were washed with PBS and stained with 1 ml of the JC-1 staining solution per ml of culture medium added to each well of the plate, with incubation at 37℃ for 20 min. The supernatant was then discarded and the cells were washed two times with JC-1 staining buffer. The fluorescence images were then obtained using a fluorescence microscope (Eclipse 80i; Nikon Corporation) in 6 random fields under a magnification of x400. ImageJ software (version 1.53; National Institutes of Health) was used to measure the fluorescence intensity.
A
B
Type
Type
10
N
DEGs
Prognostic genes
PTGS2
5
T
0
ACSF2
5
10
SLC7A11
46
7
2
FANCD2
AIFM2
ACSL4
PGD
C
D
Overall survival
pvalue
Hazard ratio
1.0
Low slc7a11 Group
…
ACSL4
<0.001 0.542 (0.389-0.753)
High slc7a11 Group
Logrank p=0.00011
FANCD2
<0.001 5.862 (3.191-10.772)
0.8
n(high)=38
n(low)=37
PTGS2
<0.001 1.609 (1.229-2.107)
Percent survival
SLC7A11
<0.001 3.848 (2.143-6.909)
0.6
AIFM2
<0.001 2.003 (1.337-3.000)
H
0.4
PGD
<0.001 2.073 (1.396-3.078)
H
ACSF2
<0.001 0.555 (0.395-0.779)
0.2
0
2
4
6
8
10
Hazard ratio
0.0
0
50
100
150
Months
Overexpression of SLC7A11 and suppression of STAT1. pCMV-SLC7A11-ZsGreen-PURO and pcDNA3.1-ZsGreen-PURO plasmids were purchased from Hanbio Biotechnology Co., Ltd. The plasmid was transfected into NCI-H295R cells to overexpress SLC7A11, while the mock plasmid was used as the negative control. Cells were seeded in a six-well plate (2x105 cells/well) and grown to 60-70% confluence at 37℃ with 5% CO2 prior to transfec- tion. Transfection was performed using Lipofectamine® 3000 reagent (Invitrogen; Themo Fisher Scientific, Inc.) and Ultra-MEM Reduced Serum Medium (BioAgrio) for 6 h at 37℃ with 5% CO2. The medium was then replaced with supplemented culture medium for a further 18 h. Subsequently, cells were treated with IFNy (10 ng/ml) for 24 h, followed by erastin (20 µM) for 48 h. The efficacy of overexpression was analyzed by RT-qPCR and western blot analysis.
Fludarabine was used to inhibit the expression of STAT1. Cells were seeded in a six-well plate (2x105 cells/well) at 37℃ with 5% CO2 overnight; after 24 h pre-treatment with 2 uM fludarabine at 37℃, NCI-H295R cells were treated with IFNy (10 ng/ml) for 24 h, followed by erastin (20 µM) for 48 h.
Statistical analysis. Values are expressed as the mean ± stan- dard deviation of three or more independent experiments. Student’s t-test was used to analyze the differences between two groups. Comparisons among multiple groups were performed using a one-way ANOVA followed by a Tukey’s post-hoc test. Statistical analyses were performed using the NCSS statistical software version 21.0.3 (NCSS, LLC). P<0.05 was considered to indicate a statistically significant difference.
