The Mechanism Of IKK? Activation Promotes Amphisome Formation And Extracellular Vesicle Secretion In Tumor Cells

Objective:As ever,the interaction between organelles has been regarded as the frontier and hot spot of research.Endosome system derived from eukaryotic endomembrane system and autophagy play an important role in maintaining cell homeostasis,and the interaction between them has gradually attracted more and more attention.It is clear that tumor cells need to constantly adapt to the harsh environment inside and outside cells during their progress,in which endosome system,autophagy and stress at the transcription level exert important effect.The interaction between endosome system and autophagy has significant role to the occurrence and development of diseases and the stress of tumor cells.The early endosome in the endosome system matures into multi vesicular body(MVB)containing ILVs under the action of endosomal sorting complex required for transport(ESCRT),which can not only fuse with lysosomes to participate in the degradation and circulation of substances,but also fuse with cell membranes to release extracellular vesicles,thus playing an important role in various pathophysiological processes.For a long time,researchers hold that the interaction process between endosome system and autophagy is reflected in the fusion of autophagosomes and MVBs to form an organelle amphisome in a hybrid transition state,and its final fusion with lysosomes completes the complete degradation of the substrate.However,recent studies have revealed that another important fate of amphisome is that it fuses with cell membranes to secrete exosomes,which contain active contents derived from autophagosomes,with the important significance in pathophysiological processes.Obviously,the exploration on the mechanism of amphisome generation and secretion can more clearly reveal the mechanism of interaction between endosome system and autophagy,and further improve our understanding of the interaction between eukaryotic organelles and the survival mechanism under tumor stress.Under stress,tumor cells will initiate stress at the transcription level to participate in the regulation of cell homeostasis.NF-?B signaling pathway plays a key role in stress at the transcription level,which is closely related to the occurrence and development of diseases including inflammation and tumor.In addition,it has been proved to be a potential effective therapeutic target.When the tumor is under stress,IKK complex acts as an important inducer of autophagy in many aspects,participates in the regulation of autophagy activity of tumor cells,increases its resistance to malignant environment,thus ensuring the "safety" of tumor cells.IKK? is an important catalytic subunit of IKK complex,which can regulate the nuclear import of NF-?B.Moreover,its kinase activity can phosphorylate proteins such as SNAP23 and ?-catenin,and regulate inflammation,apoptosis,autophagy,cell proliferation and metabolism through NF-?B-independent mechanisms.However,whether the activation of IKK complex induces amphisome production and sEVs secretion,and its possible mechanism remains to be unclear.Therefore,continuously activated IKK?(CA-IKK?)is constructed to explore the possible communication between endosome system and autophagy in tumor cells and the interaction mechanism between them in this study.Through fluorescence confocal,transmission electron microscope and immuno-electron microscope,NTA,vesicle separation and polyclonal antibody synthesis,the interaction mechanism between organelles in tumor cells with IKK? activation as the initiation point,as well as the relationship between eukaryotic endometrial system and stress caused by NF-?B at the transcription level was further clarified,so as to reveal the possible survival and adaptation mechanism of tumor cells under stress.Methods:1.Western blot was used to verify the activation of IKK?.After separating sEVs in conditioned medium by ultracentrifuge experiment,western blot was used to detect sEVs marker(CD63,TSG101 and CD81);the structure of extracellular vesicles was analyzed by transmission electron microscope and immuno-electron microscope.The number and diameter distribution of sEVs in conditioned medium were analyzed by NTA nano tracer.2.Immunofluorescence confocal was used to analyze the early endosome and MVB of IKK? tumor