Yao-Ming Wu

Mucin glycosylating enzyme GALNT2 regulates the malignant character of hepatocellular carcinoma by modifying the EGF receptor.

Extracellular glycosylation is a critical determinant of malignant character. Here, we report that N-acetylgalactosaminyltransferase 2 (GALNT2), the enzyme that mediates the initial step of mucin type-O glycosylation, is a critical mediator of malignant character in hepatocellular carcinoma (HCC) that acts by modifying the activity of the epidermal growth factor receptor (EGFR). GALNT2 mRNA and protein were downregulated frequently in HCC tumors where these events were associated with vascular invasion and recurrence. Restoring GALNT2 expression in HCC cells suppressed EGF-induced cell growth, migration, and invasion in vitro and in vivo. Mechanistic investigations revealed that the status of the O-glycans attached to the EGFR was altered by GALNT2, changing EGFR responses after EGF binding. Inhibiting EGFR activity with erlotinib decreased the malignant characters caused by siRNA-mediated knockdown of GALNT2 in HCC cells, establishing the critical role of EGFR in mediating the effects of GALNT2 expression. Taken together, our results suggest that GALNT2 dysregulation contributes to the malignant behavior of HCC cells, and they provide novel insights into the significance of O-glycosylation in EGFR activity and HCC pathogenesis.

Feasibility of salvage liver transplantation for patients with recurrent hepatocellular carcinoma

Abstract:  Background:  Recurrence is the most frequent cause of treatment failure after hepatocellular carcinoma (HCC) resection. Salvage liver transplantation is an alternative treatment for recurrent HCC. The transplantability for patients with recurrent HCC has not been well studied.

Survival in patients with recurrent hepatocellular carcinoma after primary hepatectomy: Comparative effectiveness of treatment modalities

BACKGROUND Insufficient data are available on the survival of recurrent hepatocellular carcinoma after primary hepatectomy in patients receiving different treatments. We evaluated retrospectively the effects of treatment modalities on long-term survival. METHODS Between 2001 and 2007, 435 posthepatectomy hepatocellular carcinoma patients who developed recurrence were grouped by treatment modality into re-resection, radiofrequency ablation, transarterial chemoembolization, and supportive treatment groups. Treatment strategies for both primary hepatocellular carcinoma and its recurrence were selected using the same criteria. Postrecurrence survival was estimated using the Kaplan-Meier method and compared using the Cox proportional hazard model with adjusted independent prognostic factors. Survival rates after primary resection without recurrence were also compared. RESULTS In re-resection, radiofrequency ablation, transarterial chemoembolization, and supportive treatment groups, the 2-year postrecurrence survival rates were 90%, 96%, 75%, and 20%, respectively, and the 5-year survival rates were 72%, 83%, 56%, and 0%, respectively. The adjusted hazard of death was less for the re-resection and radiofrequency ablation groups than for the transarterial chemoembolization group, and the adjusted hazard ratios for the re-resection and radiofrequency ablation groups were 0.45 (95% confidence interval, 0.20-0.98) and 0.25 (0.08-0.81), respectively. The adjusted hazard ratio (95% confidence interval) of death for the radiofrequency ablation group compared to the re-resection group was 0.64 (0.19-2.19). Survival in the single resection group did not differ from that in the re-resection and radiofrequency ablation groups. CONCLUSION Postrecurrence survival in the re-resection and radiofrequency ablation groups was significantly better than that in the transarterial chemoembolization group and similar to that of patients in the primary resection without recurrence group.

Quantitative assessment of hepatic fat of intact liver tissues with coherent anti-stokes Raman scattering microscopy.

A fatty liver might progress from being a benign fatty liver, to steatohepatitis, cirrhosis, or even hepatocellular carcinoma. The great prevalence and severe outcome have warranted much investigation of the pathology and the development of effective therapies, which involve animal studies requiring critical evaluation of the hepatic fatty change. Histological examination and wet chemical analysis of liver biopsy specimens are generally employed for this purpose despite numerous procedures being involved. Using coherent anti-Stokes Raman scattering (CARS) microscopy, we have demonstrated the specific imaging of fat droplets in intact liver tissues and extracted the hepatic fat content through image analysis while eliminating laborious procedures required by traditional histopathological examination. The content of hepatic fat measured with CARS imaging was correlated strongly with that determined by biochemical analysis (R(2) = 0.89) over a pathologically significant range of the hepatic fat (from 2% to 20% of the total mass of tissue). Our work validates the quantitative assessment of fat in intact tissue through the use of CARS microscopy. When combined with the increasingly diverse animal models of diseases related to metabolic disorders of lipids, our approach is extensible to enable acquiring important insight into the genetic, environmental, and dietary factors affecting the uptake and accumulation of fat within tissues.

