Mei-Sze Chua

Sprouty 2, an Inhibitor of Mitogen-Activated Protein Kinase Signaling, Is Down-Regulated in Hepatocellular Carcinoma

The Sprouty proteins are increasingly being recognized to be deregulated in various types of cancers. This deregulation is often associated with aberrant signaling of receptor tyrosine kinases and its downstream effectors, leading to the mitogen-activated protein kinase (MAPK) signaling pathway. In human hepatocellular carcinoma, where the MAPK activity is enhanced via multiple hepatocarcinogenic factors, we observed a consistent reduced expression of the sprouty 2 (Spry2) transcript and protein in malignant hepatocytes compared with normal or cirrhotic hepatocytes. The expression pattern of Spry2 in hepatocellular carcinoma resembles that of several potential tumor markers of hepatocellular carcinoma and also that of several angiogenic factors and growth factor receptors. In contrast to previous studies of Spry2 down-regulation in other cancers, we have ruled out loss of heterozygosity or the methylation of promoter sites, two common mechanisms responsible for the silencing of genes with tumor suppressor properties. Functionally, we show that Spry2 inhibits both extracellular signal-regulated kinase signaling as well as proliferation in hepatocellular carcinoma cell lines, whereas knocking down Spry2 levels in NIH3T3 cells causes mild transformation. Our study clearly indicates a role for Spry2 in hepatocellular carcinoma, and an understanding of the regulatory controls of its expression could provide new means of regulating the angiogenic switch in this hypervascular tumor, thereby potentially controlling tumor growth.

An integrated data analysis approach to characterize genes highly expressed in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the major causes of cancer deaths worldwide. New diagnostic and therapeutic options are needed for more effective and early detection and treatment of this malignancy. We identified 703 genes that are highly expressed in HCC using DNA microarrays, and further characterized them in order to uncover novel tumor markers, oncogenes, and therapeutic targets for HCC. Using Gene Ontology annotations, genes with functions related to cell proliferation and cell cycle, chromatin, repair, and transcription were found to be significantly enriched in this list of highly expressed genes. We also identified a set of genes that encode secreted (e.g. GPC3, LCN2, and DKK1) or membrane-bound proteins (e.g. GPC3, IGSF1, and PSK-1), which may be attractive candidates for the diagnosis of HCC. A significant enrichment of genes highly expressed in HCC was found on chromosomes 1q, 6p, 8q, and 20q, and we also identified chromosomal clusters of genes highly expressed in HCC. The microarray analyses were validated by RT–PCR and PCR. This approach of integrating other biological information with gene expression in the analysis helps select aberrantly expressed genes in HCC that may be further studied for their diagnostic or therapeutic utility.

Small molecule antagonists of Tcf4/β‐catenin complex inhibit the growth of HCC cells in vitro and in vivo

Hepatocellular carcinoma (HCC) is the 5th most common cancer worldwide. It is intrinsically resistant toward standard chemotherapy, making it imperative to develop novel selective chemotherapeutic agents. The Wnt/β‐catenin pathway plays critical roles in development and oncogenesis, and is dysregulated in HCC. Our study aims to evaluate the activity of 3 small molecule antagonists of the Tcf4/β‐catenin complex (PKF118‐310, PKF115‐584 and CGP049090) on HCC cell lines in vitro and in vivo. All 3 chemicals displayed dose‐dependent cytotoxicity in vitro against all 3 HCC cell lines (HepG2, Hep40 and Huh7), but were at least 10 times less cytotoxic to normal hepatocytes (from 3 donors) by using ATP assay. In HepG2 and Huh7 cells, treatment with the antagonists decreased Tcf4/β‐catenin binding capability and transcriptional activity, associated with downregulation of the endogenous Tcf4/ β‐catenin target genes c‐Myc, cyclin D1 and survivin. In HepG2 and Huh7 cells, treatment with the antagonists induced apoptosis and cell cycle arrest at the G1/S phase. All antagonists suppressed in vivo tumor growth in a HepG2 xenograft model, associated with apoptosis and reduced c‐Myc, cyclin D1 and survivin expressions. Our results suggest that these 3 antagonists of the Tcf4/β‐catenin complex are potential chemotherapeutic agents which may offer a pathway specific option for the clinical management of HCC.

