br Finally we further investigated
Finally, we further investigated the correlation among aPKCι, Nrf2, and Keap1 in 72 GBC specimens. IHC analysis showed that the ex-pression levels of aPKCι and Nrf2 were significantly higher in GBC tissues than in pair-matched normal tissues. There was no obvious al-teration of Keap1 expression levels in GBC samples (Fig. 7A and B). 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. However, there was no sig-nificant correlation between aPKCι and Keap1 (Fig. 7C). Consistently, the upregulation of aPKCι and Nrf2 was further examined at the protein level in representative 8 paired GBC tissues (Fig. 7D). In addition, we also found that the mRNA levels of Nrf2 target genes increased in GBC tissues (Fig. S5). The expression level of aPKCι was significantly asso-ciated with advanced TNM stage (χ2 = 19.965, P < 0.001), lymph node metastasis (χ2 = 13.125, P < 0.001), and poor tumor diﬀer-entiation (χ2 = 29.154, P < 0.001) in GBC (Fig. 7E). Kaplan-Meier analysis indicated that patients with high aPKCι expression exhibited a shorter overall survival (OS) than those with low aPKCι expression (Fig. 7F). Multivariate Cox regression analyses indicated that aPKCι expression was an independent prognostic factor for OS in patients with GBC (Supplementary Table S4). Therefore, these observations sug-gested that aPKCι was frequently upregulated and associated with poor prognosis in patients with GBC. The overexpression of Nrf2 protein and its target genes may be dependent, at least in part, on the elevation of aPKCι in GBC samples.
Chemotherapy resistance is a major obstacle to the eﬀective treat-ment of cancer. Although recent evidence has shown that the elevated expression of aPKCι is correlated with chemoresistance in various human cancers , the molecular mechanisms that drive the en-hanced tumorigenic potential and drug resistance of GBC PKF118-310 remain enigmatic. Here, we provide novel evidence that aPKCι competes with Nrf2 for binding to Keap1, which leads to Nrf2 nuclear accumulation and ROS inhibition in GBC cells (Fig. 8). Our data may provide an ex-planation of how aPKCι exerts oncogenic functions in GBC. In addition, this ability of aPKCι has been further demonstrated to be associated with resistance to gemcitabine in GBC, which is notorious for its in-sensitivity to chemotherapy.
As a distinct member of the protein kinase C family, aPKCι has been reported to be a particularly attractive therapeutic target for cancer treatment [13,35]. We and other researchers have demonstrated that aPKCι drove multiple cancer cells invasion and transformed growth in vitro and in vivo [14,36,37]. Recent studies also showed that aPKCι maintained a stem-like phenotype in lung squamous cell carcinoma
through autonomous Hedgehog (Hh) signaling and served as a potential therapeutic strategy for Hh mutations that confer resistance . All abovementioned studies focused on the regulation of cell polarization and kinase activity of aPKCι, while interestingly, we found that aPKCι functioned as an antioxidative factor in a kinase-independent manner. Our findings were supported by other studies that aPKCι promoted neuronal diﬀerentiation in a manner that did not depend on kinase activity . More importantly, we further demonstrated that this function of aPKCι was involved in drug resistance and cell growth.
Nrf2 has been regarded as one of the main orchestrators of the antioxidant response pathway. For the canonical Keap1-Nrf2 system, also known as “hinge and latch” model, previous studies have reported that Keap1 homodimer interacts with monomeric Nrf2 through DLG and ETGE motifs [40,41]. This binding model facilitates optimal posi-tioning of the lysine residues between the two ubiquitin-conjugated motifs . The modification of cysteine residues in Keap1 can be in-duced by oxidative stress, which causes a conformational change of Keap1 that further reduces the ubiquitination and degradation of Nrf2 . On the other hand, accumulating lines of evidence demonstrate that there is cross talk between Keap1-Nrf2 and other proteins, such as PALB2, P62, iASPP2 and DPP3, which can disrupt the normal Keap1-Nrf2 signaling and associate with malignant progression [16,43–45]. These proteins can bind with either Keap1 or Nrf2 through Keap1-binding motifs (DLG or ETGE motifs). In this study, we found that aPKCι interacted with Keap1 through the DLL motif, a similar set in Nrf2, which is highly conserved from poultry to human in aPKCι. Indeed, we have detected the protein complex of aPKCι-Keap1-Nrf2 in GBC cell lines. Furthermore, aPKCι can elevate Keap1-Nrf2 signaling under both basal conditions and stressed conditions. The data showed that the binding amount of aPKCι and Keap1 was dramatically elevated with the treatment of gemcitabine. It is also noteworthy that increased aPKCι-Keap1 interaction was accompanied by a significant reduction of Keap1-Nrf2 interaction under oxidative stresses. According to the “hinge and latch” model, gemcitabine-induced oxidative stress may modify the specific cysteine residues of Keap1, which leads to con-formational change of Keap1 resulting in the dissociation of Nrf2 from Keap1. It is propitious for the binding of aPKCι and Keap1, and con-sequently, newly synthesized Nrf2 proteins bypass Keap1. These ob-servations indicate that revealing the regulatory mechanisms in the Keap1-Nrf2 system may provide potential opportunities for pharma-cological intervention in diﬀerent cellular conditions.