The Effects And Mechanisms Of Mitochondrial Function-Associated Protein IDH2 And Syntaphilin

Esophageal squamous cell carcinoma(ESCC)is the most frequent histological type of esophageal cancer globally.The majority of ESCC patients are diagnosed at a locally advanced stage with long esophageal lesions and serious invasion and/or enlargement of the lymph nodes,and large prospective Phase 3 trials have established definite chemoradiotherapy(dCRT)as the standard treatment for those patients.However,the curative effects of radiotherapy differ from those in patients who have an identical malignancy grade and are homogeneously treated.In addition,a large proportion of ESCC patients suffer from recurrence and metastasis even after clinical complete response(cCR).Inherent heterogeneity caused by different gene expression may contribute to that phenomenon.Therefore,exploring predictive biomarkers for radiotherapeutic response should be emphasized.Mitochondria are essential eukaryotic organelles for energy generation,apoptosis modulation and redox balance maintaining.Recent evidence has reinforced a key role of mitochondria as a driver of radioresistance,and several processes have been associated with this phenomenon,including energy metabolism,mitochondrial apoptosis,oxidative stress modulation and calcium ion influx.Therefore,we start with the protein associated with mitochondrial function to search the key factor that modulates radioresistanceThe isocitrate dehydrogenase(IDH)family is the rate-limiting enzymes in the tricarboxylic acid cycle that reversibly convert isocitrate to ?-ketoglutarate,thereby producing NADPH.IDH2 is localized in mitochondria and critical for maintaining mitochondrial redox homoeostasis.NADPH generated by IDH2 helps to regenerate the reduced glutathione(GSH)and thioredoxin(TXN),which are key antioxidants against reactive oxygen species(ROS).Inhibiting GSH and TXN could sensitize cancer cells to radiotherapy and chemotherapy both in vitro and in vivo.At present,most cancer research on IDH2 focuses on its mutation status,such as R172 and R140 mutants in acute myeloid leukaemia and R172 mutant in glioma.After searching the cBioPortal database(https://www.cbioportal.org),we found that no IDH mutation existed in 227 ESCC samples.Although several in vitro studies show that mediating IDH expression could affect the response to ?-rays in human monocytes,glioma cells,and mouse embryonic fibroblasts,animal experiments and studies on the clinical significance remain inadequate.Syntaphilin(SNPH)is a static mitochondrial anchoring protein,which immobilizes mitochondria by docking them to the microtubule.Although originally defined as neuronal specific,this trafficking network is equally exploited in cancer cells,and SNPH is the critical regulator.During tumor progression,SNPH is downregulated or even silenced,which correlates with adverse patient outcome.SNPH deletion stimulates mitochondria redistribution from the perinuclear region to the cortical cytoskeleton,powering invadopodia dynamics,cytoskeleton remodeling and focal adhesion complex turnover,resulting in increased tumor cell migration and invasion.In addition,SNPH has been reported to act as a stress-regulated mitochondrial "rheostat"for controlling the tumor cell proliferation-motility phenotype switching,which has been identified as an alternative mechanism underlying the development of resistance to targeted therapies.However,the role of SNPH-mediated mitochondria distribution in the radioresistant(RR)phenotype of ESCC remains elusive.Based on the close correlation between mitochondrial function and radioresistance,we tried to find new biomarkers that could predict radioresistance of ESCC from mitochondrial function-associated proteins.There are two parts in this thesis,and the effects and mechanism of IDH2 and SNPH on ESCC radioresistance were successively explored.This study could enrich the knowledge about the modulation of mitochondria on radiosensitivity and provide novel predictive factor and therapeutic target for ESCC patients.Part I The role and mechanism of IDH2 in radioresistance of ESCCObjective1.To explore the effects and mechanism of IDH2 on radiotherapeutic efficacy in ESCC both in vitro and in vivo.2.To determine whether IDH2 expression could be the prognostic indicator for ESCC patients that reciced dCRTMethods1.Detection of the difference in radiosensitivity of four ESCC cell lines:Kyse140,Kyse510,Kyse150 and Eca109 cells were received different doses of radiation(2,4,6 and 8 Gy),and then the colony formation assay was performed to calculate the survival fraction.The survival curves were plotted,and the mean lethal dose(D0),quasi-threshold dose(Dq)and survival fractions at 2 Gy(SF2)were calculated by fitting the survival curves to the single-hit multi-target model.2.Detection of the difference in antioxidative protein in four ESCC