Ifi6 Participates In The Malignant Progress Of Esophageal Squamous Cell Carcinoma By Modulating The Oxidative Stress

Aim and background:Esophageal cancer is one of the most common seen gastrointestinal malignancies,with incidence and mortality ranks 10^th and 6^th worldwide,respectively.More than half of the world’s new cases and deaths of esophageal cancer every year occurred in China.The histology of esophageal cancers comprises of esophageal squamous cell carcinoma（ESCC）and esophageal adenoma.In China,approximately 80%of newly diagnosed esophageal cancer belongs to ESCC.The initiation and development of ESCC experiences successive stages from atypical hyperplasia,carcinoma in situ to invasive carcinoma.If diagnosis and intervention can be made in the early stage of ESCC,surgical resection will be effective,and most patients will have a better prognosis.However,due to the lack of obvious symptoms in its early stage,the stages of most ESCC are in the advanced stage at the time of diagnosis,and therefore patients lose the best time for receiving radical surgery.Despite receiving comprehensive treatment,the prognosis of ESCC is still poor,5-year survival rate of ESCC patients is less than 20%.The reasons underlying frequent treatment failures and cancer-associated deaths of ESCC may largely attributes to the highly proliferative potential,aggressive behavior,and repaid development of treatment resistance of ESCC.The action that explores the molecular biomarkers involved in the initiation and progression of ESCC and elucidate its underlying molecular mechanism will shed a light on effectively developing treatment strategies targeting ESCC.IFI6（Interferon Alpha Inducible Protein 6）is an interferon-inducible gene that belongs to the FAM14 protein family and is mainly localized to the inner mitochondrial membrane.IFI6 can be expressed in response to type I interferon since its promoter region contains interferon-stimulatory response elements,and because of the above-mentioned observations,early studies suggested that IFI6 is mainly involved in the regulation of innate immunity.Recently,however,the role and mechanism of IFI6underlying the initiation and development of malignancies has gradually become a research hotspot.Previous studies have proved that IFI6 is highly expressed in tumors such as gastric cancer,breast cancer,melanoma,and myeloma,and is involved in the regulation of malignant biological behaviors such as proliferation,apoptosis,and resistance to radiotherapy and chemotherapy of cancer cells.For instance,the ratio between bcl-2/bax regulated by IFI6 is crucial in maintaining tumor survival.IFI6 can also found to be able to inhibit caspase-dependent apoptosis of cancer cells due to its ability on stabilizing mitochondrial membrane potential and reducing cytochrome c release.Although IFI6 has proved to be playing an important role in the initiation and progression of tumors.Little is known regarding whether IFI6 participates in the initiation and development of esophageal squamous cell carcinoma.This study aims to elucidate the clinical significance of IFI6 in ESCC,and to explore its effect and molecular mechanism by which IFI6 contributes to the malignant progression of ESCC.Our study will shed a light on the prevention,diagnosis and treatment of ESCC.This study includes four parts:1).The expression profile of IFI6 in ESCC and its impact on biological behavior of ESCC;2).IFI6 silencing promotes the generation of mitochondrial reactive oxygen species（mt ROS）by inducing mitochondrial calcium overload in ESCC;3).IFI6 silencing promotes mt ROS generation by inhibiting mitochondrial oxidative phosphorylation;4).IFI6 silencing promotes cytoplasmic ROS generation by inducing endoplasmic reticulum stress.PART I The expression profile of IFI6 in ESCC and its impact on biological behavior of ESCCMethods:1.RT-PCR,Western blot and immunohistochemistry were employed to quantify the expression of IFI6 in ESCC tissues and ESCC cell lines.2.The relationship between IFI6 expression and clinicopathological characteristics of ESCC patients was analyzed byχ2 test.Survival curves were drawn by Kaplan-Meier method,and Cox proportional hazards regression model was used to evaluate the prognostic predictive value of IFI6 expression in ESCC.3.Gene ontology（GO）functional annotation analysis was performed on the IFI6co-expressed genes derived from the expression profile of ESCC in the GEO dataset,and Gene set enrichment