Oxidative Phosphorylation And Autophagy Synergically Promote Intracellular PD-L1 Expression In Dormant Bladder Cancer Cells

BackgroundBladder cancer(BC)is a common malignant tumor of the urinary system.Chemotherapy,immunotherapy and surgery are effective methods for the treatment of BC,but the risk of recurrence and death is still very high.Exploring the causes and mechanisms of drug resistance and recurrence of BC and finding new intervention targets and strategies are the key to further improve the efficacy of BC.Studies have shown that tumors go into dormancy after radical treatment,such as high-dose chemotherapy.Tumor dormancy is a temporary,reversible mitosis and growth arrest.Dormant tumor cells(DTC)are resistant to conventional radiotherapy/chemotherapy,which is difficult to detect in clinic.At the same time,they retain the potential of reactivation and proliferation under suitable conditions.DTC can also be found in circulating tumor cells(CTC).CTC is spread through blood,which is the main cause of distant metastasis.Therefore,DTC is the source of tumor recurrence and distant metastasis.It is well known that tumor cells have the metabolic characteristics of aerobic glycolysis(Warburg effect)to meet the needs of their rapid proliferation.Compared with ordinary tumor cells,DTC is in a static and inactive state,and tumor cells that have been in a static state for a long time are bound to senescence.What are the characteristics of the metabolic pathway of DTC in this state?What is its effect on the senescence of DTC?In addition,autophagy,as a process of self-catabolism,is a protective mechanism for cells to maintain their homeostasis and resist aging under stress conditions.what is the status of autophagy in DTC?What does it have to do with the metabolic pathway?There are few reports.DTC,especially CTC,can not only survive in the body for a long time,but also overcome the immune clearance of the body,so DTC must have the function of immune escape.Programmed death ligand 1(PD-L1)is an immune checkpoint protein,which negatively regulates immunity.It has not been reported whether PD-L1 is involved in the immune escape of DTC.In addition,the level of PD-L1 protein is regulated by intracellular energy.When energy is deficient,PD-L1 will be degraded by proteasome mediated by AMPK.So,does the choice of metabolic pathway and autophagy affect the expression of PD-L1 in DTC?It hasn’t been reported yet.In this study,we identified the metabolic characteristics and autophagy of dormant bladder cancer cells(DBC),and explored their effects on cell senescence and PD-L1 expression.Objectives1.To determine the metabolic characteristics and autophagy of DBC.2.To explore the effects of OXPHOS and autophagy on senescence of DBC.3.To explore the effects of OXPHOS and autophagy on PD-L1 expression in DBC.Methods and Results1.DBC model was successfully established.Batch culture method was used to induce cisplatin-resistant human bladder cancer cells U3 and mouse bladder cancer cells MB49.After 72 h induction with high concentration of DDP,the cells entered the dormant state and the DBC was obtained.DBC induced by paclitaxel(PTX)was obtained by the same method.2.OXPHOS and autophagy of DBC promote each other and resist senescence.In DBC,the mRNA level of OXPHOS related genes(PGC-1a,Cytc,Cox5b)increased significantly,and Seahorse results confirmed that the basic level of OCR and ATP production increased significantly,the mRNA level of autophagy related genes ATG5,ATG7,BECN1 increased significantly,the protein expression levels of P62,BECN1,LC3B increased.The ratio of LC3B Ⅱ/Ⅰ increased,indicating that the OXPHOS and autophagy of DBC were enhanced.Furthermore,inhibition of OXPHOS(Metformin and Oligomycin A)can inhibit autophagy,in turn,inhibition of autophagy(CQ,3MA)can inhibit OXPHOS,indicating that OXPHOS and autophagy can promote each other.Inhibition of OXPHOS or autophagy increased DBC senescence.Interestingly,further enhanced DBC autophagy(Rapamycin),DBC senescence also increased.3.The level of intrinsic PD-L1(not on the membrane)increased in DBC.Real-time PCR and Western Blot detection showed that the expression of PD-L1 in DBC was increased.Further Western Blot was performed by separating intracellular/membrane proteins,and flow cytometry showed that the increased PD-L1 in DBC was mainly located in the cell,not on the membrane.4.OXPHOS or autophagy in DBC inhibits PD-L1 degradation through proteasome pathway.Inhibition of OXPHOS could decrease the PD-L1 expression of DBC,but upregulate the membrane PD-L1.We further confirmed that OXPHOS could inhibit the degradation of PD-L1 in DBC.In addition,inhibition of autophagy can also effectively reduce the level of PD-L1,and the combined inhibition of OXPHOS and autophagy has a more obvious inhibitory effect on PD-L1.It has been confirmed that the activation of cellular energy receptor AMPK can promote the degradation of PD-L1 through proteasome pathway.We found that MG 132 inhibition proteasome in DBC can up-regulate PD-L1 protein level,indicating that PDL1 in DBC is degraded by proteasome pathway,while inhibition of OXPHOS or autophagy can activate AMPK.It is suggested that OXPHOS can inhibit the activation of AMPK and then inhibit the proteasome degradation of PD-L1 mediated by AMPK.5.Interference with PD-L1 can promote the senescence of DBC.Through bioinformatics methods,it was found that PD-L1 was related to agingrelated genes.After successfully interfering with the expression of PD-L1 in DBC with siRNA,DBC senescence increased significantly.Conclusion1.OXPHOS and autophagy promote each other to resist senescence of DBC.2.OXPHOS and autophagy promote the expression of intracellular PD-L1 in DBC3.Intracellular PD-L1 resists senescence of DBC.