Results
Extraction of FRGs in ACC. The majority of the FRGs (53/60, 88.33%) were differentially expressed between the ACC and normal adrenal gland tissues. Of these, 7 were identified as prog- nostic factors based on the univariate Cox regression analysis (P<0.001; Fig. 1A). These intersection genes were further evalu- ated using a heatmap and univariate Cox regression analysis. The heatmap presents the expression levels of FRGs between normal and tumor tissues; downregulated genes are indicated with blue squares and upregulated genes with red squares (Fig. 1B). Analysis of the relationship between FRGs and OS was visu- alized using a forest plot (Fig. 1C). The results suggested that
A
10 μ.Μ
20 μΜ
100
Erastin
100
Erastin
Cell viability (%)
80
IFNy+Erastin
Cell viability (%)
80
IFNy+Erastin
60
60
40
40
20
20
0
0
24 h 48 h 72 h
24 h 48 h 72 h
Time (h)
Time (h)
B
Control
IFNY
Erastin
IFNy+Erastin
Merged
40
EdU Positive (% of cells)
30
**
20
EdU
10
DAPI
0
Control IFNy Erastin IFNy+
Erastin
C
Control
IFNY
Erastin
IFNy+Erastin
Calcein-AM
50
Cell death ration (%)
40
30
PI
20
10
Merged
0
Control IFN-y Erastin IFN-Y+
Erastin
SLC7A11 was upregulated in patients with ACC and its high expression was related to poor survival. Kaplan-Meier curves suggested that low expression of SLC7A11 in patients with ACC was associated with a favorable prognosis (Fig. 1D). The results of the Kaplan-Meier curve analysis for the other intersection genes [Fanconi anemia group D2 protein (FANCD2); apoptosis inducing factor mitochondria associated 2 (AIFM2); phospho- gluconate dehydrogenase (PGD); ACSL4; acyl-CoA synthetase family member 2 (ACSF2); prostaglandin-endoperoxide synthase 2 (PTGS2)] are provided in Fig. S1; these findings demonstrated that the expression of other intersection genes was associated with the prognosis of patients with ACC (P<0.05).
IFNy sensitizes NCI-H295R cells to ferroptotic cell death. NCI-H295R cells were treated with 10 ng/ml IFNy for 24 h, followed by treatment with different concentrations of erastin
for 24, 48 and 72 h. To analyze the viability of NCI-H295R cells, a CCK-8 assay was performed. The results indicated that erastin exerted a dose- and time-dependent inhibitory effect on the viability of NCI-H295R cells compared with the control group. Pretreatment of IFNy further enhanced the inhibitory effect of erastin on NCI-H295R cell viability (Fig. 2A). The EdU assay suggested that the percentage of EdU-positive cells was decreased by erastin treatment and this was attenuated by IFNy pretreatment (Fig. 2B). Live/Dead assay staining further confirmed the cytotoxic effect of the above-mentioned treatment. Green fluorescence represents live cells and red fluorescence represents dead cells. The combination of IFNy and erastin resulted in a higher cell death ratio compared with cells treated with erastin alone (Fig. 2C). All of these results indicated that IFNy sensitized NCI-H295R cells to erastin-induced cell death.
A
Control
IFNY
Erastin
IFNy+Erastin
B
3
Non-
Oxidative form Oxidative form
C11 BODIPY (Realtive MFI)
2
1
0
Merged
Control IFN-y Erastin IFN-Y+ Erastin
C
D
1.5
Control
IFNY
Erastin
IFNy+Erastin
PGSK (Realtive MFI)
1.0
0.5
E
F
0.0
**
Relative mRNA expression of ACSL4/B-actin
2.5
Relative mRNA expression of GPX4/B-actin
1.2
Control IFN-y Erastin IFN-Y+ Erastin
**
2.0
0.8
1.5
1.0
0.4
0.5
0.0
0.0
Control IFNy Erastin IFNy+
Control IFNy Erastin IFNy+
Erastin
Erastin
IFNy enhances erastin-induced ferroptosis. To determine whether IFNy enhanced the sensitivity of NCI-H295R cells to erastin, the levels of lipid peroxidation and intracellular iron were assessed. The C11-BODIPY581/591 fluorescence probe is a lipid peroxidation sensor, capable of accessing the cell membrane. Once oxidized, the fluorescent properties shift from a non-oxidative form (red fluorescence) to an oxidative form (green fluorescence). The results suggested that IFNy pretreat- ment accelerated the process of decay of red fluorescence to green fluorescence (Fig. 3A and B). The intracellular iron content may quench PGSK fluorescence. The fluorescence intensity of the PGSK probe was decreased in the IFNy + erastin group, indicative of an accumulation of iron (Fig. 3C and D). Both ACSL4 and GPX4 are important indicators of ferroptosis. The RT-qPCR assay indicated increased mRNA levels of ACSL4 and decreased mRNA levels of GPX4 in the IFNy + erastin group compared with the control group (Fig. 3E and F).
