Mitochondrial PD-L1 Mediated Metabolic Reprogramming Involved In Ferroptosis Sensitivity Of Liver Cancer Stem Cells Regulated By Tumor Microenvironment

Liver cancer is the sixth most common cancer in the world and ranked the third highest incidence,which causes a huge global disease burden.The prevention and treatment of liver cancer is still one of the most important public health issues facing the world.Understanding the molecular mechanism of the initiation and progression of liver cancer can provide better strategies for its prevention and treatment.Liver cancer was driven by chronic inflammation,and the complex immune microenvironment in liver was an important factor that promotes the occurrence and progression of liver cancer,and it was considered that cancer stem cells(CSCs)were the main cause of tumor initiation,metastasis,recurrence,and drug resistance,and TME has been shown to play an important role in the initiation of CSCs,maintenance of stemness,and mediating drug resistance of CSCs.Ferroptosis is an iron-dependent form of regulated cell death caused by lipid peroxidation,and it was a promising way in tumor therapy by targeting to ferroptosis,which ferroptosis has been found in many cancers such as liver cancer and lung cancer.In addition,ferroptosis may be a new strategy for targetable elimination of CSCs by inducing ferroptosis in CSCs.However,at present,there are few studies on how TME regulates CSCs sensitivity to ferroptosis,and the specific mechanism is still unclear.Objectives:In this study,the SP sorting of HCC cells was used to establish a LCSCs and non-LCSCs model.The effect of cytokines in TME on LCSCs was evaluated by treating LCSCs with IFN-γ,and sorafenib(Sora)was used as a ferroptosis inducer;To explore the mechanism that TME cytokine IFN-γ regulated the sensitivity of LCSCs to ferroptosis.This study may provide a theoretical basis for the targeted intervention to eliminate LCSCs with TME and ferroptosis as targets.Methods:(1)In vitro test:HepG2,Huh7 and PLC/PRF/5(PLC)cells were selected,and SP cells and MP cells were obtained from HCC cells.These cells were treated with IFN-γ(50 ng/mL)or/and Sora(5 μmol/L)as models,PD-L1 or GPX4 intervention model was established by siCD274 or siGPX4 transient transfection.PLC-CD274 cell lines overexpressed PD-L1 and HepG2-2R5C and Huh7-2R5C cell lines overexpressed P2A-B56γ were constructed as models.Assays:①Hoechst33342 fluorescence labeled flow cytometry was used for analyzing the proportion of SP cells and SP sorting;②The expressions of LCSCs markers,GPX4,Bcl2,and PD-L1 were detected by Western blotting(WB);③The expression levels of cancer stemness markers,glycolytic metabolic enzymes and PP2A-B subunits genes were detected by real-time fluorescence quantitative PCR(qRT-PCR);④The ability of sphere formation was evaluated by sphere-forming assay cultured in ultralow attachment plates;⑤MTS Cell Proliferation Assay Kit was used to evaluate the effect of Sora on cell viability;⑥Apoptotic cells were detected by Annexin V-FITC detection kit with FCM;⑦The localization of proteins and mitochondrial morphology labeled by immunofluorescence were detected by laser confocal microscopy;⑧The lipid peroxidation levels labeled by C11-BODIPY581/591 were detected by laser confocal microscopy;⑨The total ROS and mitochondria ROS level labeling with DCFH-DA and MitoSOX respectively were determined by FCM;⑩The ATP content and lactic acid release level were detected with ATP assay kit and lactic acid assay kit,respectively;11Self-renewal ability was evaluated by clonal formation assay;12Cell mitochondria isolation kit was used for mitochondrial isolation,which used for detection of PD-L1 by WB.(2)Bioinformatics analysis:GEO database(GSE54236)was used for GSEA analysis of IFN,IL6 and TNFA related pathway between HCC(n=81)and normal(n=80)samples.TCGA database was used for analysis of the correlation between the expression of CD274 and genes related to glycolysis in liver cancer samples(n=373),and the differential expression of PP2AB subunit gene in adjacent normal tissues(n=50)and liver cancer samples(n=373),and the differential expression of PPP2R5C in matched tumors and paratumors(n=50);PDB database was used for predication of interaction proteins with PD-L1.