Chun-Ming Wong

Genome-Wide Association Study in Asian Populations Identifies Variants in ETS1 and WDFY4 Associated with Systemic Lupus Erythematosus

Systemic lupus erythematosus is a complex and potentially fatal autoimmune disease, characterized by autoantibody production and multi-organ damage. By a genome-wide association study (320 patients and 1,500 controls) and subsequent replication altogether involving a total of 3,300 Asian SLE patients from Hong Kong, Mainland China, and Thailand, as well as 4,200 ethnically and geographically matched controls, genetic variants in ETS1 and WDFY4 were found to be associated with SLE (ETS1: rs1128334, P = 2.33×10−11, OR = 1.29; WDFY4: rs7097397, P = 8.15×10−12, OR = 1.30). ETS1 encodes for a transcription factor known to be involved in a wide range of immune functions, including Th17 cell development and terminal differentiation of B lymphocytes. SNP rs1128334 is located in the 3′-UTR of ETS1, and allelic expression analysis from peripheral blood mononuclear cells showed significantly lower expression level from the risk allele. WDFY4 is a conserved protein with unknown function, but is predominantly expressed in primary and secondary immune tissues, and rs7097397 in WDFY4 changes an arginine residue to glutamine (R1816Q) in this protein. Our study also confirmed association of the HLA locus, STAT4, TNFSF4, BLK, BANK1, IRF5, and TNFAIP3 with SLE in Asians. These new genetic findings may help us to gain a better understanding of the disease and the functions of the genes involved.

Hypoxia-inducible factor 1 is a master regulator of breast cancer metastatic niche formation

Primary tumors facilitate metastasis by directing bone marrow-derived cells (BMDCs) to colonize the lungs before the arrival of cancer cells. Here, we demonstrate that hypoxia-inducible factor 1 (HIF-1) is a critical regulator of breast cancer metastatic niche formation through induction of multiple members of the lysyl oxidase (LOX) family, including LOX, LOX-like 2, and LOX-like 4, which catalyze collagen cross-linking in the lungs before BMDC recruitment. Only a subset of LOX family members was expressed in any individual breast cancer, but HIF-1 was required for expression in each case. Knockdown of HIF-1 or hypoxia-induced LOX family members reduced collagen cross-linking, CD11b+ BMDC recruitment, and metastasis formation in the lungs of mice after orthotopic transplantation of human breast cancer cells. Metastatic niche formation is an HIF-1–dependent event during breast cancer progression.

MicroRNA‐125b suppressesed human liver cancer cell proliferation and metastasis by directly targeting oncogene LIN28B2

MicroRNAs (miRNAs) are small, noncoding RNAs that can act as oncogenes or tumor suppressors in human cancer. Our previous study showed that miR‐125b was a prognostic indicator for patients with hepatocellular carcinoma (HCC), but its functions and exact mechanisms in hepatic carcinogenesis are still unknown. Here we demonstrate that miR‐125b suppressed HCC cell growth in vitro and in vivo. Moreover, miR‐125b increased p21Cip1/Waf1 expression and arrested cell cycle at G1 to S transition. In addition, miR‐125b inhibited HCC cell migration and invasion. Further studies revealed that LIN28B was a downstream target of miR‐125b in HCC cells as miR‐125b bound directly to the 3′ untranslated region of LIN28B, thus reducing both the messenger RNA and protein levels of LIN28B. Silencing of LIN28B recapitulated the effects of miR‐125b overexpression, whereas enforced expression of LIN28B reversed the suppressive effects of miR‐125b. Conclusion: These findings indicate that miR‐125b exerts tumor‐suppressive effects in hepatic carcinogenesis through the suppression of oncogene LIN28B expression and suggest a therapeutic application of miR‐125b in HCC. (HEPATOLOGY 2010)

Enhancer of zeste homolog 2 epigenetically silences multiple tumor suppressor microRNAs to promote liver cancer metastasis