Mitochondrial damage caused by erastin-induced ferrop- tosis is enhanced by IFNy. The mitochondria are the primary sites of ROS production, and ROS has important roles in ferroptosis. Therefore, the impact of IFNy on mitochondrial damage in erastin-treated cells was further analyzed. MMP assays indicated an increased green/red fluorescence ratio and loss of MMP in the erastin-treated cells after pretreatment with IFNy (Fig. 4A and B). Furthermore, fluorescence microscopy and flow cytometric analysis suggested that IFNy enhanced intracellular ROS generation in the NCI-H295R cells followed by treatment with erastin (Fig. 4C-F). In addition, the morphology of the mitochondria in the NCI-H295R cells was observed using transmission electron microscopy. Upon exposure to IFNy and erastin, the mitochondria became smaller, the membrane density increased and the area of ruptured membrane also increased (Fig. 4G-I).
A
Control
IFNY
Erastin
IFNy+Erastin
B
**
JC-1 590 nm
60
Green/Red fluorescence ratio
40
20
JC-1 530 nm
0
Control
IFNY
Erastin
IFNY+
Erastin
D
**
20
Merged
Mean fluorescence intensity (AU)
15
10
cn
0
C
Control
IFNY
Erastin
IFNy+Erastin
Control
IFNY
Erastin
IFNy+
Erastin
**
E
Control
IFNY
Erastin
IFNy+Erastin
Relative intensity (DCF)
3
Mean DCPH PITCHA
200
1613427
Hash DEFH RETC.A. 199472 5
Mean DCFH FITCHA
P2(100:00%)
253641.3
Count
P2(100.00%)
200
Count
Count
200
P2(100.00%)
200
P2(100.005%)
2
Count
100
100
100
-
0
O
102103 104 105 106 107 DCFH FITC-A
0
102103 104 105 106 107
0
0
DCFH FITC-A
102103 104105 106 107 DCFH FITC-A
102103 104 105 106 107
DCFH FITC-A
Control
IFNY
Erastin
IFNy+
Erastin
G
Control
IFNY
Erastin
IFNy+Erastin
H
Relative mitochondrial area
1.5
Relative mitochondrial density
A
**
**
1.0
5
2
0.5
T
e
-
8
0.0
Control
IFNY
Erastin
IFNy+
Erastin
0
Control
IFNY
Erastin
IFNY+
Erastin
IFNy represses SLC7A11 expression through the JAK/STAT pathway. SLC7A11 has a critical role in ferroptosis. The results of the western blot analysis suggested that the levels of SLC7A11 expression were reduced in erastin-treated NCI-H295R cells. Pretreatment with IFNy further enhanced the inhibitory effect of erastin on SLC7A11 expression in NCI-H295R cells (Fig. 5A and B). Similar results were also
confirmed by RT-qPCR and immunofluorescence analysis (Fig. 5C-E). IFNy was able to activate the JAK/STAT signaling pathway and mediate immune function and proliferation of cells. It has been demonstrated that STAT3 reduces the expression of SLC7A11 through binding to the promoter of SLC7A11 (26). To further explore the molecular mechanism of the effect of IFNy to repress SLC7A11 expression, the levels
A
B
C
Relative protein expression of SLC7A11/Actin
1.0
Relative mRNA expression
1.2
0.8
of SLC7A11/Actin
**
Một
0.9
SLC7A11
0.6
Actin
0.6
0.4
Control
IFNY
Erastin
IFNy+
Erastin
0.2
0.3
0.0
0.0
Control
IFNY
Erastin
IFNy+
Erastin
Control
IFNY
Erastin
IFNy+
Erastin
D
Control
IFNY
Erastin
IFNy+Erastin
E
SLC7A11
SLC7A11 Mean fluorescence intensity (AU)
50
40
**
30
DAPI
20
10
0
Merged
Control
IFNY
Erastin
IFNy+
Erastin
F
G
エ
p-STAT1
Relative protein expression of p-STAT1/STAT1
**
STAT1
1.5
Relative protein expression of p-STAT3/STAT3
**
Relative protein expression of IRF1/Actin
**
1.0
1.5
p-STAT3
1.0
0.8
STAT3
0.6
1.0
IRF1
0.5
C
a
0.2
Actin
0.0
0.0
0.0
Control
IFNY
Erastin
IFNy+
Erastin
Control
IFNY
Erastin
IFNy+
Erastin
Control
IFNY
Erastin
IFNy+
Erastin
Control
IFNY
Erastin
IFNy+
Erastin
of pSTAT1, pSTAT3 and IRF1 were determined. The results indicated that erastin increased the expression of pSTAT1, pSTAT3 and IRF1, which were further increased by IFNY in NCI-H295R cells (Fig. 5F-I), suggesting that IFNy may repress SLC7A11 expression through JAK/STAT pathway.
