IFI6 is an effective therapeutic target in esophageal squamous cell carcinoma via the modulation of oxidative stress

Background: Esophageal squamous cell carcinoma (ESCC) is one of the most lethal forms of adult cancer with poor prognosis. Substantial evidence indicates that reactive oxygen species (ROS) are important modulators of aggressive cancer behavior. However, the mechanism by which ESCC cells integrate redox signals to modulate carcinoma progression remains elusive. Methods: The expression of interferon alpha inducible protein 6 (IFI6) in clinical ESCC tissues and cell lines was detected by RT-PCR and Western blotting. The correlation between IFI6 expression levels and aggressive ESCC disease stage was examined by immunohistochemistry. Bioinformatic analysis was conducted to explore the potential function of IFI6 in ESCC. ESCC cell lines stably depleted of IFI6 and ectopically expressing IFI6 were established using lentiviruses expressing shRNAs and an IFI6 expression plasmid, respectively. The effects of IFI6 on ESCC cells were determined by cell-based analyses, including EdU assay, apoptotic assay, cellular and mitochondria-specic ROS detection, seahorse extracellular ux, and mitochondrial calcium ux assays. Blue native-polyacrylamide gel electrophoresis was used to determine mitochondrial supercomplex assembly. Transcriptional activation of NADPH oxidase 4 (NOX4) via ATF3 was conrmed by dual luciferase assay. In vivo tumor growth was determined in mouse xenograft models. Results: We nd that the expression of IFI6, an IFN-stimulated gene localized in the inner mitochondrial membrane, is markedly elevated in ESCC patients and a panel of ESCC cell lines. High IFI6 expression correlates with aggressive disease phenotype and poor prognosis in ESCC patients. IFI6 depletion suppresses proliferation and induces apoptosis by increasing ROS accumulation. Mechanistically, IFI6 ablation induces mitochondrial calcium overload by activating mitochondrial Ca 2+ uniporter and subsequently ROS production. Following IFI6 ablation, mitochondrial ROS accumulation is also induced by mitochondrial supercomplex assembly suppression and oxidative phosphorylation dysfunction, while uptake, which in turn led to mitochondrial calcium overload and partially promoted the accumulation of mitochondrial ROS. In addition, our rescue experiments showed that IFI6 silencing elevated ER-derived ROS accumulation by driving ER stress accompanied by a substantial increase in ATF3 expression and subsequent transcriptional activation of NOX4. We also conrmed that the function of SERCA, an ER located ATP-dependent Ca 2+ pump, was modulated by ATP production and mitochondrial OXPHOS eciency resulting from altered expression of IFI6. The induction of ER stress and concomitant upregulation of ATF3 following IFI6 silencing is possibly mediated by the decreased ER Ca 2+ pools due to the reduced activity of SERCA. Finally, via knockdown and overexpression experiments, we validated the antiproliferative effect of IFI6 depletion in a nude mouse model of ESCC. Collectively, these observations imply the potential therapeutic value of IFI6 inhibition in ESCC. cells were co-transfected with plasmids containing different NOX4 promoter constructs with or without the ATF3 expression plasmid. NOX4 transcriptional activity was measured via a dual luciferase reporter assay. The data are presented as the means and SDs (n=3). Statistical signicance was determined by two-tailed Student’s t-test. ***P<0.005. K. A dual luciferase reporter assay was used to assess NOX4 transcriptional activity in HEK293T cells cotransfected with pGL3-NOX4-WT, pGL3-NOX4-Mut #1 (AAGGACTCACT), pGL3-NOX4-Mut #2 (ACTAATGTCATG), pGL3-NOX4-Mut #3 (TATGAAGACATTT) or pGL3-NOX4-Mut #4 (AATTGCATCACC) constructs with or without the ATF3 expression plasmid. The data are presented as the means and SDs (n=3). Statistical signicance was determined by two-tailed Student’s t-test. **P<0.01.

understanding of its biological functions is limited, IFI6 was characterized as a proliferative and antiapoptotic factor [21,23]. Furthermore, IFI6 was suggested to facilitate breast cancer metastasis by modulating mitochondrial ROS production [24]. However, both the biological role of IFI6 and the mechanism underlying IFI6-mediated effects in ESCC are unknown.
In this study, we rst examined the abundance of IFI6 in ESCC tissues. We showed that IFI6 contributed to ESCC cell proliferation and survival by modulating redox homeostasis. We further established that IFI6 downregulation led to mitochondrial Ca 2+ uniporter (MCU) mediated mitochondrial calcium overload, and inhibited mitochondrial supercomplex assembly and suppressed the respiratory phosphorylation e ciency, which ultimately elevated mitochondrial ROS production. Finally, we found that IFI6 inhibition induced ATP deprivation-mediated ER stress, which in turn elevated NOX4 expression in an ATF3dependent manner and enhanced ROS production. Therefore, disrupting IFI6 is a promising therapeutic strategy for ESCC.

Methods
Reagents and antibodies. Human study subjects and cell cultures.
A total of 23 fresh ESCC samples and paired adjacent normal tissues (PANTs) were collected from patients undergoing esophagectomy at Shanghai Tongji Hospital A liated with Tongji University. Immunohistochemistry (IHC) for IFI6 was conducted on 3-μm sections of formalin-xed, para nembedded tissue samples consisting of nonpathological esophageal tissues (8 specimens), esophageal hyperplasia tissues (12 specimens) and ESCC tissues (83 specimens). The clinicopathological characteristics of the subjects are summarized in Tables S1 and S3. Informed consent was obtained from each patient before enrollment in this study. This study was approved by the Medical Ethics Committee of Shanghai Tongji Hospital.
The human ESCC cell lines Eca109, TE-1, Ec9706, Kyse150, and Kyse410 and the normal esophageal squamous epithelial cell line Het-1a were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultivated in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin and streptomycin at 37 °C in 5% CO 2 . All cell lines were authenticated by the STR method and tested for mycoplasma contamination.
In vivo study.
Four-to ve-week-old female BALB/c nude mice were purchased from Jiesijie Laboratory Animal (Shanghai, China). All animal experiments were approved by the Animal Care and Use Committee of Shanghai Tongji Hospital and conducted in accordance with ethical standards. Nude mice were randomly divided into four groups as follows (n=5 mice/group): (i) ShControl; (ii) IFI6 KD; (iii) OEControl; and (iv) IFI6 OE. For in vivo model exploring the effect of MCU inhibition on tumor growth, nude mice were randomly divided into three groups as follows (n=5 mice/group): (i) ShControl; (ii) IFI6 KD; (iii) IFI6 KD+DS16570511 (DS16570511 (1mg/kg) was administered to mice intraperitoneally every week for four weeks). For in vivo model exploring the effect of OXPHOS inhibition on tumor growth, nude mice were randomly divided into three groups as follows (n=5 mice/group): (i) OEControl; (ii) IFI6 OE; (iii) IFI6 OE+ Phenformin (Phenformin (500mg/kg) was administered to mice intraperitoneally every other day for four weeks).
The indicated Eca109 cells (2 × 10 6 cells per 0.1 ml of phosphate-buffered saline) were suspended in Matrigel (Biosciences, CA, USA) and implanted subcutaneously into the right anks of the nude mice. The tumor volume was measured using a caliper every 5 days. Four weeks after implantation, the nude mice were sacri ced, and tumor samples were harvested.
RNA extraction and qRT-PCR.
One microgram of total RNA was extracted using TRIzol obtained from Invitrogen (CA, USA) and was reverse transcribed to cDNA with SuperScript II (Invitrogen, CA, USA). qRT-PCR was conducted using SYBR Green technology (Takara Bio, Beijing, China). β-Actin was used as the housekeeping gene. Data were analyzed through the 2 -ΔCT method. The primers used for detection are shown in Table S4. Immunoblotting.
