Edmund Kwok-Kwan Tung

Deregulation of microRNA expression occurs early and accumulates in early stages of HBV-associated multistep hepatocarcinogenesis

BACKGROUND & AIMS Deregulation of microRNAs (miRNAs) plays an important role in human carcinogenesis. However, miRNA deregulation in the pre-malignant lesions and expression changes during multistep hepatocarcinogenesis remain elusive. METHODS In this study, we investigated the expression changes of seven cancer-related miRNAs during the early stages of HBV related hepatocarcinogenesis. miRNA was extracted from formalin fixed paraffin embedded (FFPE) dysplastic nodules (DN), small HCCs, and their corresponding non-tumorous livers. Expression changes of miRNAs were examined by real-time RT-qPCR. RESULTS We found that down-regulation of miR-145 and miR-199b and up-regulation of miR-224 were frequently observed in pre-malignant DNs and these changes persisted throughout HCC development. Restoration of miR-145 in both HepG2 and Hep3B HCC cells significantly inhibited cell proliferation and reduced cell migration and cell invasion. Furthermore, these inhibitory functions of miR-145 could be substantially reduced by an anti-miR-145 inhibitor. CONCLUSIONS Our results showed that miRNA deregulation was an early event and accumulated throughout the various steps of HBV-associated hepatocarcinogenesis. Our findings also suggest that miR-145 is a candidate tumor suppressive miRNA and may play an important role in HCC development.

Rho-kinase 2 is frequently overexpressed in hepatocellular carcinoma and involved in tumor invasion

Deregulation of Rho family small guanosine triphosphatases has been implicated in human carcinogenesis. Rho‐kinases are downstream effectors of Rho guanosine triphosphatases in the regulation of cytoskeletal reorganization and cell motility. However, their functions in human cancers remain elusive. In this study, we aimed to investigate the role of Rho‐kinases in hepatocellular carcinoma (HCC) tumor progression and invasion. We first examined the expression of the two Rho‐kinases (ROCK1 and ROCK2) in human HCC, and found that ROCK2 was frequently overexpressed in primary HCCs (22/41 [53.66%]). Clinico‐pathological analysis revealed that overexpression of ROCK2 was significantly associated with the presence of tumor microsatellite formation (P = 0.005), suggesting that deregulation of ROCK2 may contribute to the intrahepatic metastasis of HCC. Consistently, we demonstrated that stable overexpression of ROCK2 significantly enhanced cell motility and invasiveness in HCC cells. Conversely, stable knockdown of ROCK2 by short hairpin RNA approach remarkably reduced HCC cell migration and invasion. Moreover, orthotopic liver xenograft models provided further support that stable knockdown of ROCK2 suppressed HCC invasion in vivo. Stable knockdown of ROCK2 in HCC cells significantly inhibited Golgi reorientation, myosin phosphatase phosphorylation, and formations of stress fibers, filopodia, and lamellipodia; these molecular and cellular events are crucial for cell motility and cancer invasion. Conclusion: Our results indicate that ROCK2 was overexpressed in human HCCs, and this overexpression was associated with a more aggressive biological behavior. Our findings also demonstrate that ROCK2 played a significant role in regulating cytoskeletal events and contributed to the invasion of HCC. (HEPATOLOGY 2009.)

Tissue factor pathway inhibitor-2 as a frequently silenced tumor suppressor gene in hepatocellular carcinoma†

In HCC, inactivation of tumor suppressor genes plays a significant role in carcinogenesis. Apart from deletions and mutations, growing evidence has indicated that epigenetic alterations including aberrant promoter methylation and histone deacetylation are also implicated in inactivation of tumor suppressor genes. The goal of this study was to identify epigenetically silenced candidate tumor suppressor genes in human HCC by comparing the changes in oligonucleotide microarray gene expression profiles in HCC cell lines upon pharmacological treatment with the demethylating agent 5‐Aza‐2′‐deoxycytidine (5‐Aza‐dC). By analyzing the gene expression profiles, we selected tissue factor pathway inhibitor‐2 (TFPI‐2), a Kunitz‐type serine protease inhibitor, for validation and further characterization. Our results showed that TFPI‐2 was frequently silenced in human HCC and HCC cell lines. TFPI‐2 was significantly underexpressed in approximately 90% of primary HCCs when compared with their corresponding nontumorous livers. TFPI‐2 promoter methylation was detected in 80% of HCC cell lines and 47% of human HCCs and was accompanied by reduced TFPI‐2 messenger RNA expression. In addition, TFPI‐2 expression in HCC cell lines can be robustly restored by combined treatment with 5‐Aza‐dC and histone deacetylase inhibitor trichostatin A. These findings indicate that TFPI‐2 is frequently silenced in human HCC via epigenetic alterations, including promoter methylation and histone deacetylation. Moreover, ectopic overexpression of TFPI‐2 significantly suppressed the proliferation and invasiveness of HCC cells. Conclusion: Our findings suggest that TFPI‐2 is a candidate tumor suppressor gene in human HCC. (HEPATOLOGY 2007;45:1129–1138.)