cells that were continuously activated.After the sEVs in the culture medium were separated by ultracentrifuge experiment,the ubiquitin protein content in sEVs was detected by western blot.3.Western blot was used to detect the expression of LC3 and p62 in the control group(Ctrl),wild group(WT),activated mutation group(CA)and tumor cells treated with different drugs,judge the regulation of IKK? activity on autophagy flow.Immunofluorescence confocal experiment was used to explore the co-localization of autophagosomes(LC3)and lysosomes(LAMP1)after CA-IKK?.The stable cell line stubRFP-sensGFP-LC3 was constructed,and the autophagy flow of continuously activated IKK? tumor cells was analyzed by fluorescence confocal experiment.4.Immunofluorescence confocal was used to detect the co-localization of autophagosomes(LC3)and MVB(CD63)to explore the characteristics of amphisome.The morphology and proportion of autophagosomes,MVB and amphisome in WT and CA groups were analyzed by transmission electron microscope.ImageJ was used to count the respective diameters and the number per unit area.5.Immuno-electron microscope was used to analyze the distribution of colloidal gold labeled LC3 and CD63 in CA tumor cells.Western blot was used to detect the expression of autophagy related components LC3 and p62 in sEVs secreted by IKK?-activated cells.Immunoelectron microscopy was used to detect LC3 in sEVs.Western blot was used to detect the expression of LC3 and p62 in sEVs secreted by tumor cells in Ctrl,WT and CA groups and combined with different drugs to judge the secretion of amphisome.Western blot was used to detect the expression of ATG7,LC3 and p62 in Ctrl,WT and CA groups after stable knockdown of ATG7,and the expression of LC3 and p62 in sEVs secreted by tumor cells.6.Western blot was used to detect the expression of RAB5,RAB7,RAB11,RAB27A,RAB27B,RAB35 and ATG14 in tumor cells treated with Ctrl,WT and CA groups and combined with different drugs.The level of RAB7 mRNA in WT and CA groups was verified by real time PCR.Western blot was used to detect the levels of CD63 and ubiquitin in sEVs secreted by tumor cells after RAB7 knockdown.The morphology and diameter of MVB were analyzed by immunofluorescence confocal experiment.Western blot,immunofluorescence confocal and NTA were used to detect the levels of CD63 and ubiquitin in sEVs secreted by tumor cells after co-transfection of RAB7-Q67L in WT and CA groups,the morphology and diameter of tumor cell MVB,and the number of sEVs secreted by tumor cells.7.Immunofluorescence confocal was used to analyze the co-localization of MVB(CD63)and SNAP23,the phosphorylation level of SNAP23 in CA cells was detected by phosphorylation separation gel,and the NF-?B dependent mechanism of CA was analyzed by IKK inhibitor.Regulation of SNAP23 phosphate level.Mutation inactivated SNAP23 plasmids at 95 and 110 positions were designed and co-transfected into CA-IKK? plasmid.Western blot was used to detect the expression of CD63 and ubiquitin protein in sEVs,and NTA tracer was used to quantify the concentration of sEVs.SNAP23-Ser95 antibody was synthesized,and Western blot was used to detect the SNAP23-Ser95 protein level of tumor cells in Ctrl,WT and CA groups and combined with different drugs.Results:1.IKK activation induced tumor cells to survive and release extracellular vesicles:Transmission and immunoelectron microscopy revealed the typical sEVs structure isolated by ultracentrifugation.NTA showed that the number of secreted particles in CA-IKK?group increased twice,while the size distribution of sEVs(50-200 nm)was not affected(*P<0.05).Western blot results showed that the expression of sEV markers CD63,CD81 and TSG101 in CA group was significantly up-regulated(**P<0.001,*P<0.05,and the expression of sEV markers was inhibited after the inhibition of IKK? activity.CCK8 results revealed that IKK activation and its induced sEVs maintained tumor cell stress and survival(**P<0.001,*P<0.05).2.IKK? activation induced the fusion of MVB and autophagosomes in tumor cells to form "amphisome" by regulating RAB7:Immunofluorescence confocal experiment confirmed that a large number of MVBs with larger diameter accumulated in tumor cells after IKK? activation(***P<0.0001).However,the endosomes did not change significantly in the early stage.Western blot showed that the level of ubiquitin protein in sEVs isolated from activated IKK cells increased significantly,while the treatment of HeLa and Huh7 cells with IKK inhibitors could reduce the production of ubiquitin protein in sEVs.Western blot results showed that the expression levels of LC3-II and LC3-I in WT and CA groups were significantly higher than those in Ctrl group(*P<0.05).Compared with Ctrl group,the level of p62 in WT group was significantly lower(*P<0.05).In addition,compared with WT group,p62 was not degraded in CA cells.Immunofluorescence confocal assay confirmed that the co-localization of autophagosomes(LC3)and lysosomes(LAMP1)increased significantly after CA-IKK?