cell lines:Western Blot was performed to detect the expression of IDH1,IDH2,Peroxiredoxinl-5(Prdxl-5),Thioredoxin(TXN),TXN2 and SOD2.The ratio of NADPH to NADP was measured to evaluate the enzymatic activity of IDH23.Construction of the cell line with stable expression of IDH2 shRNA:Plasmids that expressed IDH2 shRNA were transfected into Kyse140 and Kyse510 cells.At 24h after transfection,cells were passaged at 10-times dilution.Cells were selected with 1 ?g/mL puromycin,and the cell lines with stable expression of IDH2 shRNA were acquired after 1 week.4.Detection of the effects of IDH2 on radiotherapeutic efficacy in vitro:As for the radiated cells with stable expression of IDH2 shRNA,colony formation assay was performed to calculate D0,Dq and SF2,Western Blot to detect the apoptosis-related protein Bax,Bcl-2,Cytochrome c and cleaved caspase-3,flow cytometry to detect cell apoptosis.5.Detection of the effects of IDH2 knockdown on oxidative injury after radiation:As for the radiated cells with stable expression of IDH2 shRNA,2',7'-Dichlorofluorescin diacetate(DCFH-DA)and MitoSOXTM dectection by flow cytometry were applied to detect the effects of IDH2 on intracellular ROS and mitochondrial superoxide,respectively.The measurement of intracellular MDA and protein carbonyl content was applied to assess lipid peroxidation and protein oxidation,respectively.The extent of DNA oxidation was evaluated by dectecting 8-hydroxy-2'-deoxyguanosine(8-OHdG)by immunofluorescence.6.Dection of the effects of IDH2 on mitochondrial function:As for the radiated cells with stable expression of IDH2 shRNA,JC-1 dectection by flow cytometry was peformed to assess mitochondrial function.The expression of Dynamin-Related Protein 1(DRP1)and optic atrophy 1(OPA1)was detected by Western Blot to evaluate mitochondria fission.7.Dectection of the mechanism underlying the effects of IDH2 on radiotherapeutic efficacy:The expression difference of 43 kinases between normal cells and cells with stable IDH2 shRNA expression was measured by phosphokinase array,and the results was further testified by Western Blot.Whether AKT agonist SC-79 could inhibit the radiosensitization effect of sh-IDH2 was determined by flow cytometry.8.The construction of subcutaneous xenotransplanted tumor model of human ESCC in nude mice:6-week-old male BALB/cnu mice were divided into 4 groups:sh-NC,sh-IDH2,sh-NC+R(Rdiation)and sh-IDH2+R.Kyse140 cells were resuspended in a 1:1 solution of PBS/Matrigel and injected into the right shoulder of the mice.The sh-NC+R and sh-IDH2+R groups were given 2 Gy radiation at 3,5,and 7 days after injection.Tumour volume(V)was measured every 3 days in three dimensions.9.Detection of the effects of IDH2 knockdown on radiotherapeutic efficacy in vivo:The time-volume curve was plotted based on the calculation of tumor volume.The growth rate and volume of the xenograft in each group were compared.Western Blot was applied to dectect the expression of cleaved caspase-3,and cell apoptosis was measured by TUNEL staining.10.Clinical data and sample collection:The pre-treatment biopsy specimen from 141 ESCC patients that received dCRT were collected,and the IDH2 expression was dectected by immunohistochemistry.The association between IDH2 expression and the clinicopathological characteristics were analyzed by chi-square test.The overall survival(OS)and progression free survival(PFS)were estimated by the Kaplan-Meier method and compared using the log-rank test.Multivariable analysis by Cox proportional-hazard model was used to determine the independence of the prognostic factors.Results1.Kyse510 and Kyse140 cells were more radioresistant than Eca109 and Kyse 150 cells:After radiation,the Kyse510 and Kyse140 cells had higher colony formation capacity than Eca109 and Kyse 150 cells,and the Kyse510 and Kyse 140 cells had higher value of Dq and SF2.2.Kyse510 and Kyse140 cells had higher protein level and enzymatic activity of IDH2:When compared with the Eca109 and Kyse 150 cells,Kyse510 and Kyse 140 cells had higher expression of PRDX2,PRDX3,PRDX4 and TXN.Additionally,they also had higher protein level and enzymatic activity of IDH2.3.IDH2 knockdown inhibited radioresistance of ESCC:IDH2 knockdown further exacerbated the radiation-induced decrease in colony formation capacity in Kyse 140 and Kyse150 cells.Radiation caused cell apoptosis,enhanced the ratio of Bax/Bcl-2 and promoted the expression of Cytochrome C and cleaved caspase-3,and IDH2 knockdown could further promote those effects.4.IDH2 knockdown aggravated the radiation-induced oxidative injury in ESCC cells:Radiation increased the intracellular contents of ROS,mitochondrial superoxide,MDA,protein carbonyl and 8-OHdG,whilie IDH2 knockdown further intensified the radiation-induced damage on lipid,protein and DNA.5.IDH2 knockdown further exacerbated the radiation-induced mitochondrial dysfunction:IDH2 