Cells and tissues were collected and lysed in cell/tissue lysis buffer (Solarbio, Beijing, China). Proteins were extracted, and the protein concentration was measured by the BCA method (Solarbio, Beijing, China). Proteins were separated by 10% sodium dodecyl sulfate (SDS)-PAGE and transferred to 0.45-μm polyvinylidene di uoride membranes (Invitrogen, CA, USA), which were blocked with blocking buffer (EpiZyme, Shanghai, China), incubated overnight with primary antibodies, and washed and incubated with the HRP-conjugated anti-rabbit secondary antibody (Abcam, UK) for 1 h. Peroxidase activity was visualized via the ECL method (EpiZyme, Shanghai, China). Immunohistochemistry.
Para n-embedded sections were depara nized and rehydrated and were then subjected to antigen retrieval for 20 min in citrate buffer (pH 6.0). After the sections were cooled, 3% H 2 O 2 was used to block endogenous peroxidases. The sections were blocked with 10% goat serum and incubated with the anti-IFI6 primary antibody (Invitrogen, Carlsbad, CA, USA) at a 1:1000 dilution overnight at 4 °C. After incubation with the secondary antibody, immunodetection was performed with DAB staining (Zhongshan Goldenbridge Biotechnology Company, Beijing, China). For evaluation, three staining elds in each section were randomly selected and evaluated by two independent pathologists who were blinded to patient information. The (Staining index) SI of IFI6 was determined by combining the staining intensity score (1, negative; 2, weak staining; 3, moderate staining; 4, strong staining) and the proportion of positively stained tumor cells (0, no positive cells; 1, < 10%; 2, 10%-35%; 3, 35%-75%; 4, > 75%). Samples with SI ≥ 8 were considered to exhibit high expression, samples with SI 3-7 were regarded as medium expression, while samples with SI < 3 were deemed to be low expression samples.

BN-PAGE.
Mitochondria were isolated from cells with a mitochondria extraction kit (Solarbio, Beijing, China) according to the manufacturer's instructions. BN-PAGE was performed with the NativePAGE TM system (Invitrogen, CA, USA). In brief, mitochondria were solubilized with digitonin (digitonin/protein ratio of 4 g g −1 ), incubated on ice for 30 min, and centrifuged at 20000 × g for 30 min. The solubilized protein was collected from the supernatant, and the protein concentration was determined by the BCA (Solarbio, Beijing, China) method. Protein samples were mixed with Coomassie blue G-250 (Invitrogen, CA, USA) to obtain a dye/detergent mass ratio of 1/4 and were then loaded into a 4-16% Bis-Tris gel. After electrophoresis, proteins were transferred to 0.45-μm polyvinylidene di uoride membranes (Invitrogen, CA, USA) and were then sequentially probed with antibodies against complex I (NDUFB8), complex III (RISP), complex IV (COX IV), complex II (SDHA) and complex V (ATPB). The NDUFB8, RISP and COX IV immunoblot bands with high molecular weights were used to identify the complex I-, complex III-, or complex IV-containing respiratory supercomplexes (RSCs). Signal intensities were normalized to their corresponding ATPB signals.
Cell proliferation assay.
To assess cell proliferation, the indicated Eca109 and TE-1 cells were seeded in 96-well plates and incubated under standard conditions in complete medium. Twenty-four hours after seeding, cells were incubated with 50 μM EdU (RiboBio, Guangzhou, China) for 6 h and subsequently subjected to xation, permeabilization and EdU staining, which were conducted according to the manufacturer's instructions.
Nuclei were counterstained with Hoechst 33342 (Invitrogen, CA, USA) for 10 min. The proportion of cells stained with EdU was determined via uorescence microscopy.
Detection of the mitochondrial membrane potential and apoptosis induction.
The indicated Eca109 and TE-1 cells were seeded in 6-well plates and treated as indicated. Then, the cells were detached, washed, double-stained with Annexin V-FITC and PI (BD Biosciences, CA, USA) based on the manufacturer's recommendation, and analyzed by ow cytometry (Beckman Coulter, CA, USA). FlowJo (Tree Star, OR, USA) was used for data presentation and analysis.
The mitochondrial membrane potential was assessed via the JC-1 method. In brief, the indicated cells were washed and incubated with JC-1 (Beyotime, Beijing, China) working solutions for 20 min at 37 °C in the dark. After incubation, the cells were washed with JC-1 wash buffer and observed under a uorescence microscope. In healthy cells with a Δψm indicating membrane depolarization, JC-1 forms JC-1 aggregates exhibiting red uorescence. In apoptotic cells, JC-1 remains monomeric, exhibiting green uorescence.
Measurement of ATP levels.
To assess cellular ATP production, an ATP luminescence assay (Invitrogen, CA, USA) was used for quantitative determination of ATP levels with recombinant re y luciferase and its substrate D-luciferin according to the manufacturer's recommendation. After a standard curve for a series of ATP concentrations was generated, the ATP content in the samples was calculated from the standard curve.
Analysis of cellular respiration.
The Seahorse XFe96 Bioanalyzer (Seahorse Bioscience, MA, USA) was used to determine the OCR and ECAR according to the manufacturer's instructions. ECAR and OCR were determined using the Seahorse XF Glycolysis Stress Test Kit (Seahorse Bioscience, MA, USA) and the Seahorse XF Cell Mito Stress Test Kit (Seahorse Bioscience, MA, USA), respectively. In brief, for OCR determination, 20000 of the indicated ESCC cells were seeded in a Seahorse 96-well plate, and 1 h before measurement, the culture medium was replaced with Seahorse XF medium supplemented with 1 mM sodium pyruvate, 2 mM glutamate, and 25 mM glucose. Periodic measurements of the OCR were performed at baseline and after sequential administration of oligomycin (Oligo, 1 μM), tri uoromethoxy carbonylcyanide phenylhydrazone (FCCP, 0.5 μM), and rotenone (Rot, 0.5 μM) and antimycin A (AMA, 0.5 μM) (Rot&AMA). For ECAR measurement, the culture medium was replaced with XF assay medium supplemented with L-glutamine (300 µg/ml).
The oligonucleotide sequences used to silence target genes are shown in Supplementary Table S5. IFI6, ATF3, and NOX4 lentiviruses were purchased from Genechem (Shanghai, China).
To monitor mitochondrial calcium dynamics, the indicated ESCC cells were incubated with the calciumspeci c uorescent probe Rhod-2 AM (Invitrogen, CA, USA) in Hanks' balanced salt solution at room temperature for 30 min. After washout, uorescence was recorded with an integrated spectro uorometer (Photon Technology International, NJ, USA) at excitation and emission wavelengths of 550 nm and 580 nm, respectively. After 1 min of baseline recording (basal signal, F 0 ), cells were promptly treated with the agonist Tg, and uorescence intensities were monitored at 3-s intervals for another 10 min. The relative uorescence intensity (F/F 0 ), where F represents the uorescence intensity at a given time and F 0 represents the initial uorescence intensity, was used to indicate [Ca 2+ ].
For determination of Ca 2+ concentration in endoplasmic reticulum, Eca109 and TE-1 cells were transfected with low a nity aequorin construct targeted to the ER (erAEQ) [25]. Cells expressing erAEQ were reconstituted in its active form with 1 μM coelenterazine by incubation for 1 h at room temperature in Ca 2+ -free medium containing 0.5 mM EGTA. Cells were washed with Krebs Ringer Buffer (KRB: 125 mM NaCl, 5 mM KCl, 400 mM KH 2 PO 4 , 1 mM MgSO 4 , 20 mM HEPES, pH 7.4) supplemented with 2% bovine serum albumin (Sigma) and 1mM EGTA and were then placed in KRB supplemented with 5 mM glucose and 75 mM EGTA and luminescence measurement was started. After 60 s, 1mM CaCl 2 at the nal concentration was injected to re ll the ER lumen. The measurement was carried out with an integrated spectro uorometer (Photon Technology International, NJ, USA) 3-s intervals for 800 s.