SLC7A11 overexpression or STAT1 inhibition ameliorates IFNy-enhanced erastin-induced ferroptosis. Next, it was determined whether IFN-y was able to promote ferroptosis through repressing SLC7A11 via the JAK/STAT pathway. A plasmid was used to overexpress SLC7A11 and fludarabine was used to inhibit STAT1. The expression levels of SLC7A11 at the mRNA and protein levels were significantly higher in cells transfected with the plasmid compared with the empty vector control (Fig. 6A and B). Cell death was increased in cells transfected with the mock plasmid (Fig. 6C and D). The
level of lipid peroxidation and iron accumulation was reduced in SLC7A11-overexpressing NCI-H295R cells and fludara- bine-treated NCI-H295R cells (Fig. 6E-H). Furthermore, SLC7A11 overexpression prevented mitochondrial damage in the cells, and STAT1 inhibition exerted a similar effect, although it was weaker (Fig. 6I-K). These results indicated that SLC7A11 inhibited IFNy-enhanced erastin-induced ferroptosis, and STAT1 aggravated this effect.
Discussion
ACC has a poor prognosis, with a 5-year survival rate of 15% in patients with advanced-stage disease (20). Although the use of immunotherapy for the management of malignant neoplasms has increased, the immunotherapeutic efficacy in ACC is limited owing to the intrinsic mechanisms of
A
B
C
**
SLC7A11
Relative mRNA expression of SLC7A11/Actin
10
Calcein-AM
8
Actin
6
Mock
Plasmid
4
2
PI
0
Mock
Plasmid
Merged
D
E
F
Plasmid+ IFNy+Erastin
Mock+ IFNy+Erastin
Fludarabine+ IFNy+Erastin
2.5
Cell death ration (%)
C11 BODIPY (relative MFI)
Non-Oxidative
50
2.0
form
40
1.5
30
20
1.0
0.5
Oxidative
10
form
0
0.0
Plasmid+
IFN-y+Erastin
Mock+
IFN-y+Erastin
Fludarabine+
IFN-Y+Erastin
Plasmid+
IFN-Y+Erastin
Mock+
IFN-Y+Erastin
Fludarabine+
IFN-Y+Erastin
Merged
G
Plasmid+ IFNy+Erastin
Mock+ IFNy+Erastin
Fludarabine+ IFNy+Erastin
2.0
PGSK (relative MFI)
1.5
T
1.0
0.5
0.0
Plasmid+
IFN-Y+Erastin
Mock+
IFN-Y+Erastin
Fludarabine+
IFN-Y+Erastin
Plasmid+ IFNy+Erastin
Mock+ IFNy+Erastin
Fludarabine+ IFNy+Erastin
a
K
Relative mitochondrial area
1.5
**
**
Relative mitochondrial density
4
NỘI NỘI
1.0
3
u
2
=
1
5+
0.5
1
0.0
Plasmid+
IFNy+Erastin
Mock+
IFNy+Erastin
Fludarabine+
IFNy+Erastin
0
Plasmid+
IFNy+Erastin
Mock+
IFNy+Erastin
Fludarabine+
IFNy+Erastin
Plasmid+ IFNy+Erastin
Mock+ IFNy+Erastin
Fludarabine+ IFNy+Erastin
immunoresistance. Ferroptosis has an important role in affecting the efficacy of cancer immunotherapy (27). It is well
established that ferroptosis inducers exert anti-tumor effects in various types of cancer (28). In the present study, the effect of
IFNy on the sensitivity of the ACC cell line NCI-H295R to a ferroptosis inducer, erastin, was determined.