analysis（GSEA）was performed on expression profile of ESCC from TCGA dataset.4.The lentiviral sh RNA expression vector was used to construct ESCC cell line with stably IFI6 knockdown,and the IFI6 overexpression plasmid was used to construct the ESCC cell line with IFI6 overexpression.5.The impact of IFI6 on the proliferation of ESCC cells was determined by Ed U assay.Annexin V-FITC/PI flow cytometry was used to determine the impact of IFI6 on apoptosis of ESCC cells.The impact of IFI6 on the ROS production in ESCC cells was detected by H₂DCFDA fluorescent probe.The mitochondrial membrane potentialΔΨm fluorescent probe JC-1 was used to determine the impact of IFI6 on the membrane potential and mitochondria-dependent apoptosis of ESCC cells.Results:1.IFI6 is overexpressed in ESCC:Analysis of gene expression profiles of ESCC in GEO dataset showed that the m RNA level of IFI6 in ESCC was significantly higher than that in normal esophageal tissue.RT-PCR analysis of clinically collected ESCC tissues showed that IFI6 expression in ESCC tissues was significantly higher than that in adjacent normal tissues.Western blot and RT-PCR results showed that IFI6expression was significantly higher in ESCC cell lines than that in normal human esophageal epithelial cells.2.High expression of IFI6 correlates with malignant characteristics of ESCC:immunohistochemical analysis showed that IFI6 expression was higher in advanced ESCC tissues than that in esophageal precancerous lesions.IFI6 level was significantly positively correlated with T stage,differentiation degree and TNM stage of ESCC.3.High expression of IFI6 predicts poor prognosis of ESCC:Survival analysis of ESCC data from TCGA and in our hospital showed that high level of IFI6 was associated with poor prognosis of ESCC patients.Univariate and multivariate Cox regression analysis showed that IFI6 was an independent prognostic factor for ESCC（univariate:HR=2.491,95%CI:1.806-4.145,P<0.001;multivariate:HR=2.264,95%CI:1.395-3.406,P=0.001）.4.Bioinformatics analysis suggests the biological function of IFI6 in ESCC:GO function annotation was performed on genes related to IFI6 expression screened by ESCC data in GEO,and GSEA results based on IFI6 expression value on ESCC data in TCGA suggested that the function of IFI6 may be related to the regulation of proliferation,apoptosis,and oxidative stress of ESCC cells.5.IFI6 silencing ESCC cells and IFI6 overexpressing cells were successfully constructed.6.In vitro functional exploration of the biological function of IFI6 in ESCC:Ed U assays showed that IFI6 knockdown inhibited,whereas IFI6 overexpression promoted proliferation of ESCC cells;apoptosis assays showed that IFI6 knockdown induced cell apoptosis,whereas IFI6 overexpression inhibited apoptosis of ESCC cells;ROS labeling assay showed that IFI6 knockdown promoted ROS generation,while IFI6overexpression decreased cellular ROS levels.7.IFI6 modulates proliferation and apoptosis of ESCC cells by regulating ROS levels:when scavenging cellular ROS in IFI6-silenced ESCC cells by adding antioxidants,Ed U experiments show that scavenging ROS can completely block the inhibitory effect of IFI6 silencing on proliferation of ESCC cells,JC-1 assay showed that the apoptotic-inducing effect of IFI6 knockdown could be reversed by ROS scavenging.Conclusions:1.IFI6 is overexpressed in ESCC and high expression of IFI6 predicts advanced stage and poor prognosis of ESCC.2.IFI6 promotes the proliferation,while inhibiting apoptosis and ROS production of ESCC cells.3.IFI6 knockdown inhibits proliferation and survival of ESCC cells by up-regulating cellular ROS levels and inducing oxidative stress.PART II IFI6 silencing promotes mitochondrial reactive oxygen species production by inducing mitochondrial calcium overload in ESCCMethods:1.The m RNA and protein levels of MCU,VDAC1 and NCLX were determined by RT-PCR and Western blot,respectively.2.Mitochondrial ROS（mt ROS）fluorescent probe Mito SOX was used to label mt ROS,and the impact of IFI6 on mt ROS levels were analyzed by flow detection or fluorescence microscope.3.Mitochondrial Ca²⁺fluorescent probe Rhod-2 was used to label mitochondrial Ca²⁺,and the impact of IFI6 on mitochondrial Ca²⁺concentration was monitored in real time by fluorescence spectrophotometer.Results:1.IFI6 decreases mt ROS level:Mito SOX was used to detect mt ROS level in ESCC cells,and it was found that IFI6 knockdown promotes mt ROS production,while IFI6 overexpression reduces