Overexpression of NDRG1 is an indicator of poor prognosis in hepatocellular carcinoma

Hepatocellular carcinoma is a highly lethal cancer that typically has poor prognosis. Prognostic markers can help in its clinical management and in understanding the biology of poor prognosis. Through an earlier gene expression study, we identified N-Myc downregulated gene 1 (NDRG1) to be significantly highly expressed in hepatocellular carcinoma compared to nontumor liver. As NDRG1 is a differentiation-related gene with putative metastasis suppressor activity, we investigated the clinical significance of its overexpression. Quantitative real-time polymerase chain reaction using an independent set of patient samples confirmed the significant overexpression of NDRG1 in hepatocellular carcinoma compared to nontumor liver samples (P<0.001). Additionally, high levels of NDRG1 transcript correlated with shorter overall survival (P<0.001), late tumor stage (P=0.001), vascular invasion (P=0.003), large tumor size (P=0.011), and high Edmondson-Steiner histological grade (P=0.005). Using immunohistochemistry, NDRG1 protein was found to be significantly overexpressed in hepatocellular carcinoma samples compared to nontumor liver or cirrhotic and benign liver lesions (P<0.001). Among the hepatocellular carcinoma samples, those which are moderately and poorly differentiated express higher levels of NDRG1 protein than those which are well-differentiated (P<0.005). Additionally, hepatocellular carcinomas with vascular invasion also express elevated levels of NDRG1 protein compared to those without vascular invasion (significant at P<0.005). Our results suggest NDRG1 to be a likely tumor marker for hepatocellular carcinoma, the overexpression of which is correlated with tumor differentiation, vascular invasion, and overall survival. Its significantly elevated expression in hepatocellular carcinoma could be a useful indicator of tumor aggressiveness and therefore patient prognosis.

Blockade of Wnt-1 signaling leads to anti-tumor effects in hepatocellular carcinoma cells.

BackgroundHepatocellular carcinoma (HCC) is an aggressive cancer, and is the third leading cause of cancer death worldwide. Standard therapy is ineffective partly because HCC is intrinsically resistant to conventional chemotherapy. Its poor prognosis and limited treatment options make it critical to develop novel and selective chemotherapeutic agents. Since the Wnt/β-catenin pathway is essential in HCC carcinogenesis, we studied the inhibition of Wnt-1-mediated signaling as a potential molecular target in HCC.ResultsWe demonstrated that Wnt-1 is highly expressed in human hepatoma cell lines and a subgroup of human HCC tissues compared to paired adjacent non-tumor tissues. An anti-Wnt-1 antibody dose-dependently decreased viability and proliferation of Huh7 and Hep40 cells over-expressing Wnt-1 and harboring wild type β-catenin, but did not affect normal hepatocytes with undetectable Wnt-1 expression. Apoptosis was also observed in Huh7 and Hep40 cells after treatment with anti-Wnt-1 antibody. In these two cell lines, the anti-Wnt-1 antibody decreased β-catenin/Tcf4 transcriptional activities, which were associated with down-regulation of the endogenous β-catenin/Tcf4 target genes c-Myc, cyclin D1, and survivin. Intratumoral injection of anti-Wnt-1 antibody suppressed in vivo tumor growth in a Huh7 xenograft model, which was also associated with apoptosis and reduced c-Myc, cyclin D1, and survivin expressions.ConclusionOur results suggest that Wnt-1 is a survival factor for HCC cells, and that the blockade of Wnt-1-mediated signaling may offer a potential pathway-specific therapeutic strategy for the treatment of a subgroup of HCC that over-expresses Wnt-1.

Soluble Frizzled-7 receptor inhibits Wnt signaling and sensitizes hepatocellular carcinoma cells towards doxorubicin

BackgroundThere are limited therapeutic options for hepatocellular carcinoma (HCC), the most common liver malignancy worldwide. Recent studies have identified the Frizzled-7 receptor (FZD7), important for activation of Wnt-mediated signaling, as a potential therapeutic target for HCC and other cancers.MethodsWe hypothesized that the extracellular domain of FZD7 (sFZD7) would be a clinically more relevant therapeutic modality than previously studied approaches to target FZD7. We expressed and purified sFZD7 from E. coli, and tested its functional activity to interact with Wnt3, its ability to inhibit Wnt3-mediated signaling, and its potential for combinatorial therapy in HCC.ResultssFZD7 pulled down Wnt3 from Huh7 cells, and decreased β-catenin/Tcf4 transcriptional activity in HCC cells. In vitro, sFZD7 dose-dependently decreased viability of three HCC cell lines (HepG2, Hep40, and Huh7, all with high FZD7 and Wnt3 mRNA), but had little effect on normal hepatocytes from three donors (all with low level FZD7 and Wnt3 mRNA). When combined with doxorubicin, sFZD7 enhanced the growth inhibitory effects of doxorubicin against HCC cells in vitro, and against Huh7 xenografts in vivo. Reduced expressions of c-Myc, cyclin D1, and survivin were observed in vitro and in vivo. Additionally, sFZD7 altered the levels of phosphorylated AKT and ERK1/2 induced by doxorubicin treatment in vitro, suggesting that several critical pathways are involved in the chemosensitizing effect of sFZD7.ConclusionsWe propose that sFZD7 is a feasible therapeutic agent with specific activity, which can potentially be combined with other chemotherapeutic agents for the improved management of HCC.