ATP acts via P2Y1 receptors to stimulate acetylcholinesterase and acetylcholine receptor expression: transduction and transcription control.

At the vertebrate neuromuscular junction ATP is known to stabilize acetylcholine in the synaptic vesicles and to be co-released with it. We have shown previously that a nucleotide receptor, the P2Y1 receptor, is localized at the junction, and we propose that this mediates a trophic role for synaptic ATP there. Evidence in support of this and on its mechanism is given here. With the use of chick or mouse myotubes expressing promoter–reporter constructs from genes of acetylcholinesterase (AChE) or of the acetylcholine receptor subunits, P2Y1 receptor agonists were shown to stimulate the transcription of each of those genes. The pathway to activation of the AChE gene was shown to involve protein kinase C and intracellular Ca 2+ release. Application of dominant-negative or constitutively active mutants, or inhibitors of specific kinases, showed that it further proceeds via some of the known intermediates of extracellular signal-regulated kinase phosphorylation. In both chick and mouse myotubes this culminates in activation of the transcription factor Elk-1, confirmed by gel mobility shift assays and by the nuclear accumulation of phosphorylated Elk-1. All of the aforementioned activations by agonist were amplified when the content of P2Y1 receptors was boosted by transfection, and the activations were blocked by a P2Y1-selective antagonist. Two Elk-1 binding site sequences present in the AChE gene promoter were jointly sufficient to drive ATP-induced reporter gene transcription. Thus ATP regulates postsynaptic gene expression via a pathway to a selective transcription factor activation.

Clinicopathological and prognostic significance of serum and tissue Dickkopf-1 levels in human hepatocellular carcinoma

Although Dickkopf‐1 (DKK1) is known to be a negative regulator of the Wnt/β‐catenin pathway, it has been recently found to be upregulated in cancers.

Caveolin-1 overexpression is associated with hepatocellular carcinoma tumourigenesis and metastasis†

Caveolin‐1 (Cav1) has been implicated in diverse human cancers, yet its role in hepatocellular carcinoma (HCC) tumourigenesis and metastasis remains elusive. In the current study, we aim to provide a comprehensive understanding regarding the functional role of Cav1 in HCC tumourigenesis and metastasis. Cav1 expression was examined in a panel of human HCC cell lines using western blotting analysis and quantitative RT‐PCR and human tissues by immunohistochemistry. Cav1 was not detected in normal liver cell line and all non‐tumourous liver tissues but exclusively expressed in HCC cell lines and tissues. Dramatic expression of Cav1 was found in metastatic HCC cell lines and tumours, indicating a progressive increase of Cav1 expression along disease progression. Cav1 overexpression was significantly correlated with venous invasion (p = 0.036). To investigate the functions of Cav1 in HCC, Cav1 overexpressing and knockdown stable clones were established in HCC cells and their tumourigenicity and metastatic potential were examined. Overexpression of Cav1 promoted HCC cell growth, motility, and invasiveness, as well as tumourigenicity in vivo. Conversely, knockdown of Cav1 in metastatic HCC cells inhibited the motility and invasiveness and markedly suppressed the tumour growth and metastatic potential in vivo. Collectively, our findings have shown the exclusive expression of Cav1 in HCC cell lines and clinical samples and revealed an up‐regulation of Cav1 along HCC progression. The definitive role of Cav1 in promoting HCC tumourigenesis was demonstrated, and we have shown for the first time in a mouse model that Cav1 promotes HCC metastasis. Copyright

Upregulation of the Wnt Co-Receptor LRP6 Promotes Hepatocarcinogenesis and Enhances Cell Invasion

Background Activation of the Wnt/β-catenin signaling pathway plays a crucial role in hepatocellular carcinoma (HCC). Low-density lipoprotein (LDL) receptor-related protein-6 (LRP6) is one of the co-receptors of the Wnt/β-catenin pathway and forms a signaling complex with Wnt ligand and Frizzled receptor to activate downstream signaling. However, the role of LRP6 in hepatocarcinogenesis is unclear. In this study, we examined its expression and roles in human HCC. Methodology/Principal Findings Using real-time quantitative RT-PCR, we found that LRP6 was frequently (45%) overexpressed in human HCCs (P = 0.003). In vitro studies showed that ectopic expression of LRP6 increased the protein level of β-catenin. Moreover, overexpression of the full-length and constitutively active LRP6, respectively, activated the WNT/β-catenin signaling pathway, as shown by the TCF/β-catenin reporter assay. With regard to the effects of LRP6 overexpression in HCC cells, stable overexpression of the constitutively active LRP6 in BEL-7402 HCC cells enhanced cell proliferation, cell migration, and invasion in vitro as well as tumorigenicity in nude mice. Conclusions/Significance Our findings indicate that overexpression of LRP6 contributes to the hyperactivation of the Wnt/β-catenin signaling pathway in human HCCs and suggest it may play a role in hepatocarcinogenesis.