mt ROS levels in ESCC cells.2.IFI6 silencing induces mitochondrial calcium overload:The effect of IFI6 on mitochondrial Ca²⁺uptake was detected using Rhod-2.The results showed that IFI6silencing promoted,whereas IFI6 overexpression inhibited mitochondrial Ca²⁺uptake in ESCC cells.3.IFI6 knockdown promotes mt ROS production through inducing mitochondrial calcium overload:BAPTA was used to sequester intracellular Ca²⁺or cells were cultured in Ca²⁺-free medium to simulate cellular calcium depletion,on the basis of that,Mito SOX was used to detect the effect of IFI6 on mt ROS generation.Results showed that inhibiting mitochondria Ca²⁺uptake partially reversed the effect of IFI6 silencing on mt ROS production.Conclusion:1.IFI6 maintains the calcium homeostasis of mitochondria,and IFI6 silencing induces mitochondrial calcium overload in ESCC cells.2.IFI6 silencing promotes mt ROS generation by inducing mitochondrial calcium overload.3.IFI6 influences mitochondrial calcium transport by regulating the activity of MCU calcium channel.4.Mitochondrial calcium overload is not the only mechanism by which IFI6regulates mt ROS generation in ESCC.PART III IFI6 silencing promotes mt ROS generation by inhibiting mitochondrial oxidative phosphorylationMethods:1.The impact of IFI6 on cellular ATP levels was determined by UV spectrophotometer.2.The impact of IFI6 on the oxygen consumption rate（OCR）,extracellular acidification rate（ECAR）and complex-specific OCR of ESCC cells were determined by Seahorse cell energy metabolism analyzer.3.Native blue polyacrylamide gel electrophoresis（BN-PAGE）technique was employed to analyze and compare the effects of IFI6 on the assembly and expression of each respiratory supercomplex.4.The expression of respiratory complex monomer was detected by Western blot technique.Results:1.The role of IFI6 in the regulation of energy metabolism of ESCC cells:The impact of IFI6 on the cellular ATP content was detected in ESCC cells,and it was found that IFI6 silencing reduced,while IFI6 overexpression increased cellular ATP levels;Seahorse analysis confirmed that IFI6 overexpression increased baseline OCR and maximal respiratory reserve in ESCC cells,and IFI6 silencing exhibited the opposite effect.IFI6 did not affect the rate of glycolysis of ESCC cells.2.The impact of IFI6 on component of aerobic respiratory chain:Seahorse was used to determine the impact of IFI6 on respiratory complex-specific OCR.The results showed that IFI6 overexpression increased,while IFI6 silencing inhibited the activity of mitochondrial respiratory complexes I,III,and IV,and that IFI6 had no effect on respiratory complex II activity.3.IFI6 knockdown inhibits the assembly of mitochondrial respiratory supercomplex:BN-PAGE was employed to detect the effect of IFI6 expression on the assembly and expression of mitochondrial respiratory supercomplex,the results showed that IFI6 overexpression promoted,while IFI6 silencing inhibited the assembly of supercomplex CI?+?CIII₂?+?CIVn,CI+?CIII₂ and CIII₂?+?CIVn.Conclusion:1.IFI6 promotes aerobic respiration and ATP production of ESCC cells.2.IFI6 silencing inhibits the aerobic respiration activity of mitochondrial respiratory complexes I,III and IV,and thereby promoting mt ROS production.3.IFI6 silencing reduces the aerobic respiration rate of complex I,III and IV by inhibiting the assembly of mitochondrial respiratory supercomplex（CI?+?CIII₂?+?CIVn,CI?+?CIII₂,CIII₂?+?CIVn）.PART IV IFI6 silencing promotes cytoplasmic ROS generation by inducing endoplasmic reticulum stressMethods:1.H₂ DCFDA was used to label cellular ROS,and changes of cellular ROS were determined by flow detection or fluorescence microscope.2.RT-PCR and Western blot were used to detect the m RNA and protein levels of interest molecule.3.The endoplasmic reticulum specific Ca²⁺fluorescent probe Ig H-2mut AEQ was used to label the endoplasmic reticulum Ca²⁺,and the change of endoplasmic reticulum Ca²⁺concentration was monitored in real time by a fluorescence spectrophotometer.4.The lentiviral sh RNA expression vector was used to construct ESCC cells with stably ATF3 or NOX4 knockdown.ATF3,NOX4 or MFN2 overexpression plasmids were constructed and transfected into ESCC cell lines to construct ESCC cells with ATF3,NOX4,or MFN2 overexpression.5.Constructing reporter gene plasmids containing promoter fragment of PDI,XBP1,ATF3 or NOX4,respectively.Constructing reporter gene plasmids containing truncated or deleted mutant version of