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  • br Although previous studies have reported that

    2020-08-12


    Although previous studies have reported that phosphorylation of Nrf2 by PKC facilitated the dissociation of Nrf2 from Keap1 [19,46], the main enzyme and molecular mechanism underlying the interaction were not investigated. In contrast, the kinase-independent functions of aPKC, including aPKCι, have been reported in multiple signaling pathways [39,47–49]. In this study, we have investigated that aPKCι can promote nuclear translocation of Nrf2, but not phosphorylated Nrf2, to activate cytoprotective gene expression. Mechanistically, we constructed a series of mutants to demonstrate how aPKCι disrupts the Keap1-Nrf2 system. Interestingly, we found that aPKCι may act as a “competitive” binder of Keap1. On one hand, Keap1 knockdown has no effect on the Lactacystin of aPKCι. Conversely, aPKCι depletion also does not result in the change of Keap1. This suggests that Keap1 is not a substrate of aPKCι. On the other hand, our data show that aPKCι overexpression increased, while knockdown decreased, the protein level of Nrf2. However, the mRNA level of Nrf2 has not significant al-teration with aPKCι overexpression or knockdown. Therefore, we
    Fig. 8. Schematic summary of how aPKCι induces ROS inhibition and promotes cell tumorigenesis and gemcitabine resistance in GBC. aPKCι com-petes with Nrf2 for binding to Keap1, which leads to Nrf2 nuclear accumulation and activation of its target genes; this ultimately induces ROS inhibition, tumor growth, and drug resistance.
    speculated that aPKCι might modulate Nrf2 protein did not occur at the transcriptional level. Indeed, we found that aPKCι regulated the protein stability of Nrf2 and inhibited its proteasome-mediated degradation. Although DLG motif is weaker than ETGE motif, both DLG and ETGE motifs are required for the Keap1-dependent degradation of Nrf2. In addition, a recent study has shown that the interaction between Keap1 and Nrf2 is dynamic. It contains two distinct conformations and follows a cycle: “open”, in which Nrf2 binds one Keap1 molecule through ETGE, and “closed”, in which Nrf2 interacts with the Keap1 dimer through DLG and ETGE [42,44]. Of note, it has been reported that when the ETGE motif bound with Keap1 singly, Nrf2 would not be ubiquiti-nated by the E3-ubiquitin ligase [50]. When the conformations changed from an open to a closed, Nrf2 was polyubiquitinated and then subse-quently released for degradation by the proteasome [42]. Then, aPKCι will be placed in a better position to bind with the regenerated free Keap1 dimer through the DLL motif, which leads to the newly synthe-sized Nrf2 accumulation and nuclear translocation. Thus, it is not sur-prising that aPKCι regulates the Keap1-Nrf2 system by competing with Nrf2.
    It is also noteworthy that elevated aPKCι expression was further confirmed in human gallbladder cancer specimens. Although lack of high throughput sequencing data, our results showed that the upregu-lation of Nrf2 protein and its target genes may be at least in part de-pendent on the elevation of aPKCι in GBC samples. The overexpression of aPKCι significantly correlates with poor prognosis in patients with GBC. Furthermore, it is widely accepted that sustained increase in ROS leads to develop chemoresistance, which may be presumably due to the abnormalities of the Keap1-Nrf2 pathway. Importantly, elevated aPKCι can induce drug resistance in GBC cell lines, indicating that aPKCι may be a potential and an attractive therapeutic target for GBC. However, whether aPKCι modulates mitochondrial ROS in a similar or distinct manner is yet to be delineated. Further studies are required to under-stand how to target the aPKCι-Keap1-Nrf2 pathway to improve the ef-ficacy of treatment for GBC.
    In conclusion, our study demonstrated the importance of elevated aPKCι in promoting the tumorigenesis and gemcitabine resistance through competitive interaction with Nrf2 for binding to Keap1 in GBC cells. Accordingly, our findings expand our knowledge of the functions of aPKCι in cancer, especially its role as an anti-ROS factor is kinase-independent. These results provide important insights into the devel-opment of new, effective therapeutic approaches to overcome drug resistance for the treatment of GBC. 
    Conflicts of interest
    The authors declare no potential conflicts of interest.
    Declarations of interest
    None.
    Acknowledgments
    We thank Prof. Yingbin Liu and Dr. Shanshan Xiang (Xinhua Hospital, Shanghai Jiao Tong University School of Medicine) for the generous gift of the GBC cell lines and valuable advice regarding cell culture. This study was financially supported by the National Natural Science Foundation of China [No. 81172015, 81572417] and the Huazhong University of Science and Technology “Double Top” Construction Project [No. 540- 5001540068].