RhoE is frequently down-regulated in hepatocellular carcinoma (HCC) and suppresses HCC invasion through antagonizing the Rho/Rho-Kinase/Myosin phosphatase target pathway†‡

Deregulation of Rho guanosine triphosphatase (GTPase) pathways plays an important role in tumorigenesis and metastasis of hepatocellular carcinoma (HCC). RhoE/Rnd3 belongs to an atypical subfamily of the RhoGTPase, the Rnd family, as it lacks the intrinsic GTPase activity and remains always in its active GTP‐bound form. In this study we investigated the role of RhoE in HCC. We examined the expression of RhoE in primary HCC samples from patients predominantly infected with the hepatitis B virus (HBV) and found that the RhoE messenger RNA (mRNA) level was frequently down‐regulated (83.1%, 59/71) in HCCs. Low expression of RhoE in the tumors was significantly associated with shorter disease‐free survival (P = 0.020) of the patients. Knockdown of RhoE by short‐hairpin RNA using a lentiviral approach led to increased cell motility and invasiveness in SMMC7721 and BEL7402 HCC cells. Moreover, in vivo an orthotopic liver injection model in nude mice further demonstrated that knockdown of RhoE enhanced local invasion of HCC cells in the livers, with more invasive tumor front and increased incidence of venous invasion. Mechanistically, stable knockdown of RhoE in HCC cells significantly enhanced the phosphorylation of myosin phosphatase, promoted assembly of stress fibers, and increased the formation of plasma membrane blebbings, all these changes and activities being associated with activation of the Rho/Rho‐kinase (ROCK) pathway. Conclusion: RhoE was frequently down‐regulated in predominantly HBV‐associated HCCs and this down‐regulation was associated with a more aggressive HCC phenotype. RhoE regulated the cytoskeleton remodeling and suppressed HCC motility and invasiveness by way of inhibiting the Rho/ROCK axis. (HEPATOLOGY 2013)

HAI-2 is epigenetically downregulated in human hepatocellular carcinoma, and its Kunitz domain type 1 is critical for anti-invasive functions.

Pharmacological demethylation‐based gene expression profile analysis is a useful tool to identify epigenetically silenced tumour suppressor genes. HGF activator inhibitor 2 (HAI‐2), a serine protease inhibitor, has been identified as one of the candidate tumour suppressor genes in human hepatocellular carcinoma (HCC) with this technique. In this study, we aimed to characterise the epigenetic status and tumour suppressive function of HAI‐2 in HCC. We validated that HAI‐2 expression was either absent or low in most of the HCC cell lines tested, and 5‐Aza‐2′‐deoxycytidine treatment significantly restored its expression in 9 (75%) of these 12 cell lines. HAI‐2 was found to be frequently underexpressed in human HCCs (p < 0.001). With bisulphite DNA sequencing and methylation‐specific PCR, we found that the promoter of the HAI‐2 gene was frequently hypermethylated in both HCC cell lines and human HCCs. Ectopic expression of HAI‐2 significantly inhibited cell migration and invasiveness of HCC cells in vitro and suppressed tumourigenicity in vivo. In addition, we also provided the first evidence that HAI‐2 mediated its tumour suppressor function via the Kunitz domain 1 (KD‐1), as KD‐1 but not KD‐2 inactivating mutant abolished its anti‐tumour invasiveness in vitro. Our findings suggest that HAI‐2 is a candidate tumour suppressor gene that is frequently hypermethylated and underexpressed in human HCCs, and the KD‐1 domain of HAI‐2 is the key region responsible for its anti‐invasive function.

Tensin2 variant 3 is associated with aggressive tumor behavior in human hepatocellular carcinoma

Tensins are a new family of proteins that act as an important link among extracellular matrix, actin cytoskeleton, and signal transduction and have been implicated in human cancers. Tensin2 was initially identified in a search for new tensin family members that share extensive sequence homology with tensin1. Tensin2 was highly expressed in liver tissues. A recent study reported that one of the splicing variants of tensin2, variant 3, promotes cell migration. In the present study, we aimed to elucidate the role of variant 3 in hepatocarcinogenesis by assessing the expression of variant 3 mRNA in hepatocellular carcinoma (HCC) tissue and ectopically expressing variant 3 in HCC cell lines. Analysis of variant 3 expression in human HCC tissue revealed it was overexpressed in 46% (23/50) of tumor tissues as compared with the corresponding nontumorous livers. High expression of variant 3 was significantly associated with venous invasion (P = .037), tumor microsatellite formation (P = .022), and tumor nonencapsulation (P = .049). Our ectopic expression study showed that variant 3 significantly promoted the cell growth and motility of HCC cells. The clonal transfectants of variant 3 were more closely packed and resulted in a higher saturation density than in the control vector transfectants. Variant 3 expression also enhanced the proliferation rate in culture and in vivo tumorigenicity in nude mice. In conclusion, we reveal a novel role for variant 3 in the progression of HCC and suggest the feasibility of elevated variant 3 expression as a tumor progression marker for HCC. (HEPATOLOGY 2006;44:881–90.)