Epigenetic alterations and microRNA (miRNA) deregulation are common in hepatocellular carcinoma (HCC). The histone H3 lysine 27 (H3K27) tri‐methylating enzyme, enhancer of zeste homolog 2 (EZH2) mediates epigenetic silencing of gene expression and is frequently up‐regulated in human cancers. In this study we aimed to delineate the implications of EZH2 up‐regulation in miRNA deregulation and HCC metastasis. Expressions of a total of 90 epigenetic regulators were first determined in 38 pairs of primary HCCs and their corresponding nontumorous livers. We identified EZH2 and its associated polycomb repressive complex 2 (PRC2) as one of the most significantly deregulated epigenetic regulators in primary HCC samples. Up‐regulation of EZH2 was next confirmed in 69.5% (41/59) of primary HCCs. Clinicopathologically, EZH2 up‐regulation was associated with HCC progression and multiple HCC metastatic features, including venous invasion (P = 0.043), direct liver invasion (P = 0.014), and absence of tumor encapsulation (P = 0.043). We further demonstrated that knockdown of EZH2 in HCC cell lines reduced the global levels of tri‐methylated H3K27, and suppressed HCC motility in vitro and pulmonary metastasis in a nude mouse model. By interrogating the miRNA expression profile in EZH2‐knockdown cell lines and primary HCC samples, we identified a subset of miRNA that was epigenetically suppressed by EZH2 in human HCC. These included well‐characterized tumor‐suppressor miRNAs, such as miR‐139‐5p, miR‐125b, miR‐101, let‐7c, and miR‐200b. Pathway enrichment analysis revealed a common regulatory role of these EZH2‐silenced miRNAs in modulating cell motility and metastasis‐related pathways. Our findings suggest that EZH2 exerts its prometastatic function by way of epigenetic silencing of multiple tumor suppressor miRNAs. Conclusion: Our study demonstrated that EZH2 epigenetically silenced multiple miRNAs that negatively regulate HCC metastasis. (HEPATOLOGY 2012)

Molecular pathogenesis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide. Hepatocarcinogenesis is a multistep process evolving from normal through chronic hepatitis/cirrhosis and dysplastic nodules to HCC. With advances in molecular methods, there is a growing understanding of the molecular mechanisms in hepatocarcinogenesis. Hepatocarcinogenesis is strongly linked to increases in allelic losses, chromosomal changes, gene mutations, epigenetic alterations and alterations in molecular cellular pathways. Some of these alterations are accompanied by a stepwise increase in the different pathological disease stages in hepatocarcinogenesis. Overall, a detailed understanding of the underlying molecular mechanisms involved in the progression of HCC is of fundamental importance to the development of effective prevention and treatment regimes for HCC.

Rho GTPase-Activating Protein Deleted in Liver Cancer Suppresses Cell Proliferation and Invasion in Hepatocellular Carcinoma

Deleted in liver cancer (DLC1) is a candidate tumor suppressor gene recently isolated from human hepatocellular carcinoma. Structurally, DLC1 protein contains a conserved GTPase-activating protein for Rho family protein (RhoGAP) domain, which has been thought to regulate the activity of Rho family proteins. Previous studies indicated that DLC1 was frequently inactivated in cancer cells. In the present study, we aimed to characterize the tumor suppressor roles of DLC1 in hepatocellular carcinoma. We showed that DLC1 significantly inhibited cell proliferation, anchorage-independent growth, and in vivo tumorigenicity when stably expressed in hepatocellular carcinoma cells. Moreover, DLC1 expression greatly reduced the motility and invasiveness of hepatocellular carcinoma cells. With RhoGAP-deficient DLC1 mutant (DLC1-K714E), we showed that the RhoGAP activity was essential for DLC1-mediated tumor suppressor function. Furthermore, the 292- to 648-amino acid region and the steroidogenic acute regulatory related lipid transfer domain played an auxiliary role to RhoGAP and tumor suppressor function of DLC1. Taken together, our findings showed that DLC1 functions as a tumor suppressor in hepatocellular carcinoma and provide the first evidence to support the hypothesis that DLC1 suppresses cancer cell growth by negatively regulating the activity of Rho proteins.

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.

Meta-analysis Followed by Replication Identifies Loci in or near CDKN1B, TET3, CD80, DRAM1, and ARID5B as Associated with Systemic Lupus Erythematosus in Asians

Systemic lupus erythematosus (SLE) is a prototype autoimmune disease with a strong genetic involvement and ethnic differences. Susceptibility genes identified so far only explain a small portion of the genetic heritability of SLE, suggesting that many more loci are yet to be uncovered for this disease. In this study, we performed a meta-analysis of genome-wide association studies on SLE in Chinese Han populations and followed up the findings by replication in four additional Asian cohorts with a total of 5,365 cases and 10,054 corresponding controls. We identified genetic variants in or near CDKN1B, TET3, CD80, DRAM1, and ARID5B as associated with the disease. These findings point to potential roles of cell-cycle regulation, autophagy, and DNA demethylation in SLE pathogenesis. For the region involving TET3 and that involving CDKN1B, multiple independent SNPs were identified, highlighting a phenomenon that might partially explain the missing heritability of complex diseases.

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.)