Kwan Man

MicroRNA Expression, Survival, and Response to Interferon in Liver Cancer

BACKGROUND Hepatocellular carcinoma is a common and aggressive cancer that occurs mainly in men. We examined microRNA expression patterns, survival, and response to interferon alfa in both men and women with the disease. METHODS We analyzed three independent cohorts that included a total of 455 patients with hepatocellular carcinoma who had undergone radical tumor resection between 1999 and 2003. MicroRNA-expression profiling was performed in a cohort of 241 patients with hepatocellular carcinoma to identify tumor-related microRNAs and determine their association with survival in men and women. In addition, to validate our findings, we used quantitative reverse-transcriptase-polymerase-chain-reaction assays to measure microRNAs and assess their association with survival and response to therapy with interferon alfa in 214 patients from two independent, prospective, randomized, controlled trials of adjuvant interferon therapy. RESULTS In patients with hepatocellular carcinoma, the expression of miR-26a and miR-26b in nontumor liver tissue was higher in women than in men. Tumors had reduced levels of miR-26 expression, as compared with paired noncancerous tissues, which indicated that the level of miR-26 expression was also associated with hepatocellular carcinoma. Moreover, tumors with reduced miR-26 expression had a distinct transcriptomic pattern, and analyses of gene networks revealed that activation of signaling pathways between nuclear factor kappaB and interleukin-6 might play a role in tumor development. Patients whose tumors had low miR-26 expression had shorter overall survival but a better response to interferon therapy than did patients whose tumors had high expression of the microRNA. CONCLUSIONS The expression patterns of microRNAs in liver tissue differ between men and women with hepatocellular carcinoma. The miR-26 expression status of such patients is associated with survival and response to adjuvant therapy with interferon alfa.

Prospective evaluation of Pringle maneuver in hepatectomy for liver tumors by a randomized study.

OBJECTIVE To evaluate whether vascular inflow occlusion by the Pringle maneuver during hepatectomy can be safe and effective in reducing blood loss. SUMMARY BACKGROUND DATA Hepatectomy can be performed with a low mortality rate, but massive hemorrhage during surgery remains a potentially lethal problem. The Pringle maneuver is traditionally used during hepatectomy to reduce blood loss, but there is a potential harmful effect on the metabolic function of hepatocytes. There has been no prospective randomized study to determine whether the Pringle maneuver can decrease blood loss during hepatectomy, improve outcome, or affect the metabolism of hepatocytes. METHODS From July 1995 to February 1997, we studied 100 consecutive patients who underwent hepatectomy for liver tumors. The patients were randomly assigned to liver transection under intermittent Pringle maneuver of 20 minutes and a 5-minute clamp-free interval (n = 50), or liver transection without the Pringle maneuver (n = 50). The surface area of liver transection was measured and blood loss during transection per square centimeter of transection area was calculated. Routine liver biochemistry, arterial ketone body ratio (AKBR), and the indocyanine green (ICG) clearance test were done. RESULTS The two groups were comparable in terms of preoperative liver function and in the proportion of patients having major hepatectomy. The Pringle maneuver resulted in less blood loss per square centimeter of transection area (12 mL/cm2 vs. 22 mL/cm2, p = 0.0001), a shorter transection time per square centimeter of transection area (2 min/cm2 vs. 2.8 min/cm2, p = 0.016), a significantly higher AKBR in the first 2 hours after hepatectomy, lower serum bilirubin levels in the early postoperative period, and, in cirrhotic patients, higher serum transferrin levels on postoperative days 1 and 8. The complication rate, the hospital mortality rate, and the ICG retention at 15 minutes on postoperative day 8 were equal for the two groups. CONCLUSION Performing the Pringle maneuver during liver transection resulted in less blood loss and better preservation of liver function in the early postoperative period. This is probably because there was less hemodynamic disturbance induced by the bleeding.

Twist Overexpression Correlates with Hepatocellular Carcinoma Metastasis through Induction of Epithelial-Mesenchymal Transition

