APKCι Promotes Gallbladder Cancer Tumorigenesis And Gemcitabine Resistance By Competing With Nrf2 For Binding To Keap1

Part Ⅰ aPKCι promotes Nrf2 accumulation and mediates ROS inhibitionObjective To investigate the regulation of aPKCι on intracellular ROS level under normal conditions and oxidative stress,and further to explore the correlation between aPKCι and Nrf2 protein expression in gallbladder cancer cells.Methods The lentiviral vectors that contained the human aPKCι-c DNA(aPKCι)or specifical si RNA(si-aPKCι)were used for establishing stable GBC cell lines.Western blot and qRT-PCR were used to detect the protein and m RNA expression of aPKCι.The intracellular ROS levels were measured by DCFH-DA in GBC cells with the treatment of aPKCι overexpression,knockdown,re-expression or gemcitabine.Protein levels of aPKCι and Nrf2 were tested in GBC cells after aPKCι overexpression with or without PSI treatments.Western blotting was performed to detect the expression of p-Nrf2 and T-Nrf2 in GBC cells with Flag-tagged wild-type(WT),constitutively active catalytic domain (CAT)and kinase-inactive(KI)mutants.Western blotting and qRT-PCR were used to measure the m RNA levels of Nrf2 target genes in the indicated GBC cell lines.Then,we further tested the intracellular distribution of aPKCι,Nrf2 and Keap1 proteins by using the cytoplasmic and nuclear fractions after aPKCι overexpression or knockdown in GBC cells.In addition,the relative ROS levels were detected in aPKCι overexpression GBC cells with or without Nrf2 knockdown.Results The ectopic expression of aPKCι significantly reduced the cellular ROS levels in GBC cells.Conversely,aPKCι silencing triggered the accumulation of ROS.Restoring exogenous aPKCι expression reversed the effects of aPKCι knockdown.aPKCι overexpression also attenuated gemcitabine-induced ROS production.aPKCι deletion elevated the cellular ROS levels under this condition,which was reversed by the expression of exogenous aPKCι.PSI had minimal impact on the expression levels of Nrf2 and aPKCι-mediated ROS inhibition.There was weak and no obvious alteration of phosphorylated Nrf2 after aPKCι overexpression in GBC cells.aPKCι facilitated Nrf2 accumulation,nuclear translocation and activated its target genes.However,aPKCι did not affect the protein expression of Keap1.Furthermore,aPKCι-mediated ROS inhibition can be abolished by Nrf2 knockdownConclusion aPKCι promotes Nrf2 accumulation and nuclear location in a kinase-independent manner,and lowers the cellular ROS levels through Nrf2-mediated antioxidative pathway.Part Ⅱ The molecular mechanism of aPKCι competes with Nrf2 for binding to Keap1Objective To study the molecular mechanism of how aPKCι regulates Keap1-Nrf2 pathway.Methods The protein levels of aPKCι and Nrf2 were determined by western blotting in negative control or aPKCι knockdown NOZ cells with or without MG132 treatments(20 μM for 6 hours).The ubiquitinated Nrf2(Ub-Nrf2)was measured by IP in NOZ cells after aPKCι overexpression or knockdown.The protein levels of aPKCι,Nrf2,Keap1 and p62 were examined in aPKCι overexpression GBC cells with or without Keap1 knockdown.Co-IP assays were performed to detect the interaction between Flag-tagged aPKCι and Myc-tagged Keap1 in HEK293 cells.The interaction among aPKCι,Nrf2 and Keap1 was determined by Co-IP in GBC cells with the treatment of gemcitabine for 24 hours or in vitro translation systems.Site-directed mutagenesis or deletion mutants were used to analyse the molecular recognition between aPKCι and Keap1.Results The proteasome inhibitor MG132 completely rescued aPKCι knockdown-induced reduction of Nrf2.Ectopic aPKCι expression significantly reduced the ubiquitination of the endogenous Nrf2 in GBC cells.Conversely,aPKCι knockdown increased the level of ubiqutin-Nrf2.Keap1 knockdown did not affect the protein expression of aPKCι,while abolished aPKCι-induced Nrf2 accumulation.Under normal cellular conditions,we detected the interaction of Flag-aPKCι and Myc-Keap1.aPKCι overexpression increased,while aPKCι knockdown reduced,the aPKCι-Keap1 complex in GBC cells.The Keap1-Nrf2 complex exhibited an opposite result.The binding amount of aPKCι and Keap1 proteins was significantly increased,accompanying with a decreased interaction of Keap1 and Nrf2 under gemcitabine-induced oxidative stress.In vitro translation systems,the binding amount of Nrf2 and Keap1 was gradually decreased with an increased interaction of aPKCι and Keap1.The deletion mutant and DLL257 mutant of aPKCι were unable to interact with Keap1.However,DLM341 and DLK380 mutants have no obvious effect on this interaction.The results also showed that the Keap1 N-terminal deletion mutants(D1,D2 and D3)and C-terminal deletion mutant D5 bound with aPKCι,whereas DGR deletion mutant(D4)failed to interact with aPKCι.Conclusion aPKCι competes with Nrf2 to bind with Keap1 through the DLL