Mitochondrial NOS1 Regulates Chemo-resistance And Type ? Interferon Pathway In Colon Cancer

Background and ObjectivesNitric oxide(NO),a multipotent free radical gas,plays a key role in physiological and pathological processes,such as blood pressure regulation,inhibition of platelet aggregation and immune defense.In the past decades,NO has also been recognized as an important molecule in carcinogenesis and tumor progression.It has been reported that NO regulated various cancer processes,including angiogenesis,apoptosis,invasion and metastasis.However,there is considerable controversy and confusion in understanding its role in cancer biology.Studies have shown that it has the function of inhibiting tumors and promoting tumors,depending on its concentration,location and time.Endogenous NO is synthesized by three types of nitric oxide synthase(NOS):neuronal nitric oxide synthase(nNOS,NOS1),inducible nitric oxide synthase(iNOS,NOS2)and endothelial nitric oxide synthase(eNOS,NOS3).In mammalian cells,three NOS isozymes locates in various subcellular locations,including cytoplasm,lysosome and mitochondria.The localization of NOS in cells may affect the biological role of NO produced by the enzyme.Unlike its endothelial and inducible counterparts,NOS1 displays a PDZ(PSD-95/Dlg/ZO-1)domain located at its N-terminus,which involves in subcellular targeting.It has been reported that NOS1 was translocated into mitochondria through its PDZ domain in cardiomyocyte.However,the function of mitochondrial NOS1 is not fully understood.The role of NO in colon carcinogenesis and progression has been confirmed in a large number of studies.However,many related studies focused on NOS2 and NOS3 among the three NOSs,but little attention was paid to NOS1.Although NOS1 was originally found in neuronal tissues,it is now known to be much more widely distributed.The role of NOS 1 in progression of cancer has been studied also.In the present study,we aim to investigate whether NOS1 plays a special role in colon cancer progression thought its mitochondria location.Results1.NOS1 protein expression in mitochondriaTo determine the association between NOS1 expression and the prognosis of patients with colon cancer.Survival data of CRC patients were obtained from TCGA,Kaplan-Meier analysis indicated that the patients with higher NOS1 expression(235 cases)tended to exhibit a poorer overall survival c0mpared with patients with lower NOS1 expression(268 cases).Moreover,the expression of NOS 1 increased gradually with the increase of clinical stage of colon cancer.In order to identify the role of NOS1 in colon cancer,stable overexpression of NOS1 in colon cancer cells was successfully constructed Using Lentivirus Vector.As it was reported that the alpha isoform of NOS 1 which contains PDZ domain could transported into mitochondria,we examined subcellular localization of NOS 1.we isolated mitochondria from NOS1 overexpression colon cancer cells and control.Western blot analysis showed a significantly increased NOS1 protein expression in isolated mitochondria of NOS 1-overexpressing cells.To confirm these results,We observed the co-localization of NOS 1 and mitochondria under confocal microscopy.By labeling mitochondria with red fluorescence and NOS1 with GFP,we clearly observed that NOS1 and mitochondria were co-localized.mitochondria are rich in calcium ions and NOS1 produces NO in a Ca2+-dependent status.Here,we demonstrated that nitrite levels were significantly increased in isolated mitochondria of NOS 1 overexpression colon cells.2.NOS1 inhibited mitochondrial ROS and induced apoptosis-resistance in colon cancer ceilsMitochondrial reactive oxygen species were detected by MITOSOX assay.The results showed that NOS1 overexpression inhibited mitochondrial superoxide anions.Moreover,mitochondrial superoxide anions induced by cisplatin(DDP)were attenuated by NOS1 also.Mitochondria play a central role in regulating cell survival and death signaling.The accumulation of mitochondrial reactive oxygen species(ROS)can lead to the expansion of mitochondria and increase the permeability of mitochondrial inner membrane.We found that both cytosol cytochrome C and caspase 3 activity induced by cisplatin were inhibited significantly by NOS1 overexpression.After DDP treatment,Flow cytometry analysis showed that SW480 or SW620 cells with NOS1 overexpression had much less apoptotic cells than those control cells.These