Detection of cellular and mitochondria-speci c ROS determination.
To detect mitochondrial ROS, the indicated ESCC cells were seeded in 24-well plates and incubated with the mitochondria-speci c ROS probe MitoSOX (Invitrogen, CA, USA) for 20 min at 37 °C. For cellular ROS determination, the indicated ESCC cells were seeded in 24-well plates, loaded with the cellular ROS indicator carboxy-H 2 DCFDA and further incubated at 37 °C for 20 min. Nuclei were counterstained with Hoechst 33342, and the uorescence intensity was measured by uorescence microscopy or ow cytometry.
Construction of reporter plasmids and luciferase assay.
Fragments of the human ATF3, PDI and XBP1s were synthesized and subcloned into the pGL3 vector (Promega, WI, USA). Furthermore, different fragments (containing bp -3000 to +1, -2000 to +1, or -1000 to +1) of the human NOX4 promoter were synthesized and subcloned into the pGL3 vector (Promega, WI, USA). Another set of mutant plasmids was constructed; in these plasmids, one of the potential ATF3binding motifs (AAGGACTCACT, ACTAATGTCATG, TATGAAGACATTT, or AATTGCATCACC) was deleted from the NOX4 promoter.
The above promoter reporters were transfected into Eca109, TE-1, or HEK293T cells or were cotransfected with the ATF3-pcDNA3.1 expression vector. The indicated cells were harvested, and the promoter activity of NOX4 was measured with a dual luciferase reporter assay kit (Promega, WI, USA) according to the manufacturer's instructions. In brief, the indicated cells were seeded in 24-well plates, transfected with reporter plasmids using Lipofectamine 3000 (Invitrogen, CA, USA) and incubated for 24 h. After transfection, cells were collected with passive buffer, and re y and Renilla luciferase activities were measured with a Dual Luciferase Reporter Assay System (Promega, WI, USA) and an illuminometer.
RNA-seq expression data (quanti ed by count and fragments per kilobase per million mapped reads (FPKM)) in the TCGA-ESCA dataset were downloaded from the Genomic Data Commons (GDC) portal (https://portal.gdc.cancer.gov/). TCGA dataset manipulation and analysis as well as GSEA were implemented in R 3.6.0.
For microarray data analysis, normalized data (GEO accession no. GSE20347, no. GSE23400, no. GSE45670 and no. GSE75241) and the corresponding probe annotation data were downloaded from the NCBI GEO database (https://www.ncbi.nlm.nih.gov/gds). Microarray data manipulation and analysis were performed in R 3.6.0.
IFI6 coexpression analysis was based on calculation of the Pearson correlation coe cient (r PCC ) values of the correlations between the expression levels of IFI6 and potential mRNAs in the four above GEO datasets. mRNAs for which |r PCC |>0.5 and P < 0.05 were recommended for further analysis (Table S6).
Survival analysis of ESCC patients in TCGA database were carried out with web-based analysis tool Kaplan-Meier Plotter (http://www.kmplot.com).

Results
IFI6 expression is elevated in ESCC and correlated with disease stages.
Initially, we analyzed raw microarray data of ESCC patients from several subsets of Gene Expression Omnibus (GEO) datasets by a bioinformatic approach and found that IFI6 expression was dramatically upregulated in ESCC tissues compared with non-tumorous esophageal tissues ( Figure 1A). We also validated these data by assessing IFI6 expression in 23 ESCC samples and corresponding normal esophageal tissues from patients in our hospital ( Figure 1B). Given that ESCC is heterogenous both genetically and in its clinical manifestation, we further explored the IFI6 expression pro le across a series of genetically distinct ESCC cell lines. As expected, IFI6 expression patterns were heterogeneous; however, IFI6 expression was higher in all ve ESCC cell lines tested than in the normal esophageal squamous epithelial cell lines ( Figure 1C). The high levels of IFI6 expression were further con rmed at the protein level via Western blot analysis ( Figure 1D).
To explore the clinical relevance of IFI6, we assessed the IFI6 abundance in non-cancerous esophageal tissues and in samples with progressively aggressive characteristics: esophageal hyperplasia, esophageal carcinoma in situ, low-grade ESCC, moderate-grade ESCC, and high-grade ESCC using IHC.
The image in Figure 1E shows that IFI6 staining is absent in the healthy esophageal epidermis but starts to increase in esophageal hyperplasia and remains considerably high at more advanced ESCC stages. Consistent with this observation, statistical analysis revealed that aberrant IFI6 protein levels were signi cantly correlated with tumor grade, the depth of invasion, TNM stage (Table S1). Moreover, we categorized our 83 cases of ESCC cohort into IFI6-High, IFI6-Medium and IFI6-Low groups and then analyzed the survival probability of each group via Kaplan-Meier and log-rank analyses. As shown in Figure 1F, high IFI6 expression predicted an unfavorable prognosis (IFI6-High vs. IFI6-Medium, P log rank =0.036; IFI6-Medium v.s IFI6-Low, P log rank =0.012; P log rank trend <0.001). The univariate and multivariate Cox regression analyses demonstrated that IFI6 expression was an independent prognostic factor in ESCC (Table S2). Ultimately, we analyzed the survival probability of patients categorized as IFI6-high versus patients categorized as IFI6-low in the TCGA-ESCA dataset through the web-based tool Kaplan-Meier Plotter (www.kmplot.com). Compared with patients categorized as IFI6-low, patients categorized as IFI6-high exhibited a substantially reduced survival probability ( Figure S1A). These data support a potential functional role of IFI6 in ESCC carcinogenesis and development.
Bioinformatic analyses indicate the potential role of IFI6 in ESCC is associated cell proliferation, apoptosis and ROS production.
Given that the in-depth characterization of its function in ESCC has not been investigated, we next sought to explore the potential function of IFI6 in more detail. We initially analyzed genes coexpressed with IFI6 in the abovementioned four GEO ESCC microarray datasets. According to the screening criteria (|r PCC | ≥0.5, P-value<0.05), 167 genes were coexpressed with IFI6 in all four of the above datasets (Figure 2A, B). To broadly consider the cellular pathways in which IFI6 might play a role, we performed Gene Ontology (GO) enrichment analysis via the Database for Annotation, Visualization, and Integrated Discovery (DAVID) tool (https://david.ncifcrf.gov) for the 167 coexpressed genes in terms of biological process (BP). Genes whose expression correlated with IFI6 were enriched in the following BP terms: response to starvation, response to reactive oxygen species, cell proliferation, regulation of apoptotic process, response to oxidative stress, and response to ER stress ( Figure 2C). To further consolidate the above GO analysis results, we grouped ESCC patients from The Cancer Genome Atlas (TCGA) database (TCGA-ESCA dataset) based on their IFI6 expression level. Gene set enrichment analysis (GSEA) demonstrated that high IFI6 levels led to upregulation of gene sets related to cell proliferation and negative regulation of apoptosis ( Figure 2D, E). In addition, patients with higher IFI6 levels exhibited lower levels of genes related to the oxidative stress response ( Figure 2F, G).
Collectively, the results of these bioinformatic analyses led us to hypothesize that IFI6 can promote proliferation, inhibit apoptosis and ameliorate oxidative stress.
IFI6 silencing inhibits cell growth, induces apoptosis through cellular ROS accumulation.
