Arthur K.K. Ching

Viral-Human Chimeric Transcript Predisposes Risk to Liver Cancer Development and Progression

The mutagenic effect of hepatitis B (HBV) integration in predisposing risk to hepatocellular carcinoma (HCC) remains elusive. In this study, we performed transcriptome sequencing of HBV-positive HCC cell lines and showed transcription of viral-human gene fusions from the site of genome integrations. We discovered tumor-promoting properties of a chimeric HBx-LINE1 that, intriguingly, functions as a hybrid RNA. HBx-LINE1 can be detected in 23.3% of HBV-associated HCC tumors and correlates with poorer patient survival. HBx-LINE1 transgenic mice showed heightened susceptibility to diethylnitrosamine-induced tumor formation. We further show that HBx-LINE1 expression affects β-catenin transactivity, which underlines a role in activating Wnt signaling. Thus, this study identifies a viral-human chimeric fusion transcript that functions like a long noncoding RNA to promote HCC.

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.

TOP2A overexpression in hepatocellular carcinoma correlates with early age onset, shorter patients survival and chemoresistance.

Genomic gain represents an important mechanism in the activation of proto‐oncogenes. In many instances, induced oncogenes hold clinical implications both as prognostic markers and targets for therapeutic design. In hepatocellular carcinoma (HCC), although chromosomal gains are common, information on underlying oncogenes induced remains minimal. Here, we examined 7 causal sites of HCC for overexpressed genes by array‐based transcriptional mapping. In 22 HCC cell lines and early passages of cultures studied, clusters of up‐regulated genes were indicated, where TOP2A expression ranked the highest. Distinct TOP2A transcriptions were confirmed in an independent series of HCC tumors relative to adjacent non‐tumoral liver (p = 0.0018). By tissue microarray analysis of 172 HCC, we found TOP2A expressions correlated with advance histological grading (p < 0.001), microvascular invasion (p = 0.004) and an early age onset of the malignancy (≤40 years; p = 0.007). In conjunction with P‐gp and MRP1, TOP2A were further assessed for its association with chemotherapy responsiveness and survival in 148 patients who entered our recently reported Phase III prospective randomized study. In 73 chemoresistant and 75 nonresistant patients, only TOP2A positivity correlated with chemoresistance (p = 0.029) and shorter patients survival (p < 0.0001). The potential therapeutic value in targeting TOP2A by Etoposide, as a single agent, and in combination with Doxorubicin was also explored. In vitro cytotoxic studies suggested Etoposide at IC20 readily reduced IC50 values of Doxorubicin by a magnitude of ∼3.5 to 10‐fold compared to Doxorubicin alone (p < 0.028). Our study highlighted for the first time the prognostic value of TOP2A in HCC and the potential use of TOP2A reactive agents in therapy.

Deep sequencing of small RNA transcriptome reveals novel non-coding RNAs in hepatocellular carcinoma.

BACKGROUND & AIMS Small non-coding RNAs (ncRNA) are increasingly recognized to play important roles in tumorigenesis. With the advent of deep sequencing, efforts have been put forth to profile the miRNome in a number of human malignancies. However, information on ncRNA in hepatocellular carcinoma (HCC), especially the non-microRNA transcripts, is still lacking. METHODS Small RNA transcriptomes of two HCC cell lines (HKCI-4 and HKCI-8) and an immortalized hepatocyte line (MIHA) were examined using Illumina massively parallel sequencing. Dysregulated ncRNAs were verified in paired HCC tumors and non-tumoral livers (n=73) by quantitative reverse transcription-polymerase chain reaction. Clinicopathologic correlations and in vitro functional investigations were further carried out. RESULTS The combined bioinformatic and biological analyses showed the presence of ncRNAs and the involvement of a new PIWI-interacting RNA (piRNA), piR-Hep1, in liver tumorigenesis. piR-Hep1 was found to be upregulated in 46.6% of HCC tumors compared to the corresponding adjacent non-tumoral liver. Silencing of piR-Hep1 inhibited cell viability, motility, and invasiveness, with a concomitant reduction in the level of active AKT phosphorylation. In the analysis of miRNA, we showed for the first time, the abundant expression of miR-1323 in HCC and its distinct association in tumors arising from a cirrhotic background. Furthermore, miR-1323 overexpression in cirrhotic HCC correlated with poorer disease-free and overall survivals of patients (p<0.009). CONCLUSIONS Our study demonstrated the value of next-generation sequencing in dissecting the ncRNome in cancer. The comprehensive definition of transcriptome unveils virtually all types of ncRNAs and provides new insight into liver carcinogenetic events.