The differentially expressed FRGs between normal adrenal glands and ACC samples were determined, and univariate Cox regression analysis was then performed to identify prognostic genes (ACSL4, FANCD2, PTGS2, SLC7A11, AIFM2, PGD and ACSF2). Recent research has revealed that a combination of IFNy derived from immunotherapy-activated CD8+ T cells and radiotherapy-activated ATM promote cancer ferroptosis via synergistic repression of SLC7A11 (29). Thus, it was specu- lated that SLC7A11 was a novel common gene involved in both ferroptosis and cancer immunotherapy. SLC7A11, an important subunit of cystine/glutamate antiporter system Xc-, mediates cystine uptake and GSH biosynthesis, resulting in the suppres- sion of ferroptosis and reducing oxidative stress (30). Several studies have indicated that SLC7A11 expression is upregulated in various types of cancer (31-33). Weigand et al (34) reported that SLC7A11 was upregulated in 33ACC tissues compared with 10 normal adrenal gland samples from the Gene Expression Omnibus dataset GSE10927. Furthermore, the present study determined that SLC7A11 was upregulated in 79 ACC tissues compared with 128 normal adrenal gland tissues based on the TCGA dataset and GTEx dataset. Chen et al (35) determined SLC7A11 was correlated with OS of patients with ACC from the TCGA dataset. Shi et al (36) reported that high expression of SLC7A11 was significantly associated with a shortened OS in patients with ACC according to the GEPIA2 database; thus, SLC7A11 is a candidate prognostic biomarker for ACC. The present study also indicated that SLC7A11 is a prognosis-related ferroptosis gene in patients with ACC and further explored relevant regulatory mechanisms of SLC7A11 in the ferroptosis of ACC cells. The present study suggested that targeting SLC7A11 was able to promote ferroptosis in ACC cells through IFNy. Therefore, SLC7A11 may be consid- ered a potential immunological therapeutic target in ACC.
Erastin is a classical ferroptosis inducer that inhibits the activity of System Xc-, thereby facilitating an increase in sensitivity to ferroptosis in tumor cells (37). Erastin has excellent prospects for clinical use, enhancing the sensi- tivity of various types of human cancer to chemotherapy or radiotherapy (18,19,38). Weigand et al (34) demonstrated that ACC cells were insensitive to treatment with Erastin for 24 h. However, recent research suggested that knockdown of SLC7A11 or IFNy-mediated downregulation of SLC7A11 increased the sensitivity of tumor cells to Erastin-induced ferroptosis (11). Therefore, it was hypothesized that there exists crosstalk between IFNy-mediated signaling and ferroptosis in ACC. In the present study, it was indicated that pretreatment with IFNy prolonged the period of action of erastin to inhibit the viability of the NCI-H295R ACC cells.
Ferroptosis is characterized by iron-dependent lipid peroxidation and ROS production. A higher level of ROS generation promotes cancer suppression (39,40). The results of the present study suggested that IFNy together with erastin enhanced the ferroptosis-like changes, including accumulation of intracellular iron content, elevated levels of lipid peroxida- tion, increased ROS production and mitochondrial damage. Both GPX4 and ACSL4 are essential determinants of sensi- tivity to ferroptosis. ACSL4 is involved in the formation of lipid ROS, which leads to ferroptosis, whereas GPX4, using
GSH as a substrate, protects cells from lipid peroxidation (15). The results of the present study indicated that the expression of ACSL4 was upregulated in the IFNy + erastin group, whereas the expression of GPX4 was downregulated. To summarize, these results suggested that IFNy enhanced erastin-induced ferroptosis in an ACC cell line, highlighting a potential thera- peutic approach for the management of ACC.
