IFI6 depletion inhibits esophageal squamous cell carcinoma progression through reactive oxygen species accumulation via mitochondrial dysfunction and endoplasmic reticulum stress

Lipofectamine 3000 (L3000015), MitoSOX (M36008), carboxy-H₂DCFDA (C400) and the anti-IFI6 antibody (1:1000, PA5–99824) were purchased from Invitrogen (CA, USA). RPMI-1640 (72400047), penicillin and streptomycin (10378016), and Fetal bovine serum (FBS) (16140071) were obtained from Gibco (CA, USA). N-acetyl-L-cysteine (NAC) (A7250), PEG-catalase (CAT) (C1345), dithiothreitol (DDT) (45839), MitoTEMPO (SML0737), ATP (A6559), BAPTA-AM (A4926), Phenformin (PHE) (P7045), Cyclopiazonic acid (CPA) (C1530), Tunicamycin (Tunica) (654380) and Thapsigargin (Tg) (T9033) were purchased from Sigma-Aldrich (Munich, Germany). DS16570511 (HY-115595) was obtained from MedChemExpress (NJ, USA). Antibodies against GAPDH (1:10000, ab181602), β-Actin (1:5000, ab179467), NCLX (1:1000, ab136975), MCU (1:1000, ab121499), VDAC1 (1 μg/ml, ab15895), NDUFB8 (1:5000, ab192878), SDHA (1:5000, ab137040), RISP (1 μg/ml, ab14746), COXIV (1 μg/ml, ab33985), ATPB (1:10000, ab170947), 4-HNE (1:1000, ab48506), MDA (1:1000, ab243066), ATF3 (1:2000, ab207434), ATF4 (1:1000, ab184909), ATF6 (1:1000, ab122897), XBP1 (1:5000, ab109221), PDI (1:1000, ab2792), BiP (1:1000, ab21685), NOX1 (0.5 μg/ml, ab131088), NOX2 (1 μg/ml, ab80508), NOX3 (1 μg/ml, ab81864), NOX4 (1:1000, ab133303), and NOX5 (1:1000, ab198213) were obtained from Abcam (Cambridge, UK).

A total of 23 fresh ESCC samples and paired adjacent non-tumorous tissues (PANTs) were collected from patients undergoing esophagectomy at Shanghai Tongji Hospital Affiliated with Tongji University. Immunohistochemistry (IHC) for IFI6 was conducted on 3-μm sections of formalin-fixed, paraffin-embedded 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% CO₂. All cell lines were authenticated by the STR method and tested for mycoplasma contamination.

The indicated Eca109 cells (2 × 10⁶ cells per 0.1 ml of phosphate-buffered saline) were suspended in Matrigel (Biosciences, CA, USA) and implanted subcutaneously into the right flanks of the nude mice. The tumor volume was measured using a caliper every 5 days. Four weeks after implantation, the nude mice were sacrificed, and tumor samples were harvested.

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.

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 difluoride 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).

Paraffin-embedded sections were deparaffinized and rehydrated and were then subjected to antigen retrieval for 20 min in citrate buffer (pH 6.0). After the sections were cooled, 3% H₂O₂ 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 fields 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.

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™ 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 difluoride 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.

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 fixation, 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 fluorescence microscopy.

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 flow 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 fluorescence microscope. In healthy cells with a Δψm indicating membrane depolarization, JC-1 forms JC-1 aggregates exhibiting red fluorescence. In apoptotic cells, JC-1 remains monomeric, exhibiting green fluorescence.

To assess cellular ATP production, an ATP luminescence assay (Invitrogen, CA, USA) was used for quantitative determination of ATP levels with recombinant firefly 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.

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, 20,000 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), trifluoromethoxy 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). Glucose (10 mM), Oligo (1 μM) and 2-deoxy-D-glucose (2-DG) (100 mM) were sequentially added to the cell culture, and real-time ECAR was determined with the Seahorse XFe96 Bioanalyzer. For complex-specific OCR measurement, cells were permeabilized with a solution of digitonin (500 μg per 10⁵ cells) supplemented with 1 mM MgCl₂, 1 mM ADP, 5 mM KH₂PO₄, 150 mM KCl and 5 mM Tris (pH 7.2). The substrates and inhibitors used for complex-specific OCR measurement were malate (5 mM), Rot (1 μM), AMA (1 μM), ascorbate (5 mM), TMPD (500 μM), pyruvate (5 mM), succinate (5 mM), and G3P (5 mM). All reagents and compounds were purchased from Sigma-Aldrich (Munich, Germany).