To con rm the above bioinformatic analysis results, we chose Eca109 and TE-1 as our cellular models. IFI6-shRNA was transfected into ESCC cell lines. We also constructed stable ESCC cell lines ectopically expressing IFI6 as well as OEControl cell lines. Silencing as well as overexpression e ciencies were validated via qRT-PCR and Western blot assays (Figure S1B-E). Initially, an EdU assay was conducted, which indicated that cell growth, assessed as the percentage of EdU-positive cells, was signi cantly inhibited in IFI6-silenced ESCC cells compared with ShControl ESCC cells ( Figure 3A, B). To further demonstrate whether apoptosis could also play a role in the reduction in cell viability implied by the aforementioned bioinformatic analysis results ( Figure 2C, E), we next conducted annexin V-FITC/propidium iodide (PI) staining. Consistent with the EdU assay results, IFI6 inhibition signi cantly induced ESCC cell apoptosis ( Figure 3C). In the reciprocal experiment, cell proliferation markedly increased by IFI6 overexpression, whereas ESCC cell apoptosis was signi cantly inhibited following IFI6 overexpression ( Figure S2A-C).
We next examined the mechanism underlying the reduction in cell viability. Because IFI6 can ameliorate oxidative stress, as revealed by the above bioinformatic analysis results ( Figure 2C, F, G), we then investigated cellular ROS levels following IFI6 silencing. IFI6 inhibition signi cantly enhanced the generation of cellular ROS, as represented by the live cell ROS indicator carboxy-H 2 DCFDA ( Figure 3D), and IFI6 overexpression exhibited the opposite effect ( Figure S2D). Accordingly, we sought to determine whether the augmented cellular oxidative environment caused by altered IFI6 expression is responsible for the reduction in cell viability. To test this hypothesis, we monitored the growth of IFI6-silenced Eca109 cells in the absence or presence of a reducing agent (dithiothreitol, DDT) (2 mM), the hydrogen peroxide scavenger (PEG-catalase, CAT) (100 U/ml) and an antioxidant (N-acetyl-L-cysteine, NAC) (200 μM). Treatment with all of these ROS-eliminating reagents indeed decreased the level of carboxy-H 2 DCFDA staining ( Figure 3E, F). Moreover, as shown in Figure 4A, all these ROS-eliminating reagents effectively reversed the effect of IFI6 silencing on cell viability. Our ndings were corroborated in TE-1 cells ( Figure  S3A). To further explore the effect of ROS on IFI6-mediated cell survival, we used the mitochondrial membrane potential indicator JC-1, an alternative method to detect mitochondria-mediated apoptosis as well as mitochondrial dysfunction. Consistent with the EdU assay results, treatment of ESCC cells with ROS inhibitors completely reversed IFI6 silencing-induced apoptosis ( Figure 4B, Figure S3B), which supported the role of ROS in the IFI6 silencing-induced reduction in cell viability.
IFI6 modulates mitochondrial ROS production partially by regulating calcium in ux through regulating the activity of mitochondrial Ca 2+ uniporter.
We next sought to understand how IFI6 controls ROS production and therefore tumor cell viability. We reasoned that this regulatory mechanism is likely to be related to mitochondria, given that mitochondrial respiration is the major source of cellular ROS. To explore the role of mitochondria in ROS generation after alterations in IFI6 expression, we used the mitochondrial ROS-speci c probe MitoSOX to detect mitochondrial ROS production in Eca109 and TE-1 cells. IFI6 silencing led to an elevation in mitochondrial ROS levels, whereas mitochondrial ROS production was signi cantly inhibited in cells overexpressing IFI6 ( Figure 5A, B). The above observations indicated that the expression level of IFI6 directly affects mitochondrial ROS production. As reported, mitochondrial Ca 2+ signaling profoundly in uences mitochondrial OXPHOS and, therefore, ROS generation [26]. We next sought to determine whether IFI6 impacts calcium dynamics and thus mitochondrial ROS production. To this end, we observed the effect of thapsigargin (Tg), a plant-derived sesquiterpene lactone that increases the cytosolic calcium concentration by inhibiting ER-resident ATP-dependent calcium pumps and activating store-operated Ca 2+ entry (SOCE) channels [27], on mitochondrial calcium dynamics. By using the mitochondrial calcium indicator Rhod-2 AM, we showed that IFI6-silenced ESCC cells exhibited substantially increased calcium uptake following the Tg-mediated increase in the cytosolic calcium concentration ( Figure 5C, D). Previous studies showed that calcium in ux into mitochondria leads to mitochondrial dysfunction and contributes to ROS generation as well as pathological induction of cell death [28,29]. We next sought to determine whether inhibiting calcium uptake by mitochondria would reverse IFI6 silencing-induced mitochondrial ROS production. However, suppression of mitochondrial Ca 2+ uptake by Ca 2+ chelators with 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) only partially reversed the IFI6 silencing-induced mitochondrial ROS generation ( Figure 5E). By removing Ca 2+ from the cell culture medium, we obtained similar results ( Figure 5F). Based on the above ndings, we sought to determine whether the abundance of IFI6 might modulate the expression of mitochondrial calcium channels. For this purpose, we evaluated the expression levels of voltage-dependent anion channel type 1 (VDAC1), mitochondrial Ca 2+ uniporter (MCU) and mitochondrial sodium calcium lithium exchanger (NCLX) by Western blotting and RT-PCR. However, as indicated in FigureS4A-B, no overt alteration of VDAC1, MCU, and NCLX expression occurred in TE-1 and Eca109 cells after the downregulation of IFI6. To investigate this phenomenon in more detail, we also sought to determine whether the redox status of the mitochondrial MCU complex plays a role in mitochondrial calcium uptake [30]. To this end, we used the mitochondria-targeted ROS scavenger MitoTEMPO (20 μM) to evaluate the effect of redox status on mitochondrial calcium uptake in parental and IFI6-silenced cells. As shown in Figure 5G-H, treatment with this mitochondrial ROS scavenger completely reversed the IFI6 silencinginduced increase in mitochondrial Ca 2+ uptake. Furthermore, following IFI6 ablation, pharmacological inhibition of MCU with DS16570511 partially prevented IFI6 silencing-induced mitochondrial ROS generation ( Figure 5I).
Taken together, these observations imply that after IFI6 knockdown, the increased level of mitochondrial ROS may disrupt the redox regulation of the mitochondrial MCU, which in turn leads to mitochondrial calcium overload and, subsequently, mitochondrial ROS accumulation. Given the fact that blocking mitochondrial Ca 2+ only partially attenuated IF6 silencing-induced mitochondrial ROS production, mitochondrial Ca 2+ in ux disruption plays a role in, but may not be the only cause of, elevated mitochondrial ROS production. The driving contributor to mitochondrial ROS generation following IFI6 ablation remains to be explored.
IFI6 regulates mitochondrial ATP production and mitochondrial oxidative phosphorylation.
Several lines of evidence have demonstrated that the ETC during OXPHOS constitutes the principal source of mitochondrial ROS production and that mitochondrial supercomplex formation can modulate this process [31]. Consistent with these observations, the results of our GSEA of TCGA data implied a state of energy deprivation ( Figure 6A). To clarify the alternative reason why mitochondrial ROS production was increased following IFI6 knockdown, we measured ATP levels in ESCC cell lines after IFI6 silencing or overexpression and compared them with those in the corresponding parental cell lines.
Eca109 and TE-1 cells ectopically expressing IFI6 exhibited higher ATP levels than the corresponding control cells, while cells with IFI6 silencing had lower ATP levels than the corresponding control cells ( Figure 6B).