Hepatocyte transplantation and drug‐induced perturbations in liver cell compartments

The potential for organ damage after using drugs or chemicals is a critical issue in medicine. To delineate mechanisms of drug‐induced hepatic injury, we used transplanted cells as reporters in dipeptidyl peptidase IV–deficient mice. These mice were given phenytoin and rifampicin for 3 days, after which monocrotaline was given followed 1 day later by intrasplenic transplantation of healthy C57BL/6 mouse hepatocytes. We examined endothelial and hepatic damage by serologic or tissue studies and assessed changes in transplanted cell engraftment and liver repopulation by histochemical staining for dipeptidyl peptidase IV. Monocrotaline caused denudation of the hepatic sinusoidal endothelium and increased serum hyaluronic acid levels, along with superior transplanted cell engraftment. Together, phenytoin, rifampicin, and monocrotaline caused further endothelial damage, reflected by greater improvement in cell engraftment. Phenytoin, rifampicin, and monocrotaline produced injury in hepatocytes that was not apparent after conventional tissue studies. This led to transplanted cell proliferation and extensive liver repopulation over several weeks, which was more efficient in males compared with females, including greater induction by phenytoin and rifampicin of cytochrome P450 3A4 isoform that converts monocrotaline to toxic intermediates. Through this and other possible mechanisms, monocrotaline‐induced injury in the endothelial compartment was retargeted to simultaneously involve hepatocytes over the long term. Moreover, after this hepatic injury, native liver cells were more susceptible to additional pro‐oxidant injury through thyroid hormone, which accelerated the kinetics of liver repopulation. Conclusion: Transplanted reporter cells will be useful for obtaining insights into homeostatic mechanisms involving liver cell compartments, whereas targeted injury in hepatic endothelial and parenchymal cells with suitable drugs will also help advance liver cell therapy. (HEPATOLOGY 2007.)

High-Titer Antibody to Hepatitis B Surface Antigen Before Liver Transplantation Can Prevent De Novo Hepatitis B Infection

Objective: De novo hepatitis B virus (HBV) infection is defined as infection occurring in HBV surface antigen (HBsAg)–negative patients who become HbsAg positive after organ transplantation. We assessed the incidence and risk factors of de novo HBV infection in pediatric liver transplant recipients. Patients and Methods: From 1996 to 2006, 71 Taiwanese children with non-HBV-related liver diseases underwent orthotopic liver transplantation (OLT) at the National Taiwan University Hospital. All of the surviving recipients were tested regularly for liver function, serum levels of immunosuppressant, HBsAg, titers of antibodies to hepatitis B surface antigen (anti-HBs), antibodies to hepatitis B core antigen (anti-HBc), and antibodies to hepatitis C virus (anti-HCV). HBV vaccination histories and the anti-HBs titers before OLT were recorded. No regular prophylaxis was given. Results: Fifty-nine patients (33 girls and 26 boys) were followed up for a median of 4.4 years (range 1.0–10.0). Of those, 36 (61.0%) received allografts from anti-HBc-positive and HBsAg-negative donors. De novo HBV infection was found in 9 (15.3%) patients after OLT, 8 of whom received allografts from HBsAg-negative and anti-HBc-positive donors. Forty-eight (81.4%) patients received 3 or more doses of HBV vaccine before OLT. Pre-OLT anti-HBs titers were available for 49 recipients. Of the 9 de novo HBV-infected recipients, 8 had anti-HBs titers <200 mIU/mL. No graft loss or fulminant hepatitis was noted. Conclusions: In the absence of adequate prophylaxis, the incidence of de novo HBV infection in pediatric OLT recipients is 15.3%. An anti-HBs titer of >200 mIU/mL before OLT may be sufficient to prevent de novo HBV infection in HBsAg-negative recipients.

Up-regulation of microRNA-190b plays a role for decreased IGF-1 that induces insulin resistance in human hepatocellular carcinoma.

Background & Aims Insulin-like growth factor, (IGF)-1, is produced mainly by the liver and plays important roles in promoting growth and regulating metabolism. Previous study reported that development of hepatocellular carcinoma (HCC) was accompanied by a significant reduction in serum IGF-1 levels. Here, we hypothesized that dysregulation of microRNAs (miRNA) in HCC can modulate IGF-1 expression post-transcriptionally. Methods The miRNAs expression profiles in a dataset of 29 HCC patients were examined using illumina BeadArray. Specific miRNA (miR)-190b, which was significantly up-regulated in HCC tumor tissues when compared with paired non-tumor tissues, was among those predicted to interact with 3′-untranslated region (UTR) of IGF-1. In order to explore the regulatory effects of miR-190b on IGF-1 expression, luciferase reporter assay, quantitative real-time PCR, western blotting and immunofluorecence analysis were performed in HCC cells. Results Overexpression of miR-190b in Huh7 cells attenuated the expression of IGF-1, whereas inhibition of miR-190b resulted in up-regulation of IGF-1. Restoration of IGF-1 expression reversed miR-190b-mediated impaired insulin signaling in Huh7 cells, supporting that IGF-1 was a direct and functional target of miR-190b. Additionally, low serum IGF-1 level was associated with insulin resistance and poor overall survival in HCC patients. Conclusions Increased expression of miR-190 may cause decreased IGF-1 in HCC development. Insulin resistance appears to be a part of the physiopathologic significance of decreased IGF-1 levels in HCC progression. This study provides a novel miRNA-mediated regulatory mechanism for controlling IGF-1 expression in HCC and elucidates the biological relevance of this interaction in HCC.