Purpose: Hepatocellular carcinoma (HCC) is a rapidly growing tumor associated with a high propensity for vascular invasion and metastasis. Epithelial-mesenchymal transition (EMT) is a key event in the tumor invasion process. Recently, Twist has been identified to play an important role in EMT-mediated metastasis through the regulation of E-cadherin expression. However, the actual role of Twist in tumor invasiveness remains unclear. The purpose of this study is to investigate the expression and possible role of Twist in HCC. Experimental Design: We evaluated Twist and E-cadherin expression in HCC tissue microarray of paired primary and metastatic HCC by immunohistochemical staining. The role of Twist in EMT-mediated invasiveness was also evaluated in vitro in HCC cell lines. Results: We first showed that overexpression of Twist was correlated with HCC metastasis (P = 0.001) and its expression was negatively correlated with E-cadherin expression (P = 0.001, r = −0.443) by tissue microarray. A significant increase of Twist at the mRNA level was also found in metastatic HCC cell lines MHCC-97H, MHCC-97L, and H2M when compared with nonmetastatic Huh-7, H2P, and PLC cell lines. The MHCC-97H cell line, which has a higher metastatic ability, was found to have a higher level of Twist than MHCC-97L. Accompanied with increased Twist expression in the metastatic HCC cell lines when compared with the nonmetastatic primary ones, we found decreased E-cadherin mRNA expression in the metastatic HCC cell lines. By ectopic transfection of Twist into PLC cells, Twist was able to suppress E-cadherin expression and induce EMT changes, which was correlated with increased HCC cell invasiveness. Conclusion: This study shows that Twist overexpression was correlated with HCC metastasis through induction of EMT changes and HCC cell invasiveness.

Recoding RNA editing of AZIN1 predisposes to hepatocellular carcinoma

A better understanding of human hepatocellular carcinoma (HCC) pathogenesis at the molecular level will facilitate the discovery of tumor-initiating events. Transcriptome sequencing revealed that adenosine-to-inosine (A→I) RNA editing of AZIN1 (encoding antizyme inhibitor 1) is increased in HCC specimens. A→I editing of AZIN1 transcripts, specifically regulated by ADAR1 (encoding adenosine deaminase acting on RNA-1), results in a serine-to-glycine substitution at residue 367 of AZIN1, located in β-strand 15 (β15) and predicted to cause a conformational change, induced a cytoplasmic-to-nuclear translocation and conferred gain-of-function phenotypes that were manifested by augmented tumor-initiating potential and more aggressive behavior. Compared with wild-type AZIN1 protein, the edited form has a stronger affinity to antizyme, and the resultant higher AZIN1 protein stability promotes cell proliferation through the neutralization of antizyme-mediated degradation of ornithine decarboxylase (ODC) and cyclin D1 (CCND1). Collectively, A→I RNA editing of AZIN1 may be a potential driver in the pathogenesis of human cancers, particularly HCC.

Graft injury in relation to graft size in right lobe live donor liver transplantation: a study of hepatic sinusoidal injury in correlation with portal hemodynamics and intragraft gene expression.

ObjectiveTo investigate the degree and mechanism of hepatic sinusoidal injury in different graft sizes in right lobe live donor liver transplantation (LDLT). Summary Background DataLiver grafts from living donors are likely to be small-for-size for adult recipients. Graft injury after reperfusion is common, but the mechanism and degree of injury remain unclear. The hepatic sinusoidal injury in different graft sizes and its relationship with portal hemodynamics and intragraft gene response at the early phase after reperfusion have not been studied in right lobe LDLT. MethodsFrom May 2000 to November 2001, 40 adults receiving right lobe LDLT had portal pressure measured continuously before and after reperfusion. Liver biopsies were taken before and after reperfusion for detection of vasoregulatory genes (endothelin-1 and endothelial nitric oxide synthase) and heat shock genes (heat shock protein 70 and heme oxygenase-1), and electron microscope examination. Blood samples from the portal vein and suprahepatic inferior vena cava were taken for the measurement of plasma nitric oxide level. ResultsThe recipients were grouped according to the ratio of graft weight to estimated standard liver weight: group 1 (n = 10), less than 40%; group 2 (n = 21), 40% to 60%; and group 3 (n = 9), more than 60%. The portal pressures recorded after reperfusion in group 1 were significantly higher within 30 minutes of reperfusion than those in groups 2 and 3. After reperfusion, the intragraft endothelin-1 mRNA level in group 1 increased by 161% of the basal level but decreased by 31.5% and 62% of the basal level in groups 2 and 3, respectively. The intragraft mRNA level of heme oxygenase-1 in groups 1 and 2 decreased by 75.5% and 25.3% of the basal level respectively but increased by 41% of basal level in group 3. The intragraft protein level of heat shock protein 70 decreased by 50 ng/mL after reperfusion in group 1 but increased by 12.4 ng/mL and 0.6 ng/mL in groups 2 and 3, respectively. The portal vein plasma nitric oxide level decreased more significantly after reperfusion in group 1 than in group 2. Electron microscope examination of liver biopsies in group 1 showed tremendous mitochondrial swelling as well as irregular large gaps between the sinusoidal lining cells. There were two hospital deaths in group 1 and none in the other two groups. ConclusionsPatients implanted with grafts less than 40% of standard liver weight suffered from transient portal hypertension early after reperfusion. The phenomenon was accompanied by intragraft upregulation of endothelin-1 and ultrastructural evidence of sinusoidal damage. The transient portal hypertension after reperfusion, subsequent endothelin-1 overexpression, and plasma nitric oxide level reduction, together with downregulation of heme oxygenase-1 and heat shock protein 70, may account for the small-for-size graft injury.

