Hai Feng

EZH2-Mediated Concordant Repression of Wnt Antagonists Promotes β-Catenin–Dependent Hepatocarcinogenesis

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the Polycomb-repressive complex 2 (PRC2) that represses gene transcription through histone H3 lysine 27 trimethylation (H3K27me3). Although EZH2 is abundantly present in various cancers, the molecular consequences leading to oncogenesis remain unclear. Here, we show that EZH2 concordantly silences the Wnt pathway antagonists operating at several subcellular compartments, which in turn activate Wnt/β-catenin signaling in hepatocellular carcinomas (HCC). Chromatin immunoprecipitation promoter array and gene expression analyses in HCCs revealed EZH2 occupancy and reduced expression of Wnt antagonists, including the growth-suppressive AXIN2, NKD1, PPP2R2B, PRICKLE1, and SFRP5. Knockdown of EZH2 reduced the promoter occupancy of PRC2, histone deacetylase 1 (HDAC1), and H3K27me3, whereas the activating histone marks were increased, leading to the transcriptional upregulation of the Wnt antagonists. Combinatorial EZH2 and HDAC inhibition dramatically reduced the levels of nuclear β-catenin, T-cell factor-dependent transcriptional activity, and downstream pro-proliferative targets CCND1 and EGFR. Functional analysis revealed that downregulation of EZH2 reduced HCC cell growth, partially through the inhibition of β-catenin signaling. Conversely, ectopic overexpression of EZH2 in immortalized hepatocytes activated Wnt/β-catenin signaling to promote cellular proliferation. In human HCCs, concomitant overexpression of EZH2 and β-catenin was observed in one-third (61/179) of cases and significantly correlated with tumor progression. Our data indicate that EZH2-mediated epigenetic silencing contributes to constitutive activation of Wnt/β-catenin signaling and consequential proliferation of HCC cells, thus representing a novel therapeutic target for this highly malignant tumor.

Inhibitory role of peroxisome proliferator-activated receptor gamma in hepatocarcinogenesis in mice and in vitro†

Although peroxisome proliferator‐activated receptor gamma (PPARγ) agonist have been shown to inhibit hepatocellular carcinoma (HCC) development, the role of PPARγ in hepatocarcinogenesis remains unclear. We investigated the therapeutic efficacy of PPARγ against HCC. PPARγ‐deficient (PPARγ+/−) and wild‐type (PPARγ+/+) littermates were used in a diethylnitrosamine (DEN)‐induced HCC model and treated with PPARγ agonist (rosiglitazone) or the vehicle alone for 8 months. The effects of PPARγ on HCC cell growth and apoptosis were examined using PPARγ‐expressing adenovirus (Ad‐PPARγ). PPARγ+/− mice were more susceptible to DEN‐induced HCC than PPARγ+/+ mice (94% versus 62%, P < 0.05), and rosiglitazone significantly reduced the incidence of HCC in PPARγ+/+ mice (vehicle 62% versus treatment 24%, P < 0.01), but not in PPARγ+/− mice, indicating that PPARγ suppresses hepatocellular carcinogenesis. A pronounced expression of PPARγ was observed in a HCC cell line (Hep3B) infected with Ad‐PPARγ. Such induction markedly suppressed HCC cell viability (P < 0.01). Further, Hep3B infection with Ad‐PPARγ revealed a decreased proportion of cells in S‐phase (12.92% versus 11.58%, P < 0.05), with arrest at G2/M phase (38.2% versus 55.68%, P < 0.001), and there was concomitant phosphorylation of the key G2/M phase inhibitors cdc25C and cdc2. PPARγ overexpression increased cell apoptosis (21.47% versus 35.02%, P < 0.01), mediated by both extrinsic (Fas and tumor necrosis factor‐α) and intrinsic (caspase‐9, caspase‐3, caspase‐7, and poly[ADP‐ribose] polymerase) pathways. Moreover, PPARγ directly induced a putative tumor suppressor gene, growth differentiation factor‐15. Conclusion: Loss of one PPARγ allele is sufficient to enhance susceptibility to HCC. PPARγ suppresses tumor cell growth through reducing cell proliferation and inducing G2/M phase arrest, apoptosis, and up‐regulating growth differentiation factor‐15. Thus, PPARγ acts as a tumor‐suppressor gene in the liver. HEPATOLOGY 2010

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

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.