To further assess the effect of IFI6 on cellular bioenergetics, we conducted extracellular ux analysis to assess the extracellular acidi cation rate (ECAR) as well as the oxygen consumption rate (OCR), which are assumed to be representative indicators of lactic acid generation and mitochondrial OXPHOS, respectively. Accordingly, we measured the OCR of ESCC cells in a Seahorse Bioanalyzer with the sequential addition of several mitochondrial stressors, such as Oligo, FCCP, Rot and AMA. As shown in Figure 6C, the basal cellular OCR and maximum respiration rate were signi cantly elevated in IFI6overexpressing Eca109 cells compared with parental Eca109 cells. On the other hand, IFI6 silencing via shRNA decreased both the resting OCR and the maximum OCR in Eca109 cells ( Figure 6C). Similar results were also observed in TE-1 cells ( Figure S5A). However, there was no overt difference in ECAR between ESCC cells ectopically expressing IFI6 and parental ESCC cells (Figure6D, Figure S5B).
To further understand the mechanism underlying these alterations in mitochondrial energy and ROS generation, we measured the OCR in the presence of substrates for complex I (pyruvate, malate), complex II (succinate), complex III (glyceraldehyde 3-phosphate, G3P) or complex IV (N,N,N',N'-tetramethyl-1,4phenylenediamine (TMPD)/ascorbate). The complex I-speci c OCR in IFI6-silenced cells was substantially reduced compared with that in parental cells; in contrast, IFI6 overexpression signi cantly increased the complex I-speci c OCR in both Eca109 and TE-1 cells ( Figure 6E, Figure S5C). In addition, the OCRs speci c to complexes III and IV exhibited similar patterns ( Figure 6F-G, Figure S5D-E). However, in Eca109 and TE-1 cells, the OCR supported by complex II remained unchanged ( Figure 6G, Figure S5E), which implied that IFI6 might alter respiratory complex function at multiple sites (namely, complex I, complex III and complex IV).
These observations con rmed that altered IFI6 expression levels might play a role in energy handling and mitochondrial dysfunction.
IFI6 regulates the mitochondrial redox status by modulating respiration supercomplex assembly.
Previous studies reported that mitochondrial ROS production is closely interlinked with OXPHOS, whereas impaired respiratory complex function and respirasome formation enhance ROS production and impair ATP generation [32,33]. We further assessed the contribution of IFI6 to OXPHOS complex assembly via blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by immunoblotting. We used antibodies against NDUFB8 (complex I), SDHA (complex II), RISP (complex III), COXIV (complex IV) and ATPB (complex V) to detect respiratory complexes as well as respiratory supercomplexes (RSCs) in mitochondrial preparations from Eca109 and TE-1 cells. Consistent with previous studies, in all groups tested, complex II and complex V existed mainly as individual entities, whereas complexes I, III and IV were assembled into supercomplexes ( Figure 7A, Figure S6A). Moreover, silencing IFI6 decreased the signal of NDUFB8, RISP or COXIV at the CI+CIII 2 +CIV n and CI+CIII 2 positions compared with those in parental ESCC cells. The signals of CI+CIII 2 +CIV n and CI+CIII 2 RSCs were elevated in ESCC cells ectopically expressing IFI6 compared to parental cells ( Figure 7B, Figure S6B). In addition, IFI6 ablation resulted in a decreased signal at the CIII 2 +CIV n position, while overexpressing IFI6 had the opposite effect, as detected by immunoblotting with anti-RISP and anti-COXIV antibodies ( Figure 7B, Figure S6B ). However, no consistent changes were observed in the levels of complex III dimers or complex IV monomers following alteration of IFI6 expression in either Eca109 or TE-1 cells ( Figure 7C, Figure S6C).
These observations were consistent with those of the aforementioned extracellular ux analysis. We further assessed the abundance of individual respiratory complexes and showed that the expression level of IFI6 did not affect the expression of individual respiratory complexes, as evaluated by NDUFB8 (complex I), SDHA (complex II), RISP (complex III), COXIV (complex IV) and ATPB (complex V) expression ( Figure 7D, E), indicating that the changes in mitochondrial supercomplex formation are not due to alterations in the expression of individual OXPHOS complex subunits. Taken together, these observations indicate the relevance of IFI6 in the assembly of RSCs as well as CIII 2 +CIV n in ESCC.
Collectively, our data suggested two causes of elevated mitochondrial ROS following IFI6 silencing: rst and the driving cause, impaired respiratory complex function and supercomplex assembly, which decreases the e ciency of OXPHOS and in its turn accumulates mitochondrial ROS; Secondly, the altered redox state of MCU induces mitochondrial Ca 2+ overload and subsequently adds to mitochondrial ROS production.
IFI6 depletion-mediated ATP shortage activates ER stress and subsequent cellular ROS generation through the ATF3-NOX4 axis.
We next sought to rule out a role for alternative mechanisms in the increase in cellular ROS accumulation in addition to mitochondrial ROS production following IFI6 silencing in ESCC cells. For this purpose, we used carboxy-H 2 DCFDA to measure cellular ROS levels in control and IFI6-silenced cells. As expected, IFI6 depletion led to a dramatic increase in cellular ROS levels. Intriguingly, IFI6-KD ESCC cells treated with MitoTEMPO (20 μM), a mitochondria-speci c antioxidant, exhibited only a partial reversal of IFI6 silencing-induced cellular ROS generation compared with vector-treated ESCC cells ( Figure 8A, B).
However, the combination treatment with MitoTEMPO and exogenous ATP led to a signi cantly greater decrease in cellular ROS levels than each compound alone ( Figure 8A, B), implying that in addition to mitochondria, IFI6 silencing induces ROS production via another source and that supplying cells with exogenous ATP could block this pathway.
In most cells, mitochondria and the activities of NADPH oxidases (NOXs), which are located mainly in membranes such as the ER membrane, are the two primary sources of cellular ROS generation [34,35].
We next analyzed the expression levels of the ve different NOX isoforms in ESCC cells after IFI6 knockdown. Among a panel of NOX isoforms, NOX4 was substantially upregulated following IFI6 silencing in Eca109 and TE-1 cells ( Figure 8C). To validate these observations via functional analysis, we examined the role of NOX4 in the production of cellular ROS in IFI6-silenced ESCC cells using carboxy-H 2 DCFDA. As shown previously, cellular ROS levels increased after IFI6 ablation, while this increase was mitigated after treatment with NOX4 shRNA. Intriguingly, treatment of ESCC cells with either NOX4 shRNA alone or exogenous ATP substantially suppressed ROS production, while the combination of these two factors did not further decrease cellular ROS levels in IFI6-silenced ESCC cells. Furthermore, the exogenous ATP-induced decrease in cellular ROS was reversed by ectopic NOX4 expression in IFI6silenced ESCC cells ( Figure 8D). These patterns suggest that mitochondrial dysfunction and an ATP shortage might contribute to cellular ROS production mediated by induction of NOX4 overexpression.
To explain the mechanism by which ATP deprivation leads to this substantial increase in NOX4 levels, we reasoned that this increase is likely to be correlated with induction of ER stress. Previous studies showed that ER stress can be induced by various means, including ATP deprivation [36]. Consistent with this observation, our aforementioned GSEA of TCGA data also revealed a state of energy deprivation as well ER stress induction ( Figure 8E, Figure 6A). Therefore, we tested this hypothesis by measuring the expression levels of several ER stress markers, such as the transcription factors ATF3, ATF4, ATF6, and Xbox binding protein 1 (XBP1s), as well as protein disul de isomerase (PDI) binding immunoglobulin protein (BiP), following IFI6 silencing via PCR and Western blotting. We demonstrated consistent activation of ER stress in IFI6-silenced cells and concomitant elevation of NOX4 mRNA levels in Eca109 cells. This phenomenon was corroborated in TE-1 cells ( Figure 8F). Among these ER stress mediators, the transcription factor ATF3 exhibited the most dramatic elevation following IFI6 knockdown, implying that ATF3 might play a pivotal role in IFI6 silencing-mediated ER stress. To further characterize the molecular mechanism underlying overexpression of NOX4 elicited by ER stress following energy shortage, we treated IFI6-silenced ESCC cells with or without exogenous ATP and found that ATP fully rescued the increases in ATF3 and NOX4 expression induced by IFI6 silencing ( Figure 8G). These results indicate that IFI6 silencing is the initial event and is followed by ATP shortage and ER stress induction with subsequent elevation of NOX4 expression, which nally leads to increased cellular ROS levels independent of the mitochondrial source.
