Abstract 3752: A cell cycle-related kinase (CCRK) self-regulatory circuitry in NAFLD-associated hepatocarcinogenesis

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Abstract Background and aim: Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome including obesity and diabetes which elevate the risk of hepatocellular carcinoma (HCC). Accumulating evidence has unmasked the molecular linkage between androgen receptor (AR) signaling and gender disparity in NAFLD and HCC incidence. Our previous genome-wide location and functional analysis has pinpointed cell cycle-related kinase (CCRK) as a critical mediator of AR oncogenic activity in hepatitis B virus-associated HCC through glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling (1-3). Here we investigated whether CCRK plays an oncogenic role in NAFLD-related HCC. Methods: We investigated the functional role of CCRK in a high-fat high carbohydrate diet-induced obesity model exposed to low-dose diethylnitrosamine using lentiviral-mediated gene knockdown, followed by measurement of glucose/lipid metabolism via intraperitoneal glucose tolerance test and triglyceride/non-esterified fatty acids levels, and HCC incidence. Gene regulation was assessed by chromatin immunoprecipitation, site-directed mutagenesis, luciferase reporter, co-immunoprecipitation and expression analyses. Results: Lentiviral-mediated Ccrk silencing in male mice significantly reduced obesity-promoted tumor multiplicity and size by >70%. The reduced tumorigenicity was also associated with significant restoration of circulating metabolic profiles and reduction in hepatocellular lipid level. Mechanistic studies demonstrated that the pro-inflammatory cytokine interleukin-6 (IL-6) triggered signal transducer and activator of transcription 3 (STAT3) signaling to up-regulate CCRK expression in an AR-dependent manner. Simultaneously, the phosphorylation of STAT3 by CCRK facilitated the co-occupancy of CCRK promoter by STAT3-AR complex and its subsequent transcriptional activation, thus forming a self-reinforcing circuitry. In addition, both in vitro and in vivo data suggested that CCRK phosphorylated GSK-3β to activate not only β-catenin but also mechanistic target of rapamycin complex 1 (mTORC1) signaling pathways that are crucial for cell survival, proliferation and lipogenesis. Furthermore, transgenic over-expression of CCRK in mouse liver led to concordant activation of STAT3, β-catenin and mTORC1 signaling pathways. Conclusion: Our findings highlight the critical role of CCRK in a self-reinforcing circuitry that regulates NAFLD-associated hepatocarcinogenesis and offer novel therapeutic strategies to combat this dreadful disease in an era of global obesity. Acknowledgement: This project was supported by the Collaborative Research Fund C4017-14G and General Research Fund 14102914.