Pro-inflammatory cytokines, such as IFNy, are released from immune cells and are responsible for anti-tumor immu- nity. IFNy binds to specific receptors at the cell membrane to trigger the phosphorylation of STAT1 and activation of IRF1. This results in the activation of the JAK/STAT signaling pathway, which is an essential signaling pathway involved in regulating SLC7A11 expression (11,12). Recent research confirmed that IFNy promoted STAT1-mediated suppression of SLC7A11 in HT-1080 cells (11). In addition, STAT3 and STAT5 contribute to the regulation of SLC7A11 in numerous types of cancer, such as breast cancer (41). The results of the present study suggested that dual treatment with IFNy and erastin significantly suppressed the expression of SLC7A11. Furthermore, IFNy treatment indirectly upregulated the expression of pSTAT1, pSTAT3 and IRF1. In addition, SLC7A11 overexpression or STAT1 inhibition significantly ameliorated IFNy-enhanced erastin-induced ferroptosis. Therefore, it may be hypothesized that IFNy inhibits SLC7A11 via the JAK/STAT pathway.
The present study has certain limitations. First, ferrop- tosis-inhibiting drugs, such as ferrostatin-1, were not used to explore their protective effects under IFNy-enhanced erastin-induced ferroptosis. Furthermore, the role of IFNy was not verified in vivo or in another ACC cell line. In addi- tion, a comparison of the expression status of SLC7A11 between normal adrenocortical cells and NCI-H295R was not performed due to the lack of normal adrenocortical cells. The above-mentioned factors will be investigated in future studies.
In conclusion, it was demonstrated for the first time that IFNy treatment increased the sensitivity of ACC cells to erastin-induced ferroptosis via downregulation of SLC7A11 via the JAK/STAT pathway. Furthermore, it increased the levels of lipid peroxida- tion and ROS and increased mitochondrial damage, which in turn led to ferroptosis in ACC tumor cells. The present study provides critical insight into ferroptosis and how it may be used to improve the efficacy of cancer immunotherapy in ACC.
Acknowledgements
The authors express their gratitude to Dr Lei Guo (Department of Orthopaedics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China) for providing experimental assistance for this research.
Funding
This work was supported by the Natural Science Foundation of Shanghai, China (grant no. 21ZR1440700).
Availability of data and materials
All data generated and/or analyzed during the present study are included in this published article.
Authors’ contributions
XY and DZ contributed to the design of the study, performed the study and wrote the manuscript. BL contributed to the statistical analysis of the data. WK and YC contributed to cell culture. YZ analyzed the data and edited the manuscript. All authors contributed to the article, and read and approved the final version. XY and YZ confirm the authenticity of all the raw data.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
References
1. Assié G, Letouzé E, Fassnacht M, Jouinot A, Luscap W, Barreau O, Omeiri H, Rodriguez S, Perlemoine K, René-Corail F, et al: Integrated genomic characterization of adrenocortical carci- noma. Nat Genet 46: 607-612, 2014.
2. Crona J and Beuschlein F: Adrenocortical carcinoma-towards genomics guided clinical care. Nat Rev Endocrinol 15: 548-560, 2019.
3. Le Tourneau C, Hoimes C, Zarwan C, Wong DJ, Bauer S, Claus R, Wermke M, Hariharan S, von Heydebreck A, Kasturi V, et al: Avelumab in patients with previously treated metastatic adre- nocortical carcinoma: phase 1b results from the JAVELIN solid tumor trial. J Immunother Cancer 6: 111, 2018.
4. Sperone P, Ferrero A, Daffara F, Priola A, Zaggia B, Volante M, Santini D, Vincenzi B, Badalamenti G, Intrivici C, et al: Gemcitabine plus metronomic 5-fluorouracil or capecitabine as a second-/third-line chemotherapy in advanced adrenocortical carcinoma: A multicenter phase II study. Endocr Relat Cancer 17: 445-453, 2010.
5. Henning JEK, Deutschbein T, Altieri B, Steinhauer S, Kircher S, Sbiera S, Wild V, Schlötelburg W, Kroiss M, Perotti P, et al: Gemcitabine-based chemotherapy in adre- nocortical carcinoma: A multicenter study of efficacy and predictive factors. J Clin Endocrinol Metab 102: 4323-4332, 2017.