IFI6 silencing induces ER stress and ATF3 activation by disrupting Ca 2+ storage of ER through ER Ca 2+ -ATPase pump.-To connect our observations related to ER stress and NOX4 elevation, we speci cally downregulated ATF3 expression via shRNA in Eca109 and TE-1 cells. Subsequently, cell lysates were subjected to immunoblotting to assess the expression of ATF3 and NOX4 after IFI6 silencing. As predicted, ATF3 silencing inhibited the increase in NOX4 expression induced by IFI6 silencing, indicating that ATF3 may be the predominant transcriptional regulator of NOX4 upon alteration of IFI6 expression ( Figure 9A). We next explored the molecular mechanism could be responsible for the regulation of ER stress and subsequently ATF3 activation following IFI6 alteration. For this, we constructed plasmids containing ATF3 promoter fragments and transfected them into ESCC cells with or without IFI6 overexpression. As expected, upregulation of IFI6 signi cantly inhibited ATF3 expression. However, atractyloside (Atra, 2 μM), an inhibitor of the adenine nucleotide transporter (ANT) that exports synthetized ATP from the mitochondrial matrix, fully reversed the effect of IFI6 overexpression on ATF3 transcriptional suppression even when OXPHOS was greatly enhanced following IFI6 upregulation ( Figure 9B). As reported, mitochondrial ATP pools is crucial for the function of the sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA), the activity of which was shown to be inhibited following decreased mitochondrial ATP located in the interstitial spaces of mitochondria-associated membranes (MAMs) [37,38]. Consistent with previous study, IFI6 downregulation caused a reduced ER Ca 2+ levels, whereas IFI6 overexpression in ESCC cells signi cantly elevated Ca 2+ levels in ER, which was reversed by treatment with compounds able to reduce mitochondrial ATP production (Oligomycin, 1 μM) or its export to the cytosol (Atra, 1 μM) ( Figure 9C, Figure S7A). Considering the crucial inter-talk between mitochondria and ER in Ca 2+ signaling and the fact that decreased Ca 2+ storage in ER may lead to ER stress [39][40][41], we further explored the implication of MAMs in IFI6-mediated, ATP deprivation-induced Ca 2+ dynamic change of ER. We overexpressed Mitofusin2 (MFN2) in ESCC cells as MFN2 was known to enhance the number of endoplasmic reticulum-mitochondria contact sites [42]. As shown in Figure 9D and S7B, IFI6 knockdown decreased ER Ca 2+ uptake, which was reversed by MFN2 upregulation. Remarkably, ER Ca 2+ concentration was consistently upregulated by MFN2 overexpression while cyclopiazonic acid (CPA, 10 μM), a speci c SERCA inhibitor [43], was able to attenuate the effect of MFN2 overexpression on ER Ca 2+ levels. These data indicated that IFI6 regulated ER Ca 2+ stores through regulating SERCA pump activity possibly by affecting mitochondrial ATP abundance in MAMs. Ultimately, the dual-luciferase assays demonstrated that the classical endoplasmic reticulum stress-related events as upregulation of ATF3, XBP1s and PDI transcriptional activity following IFI6 overexpression were all blocked either by addition of CPA or supplement of Tunicamycin (Tunica, 0.5 mg/mL), an ER stress inducer ( Figure 9E-G). Taken together, we identi ed an IFI6-ATF3 signaling pathway whereby a reduced mitochondrial ATP levels subsequently suppressed Ca 2+ uptake into ER, resulting in ER stress after IFI6 ablation.
IFI6 silencing promotes the expression of NOX4 through transcriptional activation by ER stress effector ATF3.
Finally, to decipher the molecular mechanism underlying the regulation of NOX4 by ATF3, we used a dual luciferase assay to investigate whether ATF3 regulated NOX4 transcriptional activity. As shown in Figure  9H, knockdown of ATF3 in ESCC cells inhibited NOX4 promoter activity, while ectopic expression of ATF3 restored the transcriptional activity of NOX4.
Moreover, we performed a screen through JASPAR (http://jaspardev.genereg.net) to determine whether ATF3 could bind the NOX4 promoter and found that the NOX4 promoter region contains 4 potential ATF3 binding sites ( Figure 9I). To further validate whether ATF3 directly targets the NOX4 promoter, we constructed plasmids containing NOX4 promoter fragments of different lengths and transfected them into HEK293T cells with or without the ATF3 plasmid. As shown in Figure 9J, NOX4 transcriptional activity was low in the absence of ATF3 expression. However, after ATF3 expression, the NOX4 promoter activity was substantially increased in HEK293T cells transfected with the full-length NOX4 promoter but not in cells transfected with the vectors containing bp -2000 to bp +1 or bp -1000 to bp +1, implying that the bp -3000 to bp -2000 region of the NOX4 promoter is responsible for the ATF3-induced transcriptional activity. Consistent with the above bioinformatic analysis results, this region contained all potential ATF3 binding sites, as illustrated in Figure 8K. Moreover, mutation of either region #1 (AAGGACTCACT) or region #2 (ACTAATGTCATG) markedly suppressed ATF3-induced transcriptional activation of NOX4 ( Figure 9K).
Collectively, all these data indicate that interference with OXPHOS and the ETC through IFI6 downregulation leads to energy deprivation and subsequent ER stress, which in turn induces cellular ROS elevation through transcriptional activation of NOX4 via ATF3.

IFI6 promotes ESCC growth in a xenograft model.
Given that IFI6 elevation was implicated in the acceleration of ESCC cell growth in vitro, we further sought to determine whether altered IFI6 expression was su cient to in uence the proliferation of ESCC cells in vivo. For this purpose, we implanted parental Eca109 cells, IFI6-expressing Eca109 cells and IFI6KD Eca109 cells into the anks of female nude mice. Notably, tumor growth was signi cantly inhibited following IFI6 knockdown, whereas upregulation of IFI6 produced the opposite effect ( Figure 10A, B). IHC was used to validate the abundance of IFI6, ATF3 and NOX4 in tumor tissues derived from xenograft model( Figure 10C). Furthermore, we detected the protein levels of ROS markers (Malondialdehyde, MDA; 4-hydroxynonenal, 4-HNE), ATF3 and NOX4 in the primary tumors by Western blotting. As shown in Figure  10D, ROS production and ATF3/NOX4 axis was upregulated in primary tumors derived from IFI6knockdown cells than tumors derived from control cells. Conversely, IFI6 overexpression markedly decreased oxidative stress and suppressed ATF3/NOX4 signaling pathway, further con rmed IFI6mediated ER stress in an in vivo model.
We further explored whether inhibition of MCU in xenograft model produced similar result as in vitro study. We observed that IFI6 knockdown compromised the growth of primary tumors in xenograft model, and this effect was partially rescued by DS16570511 administration ( Figure 10E). Moreover, the elevation of ROS markers in primary tumors derived from IFI6-knockdown was partially reversed after MCU inhibition.
Finally, phenformin (PHE), a well-known oxidative phosphorylation inhibitor, was administered to mice intraperitoneally every other day to evaluate the effect of OXPHOS inhibition after IFI6 overexpression. As expected, IFI6 overexpression promoted ESCC cells proliferation, while this effect was completely reversed after PHE treatment ( Figure 10G). Importantly, immunoblot from tumor tissues demonstrated that the suppression of ATF3/NOX4 axis in IFI6-overexpressed cells while this effect was reversed by phenformin treatment (Figure 10H), validating the effect of IFI6-mediated OXPHOS e ciency on ATF3/NOX signaling pathway.