Cell cycle-related kinase mediates viral-host signalling to promote hepatitis B virus-associated hepatocarcinogenesis

Background Androgen receptor (AR) signalling contributes to male predominance in hepatocellular carcinoma (HCC), which is more pronounced in HBV-endemic areas. Cell cycle-related kinase (CCRK) is essential for AR-induced hepatocarcinogenesis but its molecular function in HBV-associated HCC remains obscure. Objective To determine the molecular function of CCRK in HBV-associated HCC. Design Transcriptional regulation was assessed by chromatin immunoprecipitation, promoter mutation and luciferase reporter assays. Hepatocellular proliferation and tumourigenesis were examined by colony formation, soft agar assays and using HBV X protein (HBx) transgenic mice with low-dose exposure to diethylnitrosamine. Protein expressions were examined in clinical samples and correlated with patient survival by log-rank Mantel–Cox test. Results Overexpression of CCRK, but not its kinase-defective mutant, activated β-catenin/T cell factor signalling through phosphorylation of glycogen synthase kinase-3β (GSK-3β) at Ser9, led to upregulation of AR transcriptional activity and, subsequently, expression of HBx. The viral transactivator in turn induced CCRK expression through enhanced AR signalling, thus forming a positive regulatory loop. RNA interference silencing of CCRK, which suppressed the CCRK/GSK-3β/β-catenin/AR regulatory loop, significantly suppressed HBx-induced hepatocellular proliferation (p=0.001) and transformation (p<0.001) and remarkably reduced >80% diethylnitrosamine-mediated hepatocarcinogenesis in HBx transgenic mice. Finally, patients with HBV-associated HCC with concordant overexpression of CCRK, GSK-3β phosphorylation at Ser9, active dephosphorylated β-catenin and AR phosphorylation at Ser81 had poorer overall (HR=31.26, p<0.0001) and disease-free (HR=3.60, p<0.01) survival rates. Conclusions Our findings highlight the critical role of CCRK in a self-reinforcing circuitry that regulates HBV-associated hepatocarcinogenesis. Further characterisation of this intricate viral-host signalling may provide new prognostic biomarkers and therapeutic targets for HCC treatment.

A CCRK-EZH2 epigenetic circuitry drives hepatocarcinogenesis and associates with tumor recurrence and poor survival of patients

BACKGROUND & AIMS Aberrant chromatin modification is a key feature of hepatocellular carcinoma (HCC), which is characterized by strong sexual dimorphism. Both enhancer of zeste homolog 2 (EZH2) and cell cycle-related kinase (CCRK) contribute to hepatocarcinogenesis, yet whether the two oncogenic factors have functional crosstalk is unknown. METHODS Cellular proliferation and tumorigenicity upon transgenic expression and RNA interference were determined by colony formation and soft agar assays, xenograft, orthotopic and diethylnitrosamine-induced HCC models. Gene regulation was assessed by chromatin immunoprecipitation, site-directed mutagenesis, luciferase reporter, co-immunoprecipitation and expression analyses. Protein levels in clinical specimens were correlated with clinicopathological parameters and patient survival rates. RESULTS Ectopic CCRK expression in immortalized human liver cells increased EZH2 and histone H3 lysine 27 trimethylation (H3K27me3) to stimulate proliferation and tumor formation. Conversely, knockdown of CCRK reduced EZH2/H3K27me3 levels and decreased HCC cell growth, which could be rescued by EZH2 over-expression. Mechanistically, GSK-3β phosphorylation by CCRK activated a β-catenin/TCF/E2F1/EZH2 transcriptional feedback loop to epigenetically enhance androgen receptor (AR) signaling. Simultaneously, the phosphorylation of AKT/EZH2 by CCRK facilitated the co-occupancy of CCRK promoter by EZH2-AR and its subsequent transcriptional activation, thus forming a self-reinforcing circuitry. Lentiviral-mediated knockdown of CCRK, which abrogated the phosphorylation-transcriptional network, prevented diethylnitrosamine-induced tumorigenicity. More importantly, the hyperactivation of the CCRK-EZH2 circuitry in human HCCs correlated with tumor recurrence and poor survival. CONCLUSIONS These findings uncover an epigenetic vicious cycle in hepatocarcinogenesis that operates through reciprocal regulation of CCRK and EZH2, providing novel therapeutic strategy for HCC.

Overexpression of HMGA1 deregulates tumor growth via cdc25A and alters migration/invasion through a cdc25A-independent pathway in medulloblastoma

Overexpression of high mobility group AT-hook 1 (HMGA1) is common in human cancers. Little is known about the mechanisms underlying its deregulation and downstream targets, and information about its clinical and biological significance in medulloblastoma (MB) is lacking. Here, we demonstrated frequent genomic gain at 6p21.33–6p21.31 with copy number increase leading to overexpression of HMGA1 in MB. The overexpression correlated with a high proliferation index and poor prognosis. Moreover, we found that hsa-miR-124a targeted 3′UTR of HMGA1 and negatively modulated the expression in MB cells, indicating that loss/downregulation of hsa-miR-124a reported in our previous study could contribute to the overexpression. Regarding the biological significance of HMGA1, siRNA knockdown and ectopic expression studies revealed the crucial roles of HMGA1 in controlling MB cell growth and migration/invasion through modulation of apoptosis and formation of filopodia and stress fibers, respectively. Furthermore, we identified cdc25A as a target of HMGA1 and showed that physical interaction between HMGA1 and the cdc25A promoter is required for transcriptional upregulation. In clinical samples, HMGA1 and cdc25A were concordantly overexpressed. Functionally, cdc25A is involved in the HMGA1-mediated control of MB cell growth. Finally, netropsin, which competes with HMGA1 in DNA binding, reduced the expression of cdc25A by suppression of its promoter activity and inhibited in vitro and in vivo intracranial MB cell growth. In conclusion, our results delineate the mechanisms underlying the deregulation and reveal the functional significance of HMGA1 in controlling MB cell growth and migration/invasion. Importantly, the results highlight the therapeutic potential of targeting HMGA1 in MB patients.