C1GALT1 Enhances Proliferation of Hepatocellular Carcinoma Cells via Modulating MET Glycosylation and Dimerization

Altered glycosylation is a hallmark of cancer. The core 1 β1,3-galactosyltransferase (C1GALT1) controls the formation of mucin-type O-glycans, far overlooked and underestimated in cancer. Here, we report that C1GALT1 mRNA and protein are frequently overexpressed in hepatocellular carcinoma tumors compared with nontumor liver tissues, where it correlates with advanced tumor stage, metastasis, and poor survival. Enforced expression of C1GALT1 was sufficient to enhance cell proliferation, whereas RNA interference-mediated silencing of C1GALT1 was sufficient to suppress cell proliferation in vitro and in vivo. Notably, C1GALT1 attenuation also suppressed hepatocyte growth factor (HGF)-mediated phosphorylation of the MET kinase in hepatocellular carcinoma cells, whereas enforced expression of C1GALT1 enhanced MET phosphorylation. MET blockade with PHA665752 inhibited C1GALT1-enhanced cell viability. In support of these results, we found that the expression level of phospho-MET and C1GALT1 were associated in primary hepatocellular carcinoma tissues. Mechanistic investigations showed that MET was decorated with O-glycans, as revealed by binding to Vicia villosa agglutinin and peanut agglutinin. Moreover, C1GALT1 modified the O-glycosylation of MET, enhancing its HGF-induced dimerization and activation. Together, our results indicate that C1GALT1 overexpression in hepatocellular carcinoma activates HGF signaling via modulation of MET O-glycosylation and dimerization, providing new insights into how O-glycosylation drives hepatocellular carcinoma pathogenesis.

14-3-3ε Overexpression Contributes to Epithelial-Mesenchymal Transition of Hepatocellular Carcinoma

Background 14-3-3ε is implicated in regulating tumor progression, including hepatocellular carcinoma (HCC). Our earlier study indicated that elevated 14-3-3ε expression is significantly associated with higher risk of metastasis and lower survival rates of HCC patients. However, the molecular mechanisms of how 14-3-3ε regulates HCC tumor metastasis are still unclear. Methodology and Principal Findings In this study, we show that increased 14-3-3ε expression induces HCC cell migration and promotes epithelial-mesenchymal transition (EMT), which is determined by the reduction of E-cadherin expression and induction of N-cadherin and vimentin expression. Knockdown with specific siRNA abolished 14-3-3ε-induced cell migration and EMT. Furthermore, 14-3-3ε selectively induced Zeb-1 and Snail expression, and 14-3-3ε-induced cell migration was abrogated by Zeb-1 or Snail siRNA. In addition, the effect of 14-3-3ε-reduced E-cadherin was specifically restored by Zeb-1 siRNA. Positive 14-3-3ε expression was significantly correlated with negative E-cadherin expression, as determined by immunohistochemistry analysis in HCC tumors. Analysis of 14-3-3ε/E-cadherin expression associated with clinicopathological characteristics revealed that the combination of positive 14-3-3ε and negative E-cadherin expression is significantly correlated with higher incidence of HCC metastasis and poor 5-year overall survival. In contrast, patients with positive 14-3-3ε and positive E-cadherin expression had better prognostic outcomes than did those with negative E-cadherin expression. Significance Our findings show for the first time that E-cadherin is one of the downstream targets of 14-3-3ε in modulating HCC tumor progression. Thus, 14-3-3ε may act as an important regulator in modulating tumor metastasis by promoting EMT as well as cell migration, and it may serve as a novel prognostic biomarker or therapeutic target for HCC.

Overexpression of MUC15 activates extracellular signal-regulated kinase 1/2 and promotes the oncogenic potential of human colon cancer cells

Mucins play a key role in tumorigenesis. MUC15 is a membrane-bound mucin and the MUC15 messenger RNA (mRNA) has been detected in various organs. However, its role in tumor malignancy is still unclear. This study was to investigate the MUC15 expression in colorectal tumors and the role of MUC15 in colon cancer cells. We found that the mRNA expression of MUC15 was significantly higher in 70.8% (51/72) of colorectal tumors compared with their normal counterparts by real-time reverse transcription-polymerase chain reaction. Immunohistochemistry showed that MUC15 expression was increased in 82.6% (43/52) of colorectal tumors. MUC15 overexpression in HCT116 cells enhanced cell proliferation, cell-extracellular matrix adhesion, colony-forming ability and invasion. Furthermore, these effects were significantly reversed by knockdown of MUC15 with short-hairpin RNA. In nude mice models, MUC15 overexpression significantly (P < 0.01) enhanced tumor growth. In addition, treatment of PD98059 significantly (P < 0.01) inhibited MUC15-enhanced invasion, suggesting that the invasion induced by MUC15 in HCT116 cells was primarily mediated through activation of extracellular signal-regulated kinase 1/2. In conclusion, these results suggest that MUC15 is upregulated in colorectal tumors and its expression enhances the oncogenic potential of colon cancer cells.