NOX4 promoter fragments.Dual luciferase reporter system assay was used to determine the relative fluorescence in cells transfected with above indicated plasmid.6.Xenograft nude mouse model was used to compare and analyze the impacts of interest factors on tumor growth in vivo by monitoring the volume of the implanted tumors,to quantify the expression of oxidative stress markers and endoplasmic reticulum stress markers in implanted tumors through Western blot and immunohistochemistry.Results:1.IFI6 knockdown promotes cytoplasmic ROS generation by upregulating NOX4:H₂DCFDA was used to label cellular ROS,and it was found that supplementing exogenous ATP or NOX4 knockdown could partially reverse the promotive effect of IFI6 silencing on ROS generation,while NOX4 overexpression reversed the inhibitory effect of ATP on ROS production.2.IFI6 silencing induces endoplasmic reticulum stress in ESCC cells through inducing ATP depletion:RT-PCR and Western blot showed that IFI6 silencing promoted,whereas IFI6 overexpression reduced the expression of endoplasmic reticulum stress-responsive effectors in ESCC cells.Western blot showed that IFI6 silencing induced the expression of ATF3 and NOX4,while supplementation of exogenous ATP could block the regulation effect of IFI6 on ATF3 and NOX4 expression.3.IFI6 interferes with endoplasmic reticulum calcium transport by decreasing ATP levels:Western blot analysis showed that knockdown of ATF3 blocked the promotive effect of IFI6 silencing on NOX4 expression.Luciferase assays showed that IFI6overexpression inhibited the transcriptional activity of ATF3,while blocking mitochondrial ATP transport reversed the inhibitory effect of ATF3 transcription exerted by IFI6 overexpression.The endoplasmic reticulum Ca²⁺was labeled with Ig H-2mut AEQ fluorescent probe,and it was found that the IFI6 silencing reduced,whereas IFI6 overexpression increased endoplasmic reticulum Ca²⁺level.Meanwhile,inhibiting aerobic respiration by adding oligomycin or inhibiting mitochondrial ATP transport by adding atractyloside could block the regulation effect of IFI6 on endoplasmic reticulum Ca²⁺uptake.4.ATP depletion inhibits SERCA calcium pump activity and decreased endoplasmic reticulum Ca²⁺concentration induces endoplasmic reticulum stress:Ig H-2mut AEQ fluorescent probe was used to label Ca²⁺in the endoplasmic reticulum,and it was found that overexpression of MFN2 not only promoted Ca²⁺uptake of endoplasmic reticulum,but also reversed the inhibitory effect of IFI6 silencing on endoplasmic reticulum Ca²⁺uptake.SERCA inhibitor cyclopiazonic could completely block the promotive effect of MFN2 overexpression on endoplasmic reticulum Ca²⁺uptake.Luciferase assay showed that either inhibition of SERCA activity or induction of ER stress with Tunicamycin could reverse the transcriptional repressive effect of IFI6overexpression on ER stress-responsive effectors.5.ATF3 transcriptionally activates NOX4:Luciferase assay confirmed that ATF3could transcriptional up-regulating NOX4.Based on the prediction of ATF3 binding target by JASPAR,combined with the construction reporter plasmid containing truncated or deletion mutant version of NOX4 promoter fragment,the exact binding region and binding motif of ATF3 on NOX4 promoter was elucidated by luciferase assay.6.In vivo validation of the biological effect and underlying molecular mechanism of IFI6 in ESCC:A xenograft nude mouse model which ESCC cells were subcutaneously implanted was constructed,the promoting effect of IFI6 on the growth of ESCC cells as well as the relationship between IFI6/ATF3/NOX4 pathway and ROS generation was confirmed in vivo.Moreover,it is confirmed that IFI6 regulates mt ROS production by modulating mitochondrial calcium homeostasis;Ultimately,it is also confirmed that IFI6 modulates endoplasmic reticulum stress and the activity of ATF3/NOX4 pathway by regulating mitochondrial aerobic respiration.Conclusion:1.IFI6 silencing promotes cytoplasmic ROS generation by up-regulating NOX4.2.IFI6 silencing upregulates NOX4 expression by inducing endoplasmic reticulum stress.3.IFI6 silencing decreases ATP level,which in turn inhibits the activity of SERCA and endoplasmic reticulum Ca²⁺uptake,thereby inducing endoplasmic reticulum stress.4.Endoplasmic reticulum stress up-regulates ATF3 expression,which in turn transcriptionally activates NOX4.5.Xenograft nude mouse model confirmed the biological effect and underlying molecular mechanism of IFI6 in ESCC.