Supported by National Institutes of Health grants 1R01DK62277 and 1R01CA124414 to S.P.M. and by Rango's Fund for the Enhancement of Pathology Research. Potential conflict of interest: Nothing to report.
Cell cycle–related kinase links androgen receptor and β-catenin signaling in hepatocellular carcinoma: Why are men at a loss?†
Article first published online: 23 FEB 2012
Copyright © 2012 American Association for the Study of Liver Diseases
Volume 55, Issue 3, pages 970–974, March 2012
How to Cite
Awuah, P. K., Monga, S. P. (2012), Cell cycle–related kinase links androgen receptor and β-catenin signaling in hepatocellular carcinoma: Why are men at a loss?. Hepatology, 55: 970–974. doi: 10.1002/hep.24774
- Issue published online: 23 FEB 2012
- Article first published online: 23 FEB 2012
Feng H, Cheng AS, Tsang DP, Li MS, Go MY, Cheung YS, et al. Cell cycle-related kinase is a direct androgen receptor-regulated gene that drives beta-catenin/T cell factor-dependent hepatocarcinogenesis. J Clin Invest 2011;121:3159-3175. (Reprinted with permission.)
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. It is more prevalent in men than in 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.
Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third cause of cancer-related death worldwide. HCC usually occurs in the setting of chronic liver disease due to hepatitis of various etiologies. Men are at higher risk of developing HCC, and the role of male sex hormones as tumor promoters is highly relevant. 1 The androgen receptor (AR) is critical for the development and maintenance of the male sexual phenotype. 2 AR is a type of nuclear receptor that regulates gene transcription when activated by androgens. Upon activation, AR translocates to the nucleus, binds to androgen response elements (AREs) on target genes, and engages in crosstalk with transcription factors to eventually induce transcription of certain genes. Overexpression of AR has been reported in 60%-80% of human HCCs, 3 making it a formidable player in male HCC. In addition, liver-specific deletion of AR was shown to significantly reduce tumorigenicity in both carcinogen-induced and hepatitis B virus (HBV)-induced HCC mouse models. 4, 5
The mechanism of AR in HCC had remained unclear until a recent study established a novel means of crosstalk through cell cycle–related kinase (CCRK) to the Wnt/β-catenin signaling pathway, another important oncogenic pathway in HCC. 6 Abnormal activation of the Wnt/β-catenin signaling pathway due to multiple diverse mechanisms, which lead to stabilization and nuclear translocation of β-catenin, has been reported in a significant subset of HCC patients; the downstream mechanisms of how β-catenin contributes to HCC are not completely understood. 7 Although β-catenin itself does not bind DNA, it can interact with other transcription factors (especially the more studied T-cell factor/lymphoid enhancer factor [TCF/LEF] family of transcription factors) to induce target gene expression. Feng et al. have recently linked the β-catenin and AR pathways in a feed-forward loop through CCRK in HCC. 6
In an effort to identify AR-dependent mechanisms and thus address the male predominance of HCC, Feng and colleagues took advantage of chromatin immunoprecipitation (ChIP)-chip analysis and identified CCRK as a direct target of AR in two androgen-expressing HCC cell lines—Huh7 and PLC5. In fact, 212 target genes that were common between the two cell lines were identified; 21 of these 212 genes were cell cycle regulators, and CCRK was identified as having the highest binding affinity to AR. Through multiple means it was shown that AR strongly bound and transactivated the CCRK promoter. AR knockdown diminished the CCRK promoter activity, and, conversely, the AR agonist R1881 induced AR binding and transactivation of the CCRK reporter; additional validation was provided through site-directed mutagenesis. The same authors also transfected the AR gene in two cell lines with low endogenous AR (SK-Hep1 and LO2) and identified a significant increase in CCRK expression, which was also substantiated by immunofluorescence (IF). The authors then proceeded to determine the functional relevance of CCRK expression control by the AR. Whereas AR stimulation led to cell cycle progression, CCRK down-regulation under such circumstances abrogated tumor cell proliferation. Conversely, ectopic expression of CCRK was able to significantly reverse proliferation and cell cycle arrest following AR inhibition. A similar relationship was also evident in focus assays and anchorage-independent soft agar assays and further corroborated a direct relationship between AR and CCRK in promoting cellular proliferation and transformation.
To further test the biological relevance of these findings in vivo, the authors demonstrated that injection of PLC5 hepatoma cells expressing shRNA to CCRK led to significantly diminished tumor formation compared to the controls in tumor xenograft studies. Conversely, stably transfected LO2 cells expressing CCRK displayed incredible tumor growth, with 20-fold mean tumor volumes compared with empty vector controls. These results undoubtedly demonstrate the oncogenic properties of CCRK.
To elucidate the mechanism of CCRK in hepatic tumorigenesis, and based on previously published data from a protein kinase–enriched shRNA library screen illustrating regulation of β-catenin activity by another cell cycle kinase (CDK8) in colorectal cancer, 8 the authors postulated that CCRK modulated β-catenin signaling. IF studies indeed showed redistribution of β-catenin from the nucleus to the cytoplasm after CCRK knockdown compared with control in two different cell lines. The authors further demonstrated decreased protein levels of active but not of total β-catenin in this situation. Mechanistically, they determined that CCRK knockdown decreased phosphorylation of GSK3β and the ensuing decrease in β-catenin activity. Conversely, ectopic expression of CCRK led to increased phosphorylation of GSK3β culminating in enhanced β-catenin signaling. Phosphorylation of GSK3β has been shown to mediate β-catenin activation through inhibition of β-catenin degradation complex. 9 In addition, the authors demonstrated that knockdown of CCRK abrogated some known β-catenin downstream targets such as epidermal growth factor receptor (EGFR) and cyclin-D1, which was reversed by ectopic CCRK expression. These targets have independently been shown to regulate proliferation in HCC. 10, 11 Knockdown of β-catenin despite ectopic expression of CCRK led to decreased tumor cell proliferation, and additional functional studies such as soft agar assays and tumor xenograft studies further validated these observations.
