Mitophagy Enhances Oncolytic Measles Virus Replication By Mitigating RLRs Signaling And Switches Cell Death From Apoptosis To Necrosis In NSCLC Cells

Background and ObjectiveTumor is a worldwide threat to human being.Non-small cell lung cancer(NSCLC)is one of the leading causes of cancer-related morbidity and mortality.Oncolytic virotherapy is an emerging therapeutic modality that utilizes replication-competent viruses to destroy cancers.Due to its excellent safety records and therapeutic efficacy,attenuated measles virus vaccine Edmonston strain(MV-Edm)has been investigated in several clinical trials to treat advanced cancer patients.The success of future clinical trials with oncolytic measles viruses depends on the comphrehensive understanding of oncolytic mechanisms.In this study,for the first time,we investigated that oncolytic MV-Edm utilizes mitophagy to mitigate antiviral innate immune response and to block proapoptotic signaling,which results in enhanced viral replication and eventually leads to necrotic cell death in NSCLCs.Thus,we provide rationale for future improvement using oncolytic MV-Edm combined with autophagy inducers.Materials and Methods1.Autophagy detection:1)A549 and H1299 were both transfected with eGFP-LC3,the location of eGFP in cells was monitored by confocal fluorescence microscope,2)western blot was employed to evaluate the expression of LC-3? and SQSTM1 in cells infected with MV-Edm in the presence or absence of chloroquine.2.Mitophagy determination:1)evaluation of colocalization of mitochondria and autophagosomes,2)to analyze Mitotracker Green stained cells by cytometric analysis,3)to evaluate the dynamic changes of mitochondrial protein heat shock protein 60 by western bolt,4)and to determine the typic double-layered structures of mitophagosomesby Electron microscopy3.Interaction of mitophagy with RLRs and proapoptotic signaling:in autophagy-impaired cells,viral replication was determined by TCID50,RT-PCR and crystal violet staining;the anti-viral cytokines IFNB1,CXCL10,IFI27 and 2',5'-oligoadenylate synthetase 1(OAS1)were analyzed by RT-PCR and ELISA,or DDX58/IFIH1/MAVS signaling was determined by RT-PCR and western bolt;expression levels of the cleaved caspase-3,caspase-9 and PARP was evaluated by western bolt and dysfunctional mitochondria was analyzed by flow cytometry4.Identification of oncolytic mechanism of MV-Edm in NSCLCs:1)the cell viability was quantified by trypan-blue exclusion in the presence or absence of the pan-caspase inhibitor z-Vad-fmk,the inhibitor of necroptosis necrostatin-1 or antioxidant NAC;2)the production of LC3-?,SQSTM1,cytoplasmic cytochrome c,cleaved caspase-9,-3,PARP and HMGB1 were determined by western blot;3)the correlation between ATP generation and viral replication was analyzed;4)the correlation between viral replication and HMGB1 release was analy-zed,and 5)ROS generation was determined by flow cytometry.Results1.MV-Edm induced autophagy and autophagic flux in NSCLCs.MV-Edm infection resulted in EGFP-MAP1LC3B from a homogenous to a punctuate distribution in the cytosol of the transfected cells,increased levels of LC3-II,decreased levels of SQSTM1 which was reversed in the presence of lysosomal inhibitor chloroquine2.MV-Edm induces mitochondrial degradation by mitophagy in NSCLCs.After MV-Edm infection,the colocalization of mitochondria and autophagosomes was significantly increased,as a quantity of double-layered structures characteristic for autophagosomes enveloped mitochondria were observed,mitochondrial mass per cell was decreased,and of the mitochondrial conservative protein HSP60 was significantly decreased.3.MV-Edm exploited selective autophagy coined mitophagy to mitigate DDX58/RIG-? like receptors(RLRs)innate immune response against virus.in NSCLCs.Both RNAi and forced overexpression of autophagy related genes demonstrated that autophagy enhanced viral replication and inhibited the production of type-? interferon.The mitophagic degradation mediated bySQSTM1/p62 contributed to decrease mitochondrion-tethered MAVS and subsequently weakenedinnate immune response.4.The SQSTM1-mediated mitophagy contributed to clearance of damaged mitochondria leading to decreased cytochrome c release,and thus blocked the proapoptotic cascade in NSCLCs.The blocked apoptosis by mitophagy favored viral replication.Persistent viral replication sustained by autophagy ultimately resulted in necrotic cell death due to ATP exhaustion.Importantly,when autophagy was impaired in NSCLCs,MV-Edm-induced cell death was significantly abrogated despite of increased apoptosis.Conclusions1.MV-Edm usurps mitophagy to mitigate the antiviral innate immune response.2.Mitophagy switches cell death from apoptosis to more efficient necrosis in NSCLCs treated with MV-Edm.3.These findings provide a rationale to modulate autophagy in oncolytic virotherapy,and provide a foundation for future improvement of oncolytic virotherapy or antiviral therapy.