br Introduction br Gallbladder cancer GBC a primary malignan
Gallbladder cancer (GBC), a primary malignancy of the biliary tract, is the sixth most common gastrointestinal cancer and has a 5-year survival rate of 5% [1,2]. Although radical resection is the most pro-mising potential curative approach for patients, less than 10% of pa-tients are considered candidates for resection because of advanced stage disease [3,4]. Gemcitabine has been widely used for the treatment of GBC; however, the response rate of only 36% leaves much to be desired . Therefore, it is urgently required to identify the molecular me-chanisms responsible for chemotherapy resistance in GBC.
Maintenance of reactive oxygen species (ROS) homeostasis is es-sential for cell survival. In recent years, growing evidence shows that ROS dysregulation is involved in the development of multiple diseases,
including cancers [6,7]. In order to survive in complex internal and external environments, cancer HG 9 91 01 have developed eﬀective anti-oxidant system to limit the excessive accumulation of ROS . Indeed, conventional chemotherapeutic drugs often utilize elevated ROS to eliminate cancer cells . The upregulation of antioxidant capacity in cancer cells can confer drug resistance. Thus, dissecting such drug-re-sistant molecular mechanisms may be potential therapeutic targets to overcome the resistance.
The Kelch-like ECH-associated protein 1 (Keap1)–nuclear factor erythroid-2-related factor 2 (Nrf2) complex is the major regulator of cytoprotective responses to endogenous and exogenous stresses caused by ROS and electrophiles . Nrf2, as the key signaling protein of the pathway, mediates the expression of a series of oxidative stress-related genes that maintain cellular redox balance . Under normal
Abbreviations: AJCC, American joint committee on cancer; ARE, antioxidant response element; aPKC, atypical protein kinase C; Bax, Bcl-2-associated X; Bcl2, B-cell lymphoma 2; CCA, Cholangiocarcinoma; CCK-8, Cell Counting Kit-8; Co-IP, Co-Immunoprecipitation; Ect2, Epithelial cell transforming sequence 2; GBC, Gallbladder cancer; IHC, Immunohistochemistry; Keap1, Kelch-like ECH-associated protein 1; Nrf2, Nuclear factor erythroid 2-related factor 2; OS, overall survival; ROS, Reactive oxygen species
∗ Corresponding author. E-mail address: [email protected] (J. Wang).
physiological conditions, Nrf2 activity is tightly regulated by Keap1 and promoted its ubiquitin-dependent proteasomal degradation in the cy-toplasm; however, oxidative stresses prevent the Keap1-induced de-gradation of Nrf2, which leads to Nrf2's accumulation, nuclear trans-location and subsequently transcriptional activation of its downstream target genes such as heme oxygenase 1 (HMOX1), NADPH quinone oxidoreductase 1 (NQO1), and glutamate-cysteine ligase (GCL) . Recent studies suggest that the dysfunctional Keap1-Nrf2 interaction may be involved in drug metabolism and increasing the resistance to chemotherapy . However, the precise molecular mechanisms un-derlying the Keap1-Nrf2 pathway and chemoresistance remain largely unknown.
Atypical protein kinase Cι (aPKCι), one of the protein kinase C (PKC) isozymes, has emerged as an important oncogene in various cancers . We previously demonstrated that aPKCι was overexpressed and correlated with poor clinical outcome in cholangiocarcinoma (CCA) and drove CCA cells invasion and metastasis in vitro and in vivo . Moreover, aPKCι was also found to function in conferring resistance to chemotherapy of human leukemia cells . The above reports have expanded the concept that aPKCι is a particularly attractive therapeutic target for cancer treatment. Unfortunately, how aPKCι functions in GBC has not been extensively studied.
In this study, we found that a previously unknown fundamental function of aPKCι is to compete with Nrf2 for binding to Keap1. We further demonstrated that aPKCι-Keap1-Nrf2 signaling is critical for promoting cell tumorigenesis and gemcitabine resistance in GBC.
2. Materials and methods
Human GBC cell lines NOZ and GBC-SD were generously provided by Prof. Yingbin Liu, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, China. These cells were maintained in William's medium or RPMI 1640 (both from Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco), respectively. All cell lines were authenticated, mycoplasma-free and cultured at 37 °C in a humidified incubator containing 5% CO2.
2.2. Plasmids construction
The pFlag-PRKCI and pMyc-Keap1 plasmids were purchased from ViGene Biosciences (Shandong, China). All 5 truncated Keap1 mutants were constructed as previously described . The PRKCI and Keap1 deletion mutants were subcloned into the pENTER vector using an
appropriate restriction enzyme. Site-directed mutagenesis was per-formed to generate DLL257, DLM341 and DLK380 mutants of PRKCI by