Nathalie Wong

Nasopharyngeal carcinoma cell line (C666-1) consistently harbouring Epstein-Barr virus

We have established a cell line (C666‐1) from undifferentiated nasopharyngeal carcinoma (NPC). This cell line consistently carries the Epstein‐Barr virus (EBV) in long‐term cultures. C666‐1 is a subclone of its parental cell line, C666, derived from an NPC xenograft of southern Chinese origin. It grows as an adherent culture and lacks contact inhibition. In addition, it is tumorigenic in athymic nude mice. The cells consistently express EBV‐encoded RNAs and are positively stained for cytokeratin, an epithelial marker. In addition, they express EBNA1 protein, LMP1 and LMP2 transcripts and thus resemble the EBV latency II pattern. The virus genotype is EBV‐1 with the latent membrane protein 1 gene showing a 30‐bp deletion at the carboxyl terminus, both consistent with findings in southern Chinese NPC tumours. Cytogenetic analysis revealed a sub‐diploid status with a chromosomal modal number of 45. C666‐1 is unique among NPC cell lines in that it carries EBV. These cells may serve as a good investigative tool as the viral latency pattern and genotype are observed in the majority of primary NPC biopsies from Chinese patients. Int. J. Cancer 83:121–126, 1999.

miR-130b Promotes CD133+ Liver Tumor-Initiating Cell Growth and Self-Renewal via Tumor Protein 53-Induced Nuclear Protein 1

A novel paradigm in tumor biology suggests that cancer growth is driven by stem-like cells within a tumor, called tumor-initiating cells (TICs) or cancer stem cells (CSCs). Here we describe the identification and characterization of such cells from hepatocellular carcinoma (HCC) using the marker CD133. CD133 accounts for approximately 1.3%-13.6% of the cells in the bulk tumor of human primary HCC samples. When compared with their CD133⁻ counterparts, CD133(+) cells not only possess the preferential ability to form undifferentiated tumor spheroids in vitro but also express an enhanced level of stem cell-associated genes, have a greater ability to form tumors when implanted orthotopically in immunodeficient mice, and can be serially passaged into secondary animal recipients. Xenografts resemble the original human tumor and maintain a similar percentage of tumorigenic CD133(+) cells. Quantitative PCR analysis of 41 separate HCC tissue specimens with follow-up data found that CD133(+) tumor cells were frequently detected at low quantities in HCC, and their presence was also associated with worse overall survival and higher recurrence rates. Subsequent differential microRNA expression profiling of CD133(+) and CD133⁻ cells from human HCC clinical specimens and cell lines identified an overexpression of miR-130b in CD133(+) TICs. Functional studies on miR-130b lentiviral-transduced CD133⁻ cells demonstrated superior resistance to chemotherapeutic agents, enhanced tumorigenicity in vivo, and a greater potential for self renewal. Conversely, antagonizing miR-130b in CD133(+) TICs yielded an opposing effect. The increased miR-130b paralleled the reduced TP53INP1, a known miR-130b target. Silencing TP53INP1 in CD133⁻ cells enhanced both self renewal and tumorigenicity in vivo. Collectively, miR-130b regulates CD133(+) liver TICs, in part, via silencing TP53INP1.

Assessment of Genetic Changes in Hepatocellular Carcinoma by Comparative Genomic Hybridization Analysis : Relationship to Disease Stage, Tumor Size, and Cirrhosis

Hepatocellular carcinoma (HCC) is a common and highly malignant tumor that is prevalent in Southeast Asia. Although the etiological factors associated are now well recognized, the interactions between individual factors and the molecular mechanisms by which they lead to cancer remain unclear. Cytogenetic analysis on HCC has been limited because of poor hepatocyte growth in vitro. The recently developed technique of comparative genomic hybridization (CGH), however, permits screening of the entire genome without the need of cell culture. CGH was applied to the study of genomic aberrations in 67 surgically resected samples of HCC, 3 of adenomatous hyperplasia (AH), and 12 of nontumorous cirrhotic liver surrounding the tumors. All samples were from patients of a racially and etiologically homogeneous population in Southern China, where chronic hepatitis B virus infection is the main etiological factor. CGH analysis of the HCC samples revealed frequent copy number gain of 1q (48/67 cases, 72%), 8q (32/67 cases, 48%), 17q (20/67 cases, 30%), and 20q (25/67 cases, 37%) and common losses on 4q (29/67 cases, 43%), 8p (25/67 cases, 37%), 13q (25/67 cases, 37%), and 16q (20/67 cases, 30%). Our finding of a high incidence of 1q gain strongly suggested this aberration was associated with the development of HCC. Genomic abnormalities were detected in 1 of the 3 AH specimens but absent in all 12 cirrhotic tissues surrounding the tumor. Clinical staging classified 3/67 HCC cases as T1, 53 cases as T2, and 11 cases as T3. No significant difference in the pattern of genomic imbalances was detected between stages T2 and T3. A significant copy number loss of 4q11-q23 was, however, identified in those tumors larger than 3 cm in diameter. Of particular interest was the identification of 8q copy number gain in all 12 cases of HCC that arose in a noncirrhotic liver, compared with only 20/55 cases in HCC arising in a cirrhotic liver. We suggest that 8q over-representation is likely associated with a growth advantage and proliferative stimulation that have encouraged malignant changes in the noncirrhotic human liver.

