Cell cycle–related kinase is a direct androgen receptor–regulated gene that drives β-catenin/T cell factor–dependent hepatocarcinogenesis
Hai Feng, … , Ka F. To, Joseph J.Y. Sung
Hai Feng, … , Ka F. To, Joseph J.Y. Sung
Published July 11, 2011
Citation Information: J Clin Invest. 2011;121(8):3159-3175. https://doi.org/10.1172/JCI45967.
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Research Article Oncology

Cell cycle–related kinase is a direct androgen receptor–regulated gene that drives β-catenin/T cell factor–dependent hepatocarcinogenesis

Abstract

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. It is more prevalent in men than women. Related to this, recent genetic studies have revealed a causal role for androgen receptor (AR) in hepatocarcinogenesis, but the underlying molecular mechanism remains unclear. Here, we used genome-wide location and functional analyses to identify a critical mediator of AR signaling — cell cycle–related kinase (CCRK) — that drives hepatocarcinogenesis via a signaling pathway dependent on β-catenin and T cell factor (TCF). Ligand-bound AR activated CCRK transcription and protein expression via direct binding to the androgen-responsive element of the CCRK promoter in human HCC cell lines. In vitro analyses showed that CCRK was critical in human cell lines for AR-induced cell cycle progression, hepatocellular proliferation, and malignant transformation. Ectopic expression of CCRK in immortalized human liver cells activated β-catenin/TCF signaling to stimulate cell cycle progression and to induce tumor formation, as shown in both xenograft and orthotopic models. Conversely, knockdown of CCRK decreased HCC cell growth, and this could be rescued by constitutively active β-catenin or TCF. In primary human HCC tissue samples, AR, CCRK, and β-catenin were concordantly overexpressed in the tumor cells. Furthermore, CCRK overexpression correlated with the tumor staging and poor overall survival of patients. Our results reveal a direct AR transcriptional target, CCRK, that promotes hepatocarcinogenesis through the upregulation of β-catenin/TCF signaling.

Authors

Hai Feng, Alfred S.L. Cheng, Daisy P. Tsang, May S. Li, Minnie Y. Go, Yue S. Cheung, Gui-jun Zhao, Samuel S. Ng, Marie C. Lin, Jun Yu, Paul B. Lai, Ka F. To, Joseph J.Y. Sung

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Figure 1

Genome-wide location analysis of AR-binding sites identifies cell cycle–related target genes in HCC cells.

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Genome-wide location analysis of AR-binding sites identifies cell cycle–...
(A) Identification of AR direct target genes using ChIP-chip. In Huh7 and PLC5 HCC cells, AR expression was localized in the nuclei, which were counterstained by DAPI. Venn diagram showing the significant overlap of AR target genes between the 2 cell lines. Original magnification, ×400. (B) Confirmation of 10 randomly selected AR target genes by ChIP-PCR using anti-AR antibody or irrelevant antibody against IgG (negative control) in Huh7 and PLC5 cells. Input (2%) represents the genomic DNA. PEG10 and GAPDH were included as positive and negative controls, respectively. (C) Enrichment of cell cycle regulators in AR target genes as denoted by their highly significant binding P values. (D) Quantitative RT-PCR analysis of the AR-bound cell cycle regulator expressions in Huh7 and PLC5 cells treated with the AR agonist R1881 (100 nM) for 6 hours relative to the untreated cells. GAPDH was used as an internal control. (E) Silencing AR expression retarded HCC cell growth. Western blot analysis of AR following RNA interference. β-actin was used as a loading control. Cell growth was inhibited in Huh7 and PLC5 cells treated with siAR compared with siCtrl-treated cells. (F) G1/S cell cycle progression was inhibited after knockdown of AR expression in both HCC cell lines. *P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as mean + SD of 3 independent experiments.