knockdown further exacerbated the radiation-induced decrease in mitochondrial membrane potential in Kyse510 and Kyse140 cells.Radiation increased the DRP1 level and decrased the OPA1 level,while IDH2 knockdown futher promoted that effect.6.IDH2 knockdown mediated radiosensitization via inhibiting AKT phosphorylation:Phosphokinase array showed that the phosphorylation of AKT and P53 were inhibited after IDH2 knockdown,and the inhibition in AKT phosphorylation was the most significant.The attenuation in AKT activation after IDH2 knockdown was further comfirmed by Western Blot.In both the steady state and after ROS induction by radiation,the mitochondrial ROS scavenger TEMPO could alleviate the IDH2 knockdown-induced decrease in AKT phosphorylation.Additionally,the selective AKT agonist SC-79 and TEMPO could inhibit the IDH2 knockdown-induced radiosensitization.7.IDH2 knockdown enhanced the radiotherapeutic efficacy in vivo:Mice in the sh-IDH2+R group had lower tumor growth rate and volume than that in the sh-NC+R gourp.Additionlly,the sh-IDH2+R group had higher cleaved caspase-3 level and more TUNEL-positive cells than the sh-NC+R gourp.8.The correlation between IDH2 expression and therapeutic effect and survival time of ESCC patients that received dCRT:Chi-square analysis showed that IDH2 exprssion was significantly correlated with dCRT response,in which low IDH2 expression was observed more frequently in the cCR group than in the non-cCR group(59%vs.34%).Kaplan-Meier curve showed that patients with lower IDH2 expression had better OS and PFS.Multivariate analysis confirmed that IDH2 expression was the independent prognostic indicator for OS and PFS of the ESCC patients that received dCRT.Conclusion1.IDH2 induced ESCC radioresistance via maintaining mitochonrail function and regulating ROS/AKT signaling.2.IDH2 expression was considered as an independent prognostic factor for ESCC patients that received dCRT.Part ? The role and mechanism of SNPH in radioresistance of ESCCObjective1.To explore the effects of radiation on mitochondria distribution in ESCC cells,and compare the difference of mitochondria distribution between normal and radioresistant ESCC cells.2.To determine the effects and mechanism of SNPH on proliferation,migration and radiosensitivity of ESCC cells.3.To explore the prognostic value of SNPH expression for ESCC patients that received dCRT.Methods1.Dectection of the mitochondrial distribution:The F-actin and mitochondria were labelled by Acti-stain 488 Phalloidin and CellLightTMMitochondria-RFP,respectively.The image was captured by fluorescence microscope.The cell boundary(line 1)and microtubule organizing center(line 2)were labeled according to the F-actin channel and mitochondria channel,respectively.The zone between line 1 and 2 was designated as the "cortical region".The percentage of cortical mitochondria to total mitochondria in each cell was calculated.2.The establishment of radioresistant(RR)ESCC subtype cell line(RR Eca109,Eca109R):Eca109 cells were grown to approximately 50%confluence and exposed to 2 Gy radiation.Once the cells reached 90%confluence,they were trypsinized and subcultured.This procedure was repeated until cells had received a cumulative dose of 60 Gy.The radioresistance was confirmed using flow cytometry and colony formation assay.3.Construction of the cell line with stable expression of SNPH:ESCC cells were infected with lentiviral vectors expressing SNPH.At 16h after transfection,cells were passaged at 10-times dilution.Cells were selected with 1 ?g/mL puromycin,and the cell lines with stable expression of SNPH were acquired after 1 week.4.Detection of the effects of radiation on mitochondrial distribution in ESCC cells:The Eca109,Kyse150,Kyse510 and Kyse140 cells were exposed to 8Gy radiation,and the effects of radiation on mitochondrial distribution was detected.The effects of radiation on the expression of the key mitochondrial trafficking protein(SNPH,SYBU,TRAK,RHOT,KIF5B)was evaluated by Western Blot.5.Comparison of the difference between Eca109 and Eca109R cells in mitochondrial distribuation,proliferation and migration:Fluorescence labeling was used to detect mitochondrial distribution,Edu experiment to detect proliferation and Trasnwell test to measure migration.6.Detection of the effects of SNPH on proliferation,migration and mitochondrial distribuation in RR ESCC cells:Measure the effects of SNPH re-expression on proliferation,migration and mitochondrial distribuation in Eca109R cells.7.Detection of the effects of SNPH on radiotherapeutic efficiency in RR ESCC cells:For radiated Eca109R and Eca109R with stable expression of SNPH,cell apoptosis was determined by flow cytometry.DCFH-DA and MitoSOXTM staining were applied to dectect intracellular ROS and mitochondrial superoxide.The content of MDA and protein carbonyl were detected to assess lipid peroxidation and