Dissecting The Oncogenic Function Of A Novel Androgen Receptor-Dependent Direct Target, Cell Cycle-Related Kinase (Ccrk), In Hepatocellular Carcinoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide with a gender prevalence observed in men. Recent genetic studies using knockout mouse models have revealed the causal role of androgen receptor (AR) in hepatocarcinogenesis but the underlying molecular mechanism remains unclear. Here we used genome-wide location and functional analyses to show that cell cycle-related kinase (CCRK) is a direct critical mediator of AR signaling. Chromatin immunoprecipitation microarray identified over 200 high-confidence AR direct target genes in HCC cells of which cell cycle regulators were significantly enriched (n = 21; p < 0.0001). Because CCRK has the highest AR binding affinity amongst the identified cell cycle regulators, its regulation, function and expression in HCC were further investigated. Ligand-activated AR was recruited to the CCRK promoter and increased CCRK expression. AR-induced cell cycle progression was abrogated by siRNA-mediated knockdown of CCRK. On the contrary, over-expression of CCRK rescued the G1 arrest induced by AR knockdown. Ectopic CCRK expression in human immortal liver cells induced anchorage-dependent and -independent growth and tumor formation in immunodeficient mice, whereas CCRK inhibition decreased HCC cell growth in vitro and in vivo; demonstrating the strong oncogenic capacity of CCRK in HCC. Mechanistically, CCRK activated β-catenin/T-cell factor (TCF) signaling through phosphorylation of glycogen synthase kinase-3β to increase the expression of downstream pro-proliferative genes, cyclin D1 and epidermal growth factor receptor. Inhibition of β-catenin/TCF signaling significantly attenuated CCRK-induced cell cycle progression, colony formation and tumorigenicity. Conversely, HCC cell growth inhibition by CCRK knockdown was rescued by constitutively active form of β-catenin or TCF. Importantly, AR, CCRK, and active β-catenin were markedly over-expressed and positively correlated among each other in HCC specimens (p < 0.001). Furthermore, CCRK over-expression was significantly associated with tumor staging and poor disease-free survival of patients (p < 0.05), emphasizing the clinical importance of CCRK in HCC. In conclusion, our findings reveal a novel interplay between AR and β-catenin where activated AR transcriptionally up-regulates CCRK expression, thereby activating β-catenin/TCF signaling to induce aberrant cell proliferation. Dissection of this highly activated AR-CCRK-β-catenin/TCF axis sheds new mechanistic insight into hepatocarcinogenesis and provides novel therapeutic targets for the treatment of this and other male-predominant cancers. Acknowledgements: This study was partially supported by the Research Grant Council General Research Fund (462710) and the Direct Grant from the Chinese University of Hong Kong. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2170. doi:10.1158/1538-7445.AM2011-2170