For lentiviral-mediated transduction of shRNA, cells were initially treated with polybrene (Sigma-Aldrich, Munich, Germany) and selected with puromycin (Sigma-Aldrich, Munich, Germany). ShRNA-IFI6, shRNA-ATF3, shRNA-NOX4, and negative control shRNAs were purchased from Genechem (Shanghai, China). 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).

For construction of overexpression plasmids, the pcDNA3.1 (V79020) expression plasmid was obtained from Invitrogen (CA, USA). Human IFI6, ATF3, and NOX4 cDNA from Eca109 cells were cloned by PCR and subcloned into the pcDNA3.1 expression vector to generate IFI6-pcDNA3.1, ATF3-pcDNA3.1, and NOX4-pcDNA3.1. After construction, the IFI6-, ATF3-, or NOX4-pcDNA3.1 plasmid was transfected into Eca109 and TE-1 cells using Lipofectamine 3000 and selected with G418.

To monitor mitochondrial calcium dynamics, the indicated ESCC cells were incubated with the calcium-specific fluorescent probe Rhod-2 AM (Invitrogen, CA, USA) in Hanks’ balanced salt solution at room temperature for 30 min. After washout, fluorescence was recorded with an integrated spectrofluorometer (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, F₀), cells were promptly treated with the agonist Tg, and fluorescence intensities were monitored at 3-s intervals for another 10 min. The relative fluorescence intensity (F/F₀), where F represents the fluorescence intensity at a given time and F₀ represents the initial fluorescence intensity, was used to indicate [Ca²⁺].

For determination of Ca²⁺ concentration in endoplasmic reticulum, Eca109 and TE-1 cells were transfected with low affinity 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 Ca²⁺ − free medium containing 0.5 mM EGTA. Cells were washed with Krebs Ringer Buffer (KRB: 125 mM NaCl, 5 mM KCl, 400 mM KH₂PO₄, 1 mM MgSO₄, 20 mM HEPES, pH 7.4) supplemented with 2% bovine serum albumin (Sigma) and 1 mM EGTA and were then placed in KRB supplemented with 5 mM glucose and 75 mM EGTA and luminescence measurement was started. After 60 s, 1 mM CaCl₂ at the final concentration was injected to refill the ER lumen. The measurement was carried out with an integrated spectrofluorometer (Photon Technology International, NJ, USA) 3-s intervals for 800 s.

To detect mitochondrial ROS, the indicated ESCC cells were seeded in 24-well plates and incubated with the mitochondria-specific 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-H₂DCFDA and further incubated at 37 °C for 20 min. Nuclei were counterstained with Hoechst 33342, and the fluorescence intensity was measured by fluorescence microscopy or flow cytometry.

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 ATF3-binding 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 firefly and Renilla luciferase activities were measured with a Dual Luciferase Reporter Assay System (Promega, WI, USA) and an illuminometer.

RNA-seq expression data (quantified 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 coefficient (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).

Transcription factor-promoter binding predictions were performed with JASPAR (http://​jaspar.​genereg.​net).

Survival analysis of ESCC patients in TCGA database were carried out with web-based analysis tool Kaplan-Meier Plotter (http://​www.​kmplot.​com).

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 (Fig. 1a). We also validated these data by assessing IFI6 expression in 23 ESCC samples and corresponding normal esophageal tissues from patients in our hospital (Fig. 1b). Given that ESCC is heterogenous both genetically and in its clinical manifestation, we further explored the IFI6 expression profile across a series of genetically distinct ESCC cell lines. As expected, IFI6 expression patterns were heterogeneous; however, IFI6 expression was higher in all five ESCC cell lines tested than in the normal esophageal squamous epithelial cell lines (Fig. 1c). The high levels of IFI6 expression were further confirmed at the protein level via Western blot analysis (Fig. 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 Fig. 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 significantly 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 Fig. 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.