MiR-222 Overexpression Confers Cell Migratory Advantages in Hepatocellular Carcinoma through Enhancing AKT Signaling

Purpose: This study aims to profile the expressions of 156 microRNAs (miRNA) in hepatocellular carcinoma (HCC) and to characterize the functions of miR-222, the most significantly upregulated candidate identified. Experimental Design: miRNA expression profile in HCC tumors, matching adjacent cirrhotic livers, and cell lines was conducted using quantitative PCR. Common miR-222 upregulations were further validated in a larger cohort of tumors. The functional effects of miR-222 inhibition on HCC cell lines were examined. The downstream modulated pathways and target of miR-222 were investigated by coupling gene expression profiling and pathway analysis, and by in silico prediction, respectively. Luciferase reporter assay was done to confirm target interaction. Results: We identified a 40-miRNA signature that could discriminate tumors from adjacent cirrhotic liver tissue, and further corroborated common miR-222 overexpression in tumors relative to its premalignant counterpart (55.3%; P < 0.0001). Increased miR-222 expression correlated significantly with advanced stage HCC and with the shorter disease-free survival of patients (P ≤ 0.01). Inhibition of miR-222 in Hep3B and HKCI-9 significantly retarded cell motility (P < 0.05). Further investigations suggested that AKT signaling was the major pathway influenced by miR-222. A consistent reduction of AKT phosphorylation in Hep3B and HKCI-9 was shown following miR-222 suppression. The protein phosphatase 2A subunit B (PPP2R2A) was predicted as a putative miR-222 target in silico. We found that miR-222 inhibition could augment the tumor protein level and restore luciferase activity in reporter construct containing the PPP2R2A 3′ untranslated region (P = 0.0066). Conclusions: Our study showed that miR-222 overexpression is common in HCC and could confer metastatic potentials in HCC cells, possibly through activating AKT signaling. Clin Cancer Res; 16(3); 867–75

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.

Detection of recurrent chromosomal gains and losses in primary nasopharyngeal carcinoma by comparative genomic hybridisation

Nasopharyngeal carcinoma (NPC) is a common cancer in Southern China but rare in Western countries. To search for genetic alterations in NPC, we examined a series of 20 primary tumours with comparative genomic hybridisation. The identified common chromosomal alterations included gain of chromosomes 1q, 8, 12, 19 and 20 as well as loss of chromosomes 1p, 3p, 9p, 9q, 11q, 13q, 14q and 16q. In concordance with our previous loss of heterozygosity studies in primary NPC, a high incidence of loss was detected on chromosomes 3p (75%), 11q (70%) and 14q (65%). Losses of 9q (60%), 13q (50%) and 16q (40%) were also identified. Novel chromosomal gains were observed on chromosome 12, with a high frequency (70%). Current analysis has revealed a comprehensive profile of the chromosomal regions showing losses and gains in primary NPC. Our findings may provide an entry point for conducting further investigations to locate the putative tumour‐suppresser genes and oncogenes that may be involved in the tumourigenesis of NPC. Int. J. Cancer 82:498–503, 1999.