6. Cosentini D, Grisanti S, Dalla Volta A, Laganà M, Fiorentini C, Perotti P, Sigala S and Berruti A: Immunotherapy failure in adrenocortical cancer: Where next? Endocr Connect 7: E5-E8, 2018.
7. Jorgovanovic D, Song M, Wang L and Zhang Y: Roles of IFN-Y in tumor progression and regression: A review. Biomark Res 8: 49, 2020.
8. Ni L and Lu J: Interferon gamma in cancer immunotherapy. Cancer Med 7: 4509-4516, 2018.
9. Ligocki AJ, Brown JR and Niederkorn JY: Role of interferon-y and cytotoxic T lymphocytes in intraocular tumor rejection. J Leukoc Biol 99: 735-747, 2016.
10. Zhou J, Ma P, Li J, Cui X and Song W: Improvement of the cyto- toxic T lymphocyte response against hepatocellular carcinoma by transduction of cancer cells with an adeno-associated virus carrying the interferon-y gene. Mol Med Rep 13: 3197-3205, 2016.
11. Wang W, Green M, Choi JE, Gijon M, Kennedy PD, Johnson JK, Liao P, Lang X, Kryczek I, Sell A, et al: CD8(+) T cells regulate tumour ferroptosis during cancer immunotherapy. Nature 569: 270-274, 2019.
12. Kong R, Wang N, Han W, Bao W and Lu J: IFNy-mediated repres- sion of system xc drives vulnerability to induced ferroptosis in hepatocellular carcinoma cells. J Leukoc Biol 110: 301-314, 2021.
13. Mou Y, Wang J, Wu J, He D, Zhang C, Duan C and Li B: Ferroptosis, a new form of cell death: Opportunities and chal- lenges in cancer. J Hematol Oncol 12: 34, 2019.
14. Zhang X and Li X: Abnormal iron and lipid metabolism medi- ated ferroptosis in kidney diseases and its therapeutic potential. Metabolites 12: 58, 2022.
15. Li D and Li Y: The interaction between ferroptosis and lipid metabolism in cancer. Signal Transduct Target Ther 5: 108, 2020.
16. Tang X, Chen W, Liu H, Liu N, Chen D, Tian D and Wang J: Research progress on SLC7A11 in the regulation of cystine/cysteine metabolism in tumors. Oncol Lett 23: 47, 2022.
17. Wang Y, Wei Z, Pan K, Li J and Chen Q: The function and mech- anism of ferroptosis in cancer. Apoptosis 25: 786-798, 2020.
18. Cobler L, Zhang H, Suri P, Park C and Timmerman L: xCT inhi- bition sensitizes tumors to y-radiation via glutathione reduction. Oncotarget 9: 32280-32297, 2018.
19. Chen L, Li X, Liu L, Yu B, Xue Y and Liu Y: Erastin sensitizes glioblastoma cells to temozolomide by restraining xCT and cystathionine-y-lyase function. Oncol Rep 33: 1465-1474, 2015.
20. Roh JL, Kim EH, Jang HJ, Park JY and Shin D: Induction of ferroptotic cell death for overcoming cisplatin resistance of head and neck cancer. Cancer Lett 381: 96-103, 2016.
21. Liang JY, Wang DS, Lin HC, Chen XX, Yang H, Zheng Y and Li YH: A novel ferroptosis-related gene signature for overall survival prediction in patients with hepatocellular carcinoma. Int J Biol Sci 16: 2430-2441, 2020.
22. Team C: Team RDC.R: A Language And Environment For Statistical Computing. R Foundation for Statistical Computing, Vienna, 2012.
23. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W and Smyth GK: Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 43: e47, 2015.
24. Tang Z, Li C, Kang B, Gao G, Li C and Zhang Z: GEPIA: A web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res 45: W98-W102, 2017.
25. Livak KJ and Schmittgen TD: Analysis of relative gene expres- sion data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402-408, 2001.
26. Linher-Melville K, Haftchenary S, Gunning P and Singh G: Signal transducer and activator of transcription 3 and 5 regulate system Xc- and redox balance in human breast cancer cells. Mol Cell Biochem 405: 205-221, 2015.