(3)In vivo test:The nude mice of 4-6 weeks of age were selected for subcutaneous tumorforming experiment with 5 × 106 cells per mouse.When the longest tumor diameter reached 1 cm,the mice were treated with Sora(30 mg/kg/time),IFN-γ(0.1 μg/time)or/and siCD274(5 nmol/time).The mice were divided into five groups:Ctrl,IFN-γ,Sora,IFN-γ/Sora,IFNy/Sora/siCD274 group.Assays:①Tumor volume was measured every two days with a Vernier caliper,and calculated in a formula as:V=L × W × W/2,tumor weight was measured at the end point and tumor were collected for assays;②The expression levels of LCSCs markers,GPX4,and PD-L1 were detected by WB,IHC and IF assays;③The lipid peroxidation and total glutathione levels were detected with lipid peroxidation MDA assay kit and total glutathione assay kit,respectively,in tumors;④The ATP content and lactic acid levels were detected with ATP assay kit and lactic acid assay kit,respectively,in tumors.Results:(1)In vitro test:①Gene enrichment analysis showed that IFN related signaling pathways were enriched in HCC tissues,but not IL6 and TNFA signaling pathways;IFN-γpromoted the expression of CD 133 and CD44 in PLC and Huh7 cells,and increased SP proportion in PLC cells and partially alleviated the inhibition effect of Sora on SP proportion;Tumor sphere assay showed that sphere-forming ability of PLC and Huh7 cells was significantly stronger than that of HepG2 cells;②In LCSCs model constructed by SP sorting,compared with MP cells,the expressions of CD 133 and CD44 in SP cells were much higher,the sphere-forming ability of SP cells was significantly stronger,and the inhibition of cell viability in SP cells induced by sora was significantly weaker;③In the sora-induced cell death model,IFN-γ slightly inhibited the apoptosis induction of Sora in SP cells but significantly increased the apoptotic cells induced by Sora in MP cells.interestingly,compare with MP cells,SP cells had more fragmented mitochondrial and lower intracellular ROS levels,and IFN-γinhibited the sora-induced lipid peroxidation and intracellular total ROS production,upregulated the expression of GPX4 and Bcl2 in SP cells,and increased the clone formation ability of SP cells.④In the assays of LCSCs metabolism,compared with MP cells,the expressions of cancer stemness related genes and glycolysis-related genes in SP cells were up-regulated,and the up-regulation of PD-L1 in SP cells induced by IFN-γ was more obvious;TCGA database analysis showed that CD274 encoding PD-L1 was positively correlated with the expression of genes related to glycolysis metabolism;In the PD-L1 overexpressing cells,PD-L1 upregulated the expression of CD 133,CD44 and GPX4,increased extracellular lactic acid release and inhibited intracellular ATP content.In addition,by treating with IFN-γ,IFN-γ promoted the release of lactic acid in SP cells without affecting the ATP content,but increased the ATP content in MP cells.⑤In IFN-γ treated and PD-L1 overexpressed cell models,PD-L1 expressions in mitochondria were upregulated,and p53 interacted with PD-L1 in mitochondria IP assay;moreover,compared with MP cells,IFN-γ induced more PD-L1 mitochondrial translocations in SP cells;inhibiting the mitochondrial translocation of PD-L1 by knockdown of PD-L1 with siCD274 increased the production of mitochondrial ROS as well as total ROS,and inhibited the extracellular lactic acid release and the up-regulation of GPX4 induced by IFN-γ in SP cells.In addition,knockdown of GPX4 with siGPX4 increased total ROS levels and inhibited extracellular lactic acid release,but increased ATP content in SP cells.⑥ In B56y overexpression cell model,B56γ inhibited lactic acid release and increased ATP content,increased mitochondrial ROS production,inhibited clonal formation and sphere formation in 3D culture,and inhibited IFN-γ-induced mitochondrial translocation of PD-L1.(2)In vivo test:①Compared with the control group,Sora inhibited tumor growth,increased the level of lipid peroxide MDA and decreased the level of glutathione in tumor tissue,and inhibited the expression of GPX4 to promote ferroptosis,while IFN-γ upregulated the level of glutathione and GPX4,and decreased the level of MDA.Moreover,IFN-γ increased the expression of cancer stemness markers in HCC and promoted the cancer stemness phenotype;②compared with Sora group,IFN-γ combined with Sora inhibited the increase of MDA induced by Sora and rescued the inhibitory effect of glutathione and GPX4;③in addition,siCD274 intervention attenuated the inhibition of IFN-γ on Sora-induced ferroptosis and inhibited tumor growth,increased MDA levels and inhibited glutathione levels and GPX4 expression compared to the IFN-γ combined Sora group.Conclusions:In this study,we proved that LCSCs(SP cells)in liver cancer were more tolerant to Sora than non-LCSCs(MP cells),and we found that PD-L1 could translocated to mitochondrial by interacted with p53,which resulted in inhibition of mitochondrial function,thereby promoting the glycolysis and the expression of GPX4 in LCSCs,so that mediating IFNy-induced ferroptosis tolerance of LCSCs.In addition,we also demonstrated that B56γ inhibits IFN-γ-induced mitochondrial translocation of PD-L1,thereby inhibiting glycolysis metabolism and cancer stemness in HCC cells.