Thus, these xenograft models demonstrated that IFI6 promotes ESCC tumor growth in vivo, and again demonstrated a critical role of IFI6 in mitochondrial Ca 2+ , OXPHOS e ciency and ATF3/NOX4 axis.

Discussion
The current study initially aimed to unambiguously determine the role of IFI6 in ESCC. Here, we identi ed that IFI6 was overexpressed in clinical ESCC samples and cell lines. Cancer cell-based experiments demonstrated that the activity of IFI6 controls the growth and survival of ESCC cells through the modulation of cellular ROS production. Mechanistically, we showed that IFI6 ablation inhibited OXPHOS e ciency and mitochondrial supercomplex assembly, which appeared to contribute to the decreased mitochondrial ROS generation in ESCC cells. Furthermore, the oxidative stress induced after IFI6 downregulation enhanced Tg-induced mitochondrial Ca 2+ uptake, which in turn led to mitochondrial calcium overload and partially promoted the accumulation of mitochondrial ROS. In addition, our rescue experiments showed that IFI6 silencing elevated ER-derived ROS accumulation by driving ER stress accompanied by a substantial increase in ATF3 expression and subsequent transcriptional activation of NOX4. We also con rmed that the function of SERCA, an ER located ATP-dependent Ca 2+ pump, was modulated by ATP production and mitochondrial OXPHOS e ciency resulting from altered expression of IFI6. The induction of ER stress and concomitant upregulation of ATF3 following IFI6 silencing is possibly mediated by the decreased ER Ca 2+ pools due to the reduced activity of SERCA. Finally, via knockdown and overexpression experiments, we validated the antiproliferative effect of IFI6 depletion in a nude mouse model of ESCC. Collectively, these observations imply the potential therapeutic value of IFI6 inhibition in ESCC.
Our analysis of IFI6 expression in a panel of ESCC cell lines and patient samples indicated that a high abundance of IFI6 might be correlated with biological aggressiveness in ESCC. While ESCC is notorious for its heterogeneity, our research indicated the existence of an IFI6-positive patient subgroup, which was notably predominant among the cohort with ESCC at more advanced stages. These ndings were supported by analysis of data in public databases, including TCGA and GEO. Furthermore, consistent with our results, IFI6 overexpression is implicated in multiple malignant diseases [22,44,45], and the increase in ATF3 expression was associated with a favorable prognosis in patients with ESCC [46,47].
IFI6 silencing promoted ROS production, consistent with previous ndings by another group. However, our functional study did not show an overt IFI6-mediated change in metastasis potential, as previously shown for breast cancer [24]. In addition, the molecular mechanism underlying IFI6-mediated mitochondrial ROS production remains elusive, prompting us to further investigate the exact role of IFI6 in ESCC.
Previous reports indicate that mitochondrial supercomplex assembly modulates the generation of mitochondrial ROS, which is produced principally by OXPHOS [31]. As reported, the substantial accumulation of mitochondrial ROS can be induced by the loss of supercomplex organization [48,49]. Jang and Javadov recently showed that complex I and II subunit depletion elevated mitochondrial ROS production in the heart but impaired respirasome formation and ATP production [32]. A As altered IFI6 expression can alter cellular ATP production and OXPHOS e ciency, we hypothesized that the decreased mitochondrial supercomplex assembly following IFI6 silencing may enhance mitochondrial ROS production, re ecting the e ciency of the OXPHOS complex. Although IFI6 is not a component of the ETC, our research provides evidence that IFI6 may act as a bona de regulator of mitochondrial supercomplex formation. Consistent with this hypothesis, IFI6 depletion decreased the content of complexes I, III and IV in the mitochondrial supercomplex, while the expression of the individual respiratory complexes did not change. In contrast, overexpression of IFI6 effectively enhanced mitochondrial supercomplex formation. Although the exact mechanism underlying IFI6-mediated mitochondrial supercomplex assembly remains to be explored, we hypothesized that IFI6 might facilitate mitochondrial supercomplex assembly directly by interacting with respiratory complex subunits or indirectly by enhancing weak interactions between mitochondrial complexes.
Previous studies have shown that mitochondria can take up calcium from the cytosol (through ORAI channels) or via the ER (through channels ranging from IP 3 R to ER calcium leak channels) [50,51]. As calcium in ux into mitochondria leads to the induction of programmed cell death [52], we assumed that dysregulated mitochondrial calcium dynamics could lead to mitochondrial dysfunction, contribute to ROS production, and ultimately induce cell apoptosis following IFI6 depletion. Indeed, we observed a signi cant increase in the Tg-induced mitochondrial Ca 2+ uptake after IFI6 depletion. Furthermore, addition of the calcium chelator BAPTA or removal of calcium from the culture medium reduced mitochondrial ROS generation upon IFI6 silencing. Calcium released from the ER and that present in the cytosol is transported into mitochondria via VDAC1, MCU, and NCLX [53][54][55][56]; however, our expression analysis revealed no overt alterations in these components of the mitochondrial calcium uptake machinery upon IFI6 knockdown. Mitochondrial oxidative stress and calcium signaling are two functional entities that often coexist and are interconnected to maintain appropriate cellular physiology [57,58].
Moreover, Dong et al. showed that the redox regulation of MCU plays a role in mitochondrial calcium dynamics and oxidation; oxidation of MCU facilitated higher-order MCU oligomer formation and increased the MCU calcium uptake rate, mitochondrial ROS accumulation, and calcium overload-induced cell death [59]. Consistent with this observation, pharmacological inhibition of MCU with DS16570511 partially prevented IFI6 silencing-induced mitochondrial ROS elevation. Taken together, these data suggest that the insu cient OXPHOS resulting from IFI6 silencing causes the accumulation of excess mitochondrial ROS, which promotes the oxidation of MCU, which in turns leads to increased MCU Ca 2+ uptake and concomitant mitochondrial Ca 2+ overload, disruption of mitochondrial Ca 2+ dynamics further adds to ROS production. In this process, disrupted respiratory complexes formation and OXPHOS insu ciency are the leading and primary causes, while concomitant mitochondrial Ca 2+ overload is the secondary cause.
We could not rule out the possibility that other sources of ROS contribute to the oxidative stress generated upon IFI6 downregulation. The ER is a large membrane-like organelle with various important cellular functions, ranging from maintaining proper protein folding to modulating calcium homeostasis to hosting components of intracellular signaling pathways [60,61]. The unfolded protein response (UPR), speci cally activated in response to ER stress, can be induced by multiple factors, including nutrient de ciency, and produces endogenous or exogeneous damage to cellular functions, leading to impaired intracellular calcium dynamics and redox homeostasis [62,63]. Here, we demonstrated that IFI6 depletion led to induction of ER stress possibly mediated by an ATP shortage through interference with mitochondrial OXPHOS. Moreover, a reduction in the activity of SERCA upon decreased ATP supplied by mitochondria leads to ER stress, which is consistent with previous study [64]. In our experiment, we found that ER Ca 2+ was signi cantly decreased even when IFI6 was upregulated, but ATP export from mitochondrial was blocked using Atra. On the other hand, overexpressing MFN2 promoted ER Ca 2+ uptake by increasing mitochondrial-ER contacting sites. Our rescue experiments demonstrated that pharmaceutical inhibition of SERCA could block the effect of IFI6 overexpression upon ER stress suppression, this phenomenon was also mimicked by ER stress inducer tunica, which further corroborate our hypothesis. However, additional studies were necessary to decipher how ER Ca 2+ depletion mediated by IFI6 silencing contributes to ER stress and especially, the transcriptional activation of ATF3. We also revealed that NOX4, which was identi ed as a key player in cellular ROS production following IFI6 knockdown, was regulated by ER stress and the associated transcription factor ATF3. These ndings ultimately suggest that IFI6 may serve as a cellular antioxidant in ESCC by suppressing NOX4.
