Dorothy Ngo-Yin Fan

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)

Sequential alterations of microrna expression in hepatocellular carcinoma development and venous metastasis

Hepatocellular carcinoma (HCC) is a prevalent cancer with an extremely high mortality rate attributed to HCC metastasis, which is the major cause of tumor recurrence and organ failure. Presence of tumor thrombi in the portal veins (venous metastases) is a clinicopathological feature of metastatic HCCs. In this study, we analyzed the microRNA (miRNA) expression profiles of nontumorous livers, primary HCCs, and venous metastases in the same livers from 20 HCC patients by way of TaqMan low‐density array (TLDA) and identified the precise alterations of miRNA expression from nontumorous livers to primary HCCs and venous metastases globally. By unsupervised clustering analysis, nontumorous livers were distinctly segregated from primary HCCs and venous metastases, whereas no discernible difference in the expression pattern could be found between primary HCCs and venous metastases. However, a marked global reduction of miRNA expression levels was detected in venous metastases, as compared with primary HCCs. These data suggest that miRNA deregulation is an early event in liver carcinogenesis and the later global miRNA down‐regulation aggravates the preexisting miRNA deregulation to further promote HCC metastasis. Conclusion: Our study has enriched the current understanding of the deregulation of miRNAs in HCC progression and highlighted the sequential and distinctive alterations of miRNA expression in primary HCC and venous metastasis formation. (HEPATOLOGY 2012;)

Long non-coding RNA HOTTIP is frequently up-regulated in hepatocellular carcinoma and is targeted by tumour suppressive miR-125b

Hepatocellular carcinoma (HCC) is one of the most common human cancers. Recently, emerging evidence has suggested the role of long non‐coding RNAs (lncRNAs) in human carcinogenesis. In this study, we aimed to investigate the expression and functional implications of lncRNAs in human HCC.

Switching of Pyruvate Kinase Isoform L to M2 Promotes Metabolic Reprogramming in Hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is an aggressive tumor, with a high mortality rate due to late symptom presentation and frequent tumor recurrences and metastasis. It is also a rapidly growing tumor supported by different metabolic mechanisms; nevertheless, the biological and molecular mechanisms involved in the metabolic reprogramming in HCC are unclear. In this study, we found that pyruvate kinase M2 (PKM2) was frequently over-expressed in human HCCs and its over-expression was associated with aggressive clinicopathological features and poor prognosis of HCC patients. Furthermore, knockdown of PKM2 suppressed aerobic glycolysis and cell proliferation in HCC cell lines in vitro. Importantly, knockdown of PKM2 hampered HCC growth in both subcutaneous injection and orthotopic liver implantation models, and reduced lung metastasis in vivo. Of significance, PKM2 over-expression in human HCCs was associated with a down-regulation of a liver-specific microRNA, miR-122. We further showed that miR-122 interacted with the 3UTR of the PKM2 gene. Re-expression of miR-122 in HCC cell lines reduced PKM2 expression, decreased glucose uptake in vitro, and suppressed HCC tumor growth in vivo. Our clinical data and functional studies have revealed a novel biological mechanism involved in HCC metabolic reprogramming.

Abstract 145: HOTTIP, an oncogenic long non-coding RNA, is frequently up-regulated in hepatocellular carcinoma and is negatively regulated by tumor suppressive microRNA miR-125b

Hepatocellular carcinoma (HCC) is one of the most common human cancers. Due to the lack of early diagnostic and effective treatment modalities, the mortality of HCC is high. The development and progression of HCC is a multistep process driven by the accumulation of genetic and epigenetic alterations. Recently, emerging evidence has suggested the role of long non-coding RNAs (lncRNAs) in human carcinogenesis. Herein, by profiling the expression of 88 well-annotated lncRNAs, we identified that HOTTIP was the most significantly up-regulated lncRNA (81%, 57/70) in human HCCs, even in early stage of HCC formation. Functionally, knockdown of HOTTIP attenuated HCC cell proliferation in vitro and markedly abrogated tumorigenicity in vivo. In addition, knockdown of HOTTIP also inhibited migratory ability of HCC cells and significantly abrogated lung metastasis in orthotopic implantation model in nude mice. HOTTIP is an antisense lncRNA mapped to the distal end of the HOXA gene cluster. Knockdown of HOTTIP significantly suppressed the expression of a number of HOXA genes. Furthermore, we identified miR-125b as a post-transcriptional regulator of HOTTIP. Ectopic expression of miR-125b abolished HOTTIP-coupled luciferase activity and suppressed the endogenous level of HOTTIP. Moreover, in human HCCs, HOTTIP expression negatively correlated with that of miR-125b. In Summary, our findings suggest that HOTTIP is a novel oncogenic lncRNA, with miR-125b being its negative regulator. It contributes to hepatocarcinogenesis by regulating the expression of its neighboring protein-coding genes. Citation Format: Felice H. Tsang, Sandy L. Au, Lai Wei, Dorothy N. Fan, Joyce M. Lee, Carmen C. Wong, Irene O. Ng, Chun-Ming Wong. HOTTIP, an oncogenic long non-coding RNA, is frequently up-regulated in hepatocellular carcinoma and is negatively regulated by tumor suppressive microRNA miR-125b. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 145. doi:10.1158/1538-7445.AM2015-145