Toll-like receptor-4 mediates obesity-induced non-alcoholic steatohepatitis through activation of X-box binding protein-1 in mice

Background Non-alcoholic fatty liver disease is an obesity-related chronic liver disorder ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), which may progress to liver fibrosis and cirrhosis. Objective Tto investigate the role of Toll-like receptor (TLR) 4 in mediating the transition from steatosis to inflammation. Methods ApoE−/−/TLR4mut mice and ApoE−/−/TLR4 wild-type mice (ApoE−/−/TLR4-WT) were generated by cross-breeding an ApoE-deficient (ApoE−/−) strain with TLR4-mutant (TLR4mut) mice, which were fed with high-fat, high-cholesterol (HFHC) diet to induce obesity. Results ApoE−/−/TLR4-WT mice fed with an HFHC diet for 12 weeks developed typical pathological features of NASH, which is associated with obesity and the metabolic syndrome. By contrast, ApoE−/−/TLR4mut mice lacking functional TLR4 were resistant to HFHC diet-induced liver inflammation and injury and were less susceptible to the diet-induced production of reactive oxygen species (ROS) and proinflammatory cytokines. In ApoE−/−/TLR4-WT mice, X-box binding protein-1 (XBP-1), a transcription factor involved in the unfolded protein responses, was activated in the liver by an HFHC diet, whereas XBP-1 activation was abrogated in ApoE−/−/TLR4mut mice. In primary rat Kupffer cells, endotoxin induced XBP-1 activation through ROS production, whereas siRNA-mediated knockdown of XBP-1 expression resulted in a marked attenuation in endotoxin-evoked NF-κB activation and cytokine production. Furthermore, adenovirus-mediated expression of dominant negative XBP-1 led to a significant attenuation in HFHC diet-induced liver inflammation and injury in mice. Conclusions These findings support the key role of TLR4 in Kupffer cells in mediating the progression of simple steatosis to NASH, by inducing ROS-dependent activation of XBP-1.

Suppression of Liver Tumor Growth and Metastasis by Adiponectin in Nude Mice through Inhibition of Tumor Angiogenesis and Downregulation of Rho Kinase/IFN-Inducible Protein 10/Matrix Metalloproteinase 9 Signaling

Purpose: We aimed to investigate the effects of adiponectin on liver cancer growth and metastasis and explore the underlying mechanisms. Experimental Design: An orthotopic liver tumor nude mice model with distant metastatic potential was applied. Either Ad-adiponectin (1 × 108; treatment group) or Ad-luciferase (control group) was injected via portal vein after tumor implantation. Tumor growth and metastasis were monitored by Xenogen In vivo Imaging System. Hepatic stellate cell activation by α-smooth muscle actin staining, microvessel density by CD34 staining, macrophage infiltration in tumor tissue, and cell signaling leading to invasion, migration [Rho kinase (ROCK), IFN-inducible protein 10 (IP10), and matrix metalloproteinase 9], and angiogenesis [vascular endothelial growth factor (VEGF) and angiopoietin 1] were also compared. Tumor-nontumor margin was examined under electron microscopy. Direct effects of adiponectin on liver cancer cells and endothelial cells were further investigated by a series of functional studies. Results: Tumor growth was significantly inhibited by adiponectin treatment, accompanied by a lower incidence of lung metastasis. Hepatic stellate cell activation and macrophage infiltration in the liver tumors were suppressed by adiponectin treatment, along with decreased microvessel density. The treatment group had less Ki-67–positive tumor cells and downregulated protein expression of ROCK1, proline-rich tyrosine kinase 2, and VEGF. Tumor vascular endothelial cell damage was found in the treatment group under electron microscopy. In vitro functional study showed that adiponectin not only downregulated the ROCK/IP10/VEGF signaling pathway but also inhibited the formation of lamellipodia, which contribute to cell migration. Conclusion: Adiponectin treatment significantly inhibited liver tumor growth and metastasis by suppression of tumor angiogenesis and downregulation of the ROCK/IP10/matrix metalloproteinase 9 pathway. Clin Cancer Res; 16(3); 967–77