Expression of a cyclo-oxygenase-2 transgene in murine liver causes hepatitis

Background: It has been proved that cyclo-oxygenase-2 (COX-2) is rapidly induced by inflammatory mediators. However, it is not known whether overexpression of COX-2 in the liver is sufficient to promote activation or secretion of inflammatory factors leading to hepatitis. Aim: To investigate the role forced expression of COX-2 in liver by using inducible COX-2 transgenic (TG) mice. Methods: TG mice that overexpress the human COX-2 gene in the liver using the liver-specific transthyretin promoter and non-TG littermates were derived and fed the normal diet for up to 12 months. Hepatic prostaglandin E2 (PGE2) content was determined using enzyme immunoassay, nuclear factor kappaB (NF-κB) activation by electrophoretic mobility shift assays, apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labelling and proliferation by Ki-67 immunohistochemistry. Results: COX-2 TG mice exhibited strongly increased COX-2 and PGE2, elevated serum alanine aminotransferase level and histological hepatitis. Hepatic COX-2 expression in the TG mice resulted in activation of NF-κB and inflammatory cytokine cascade, with a marked expression of the proinflammatory cytokines tumour necrosis factor (TNF)-α (9.4-fold), interleukin (IL)-6 (4.4-fold), IL-1β (3.6-fold), and of the anti-inflammatory cytokine IL-10 (4.4-fold) and chemokine macrophage inflammatory protein-2 (3.2-fold). The inflammatory response of the COX-2 TG mice was associated with infiltration macrophages and lymphocytes, increased cell proliferation and high rates of cell apoptosis. Administration of the COX-2 inhibitor celecoxib in TG mice restored liver histology to normal. Conclusion: Enhanced COX-2 expression in hepatocytes is sufficient to induce hepatitis by activating NF-κB, stimulating the secretion of proinflammatory cytokines, recruiting macrophage and altering cell kinetics. Inhibition of COX-2 represents a mechanism-based chemopreventive approach to hepatitis.

Novel therapeutic potential in targeting microtubules by nanoparticle albumin-bound paclitaxel in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) shows low response to most conventional chemotherapies. To facilitate target identification for novel therapeutic development, we deployed gene expression profiling on 43 paired HCC tumors and adjacent non-tumoral liver, which is also considered as the pre-malignant liver lesion. In conjunction with ontology analysis, a major functional process found to play a role in the malignant transformation of HCC was microtubule-related cellular assembly. We further examined the potential use of microtubule targeting taxane drugs, including paclitaxel and docetaxel, and compared with findings to results from doxorubicin, a common chemotherapeutic agent used in HCC. Recent studies showed that drug delivery by nanoparticles have enhanced efficacy with reduced side effects. In this regard, the nanoparticle albumin-bound (nab)-paclitaxel was also examined. In a panel of HCC cell lines studied, a high sensitivity towards taxane drugs was generally found, although the effect from nab-paclitaxel was most profound. The nab-paclitaxel showed an effective IC50 dose at 15-fold lower than paclitaxel alone or the derivative analogue docetaxel, and ~450-fold less compared to doxorubicin. Flow cytometric analysis confirmed a cell cycle blockade at the G2/M phase and increased apoptosis following nab-paclitaxel treatment. In vivo animal studies also showed that nab-paclitaxel readily inhibited xenograft growth with less toxicity to host cells compared to other anti-microtubule drugs and doxorubicin. Gene silencing of the microtubule regulatory gene STMN1 by RNAi suggested a distinct synergistic effect in the combined treatment with nab-paclitaxel. Our findings in this study highly suggest that the microtubule assembly represents a promising therapeutic target development in HCC.

Transgenic cyclooxygenase-2 expression and high salt enhanced susceptibility to chemical-induced gastric cancer development in mice

Cyclooxoygenase (COX)-2 overexpression is involved in gastric carcinogenesis. While high-salt intake is a known risk factor for gastric cancer development, we determined the effects of high salt on gastric chemical carcinogenesis in COX-2 transgenic (TG) mice. COX-2 TG mice were developed in C57/BL6 strain using the full-length human cox-2 complementary DNA construct. Six-week-old COX-2 TG and wild-type (WT) littermates were randomly allocated to receive alternate week of N-methyl-N-nitrosourea (MNU, 240 p.p.m.) in drinking water or control for 10 weeks. Two groups of mice were further treated with 10% NaCl during the initial 10 weeks. All mice were killed at the end of week 50. Both forced COX-2 overexpression and high-salt intake significantly increased the frequency of gastric cancer development in mice as compared with WT littermates treated with MNU alone. However, no additive effect was observed on the combination of high salt and COX-2 expression. We further showed that MNU and high-salt treatment increased chronic inflammatory infiltrates and induced prostaglandin E(2) (PGE(2)) production in the non-cancerous stomach. Whereas high-salt treatment markedly increased the expression of inflammatory cytokines (tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-1 beta and IL-6) in the gastric mucosa, COX-2 overexpression significantly altered the cell kinetics in the MNU-induced gastric cancer model. In conclusion, both high salt and COX-2 overexpression promote chemical-induced gastric carcinogenesis, possibly related to chronic inflammation, induction of PGE(2), disruption of cell kinetics and induction of inflammatory cytokines.

Overexpression of ZFX confers self-renewal and chemoresistance properties in hepatocellular carcinoma.