protein oxidation,respectively.8-OHdG detection by immunofluorescence was performed to evaluate the oxidation of DNA.8.Detection of the mechanism underlying SNPH downregulation in RR ESCC cells:The effects of proteasomal inhibitor MG-132 and lysosomal inhibitor E-64 on the protein level of SNPH were determined by Western Blot.In order to clarify whether ubiquitin-proteasome-mediated protein degradation contributed to SNPH downregulation in Eca109R cells,co-immunoprecipitation was applied to campare the content of SNPH-ubiquitin complex between Eca109 and Eca109R cells.To figure out whether histone mediation contributed to SNPH downregulation in Eca109R cells,chromatin immunoprecipitation was applied to measure the protein content of H3Ac(Acetyl-Histone H3),H3K4me3(Trimethyl K4 of Histone H3),H3K9me3(Trimethyl K9 of Histone H3)and H3K27me3(Trimethyl K27 of Histone H3)in SNPH promoter.9.Research on the mechanism underlying the radiosensitization effect of SNPH re-expression in Eca109R cells:The expression difference of 43 kinases between radiated Eca109R and Eca109R with stable SNPH expression was measured by phosphokinase array.10.Detection of the effects of SNPH on radiotherapeutic efficacy in vivo:6-week-old male BALB/c-nu laboratory mice were randomly divided into 8 groups:sh-NC,sh-SNPH,sh-NC+R,sh-SNPH+R,Ctrl,SNPH,Ctrl+R and SNPH+R.The first 4 groups were implanted with transfected Eca109 cells,while the other 4 groups were implanted with transfected Eca109R cells.The animal model was established as described in Part I.The sh-NC+R,sh-SNPH+R,Ctrl+R and SNPH+R groups were given a 2 Gy radiation at 3,6 and 9 days after injection.Tumor volume was measured every 3 days in three dimensions.TUNEL staining was performed to evaluate cell apoptosis.11.Clinical data and sample collection:The pre-treatment biopsy specimen from 156 ESCC patients that received dCRT were collected,and the SNPH expression was dectected by immunohistochemistry.The association between SNPH expression and the clinicopathological characteristics were analyzed by chi-square test.The OS and PFS were estimated by the Kaplan-Meier method and compared using the log-rank test.Multivariable analysis by Cox proportional-hazard model was used to determine the independence of the prognostic factors.Results1.Radiation induced mitochondrial re-distribution to cortical region in ESCC cells:Most mitochondria were located around the microtubule organizing center in ESCC cells.When compared with the normal ESCC cells,radiated cells had more cortical mitochondria.Radiation downregulated SNPH,but had no effects on the protein level of KIF5B,TRAK,RHOT and SYBU.2.When compared with Eca109 cells,Eca109R cells had lower proliferation capacity,higher migration ability and more cortical mitochondria.Additionally,Eca109R cells had lower protein level of SNPH,indicating SNPH-mediated mitochondrial trafficking might facilitate migration of Eca109R cells.3.Re-expression of SNPH inhibited proliferation,promoted migration and reduced the quantity of cortical mitochondria.4.Re-expression of SNPH aggravated the radiation-induced oxidative injury and promoted radiosensitization:Radiation could enhance the intracellular content of ROS,mitochondrial superoxide,protein carbonyl,MDA and 8-OHdG,while SNPH re-expression could further promote those effects.Additionally,SNPH re-expression could aggravate the radiation-induced cell apoptosis.5.Ubiquitin-proteasomal protein degradation and histone mediation contributed to the SNPH downregulation in Eca109R:MG132 incubation increased the SNPH protein level in Eca109R cells.There was more SNPH-ubiquitin complex in Eca109R than Eca109 cells.Chromatin immunoprecipitation showed that there were more transcription-silencing modifications H3K9me3,H3K27me3 and less activating modifications H3Ac in the SNPH promoter region of Eca109R than those in Eca109 cells.6.SNPH re-expression enhanced radiosensitivity in vivo:Mice in the SNPH+R group had lower tumor volume and growth rate.Additionally,this group had more TUNEL-positive cells.7.The correlation between SNPH expression and therapeutic effect and survival time of ESCC patients that received dCRT:Chi-square analysis showed that SNPH exprssion was significantly correlated with dCRT efficiency.Kaplan-Meier analysis showed that patients with higher SNPH expression had better OS and PFS.Multivariate analysis proved that SNPH expression was the independent prognostic factor for OS and PFS of the ESCC patients that received dCRT.Conclusion1.Radiation downregulated SNPH,promoted mitochondria re-distribution to cell periphery and enhanced cell migration ability in ESCC cells.2.SNPH downregulation-mediated mitochondrial aggregation induced radiosensitization via exacerbating the radiation-induced oxidative injury.3.SNPH expression can be an independent prognostic indicator for ESCC patients that received dCRT.