Signal Transducers and Activators of Transcription 5b Activation Enhances Hepatocellular Carcinoma Aggressiveness through Induction of Epithelial-Mesenchymal Transition

Poor prognosis of hepatocellular carcinoma (HCC) is associated with a high potential of vascular invasion and metastasis. Epithelial-mesenchymal transition (EMT) is a key event in the tumor invasion process. Recently, signal transducers and activators of transcription 5 (STAT5) has been linked to tumor progression by EMT induction. However, the precise roles of STAT5 genes (STAT5a and STAT5b) in human epithelial cancers have not been elucidated clearly. The aim of this study is to analyze the roles of STAT5 isoforms in HCC progression using HCC clinical samples. We showed that activation of STAT5b, but not STAT5a, was found in HCC clinical samples and its expression was significantly associated with younger age (P = 0.037), advanced tumor stages (P = 0.003), venous infiltration (P = 0.016), microsatellite formation (P = 0.024), multiple tumor nodules (P = 0.02), and poor patient survival. To specifically investigate the mechanism underlying constitutive activation of STAT5b in HCC, EGFP-HBX was introduced into Huh-7 cells. STAT5b activation in HCC is at least partially mediated by HBX activation. Ectopic STAT5b transfection conferred increased HCC cell motility and invasiveness by induction of EMT changes. In conclusion, STAT5b activation enhanced HCC aggressiveness by induction of EMT, which was possibly mediated by HBX activation. STAT5b could serve as a novel molecular target for HCC treatment.

PIN1 overexpression and β-catenin gene mutations are distinct oncogenic events in human hepatocellular carcinoma

The peptidyl-proplyl-isomerase, PIN1, upregulates β-catenin by inhibiting its interaction with APC. β-catenin accumulation occurs in about 70% of hepatocellular carcinoma (HCC), of which only 20% are due to β-catenin mutations. The role of PIN1 in β-catenin upregulation in HCC was investigated. PIN1 was shown to be overexpressed in more than 50% of HCC. All cases with PIN1 overexpression also showed β-catenin accumulation, with 68% of cases showing concomitant β-catenin and cyclin D1 accumulation. PIN1 was shown to contribute to β-catenin and cyclin D1 overexpression directly by in vitro cell-line transfection experiments. Finally, we showed that PIN1 overexpression and β-catenin gene mutations appeared to be mutually exclusive events, leading to β-catenin accumulation in HCC. These results showed that PIN1 overexpression leading to β-catenin accumulation might be a critical event in hepatocarcinogenesis, and that PIN1 is a potential target for therapeutic intervention in HCC.

Proline-rich tyrosine kinase 2 (Pyk2) promotes proliferation and invasiveness of hepatocellular carcinoma cells through c-Src/ERK activation

The aim of the current study is to elucidate the mechanism of proline-rich tyrosine kinase 2 (Pyk2)-mediated cell proliferation and invasiveness in hepatocellular carcinoma (HCC) cells. Human HCC cell lines PLC and MHCC97L were stably transfected with either full-length Pyk2 or C-terminal non-kinase region of Pyk2 (PRNK). Functional studies on cell proliferation and invasion were conducted in vitro by colony formation assay, adhesion assay, migration assay and wound-healing assay. For the in vivo study, an orthotopic nude mice liver tumor model was applied to investigate the effects of Pyk2 overexpression on tumor growth and metastasis. Overexpression of Pyk2 in PLC cells resulted in an upregulation of colony formation (P = 0.021) and adhesion toward laminin (P = 0.018). Pyk2 promoted wound recovery by stimulation of actin stress fiber polymerization. In the in vivo study, transfection of PRNK in MHCC97L cells significantly decreased tumor volume (P = 0.001) and the incidence of lung metastasis (P = 0.014). Overexpression of Pyk2 promoted the activation of c-Src, formation of Pyk2/c-Src complex and activated the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK)-signaling pathway. Pyk2 upregulated the activation of ERK1/2 that is insensitive to MAPK/ERK kinase (MEK)1/2 inhibition. On the contrary, PRNK overexpression downregulated the activation of c-Src and ERK/MAPK-signaling pathways. Immunofluorescence staining showed that the focal adhesion localization of Pyk2 is a major determinant for c-Src and ERK/MAPK activation. In conclusion, our results showed that Pyk2 promoted cell proliferation and invasiveness by upregulation of the c-Src and ERK/MAPK-signaling pathways.