Molecular Profiling of Anemia in Acute Renal Allograft Rejection Using DNA Microarrays

Compromised renal function after renal allograft transplantation often results in anemia in the recipient. Molecular mechanisms leading to anemia during acute rejection are not fully understood; inadequate erythropoietin production and iron deficiency have been reported to be the main contributors. To increase our understanding of the molecular events underlying anemia in acute rejection, we analyzed the gene expression profiles of peripheral blood lymphocytes (PBL) from four pediatric renal allograft recipients with acute rejection and concurrent anemia, using DNA microarrays containing 9000 human cDNA clones (representing 7469 unique genes). In these anemic rejecting patients, an ‘erythropoiesis cluster’ of 11 down‐regulated genes was identified, involved in hemoglobin transcription and synthesis, iron and folate binding and transport. Additionally, some alloimmune response genes were simultaneously down‐regulated. An independent data set of 36 PBL samples, some with acute rejection and some with concurrence of acute rejection and anemia, were analyzed to support a possible association between acute rejection and anemia. In conclusion, analysis using DNA microarrays has identified a cluster of genes related to hemoglobin synthesis and/or erythropoeisis that was altered in kidneys with renal allograft rejection compared with normal kidneys. The possible relationship between alterations in the expression of this cluster, reduced renal function, the alloimmune process itself, and other influences on the renal transplant awaits further analysis.

Increased expression of cytotoxic effector molecules: Different interpretations for steroid‐based and steroid‐free immunosuppression

Abstract: Cytotoxic T lymphocyte (CTL) effector molecules have been studied as markers of acute rejection in renal allograft recipients on steroid‐based immunosuppression. We hypothesized that basal CTL gene expression may vary with time post‐transplantation as well as with different immunosuppression protocols (steroid‐based or steroid‐free). Variations in CTL gene expression may thus impact on the ability to predict acute allograft rejection. We used the non‐invasive method of quantitative competitive‐reverse transcription‐polymerase chain reaction (QC‐RT‐PCR) to quantify the amounts of CTL effector molecules (granulysin, GL; perforin, P; granzyme B, GB) in serial peripheral blood lymphocyte (PBL) samples from steroid‐free and steroid‐based adult and pediatric renal allograft recipients. Patients on both protocols were clinically monitored by protocol biopsies at 1, 3, 6, and 12 months post‐transplantation and for graft function at 1 yr post‐transplantation in a separate clinical study. Steroid‐free patients with stable graft function showed an increase in GL, P, and GB gene expression over time post‐transplantation with the increase being seen largely by the first post‐transplant month. A further increase in GL expression was noted at the end of the first post‐transplant year in the absence of acute rejection, whereas GB and P levels were unchanged. At comparative time‐points post‐transplantation, CTL genes were found to be higher in steroid‐free patients with stable graft function, compared to steroid‐based recipients with either clinically stable graft function or acute rejection. This study suggests that levels of CTL gene expression, although important in a steroid‐based regimen to monitor the risk of acute rejection, may not be similarly applied in patients on steroid‐free immunosuppression. The early increase in levels seen in steroid‐free patients appears to correlate with the total absence of steroids. As steroid‐free patients seem to have a lower incidence of acute rejection and better long‐term graft function at 1 yr, the early increase in CTL genes in the absence of acute rejection may suggest an early adaptive immune activation response, promoting early graft acceptance in this protocol.

Epigenetics in hepatocellular carcinoma: An update and future therapy perspectives

Hepatocellular carcinoma (HCC), the predominant form of adult liver malignancies, is a global health concern. Its dismal prognosis has prompted recent significant advances in the understanding of its etiology and pathogenesis. The deregulation of epigenetic mechanisms, which maintain heritable gene expression changes and chromatin organization, is implicated in the development of multiple cancers, including HCC. This review summarizes the current knowledge of epigenetic mechanisms in the pathogenesis of HCC, with an emphasis on HCC mediated by chronic hepatitis B virus infection. This review also discusses the encouraging outcomes and lessons learnt from epigenetic therapies for hematological and other solid cancers, and highlights the future potential of similar therapies in the treatment of HCC.

N-Myc down-regulated gene 1 mediates proliferation, invasion, and apoptosis of hepatocellular carcinoma cells

The over-expression of N-myc down-regulated gene 1 (NDRG1) in hepatocellular carcinoma (HCC) was previously reported to correlate with vascular invasion and patient survival. Our current study aims to elucidate its functions in HCC. We found that it lacked the tumorigenic ability to promote soft agar colony formation or serum-independent growth of NIH3T3 cells. We used specific small interfering RNA (siRNA) oligos to suppress the expression of NDRG1 in human HCC (Hep3B and HepG2) cell lines, and found that this significantly reduced cell proliferation and invasion, and induced apoptosis. Additionally, NDRG1-specific siRNA inhibited the HepG2 xenograft growth in nude mice. These results are consistent with our earlier clinical observations that NDRG1 is associated with more aggressive tumor behavior, and suggest that NDRG1 may be a potential therapeutic target for HCC.