The authors then asked whether the modulation of β-catenin activity by CCRK had any impact on AR expression and function because β-catenin–AR crosstalk has been previously reported. 12 Indeed, the authors determined that ectopic CCRK expression led to increased total and Ser81-phosphorylated AR, which has independently been shown to induce AR promoter activity and in turn tumor cell growth. 13 This effect was abrogated upon β-catenin silencing, suggesting that CCRK-induced β-catenin activation indeed regulates AR expression and its biological effects. Lastly, knockdown of AR affected β-catenin activity, which could be rescued by CCRK overexpression, and ectopic AR expression could increase active-β-catenin levels, which could be blocked by inhibition of CCRK. Thus, the authors established a unique AR/CCRK/β-catenin feed-forward circuitry (Fig. 1), which was also evident in a significant subset of HCC patients as concomitant up-regulation of AR/CCRK/β-catenin using both western blot analysis and immunohistochemistry. Further examination also revealed a significant correlation between overexpression of CCRK and advanced tumor stage reflected by shorter overall survival.
The current study has identified a novel circuitry that consists of an AR/CCRK/β-catenin axis that may provide at least one major mechanism of hepatocarcinogenesis in this male-predominant tumor type, thus presenting unique molecular interactions that may be exploited for therapeutic intervention. This study also suggests at least one additional mechanism by which Wnt/β-catenin signaling may in fact cause tumor progression in males. It is plausible that β-catenin activation in HCC in males would lead to higher expression and activation of AR that in turn would result in higher CCRK expression and thus incite a vicious circle of cell growth and proliferation. Traditionally, many modes of β-catenin activation have been reported in HCC. 14 It is unclear, however, whether various mechanisms of β-catenin activation in HCC will have similar and robust growth-promoting effects on tumor cells. Contrary to expectations, a study found that β-catenin/TCF activation was not equivalent when β-catenin stabilization was a consequence of CTNNB1 versus AXIN1 mutations. 15 It would have been useful to determine the cause of β-catenin activation in patients examined in the current study.
Would β-catenin activation due to the AR/CCRK axis be even more pronounced regardless of preexisting mutations in CTNNB1? Similar studies in vitro examining the effect of AR/CCRK on tumor cells expressing β-catenin with point mutations affecting key serine and threonine residues in exon-3 would be relevant in the future, because such mutations may be predicted to be autonomous of any upstream feed-forward regulation. In other words, mutations in CTNNB1 that are observed in 20%-40% of all HCC patients may be free of GSK3β-dependent β-catenin phosphorylation and degradation and may in fact introduce a break in the proposed positive regulatory circuit.
The authors elegantly unveil one of the mechanisms of sex-related disparity of HCC and extend the existing findings that AR and testosterone contribute to HCC predominance in males. The major conclusion drawn from the study is that the presence of androgens in males engages the AR to stimulate the CCRK expression, which activates β-catenin signaling, which in turn would enhance expression of EGFR and cyclin-D1 (thus promoting cell proliferation) and at the same time would up-regulate AR expression and activity and thus establish a positive regulatory cycle (Fig. 1). CCRK belongs to the mammalian cyclin-dependent kinase (CDK) family and although it has been shown to be up-regulated in several cancers, its role and regulation are not fully understood. In fact, it has been reported elsewhere that CCRK does not have an intrinsic CDK-activating kinase (CAK) activity, but that it does enhance cell proliferation. 16 It is likely that through additional, as yet uncharacterized interactions, CCRK may be influencing Thr390 phosphorylation of GSK3β. Significant studies will be necessary to extrapolate these interactions, especially because p38 mitogen-activated protein kinase (MAPK) is known to induce GSK3β inactivation through this specific event. 17
AR signaling has been attributed to induction of cellular oxidative stress both in vivo and in vitro. Intriguingly, β-catenin has been shown to also regulate the redox state of the cell. In fact, recent studies have shown that β-catenin can switch from binding to TCF4 to other transcription factors like hypoxia-inducible factor 1α (HIF1α) or FOXO and can mount an antioxidant response. 18, 19 It was also recently shown that loss of β-catenin in hepatocytes led to enhanced liver tumorigenesis after exposure to a chemical carcinogen such as diethylnitrosamine with or without phenobarbitol, and this was accompanied by enhanced oxidative stress, fibrosis, inflammation, and regeneration. 20, 21 Thus, it will be critical to determine in the context of AR signaling whether enhanced CCRK-driven β-catenin activation is globally contributing to tumorigenesis or in some cases may in fact be an oxidative stress-driven response promoting cell proliferation and regeneration in the setting of chronic liver injury and fibrosis. 10
- 21Conditional beta-catenin loss in mice promotes chemical hepatocarcinogenesis: role of oxidative stress and platelet-derived growth factor receptor alpha/phosphoinositide 3-kinase signaling. HEPATOLOGY 2010; 52: 954-965., , , , , , et al.