Abstract 2170: 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

Abstract 4881: Polycomb protein EZH2 activates Wnt/β-catenin signaling to promote hepatocellular carcinoma development

Aberrant activation of the canonical Wnt pathway due to accumulation of β-catenin occurs in approximately 70% of hepatocellular carcinomas (HCCs) and contributes to their initiation, development and progression. Genetic mutations in the components of this pathway account for only a subset of HCCs with β-catenin accumulation, suggesting that an alternative mechanism for Wnt/β-catenin activation is prevailing. Enhancer of zeste homolog 2 (EZH2) is known to promote tumorigenesis by down-regulating tumor-suppressor genes; however, whether EZH2 regulates oncogenic pathways is unclear. Using chromatin immunoprecipitation microarray, we uncovered 12 Wnt/β-catenin signal antagonists whose promoters were concordantly occupied by EZH2 and repressive histone modifications in hepatocellular HCC cells. EZH2 over-expressed in 42% (75/179) of human HCCs and significantly associated with β-catenin accumulation. Concomitant inhibition of EZH2 and histone deacetylase transcriptionally activated Wnt/β-catenin signal antagonists, suppressed T-cell factor-dependent transcriptional activity and down-regulated β-catenin transcriptional targets in HCC cells. Conversely, ectopic EZH2 over-expression in nontumorigenic hepatocytes increased cellular proliferation in a β-catenin-dependent manner and promoted tumorigenicity. This study uncovers an EZH2-mediated epigenetic mechanism that leads to Wnt/β-catenin signaling dyregulation in human HCC and suggests that therapeutic interventions targeting EZH2 may confer a clinical benefit in β-catenin-driven malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4881.

691 The Role of PPARγ Modulation in the Growth Inhibition of Hepatocellular Carcinoma Cells

Background: It has been reported that weakly acidic pharyngeal gas reflux is significantly increased in patients with reflux laryngitis. However, the diagnosis of reflux laryngitis is controversial. Therefore, we studied patients with chronic cough who responded to fulldose PPI therapy, in whom 24-hr pharyngoesophageal impedance and pH monitoring was performed. Methods: Ten patients with chronic cough responding to PPIs (CC)(24-74 yrs; 3F), 10 with GERD (GE)(20-73; 2F), and 10 healthy controls (C)(22-72; 4F) underwent ambulatory 24-hr concurrent pharyngoesophageal impedance and pH monitoring. Three types of new impedance/pH catheters (ZAI-S62C12E-14E, Sandhill Scientific Inc., CO) were used to detect esophagopharyngeal reflux with a Sleuth GER monitoring system. The subjects ate ad libitum and were instructed to record events such as coughing by using an event marker. Weakly acidic reflux was defined as a pH drop of more than 1 unit with a nadir pH > 4. The symptom association probability (SAP) method was used to analyze the relation between gastroesophageal reflux and cough. In 6 CC patients, 24-hr monitoring was repeated on treatment after 8 weeks full-dose PPI therapy. Symptoms were evaluated using Frequency scale for the symptoms of GERD (FSSG). Results: Weakly acidic pharyngeal gas reflux events were observed in 8 out of 10 CC patients, and were significantly increased in the CC versus GE or C groups (p<0.05). Acidic and weakly acidic esophagaopharyngeal reflux (EPR) events were only observed in CC patients and were always induced by swallowing and occurred during acid liquid or mixed reflux events in the proximal esophagus. Swallow-induced acidic and weakly acidic EPR was significantly increased in the CC versus GE or C groups (p<0.05). Among 10 CC patients, 9 had weakly acidic and/or acidic EPR events, while only one had pure non-acidic EPR events. This patient and 2 other CC patients showed a positive association between gastroesophageal reflux and cough on SAP analysis. In 6 CC patients, PPIs abolished swallow-induced weakly acidic and/or acidic reflux, while weakly acidic pharyngeal gas reflux events decreased significantly and symptoms improved significantly compared with before therapy (p<0.05). Conclusion: Most patients with chronic cough responding to PPIs had minor acidic gas reflux and/or swallow-induced weakly acidic and/or acidic reflux in the pharynx. A direct effect of acidic mist or acidic liquid refluxing into the pharynx was suggested to be the cause of their chronic cough. In some patients, symptoms may arise indirectly from gastroesophageal reflux via a vago-vagal reflex.