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 (Fig. 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 (Fig. 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 (Fig. 2d, e). In addition, patients with higher IFI6 levels exhibited lower levels of genes related to the oxidative stress response (Fig. 2f, g).

Collectively, the results of these bioinformatic analyses led us to hypothesize that IFI6 can promote proliferation, inhibit apoptosis and ameliorate oxidative stress.

To confirm 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 efficiencies 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 significantly inhibited in IFI6-silenced ESCC cells compared with ShControl ESCC cells (Fig. 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 (Fig. 2c, e), we next conducted annexin V-FITC/propidium iodide (PI) staining. Consistent with the EdU assay results, IFI6 inhibition significantly induced ESCC cell apoptosis (Fig. 3c). In the reciprocal experiment, cell proliferation markedly increased by IFI6 overexpression, whereas ESCC cell apoptosis was significantly 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 (Fig. 2c, f, g), we then investigated cellular ROS levels following IFI6 silencing. IFI6 inhibition significantly enhanced the generation of cellular ROS, as represented by the live cell ROS indicator carboxy-H₂DCFDA (Fig. 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-H₂DCFDA staining (Fig. 3e, f). Moreover, as shown in Fig. 4a, all these ROS-eliminating reagents effectively reversed the effect of IFI6 silencing on cell viability. Our findings 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 (Fig. 4b, Figure S3B), which supported the role of ROS in the IFI6 silencing-induced reduction in cell viability.

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-specific 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 significantly inhibited in cells overexpressing IFI6 (Fig. 5a, b). The above observations indicated that the expression level of IFI6 directly affects mitochondrial ROS production. As reported, mitochondrial Ca²⁺ signaling profoundly influences 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 Ca²⁺ 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 (Fig. 5c, d). Previous studies showed that calcium influx 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 Ca²⁺ uptake by Ca²⁺ 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 (Fig. 5e). By removing Ca²⁺ from the cell culture medium, we obtained similar results (Fig. 5f). Based on the above findings, 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 Ca²⁺ uniporter (MCU) and mitochondrial sodium calcium lithium exchanger (NCLX) by Western blotting and RT-PCR. However, as indicated in Figure S4A-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 Fig. 5g-h, treatment with this mitochondrial ROS scavenger completely reversed the IFI6 silencing-induced increase in mitochondrial Ca²⁺ uptake. Furthermore, following IFI6 ablation, pharmacological inhibition of MCU with DS16570511 partially prevented IFI6 silencing-induced mitochondrial ROS generation (Fig. 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 Ca²⁺ only partially attenuated IF6 silencing-induced mitochondrial ROS production, mitochondrial Ca²⁺ influx 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.

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 (Fig. 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 (Fig. 6b).

To further assess the effect of IFI6 on cellular bioenergetics, we conducted extracellular flux analysis to assess the extracellular acidification 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 Fig. 6c, the basal cellular OCR and maximum respiration rate were significantly elevated in IFI6-overexpressing 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 (Fig. 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 (Fig. 6d, 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-1,4-phenylenediamine (TMPD)/ascorbate). The complex I-specific OCR in IFI6-silenced cells was substantially reduced compared with that in parental cells; in contrast, IFI6 overexpression significantly increased the complex I-specific OCR in both Eca109 and TE-1 cells (Fig. 6e, Figure S5C). In addition, the OCRs specific to complexes III and IV exhibited similar patterns (Fig. 6f-g, Figure S5D-E). However, in Eca109 and TE-1 cells, the OCR supported by complex II remained unchanged (Fig. 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 confirmed that altered IFI6 expression levels might play a role in energy handling and mitochondrial dysfunction.

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 (Fig. 7a, Figure S6A). Moreover, silencing IFI6 decreased the signal of NDUFB8, RISP or COXIV at the CI + CIII₂ + CIV_n and CI + CIII₂ positions compared with those in parental ESCC cells. The signals of CI + CIII₂ + CIV_n and CI + CIII₂ RSCs were elevated in ESCC cells ectopically expressing IFI6 compared to parental cells (Fig. 7b, Figure S6B). In addition, IFI6 ablation resulted in a decreased signal at the CIII₂ + CIV_n position, while overexpressing IFI6 had the opposite effect, as detected by immunoblotting with anti-RISP and anti-COXIV antibodies (Fig. 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 (Fig. 7c, Figure S6C). These observations were consistent with those of the aforementioned extracellular flux 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 (Fig. 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 CIII₂ + CIV_n in ESCC.