Zinc finger protein X‐linked (ZFX) is a zinc finger protein of Zfy family, which is highly conserved in vertebrates. This transcriptional regulator is not only highly expressed in embryonic stem cells (ESC) and hematopoietic stem cells, but is also upregulated in a number of human cancers where it is functional related to cell proliferation and survival. Hepatocellular carcinoma (HCC) is highly aggressive cancer that commonly resistant to most chemotherapies and displays stemness characteristics. In this study, we examined the expression of ZFX in HCC and its possible functional implications in liver tumorigenesis. Quantitative RT‐PCR analysis showed common overexpressions of ZFX in 51.8% HCC tumors when compared with their adjacent nonmalignant liver (n = 43/83; p = 0.004). Inline with the pluripotency role of ZFX, we found silencing of ZFX readily inhibited self‐renewal capability (p = 0.0022), colony formation ability (p < 0.0001) and cell proliferation (p < 0.0001) through G0/G1 cell cycle arrest of HCC cells (p = 0.0038). In addition, suppression of ZFX sensitized HCC cells to chemotherapeutic agent cisplatin (p < 0.0001). Further investigations suggested that ZFX bind on the promoter of two important mediators, namely Nanog and SOX‐2, activating their expressions in HCC (p < 0.0001). Moreover, in vivo xenograft study demonstrated that overexpression of ZFX would promote the tumor growth (p = 0.031). Taken together, our results show, for the first time, commonly overexpressions of ZFX in HCC, where it likely contributes to the stemness and pluripotent behavior of this highly malignant cancer.

Block of proliferation 1 (BOP1) plays an oncogenic role in hepatocellular carcinoma by promoting epithelial‐to‐mesenchymal transition

Genomic amplification of regional chromosome 8q24 is a common event in human cancers. In hepatocellular carcinoma (HCC), a highly aggressive malignancy that is rapidly fatal, recurrent 8q24 gains can be detected in >50% of cases. In this study, attempts to resolve the 8q24 region by way of array comparative genomic hybridization for affected genes in HCC revealed distinctive gains of block of proliferation 1 (BOP1). Gene expression evaluation in an independent cohort of primary HCC (n = 65) revealed frequent BOP1 up‐regulation in tumors compared with adjacent nontumoral liver (84.6%; P < 0.0001). Significant associations could also be drawn between increased expressions of BOP1 and advance HCC staging (P = 0.004), microvascular invasion (P = 0.006), and shorter disease‐free survival of patients (P = 0.02). Examination of expression of C‐MYC, a well‐known oncogene located in proximity to BOP1, in the same series of primary HCC cases did not suggest strong clinicopathologic associations. Functional investigations by small interfering RNA–mediated suppression of BOP1 in HCC cell lines indicated significant inhibition on cell invasion (P < 0.005) and migration (P < 0.05). Overexpression of BOP1 in the immortalized hepatocyte cell line L02 showed increase cellular invasiveness and cell migratory rate (P < 0.0001). In both gene knockdown and ectopic expression assays, BOP1 did not exert an effect on cell viability and proliferation. Evident regression of the epithelial‐mesenchymal transition (EMT) phenotype was readily identified in BOP1 knockdown cells, whereas up‐regulation of epithelial markers (E‐cadherin, cytokeratin 18, and γ‐catenin) and down‐regulation of mesenchymal markers (fibronectin and vimentin) were seen. A corresponding augmentation of EMT was indicated from the ectopic expression of BOP1 in L02. In addition, BOP1 could stimulate actin stress fiber assembly and RhoA activation. Conclusion: Our findings underline an important role for BOP1 in HCC invasiveness and metastasis potentials through inducing EMT and promoting actin cytoskeleton remodeling. (HEPATOLOGY 2011;)

Elucidation of the role of COX-2 in liver fibrogenesis using transgenic mice

Hepatic COX-2 overexpression is sufficient to induce hepatitis, but its role on liver fibrosis remains unknown. We aim to elucidate possible biological effects of COX-2 in liver fibrosis using both gain-of-function and loss-of-function mouse models. COX-2 transgenic (TG) mice that specifically overexpress the human COX-2 cDNA in the liver, knockout (KO), and wild type (WT) mice were studied in two different murine fibrosis models induced by carbon tetrachloride (CCl(4)) injection or methionine and choline-deficient (MCD) diet. Liver injury was assessed by serum ALT and bilirubin levels and histological examination. Hepatic collagen content was determined by picrosirius red stain morphometry assay and quantitation of hydroxyproline. Hepatic stellate cell (HSC) activation was determined by immunohistochemical analysis of alpha-smooth muscle actin (alpha-SMA). mRNA expression of fibrogenic genes was assayed by real-time quantitative PCR. COX-2 protein was overexpressed in the liver of TG mice compared with WT littermates. CCl(4) or MCD-induced liver fibrotic injury was equally severe in TG and WT mice, as demonstrated by similar elevated levels of hepatic collagen contents. Enhanced COX-2 expression in TG liver did not affect HSC activation and fibrogenic gene expression upon CCl(4) or MCD treatment. Importantly, CCl(4)-treated KO mice did not show significant difference in liver fibrotic damage and fibrogenic gene expression compared with the WT counterparts. This is the first report on the effect of COX-2 in liver fibrosis based on genetic mouse models. The results suggest that COX-2 does not appear to mediate the development of liver fibrosis.