27. Sun L, Linghu D and Hung M: Ferroptosis: A promising target for cancer immunotherapy. Am J Cancer Res 11: 5856-5863, 2021.
28. Xia X, Fan X, Zhao M and Zhu P: The Relationship between ferroptosis and tumors: A novel landscape for therapeutic approach. Curr Gene Ther 19: 117-124, 2019.
29. Lang X, Green MD, Wang W, Yu J, Choi JE, Jiang L, Liao P, Zhou J, Zhang Q, Dow A, et al: Radiotherapy and immunotherapy promote tumoral lipid oxidation and ferroptosis via synergistic repression of SLC7A11. Cancer Discov 9: 1673-1685, 2019.
30. Koppula P, Zhuang L and Gan B: Cystine transporter SLC7A11/xCT in cancer: Ferroptosis, nutrient dependency, and cancer therapy. Protein Cell 12: 599-620, 2021.
31. Jiang L, Kon N, Li T, Wang SJ, Su T, Hibshoosh H, Baer R and Gu W: Ferroptosis as a p53-mediated activity during tumour suppression. Nature 520: 57-62, 2015.
32. Shiozaki A, Iitaka D, Ichikawa D, Nakashima S, Fujiwara H, Okamoto K, Kubota T, Komatsu S, Kosuga T, Takeshita H, et al: xCT, component of cysteine/glutamate transporter, as an inde- pendent prognostic factor in human esophageal squamous cell carcinoma. J Gastroenterol 49: 853-863, 2014.
33. Takeuchi S, Wada K, Toyooka T, Shinomiya N, Shimazaki H, Nakanishi K, Nagatani K, Otani N, Osada H, Uozumi Y, et al: Increased xCT expression correlates with tumor invasion and outcome in patients with glioblastomas. Neurosurgery 72: 33-41; discussion 41, 2013.
34. Weigand I, Schreiner J, Rohrig F, Sun N, Landwehr LS, Urlaub H, Kendl S, Kiseljak-Vassiliades K, Wierman ME, Angeli JPF, et al: Active steroid hormone synthesis renders adrenocortical cells highly susceptible to type II ferroptosis induction. Cell Death Dis 11: 192, 2020.
35. Chen X, Yan L, Jiang F, Lu Y, Zeng N, Yang S and Ma X: Identification of a ferroptosis-related signature associated with prognosis and immune infiltration in adrenocortical carcinoma. Int J Endocrinol 2021: 4654302, 2021.
36. Shi Z, Tao H, Fan Z, Song S and Bai J: Prognostic and immuno- logical role of key genes of ferroptosis in pan-cancer. Front Cell Dev Biol 9: 748925, 2021.
37. Tang R, Xu J, Zhang B, Liu J, Liang C, Hua J, Meng Q, Yu X and Shi S: Ferroptosis, necroptosis, and pyroptosis in anticancer immunity. J Hematol Oncol 13: 110, 2020.
38. Yamaguchi H, Hsu JL, Chen CT, Wang YN, Hsu MC, Chang SS, Du Y, Ko HW, Herbst R and Hung MC: Caspase-independent cell death is involved in the negative effect of EGF receptor inhibitors on cisplatin in non-small cell lung cancer cells. Clin Cancer Res 19: 845-854, 2013.
39. Belavgeni A, Bornstein SR, von Mässenhausen A, Tonnus W, Stumpf J, Meyer C, Othmar E, Latk M, Kanczkowski W, Kroiss M, et al: Exquisite sensitivity of adrenocortical carcinomas to induction of ferroptosis. Proc Natl Acad Sci USA 116: 22269-22274, 2019.
40. Wu S, Li T, Liu W and Huang Y: Ferroptosis and cancer: Complex relationship and potential application of exosomes. Front Cell Dev Biol 9: 733751, 2021.
41. Linher-Melville K and Singh G: The complex roles of STAT3 and STAT5 in maintaining redox balance: Lessons from STAT-mediated xCT expression in cancer cells. Mol Cell Endocrinol 451: 40-52, 2017.
CC
BY NO ND This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) License.