Hypomethylation of Chromosome 1 Heterochromatin DNA Correlates with q-Arm Copy Gain in Human Hepatocellular Carcinoma

Using comparative genomic hybridization (CGH) analysis, we, and others, have shown that there is a high and consistent incidence of chromosome 1q copy gain in human hepatocellular carcinoma (HCC). Chromosome 1 rearrangements, that involved peri-centromeric breakpoints, have also been frequently reported in karyotypic studies of HCC. Satellite DNA hypomethylation has been postulated as the mechanism underlying the induction of chromosome 1 peri-centromeric instability in many human cancers and in individuals with the rare recessive disorder ICF (immunodeficiency, centromeric heterochromatin instability, facial anomalies). In this study, we have investigated the role of DNA hypomethylation in 1q copy gain in HCC by examining the methylation status of chromosome 1 heterochromatin DNA (band 1q12). Thirty-six histologically confirmed samples of HCC were studied (24 paired tumor and adjacent nontumorous liver tissues, and 12 tumor only). Hypomethylation of satellite 2 (Sat2) DNA in 1q12 was analyzed by Southern blotting using methyl-sensitive enzyme digestion. In parallel, all cases were analyzed by CGH. A strong correlation between hypomethylated Sat2 sequences and 1q copy gain with a 1q12 breakpoint was found (P < 0.001). We postulate that such hypomethylation alters the interaction between the CpG-rich satellite DNA and chromatin proteins, resulting in heterochromatin decondensation, breakage and aberrant 1q formation. Spectral karyotyping further supported the presence of fragile 1q12 in HCC. Of particular interest was the finding of Sat2 DNA hypomethylation in 5 of 24 adjacent nontumorous liver tissues examined. These tissues showed no evidence of malignancy on histological examination nor did they display any CGH abnormalities. Our findings suggest a role for Sat2 demethylation in the early stages of the stepwise progression of liver carcinogenesis.

Sirtuin 1 Is Upregulated in a Subset of Hepatocellular Carcinomas where It Is Essential for Telomere Maintenance and Tumor Cell Growth

Hepatocellular carcinoma (HCC) is a highly malignant tumor with a poor prognosis. Treatment of HCC is complicated by the fact that the disease is often diagnosed at an advanced stage when it is no longer amenable to curative surgery, and current systemic chemotherapeutics are mostly inefficacious. Sirtuin 1 (SIRT1) is a class III histone deacetylase that is implicated in gene regulations and stress resistance. In this study, we found that SIRT1 is essential for the tumorigenesis of HCC. We showed that although SIRT1 was expressed at very low levels in normal livers, it was overexpressed in HCC cell lines and in a subset of HCC. Tissue microarray analysis of HCC and adjacent nontumoral liver tissues revealed a positive correlation between the expression levels of SIRT1 and advancement in tumor grades. Downregulation of SIRT1 consistently suppressed the proliferation of HCC cells via the induction of cellular senescence or apoptosis. SIRT1 silencing also caused telomere dysfunction-induced foci and nuclear abnormality that were clearly associated with reduced expressions of telomerase reverse transcriptase (TERT), and PTOP, which is a member of the shelter in complex. Ectopic expression of either TERT or PTOP in SIRT1-depleted cells significantly restored cell proliferation. There was also a positive correlation between the level of induction of SIRT1 and TERT [corrected] in human HCC. Finally, SIRT1-silencing sensitized HCC cells to doxorubicin treatment. Together, our findings reveal a novel function for SIRT1 in telomere maintenance of HCC, and they rationalize the clinical exploration of SIRT1 inhibitors for HCC therapy.

Emerging roles of microRNA in the intracellular signaling networks of hepatocellular carcinoma

MicroRNAs (miRNAs) are small non‐coding RNAs of 19–23 nucleotides that negatively regulate gene expression through binding to the 3′‐untranslated regions of target messenger RNAs (mRNAs). Although the miRNA family constitutes only a minor fraction of the human genome, they hold fundamental importance in diverse physiological and developmental processes due to their pleiotropic effects on the post‐transcriptional regulation of many vital genes. This class of regulatory RNAs has also emerged as important players in carcinogenesis; most, if not all, cancer types have abnormal miRNA expression patterns. In hepatocellular carcinoma (HCC), miRNA dysregulation plays a key role in mediating the pathogenicity of several etiologic risk factors and, more importantly, they promote a number of cancer‐inducing signaling pathways. Recent studies have also demonstrated their potential values in the clinical management of HCC patients as some miRNAs may be used as prognostic or diagnostic markers. The significance of miRNAs in liver carcinogenesis emphasizes their values as therapeutic targets, while technological advances in the delivery of miRNA has shed new possibilities for their use as novel therapeutic agents against HCC.