motif,which leads to Nrf2 accumulation and reduces the ubiquitination of Nrf2.Part Ⅲ aPKCι-mediated ROS inhibition promotes GBC cells tumorigenesis and gemcitabine resistanceObjective: To investigate the effect of aPKCι on cell growth and gemcitabine resistance in GBC cells.Methods Cell proliferation ability was analyzed by the CCK-8 assay in GBC cells with indicated treatments.Anchorage-independent growth was evaluated by soft agar growth assay in the indicated GBC cells.To investigate the effect of aPKCι on cell tumorigenesis in vivo,NOZ cells transfected with lentivirus empty vector,aPKCι overexpression,si-neg,or si-aPKCι vectors were injected subcutaneously of mice.Then,the cell viability was measured by the CCK-8 assay in GBC cells with different doses of gemcitabine for different time.Relative ROS levels were determined by DCFH-DA in the indicated GBC cells in the presence or absence of gemcitabine.Western blot and qRT-PCR were used to measure the expression levels of Nrf2 and its target genes in GBC cells with or without gemcitabine treatment.In vivo,negative control and aPKCι knockdown NOZ cells were subcutaneously injected into nude mice with or without gemcitabine treatment.aPKCι-WT,aPKCι-DLL257 deletion mutant(M1)and aPKCι-DLL257 mutant(M2)vectors were transfected into aPKCι-deficient cells to validate whether the aPKCι-Keap1 interaction through the DLL motif is associated with these biological functions.Results CCK-8 assay revealed that ectopic aPKCι expression dramatically enhanced the GBC cells proliferation compared with the cells transfected with empty vector.Conversely,aPKCι silencing significantly suppressed the cells proliferation ability,which was reversed by re-expression of aPKCι.Consistently,aPKCι overexpression increased,while aPKCι knockdown inhibited,GBC cells growth as demonstrated by a soft agar growth assay.Recovery of exogenous aPKCι expression eliminated the effect of aPKCι deficiency.The xenograft tumors grew more rapidly in the aPKCι overexpression group than that in the empty vector group.When aPKCι was knocked down,the cells exhibited more sensitivity to gemcitabine with a dramatically decreased of IC50.aPKCι or Nrf2 depletion led to ROS accumulation in gemcitabine-treated cells.aPKCι deficiency decreased the Nrf2 protein and its target genes expression levels in cells treated with gemcitabine.In vivo,the tumors were significantly repressed in the presence of gemcitabine after aPKCι knockdown.Mutant M1 or M2 could not rescue the defects of cell proliferation in aPKCι-deficient cells.Conclusion The aPKCι-Keap1 interaction through the DLL motif is required for cell growth,ROS inhibition,and chemoresistance in GBC cells.Part Ⅳ The expression and clinical significance of aPKCι,Nrf2 and Keap1 in GBCObjective To investigate the correlation of aPKCι,Nrf2 and Keap1,and clinical significance in human gallbladder cancer.Methods Immunohistochemistry was used to examine the expression of aPKCι,Nrf2 and Keap1 in 72 GBC samples and pair-matched normal tissues.The protein and m RNA levels of aPKCι,Keap1,Nrf2 and its target genes in 8 representative GBC samples and pair-matched normal tissues were evaluated by western blot and qRT-PCR.Correlation between aPKCι and clinicopathologic characteristics in patients with GBC was analyzed by Chi-square test.Survival analysis was conducted by the Kaplan-Meier method with log-rank tests.Cox regression model was used to analyse whether aPKCι was an independent prognostic factor for patients with GBC.Results Immunohistochemistry analysis showed that the expression levels of aPKCι and Nrf2 were significantly higher in GBC tissues than in pair-matched normal tissues.77.6%(38 cases)of specimens with higher aPKCι(49 cases)tended to express higher Nrf2,while 65.2%(15 cases)of specimens with lower aPKCι(23 cases)exhibited lower Nrf2(P<0.01).There was no obvious alteration of Keap1 expression levels and no significant correlation between aPKCι and Keap1 in GBC samples.Consistently,the upregulation of aPKCι and Nrf2 was further examined at the protein level in representative 8 paired GBC tissues.The m RNA levels of Nrf2 target genes increased in GBC tissues.The expression level of aPKCι was significantly associated with advanced TNM stage(χ2 = 19.965,P < 0.001),lymph node metastasis(χ2 = 13.125,P<0.001),and poor tumor differentiation(χ2 = 29.154,P<0.001)in GBC.Kaplan-Meier analysis indicated that patients with high aPKCι expression exhibited a shorter overall survival(OS)than those with low aPKCι expression.Multivariate Cox regression analyses indicated that the expression of aPKCι was an independent prognostic factor for OS in patients with GBC.Conclusion The overexpression of Nrf2 protein and its target genes may be dependent,at least in part,on the elevation of aPKCι in GBC samples.aPKCι expression was an independent prognostic factor for patients with GBC.