(*P<0.05).Analysis of double fluorescence StubRFP-SensGFP-LC3 stable cell line tumor cells autophagy and small volume aggregation after continuous activation of IKK.Confocal fluorescence results showed that the co-localization of CD63 positive MVB and LC3 in CA cells was significantly up-regulated(*P<0.05).TEM confirmed the fusion of autophagosomes and MVB in CA cells at the ultrastructural level.The results showed that a large number of heterogeneous vesicle structures accumulated,including autophagy bodies,classical MVB containing ILVs in the lumen,and amphisome containing both ILVs and autophagy structures.Further analysis showed that the number of MVB and amphisome per unit area in CA increased significantly(*P<0.05).Western blot showed that RAB7 decreased significantly in CA-IKK? cells(**P<0.001,*P<0.05).Inhibitor BAY11-7082 restored RAB7 protein level in CA-IKK? cells.The results of real time PCR showed that CA significantly inhibited the level of RAB7 mRNA in tumor cells(*P<0.05).Western blot and immunofluorescence confocal assay confirmed that stable knockdown of RAB7 could significantly increase the diameter of MVB(***P<0.0001)and induce the production of ubiquitin-rich sEVs.Immunofluorescence confocal assay confirmed that RAB7-Q67L attenuated the change of MVB diameter caused by CA-IKK?(*P<0.05),and western blot showed that RAB7-Q67L affected the secretion of sEV and the content of ubiquitin protein at the same time.NTA results showed that RAB7-Q67L attenuated the secretion of sEVs induced by CA-IKK?(*P<0.05).3.The phosphorylation of SNAP23-Ser95 induced by IKK? activation promoted the secretion of extracellular vesicles in the amphisome-dependent manner:Immunoelectron microscopy labeling confirmed that p62 resides in CD63-labeled MVB in CA-IKK?transfected HeLa cells,but not in WT group.Immunoelectron microscopy of sEVs derived from CA-IKK? confirmed that colloidal gold labeled LC3 antibody could label sEVs.Western blot showed that LC3-? and p62 were highly enriched in sEV in CA-IKK?transfected Huh7 and HeLa cells.The LC3-? and p62 levels in CA-IKK? transfected cells treated with BAY and 3-MA decreased,while the LC3-? and p62 levels were moderately increased by BafAl treatment.In HeLa cells with stable ATG7 knockdown,Western blot found that IKK?-activated cell-derived sEV had no significant expression of LC3-II and p62.Confocal analysis showed that the co-localization of CD63 and SNAP23 in CA-IKK?transfected cells was significantly up-regulated.Phosphorylation agents confirmed that CA-IKK? significantly increased the ratio of p-SNAP23/SNAP23(*P<0.05),and BAY treatment could significantly inhibit CA-IKK?-induced SNAP23 phosphorylation.Western blot results showed that the expression of SNAP23-S95A significantly eliminated the secretion of sEVs containing ubiquitin protein induced by CA-IKK?,while SNAP23-S110A did not change significantly.Antibodies that recognize phosphorylated SNAP23-Ser95 was synthesized.The phosphorylation level of SNAP23 was detected in tumor cells using phosphorylated SNAP23-Ser95 antibody(*P<0.05),and the inhibitor BAY blocked the phosphorylation of SNAP23 at the 95th Ser residue in CA-IKK? cells.Conclusion:It is demonstrated that CA-IKK? significantly weakens the lysosomal degradation pathway of endosome and autophagy by inhibiting RAB7 expression,and then induces MVB to fuse with autophagosomes to form "amphisome".In addition,the phosphorylation of SNAP23-Ser95 by CA-IKK? induces MVB or amphisome to fuse with cells to release vesicles containing autophagy-related components.In this study,how IKK?-activated tumor cells regulate the interaction between autophagy and endosome system is clarified,and a new survival adaptation mechanism of tumor cells under stress is revealed.