results showed that NOS1 inhibits apoptosis via attenuating mitochondrial superoxide.3.SIRT3 activation is enhanced by NOS 1Under physiological conditions,cells control the level of reactive oxygen species by balancing the production and the removal of reactive oxygen species.Among antioxidant enzymes,such as superoxide dismutase-SOD,reduced glutathione-GSH,and catalase-CAT,SOD2 is thought to have an essential role in controlling the level of mitochondrial superoxide.Because SOD2 is acetylated at Lys 68,which decreases its activity,the total protein and acetylation of SOD2 influences the level of ROS in.mitochondria.Thus,we investigated whether total protein or acetylation of SOD2 is regulated by NOS1.The result showed that SOD2 total protein was not influenced by NOS 1,but acetylation of SOD2(acetyl K68)was significantly decreased by NOS 1.As a result,activity of SOD2 was also increased by NOS1.SIRT3 has been recognized as a major deacetylase in mitochondria.SIRT3 deacetylates and activates SOD2 to scavenge mitochondrial ROS.The acetylation level of total mitochondrial protein was detected to determine whether NOS1 increases SIRT3 activity.Our results showed that the acetylation level of total mitochondrial protein was reduced obviously by NOS1 overexpression.NOS1 did not influence the protein level of SERT3,which indicated that NOS1 could increase SIRT3 activity.Furthermore,NOS1-induced mROS inhibition and apoptotic resistance was reversed by knocking down SIRT3.4.HSP90 inhibitor impedes NOS1 mitochondrial translocation and reverses NOSl-induced apoptosis resistanceHSP90 is a chaperone protein that assists other proteins to fold properly,.Previous studies have shown there is an interaction between HSP90 and NOS1.To determine whether the mitochondrial translocation of NOS1 depends on HSP90,Sylibum,the C-terminal HSP90 inhibitor,was used to treat NOS1 overexpression colon cells.Mitochondria isolation result showed that mitochondrial NOS1 was decreased obviously after sylibum treatment(50?g/ml,48h).The similar change was confirmed by confocal microscopy.Furthermore,the interaction between NOS1 and HSP90 was analyzed by Co-IP assay.The result showed that sylibum inhibited the interaction between NOS1 and HSP90.To investigate whether sylibum attenuates NOS1-induced chemotherapy resistance,SW480-NC/OE NOS1 cells were treated with cisplatin together with sylibum.After treatment with cisplatin and sylibum,apoptosis significantly increased in SW480-OE NOS1 cells,but not in the control cells,and two-way ANOVA analysis showed that there was an interaction effect between NOS 1 and sylibimi.These results indicate that mitochondrial translocation of NOS1 depends on HSP90.NOSl-induced chemotherapy resistance could be reversed by inhibiting mitochondrial translocation of NOS1.5.Mitochondrial NOS1 inhibits type ? interferon pathway in colon cancer cells by increasing hydrogen peroxideType ? interferon initiates innate and adaptive immunity in tumor microenvironment,and it has been found that disfunction of type ? interferon pathway exists in many difference types of tumor.To determine whether type ? interferon pathway is regulated by NOS1.Flow cytometry was used to determined IFN a-induced STAT1 phosphorylation(IFN ?-p-STAT1).The result showed that IFN a-p-STATlwas inhibited by NOS1-overexpression,and was increased by NOS1 inhibitor(NPLA)in colon cancer cells.Moreover,we found that NOS1 increased the production of hydrogen peroxide,and catalase reversed NOSl-induced IFN ?-p-STATl inhibition.RNA-seq also confirmed that NOS1 inhibited IFN a-induced downstream gene(ISGs)expression.These results showed that NOS1 inhibits type ?interferon pathway in colon cancer cells by increasing hydrogen peroxide.ConclusionNOS1 in colon cancer cells is transferred to mitochondria with the assistance of HSP90.On the one hand,mitochondrial NOS1 enhances mitochondrial anti-oxidant stress through mtNOSl-SIRT3-SOD2 pathway,which leads to chemotherapy resistance in colon cancer.NOS1-induced chemotherapy resistance was reversed by knocking down SIRT3 or HSP90 inhibitors.On the other hand,mitochondrial NOS1 induces disfunction of type I IFN pathway in colon cancer cells by increasing the production of hydrogen peroxide.This study provides a clue for the reversal of chemotherapeutic resistance of colon cancer by targeting NOS1.However,the effect of NOS 1 on tumor immune escape remains to study in vivo.