Collectively, our data suggested two causes of elevated mitochondrial ROS following IFI6 silencing: first and the driving cause, impaired respiratory complex function and supercomplex assembly, which decreases the efficiency of OXPHOS and in its turn accumulates mitochondrial ROS; Secondly, the altered redox state of MCU induces mitochondrial Ca²⁺ overload and subsequently adds to mitochondrial ROS production.

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-H₂DCFDA 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-specific antioxidant, exhibited only a partial reversal of IFI6 silencing-induced cellular ROS generation compared with vector-treated ESCC cells (Fig. 8a, b). However, the combination treatment with MitoTEMPO and exogenous ATP led to a significantly greater decrease in cellular ROS levels than each compound alone (Fig. 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 five 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 (Fig. 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-H₂DCFDA. 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 IFI6-silenced ESCC cells (Fig. 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 (Figs. 8e, 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 X-box binding protein 1 (XBP1s), as well as protein disulfide 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 (Fig. 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 (Fig. 8f). 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 finally leads to increased cellular ROS levels independent of the mitochondrial source.

IFI6 silencing induces ER stress and ATF3 activation by disrupting Ca²⁺ storage of ER through ER Ca²⁺-ATPase pump.-To connect our observations related to ER stress and NOX4 elevation, we specifically 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 (Fig. 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 significantly 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 (Fig. 9b). As reported, mitochondrial ATP pools is crucial for the function of the sarcoplasmic/endoplasmic reticulum Ca²⁺-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 Ca²⁺ levels, whereas IFI6 overexpression in ESCC cells significantly elevated Ca²⁺ 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) (Fig. 9c, Figure S7A). Considering the crucial inter-talk between mitochondria and ER in Ca²⁺ signaling and the fact that decreased Ca²⁺ storage in ER may lead to ER stress [39‐41], we further explored the implication of MAMs in IFI6-mediated, ATP deprivation-induced Ca²⁺ 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 Fig. 9d and S7B, IFI6 knockdown decreased ER Ca²⁺ uptake, which was reversed by MFN2 upregulation. Remarkably, ER Ca²⁺ concentration was consistently upregulated by MFN2 overexpression while cyclopiazonic acid (CPA, 10 μM), a specific SERCA inhibitor [43], was able to attenuate the effect of MFN2 overexpression on ER Ca²⁺ levels. These data indicated that IFI6 regulated ER Ca²⁺ 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 (Fig. 9e-g). Taken together, we identified an IFI6-ATF3 signaling pathway whereby a reduced mitochondrial ATP levels subsequently suppressed Ca²⁺ uptake into ER, resulting in ER stress after IFI6 ablation.

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 Fig. 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 (Fig. 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 Fig. 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 Fig. 8k. Moreover, mutation of either region #1 (AAGGACTCACT) or region #2 (ACTAATGTCATG) markedly suppressed ATF3-induced transcriptional activation of NOX4 (Fig. 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.

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 sufficient to influence 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 flanks of female nude mice. Notably, tumor growth was significantly inhibited following IFI6 knockdown, whereas upregulation of IFI6 produced the opposite effect (Fig. 10a, b). IHC was used to validate the abundance of IFI6, ATF3 and NOX4 in tumor tissues derived from xenograft model(Fig. 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 Fig. 10d, ROS production and ATF3/NOX4 axis was upregulated in primary tumors derived from IFI6-knockdown cells than tumors derived from control cells. Conversely, IFI6 overexpression markedly decreased oxidative stress and suppressed ATF3/NOX4 signaling pathway, further confirmed IFI6-mediated 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 (Fig. 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 (Fig. 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 (Fig. 10h), validating the effect of IFI6-mediated OXPHOS efficiency 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 Ca²⁺, OXPHOS efficiency and ATF3/NOX4 axis.