Finally, we demonstrated that IFI6 exerts pro-carcinogenic activity in vivo in an ESCC xenograft model in nude mice. These observations further con rm the in vitro ndings and suggest that the speci c inhibition of IFI6 activity might have implications for patients with ESCC. Several studies report that antioxidants can promote aggressive behavior and drug resistance in ESCC [65][66][67]. Our ndings further highlight the importance of better understanding redox homeostasis in malignant diseases and the possible clinical applicability of IFI6.
In summary, we identify what we believe to be a new IFI6-mediated pathway, manifest its hyperactivation in ESCC. Mechanically, inhibition of IFI6 suppresses mitochondrial supercomplex formation, which leads to OXPHOS de ciency and concomitant mitochondrial ROS overproduction. Excess ROS causes mitochondrial calcium overload, which in its turn further adds to mitochondrial ROS generation. The ATP shortage resulting from insu cient OXPHOS induces ER stress due to a decrease in SERCA function, leading to the upregulation of ATF3 expression and the transcriptional activation of NOX4, which enhances ER-mediated ROS production. All of these mechanisms ultimately cause oxidative stress. This circuit is illustrated in Figure 11.

Conclusions
Taken together, our data indicated the carcinogenesis of IFI6 in ESCC and revealed that knockdown of IFI6 suppressed proliferation and induced apoptosis by increasing ROS accumulation. We further found that mitochondrial ROS accumulation is induced by the suppression of mitochondrial supercomplex assembly and mitochondrial calcium overload; IFI6 inhibition also upregulated NOX4-derived ROS production in an ATF3-dependent manner through ER stress induction. All these observations would implicate a novel therapeutic avenue for the treatment of ESCC. Declarations Ethics approval and consent to participate. This study was approved by the Medical Ethics Committee of Shanghai Tongji Hospital. Informed consent was obtained from each patient before enrollment in this study. All animal experiments were approved by the Animal Care and Use Committee of Shanghai Tongji Hospital and conducted in accordance with ethical standards.

Consent for publication.
Not applicable.
Availability of data and materials.
All data from this study can be requested directly from the corresponding author upon reasonable request.
The authors declare that they have no competing interests.        presented as the means and SDs (n=3). Statistical signi cance was determined by two-tailed Student's ttest. **P<0.01. D. Representative plots (upper) and quantitative results (bottom) of the real-time ECAR, glycolysis and glycolytic capacity assays in the indicated ESCC cells. The ECAR was determined following sequential addition of glucose (10 mM), oligomycin (1 μM) and 2-DG (100 mM). Glycolysis was measured by subtracting the ECAR after glucose addition from the ECAR before glucose addition. The glycolytic capacity was calculated by subtracting the ECAR after oligomycin treatment from the ECAR before glucose addition. The data are presented as the means and SDs (n=3). Statistical signi cance was determined by a two-tailed Student's t-test. E. Representative plots (left) and quantitative results (right) of the complex I-dependent OCR in the different groups. Pyruvate (Pyr) (5 mM) and malate (Mat) (5 mM) were added to digitonin (Dig)-permeabilized cells, and the OCR was monitored. The data are presented as the means and SDs (n=3). Statistical signi cance was determined by two-tailed Student's t-test. **P<0.01.
F. Representative plots (left) and quantitative results (right) of the complex III-dependent OCR in the different groups. Rotenone was added to digitonin-permeabilized cells to inhibit complex I, after which G3P (5 mM) was added, and the OCR was monitored. The data are presented as the means and SDs (n=3). Statistical signi cance was determined by two-tailed Student's t-test. **P<0.01. G. Representative plots (left) and quantitative results (right) of the complex II-, and complex IV-dependent OCRs in the different groups. Rotenone (1 µM) was added to inhibit complex I; succinate (Suc) (5 mM), Antimycin (AMA) (1 µM) and TMPD/ascorbate (500 µM and 5 mM, respectively) were then added to digitoninpermeabilized cells, and the OCR was monitored. The data are presented as the means and SDs (n=3).
Statistical signi cance was determined by two-tailed Student's t-test. **P<0.01.  IFI6 silencing-induced ATP shortage activates ER stress by disrupting Ca2+ storage of ER and subsequently induces NOX4 up-regulation in an ATF3-dependent manner. A. Protein lysates were collected from the indicated Eca109 and TE-1 cells and subjected to immunoblotting to assess the expression of IFI6, ATF3 and NOX4. GAPDH was used as the loading control. B. ATF3 transcriptional activity was measured via a dual luciferase reporter assay in indicated ESCC cells. The data are presented as the means and SDs (n=3). Statistical signi cance was determined by two-tailed Student's ttest. ***P<0.005. C-D. Representation (left) and statistical analysis (right) of the Endoplasmic reticulum Ca2+ in the indicated ESCC cells. Endoplasmic reticulum-targeted aequorin was exploited to monitor dynamic changes in free Ca2+ concentration in ER. The uorescence intensity at each time point was recorded with an integrated spectro uorometer. The data are presented as the means and SDs (n=3).
Statistical signi cance was determined by two-tailed Student's t-test. **P<0.01. E-G. Transcriptional activity of ATF3 (E), XBP1s (F) and PDI (G) was measured via a dual luciferase reporter assay in indicated ESCC cells. The data are presented as the means and SDs (n=3). Statistical signi cance was determined by two-tailed Student's t-test. ***P<0.005. H. A dual luciferase reporter assay was used to determine NOX4 transcriptional activity. The relative luciferase activity was normalized to that in ATF3 KD ESCC cells. The data are presented as the means and SDs (n=3). Statistical signi cance was determined by two-tailed Student's t-test. ***P<0.005. I. Schematic of potential ATF3 binding sites in the NOX4 promoter, as predicted by JASPAR. J. HEK293T cells were co-transfected with plasmids containing different NOX4 promoter constructs with or without the ATF3 expression plasmid. NOX4 transcriptional activity was measured via a dual luciferase reporter assay. The data are presented as the means and SDs (n=3).
Statistical signi cance was determined by two-tailed Student's t-test. ***P<0.005. K. A dual luciferase reporter assay was used to assess NOX4 transcriptional activity in HEK293T cells cotransfected with pGL3-NOX4-WT, pGL3-NOX4-Mut #1 (AAGGACTCACT), pGL3-NOX4-Mut #2 (ACTAATGTCATG), pGL3-NOX4-Mut #3 (TATGAAGACATTT) or pGL3-NOX4-Mut #4 (AATTGCATCACC) constructs with or without the ATF3 expression plasmid. The data are presented as the means and SDs (n=3). Statistical signi cance was determined by two-tailed Student's t-test. **P<0.01.  Schematic of IFI6-modulated ROS generation in ESCC cells. (Left panel) In IFI6low ESCC cells, mitochondrial supercomplex formation is suppressed, leading to OXPHOS de ciency and subsequent mitochondrial ROS overproduction, which in turn causes mitochondrial calcium overload. The ATP shortage resulting from insu cient OXPHOS induces ER stress, leading to the upregulation of ATF3 expression and the transcriptional activation of NOX4, which enhances ER-mediated ROS production. All of these mechanisms ultimately cause oxidative stress. Elevated ROS levels suppress tumor cell proliferation and induce apoptosis. (Right panel) In IFI6high ESCC cells, undisturbed mitochondrial supercomplex assembly allows for stable mitochondrial calcium in ux, optimal mitochondrial function, and inhibition of ER stress. Under these conditions, mitochondria and NOX4 produce low or moderate levels of ROS, which can be eliminated by cellular antioxidants, ultimately promoting cancer cell proliferation and suppressing cell apoptosis.

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