Abstract 5215: miR-200b/c/429 sub-family negatively regulating Rho/ROCK signaling pathway and suppresses HCC metastasis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA MicroRNAs (miRNAs) are a class of small (∼22 nt) noncoding regulatory RNA molecules that can regulate gene expression by either cleaving or repressing the translation of their mRNA targets. Deregulation of miRNA expression is common in human cancers, including hepatocellular carcinoma (HCC). Recent studies have indicated that the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) negatively regulates epithelial-mesenchymal transition (EMT) and cell motility by targeting E-cadherin repressors, ZEB1 and ZEB2. However, the roles of the miR-200 family in HCC remain elusive. In this study, we found that miR-200 family was frequently down-regulated in human HCC, which was contributed by the frequently CpG hypermethylation of miR-200 promoters. Functionally, although all miR-200 members suppressed ZEB1/2 expression in HCC, only overexpression of miR-200b/c/429 sub-family impeded HCC cell migration. These findings implied that, in addition to ZEB1/2, other yet unidentified miR-200b/c/429 specific targets may play an important role in HCC metastasis. In silico analyses revealed that miR-200b/c/429 sub-family may have a profound role in modulating cell cytoskeletal reorganization. Along with this notion, we further identified RhoA and ROCK2 as down-stream targets of miR-200b/c/429 sub-family. By using immunofluorescence staining, scanning electron microscope and live cell imaging, we demonstrated that overexpression of miR-200b/c/429 subfamily but not miR-200a/141 sub-family suppressed Rho/ROCK signaling mediated stress fiber and focal adhesion formations, which in turn led to a dramatic cell morphology change and loss of cell migration ability. Furthermore, by employed an orthotopic liver implantation model, we showed that overexpression of miR-200b was sufficient to suppress HCC growth and metastasis in nude mice. Our results together suggest that miR-200b/c/429 sub-family functions as a metastatic suppressor in human HCC by negatively regulating Rho/ROCK signaling pathway. Citation Format: Chun-Ming Wong, Lai Wei, Sandy Leung-Kuen Au, Sandy Leung-Kuen Au, Dorothy Ngo-Yin Fan, Joyce Man-Fong Lee, Carmen Chak-Lui Wong, Irene Oi-Lin Ng. miR-200b/c/429 sub-family negatively regulating Rho/ROCK signaling pathway and suppresses HCC metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5215. doi:10.1158/1538-7445.AM2014-5215

Abstract 1059: SUV39H1 promotes HCC tumorigenesis and is targeted by tumor suppressive miRNA-125b

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Epigenetic alternation is a common dysregulated event in hepatocellular carcinoma (HCC) development. Through interrogating the expression of 90 major epigenetic regulating genes, we identified SUV39H1, the prototype histone methyltransferase as one of the most frequently up-regulated epigenetic regulators in human HCC (61%, 23/38). SUV39H1 is responsible for H3K9 trimethylation establishment and essentially involves in heterochromatin formation and transcriptional repression. We found that SUV39H1 up-regulation in human HCC was significantly associated with increased Ki67 expression (P < 0.001) and the presence of venous invasion (P = 0.020), suggesting the importance of SUV39H1 in HCC tumor growth and metastasis. To investigate the functions of SUV39H1 in human HCC, both gain- and loss-of-function models were established. Ectopic expression of SUV39H1 remarkably enhanced cell clonogenicity and migratory ability of immortalized hepatocytes. Conversely, stable knockdown of SUV39H1 by shRNA substantially reduced cell proliferation, colony formation and migration of HCC cells. In addition, an elevated lysosomal β-galactosidase activity in SUV39H1 knockdown cells was observed and suggested the senescence-suppressive role of SUV39H1 in cancer progression. Consistent with previous observations, treatment of SUV39H1 inhibitor (Chaetocin) drastically suppressed cell proliferation and induced apoptosis in HCC cells. The oncogenic function of SUV39H1 was further demonstrated by in vivo experiments, knockdown of SUV39H1 significantly inhibited tumorigenicity of HCC cells in both subcutaneous and orthotopic mouse model. Most importantly, SUV39H1 knockdown abolished pulmonary metastasis of HCC cells in orthotopic tumor implantation model. To further investigate the underlying mechanism of SUV39H1 up-regulation in HCC, we explored the potential regulation of SUV39H1 by microRNA. In silico analysis suggested SUV39H1 as a putative target of miR-125b. Ectopic expression of miR-125b precursor in HCC cells significantly inhibited SUV39H1 3′UTR luciferase reporter activity and suppressed endogenous SUV39H1 expression at both mRNA and protein levels. Interestingly, down-regulation of miR-125b was frequently observed in human HCC (70%, 38/54) and was inversely correlated with SUV39H1 expression (P = 0.001). Taken together, we demonstrated that SUV39H1 was frequently up-regulated in human HCC and functioned as an oncogene in promoting HCC progression and metastasis. miR-125b negatively regulated SUV39H1 expression and downregulation of miR-125b may account for SUV39H1 up-regulation in human HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1059. doi:1538-7445.AM2012-1059