Non-neoplastic Liver Tissue Research Articles

Glypican-3 expression and its relationship with recurrence of HCC after liver transplantation

To investigate the diagnostic value of glypican-3 (GPC3) and its relationship with hepatocellular carcinoma (HCC) recurrence after liver transplantation. HCC tissue samples (n = 31) obtained from patients who had undergone liver transplantation were analyzed. GPC3 mRNA and protein expression were analyzed by TaqMan real-time reverse transcription-polymerase chain reaction and immunohistochemistry. Correlation between the GPC3 expression and clinicopathological features was analyzed. The potential prognostic value of GPC3 was investigated by comparing recurrence-free survival between HCC patients with and without GPC3 expression. Using a cutoff value of 3.5 × 10⁻², 20 of 31 cancerous tissues had expression values of > 3.5 × 10⁻², whereas 3 of 31 adjacent non-neoplastic parenchyma and 0 of 20 control liver tissues had expression values of > 3.5 × 10⁻² (P < 0.001). GPC3 protein was immunoexpressed in 68% of cancerous tissues, but not in adjacent non-neoplastic parenchyma and control liver tissues. Vascular invasion was significantly related to GPC3 expression (P < 0.05). Recurrence-free survival was significantly longer for patients without GPC3 mRNA overexpression (> 3.5 × 10⁻²) and those without vascular invasion (P < 0.05 for both). GPC3 expression may serve as a valuable diagnostic marker for HCC. GPC3 mRNA overexpression may be an adverse indicator for HCC patients after liver transplantation.

P52 Macrophage inflammatory protein-1 α/CC chemokine ligand 3 and tumour-associated macrophages in hepatitis C virus-related hepatocellular carcinoma: relation to tumour progression and angiogenesis

IntroductionHepatitis C virus (HCV) is a major risk factor for development of hepatocellular carcinoma (HCC), however, the mechanism of hepatocarcinogenesis in HCV infection is still undefined. Chemokines, which can induce...

Study on the expression of Glut1, HIF-1α and Ki-67 in hepatocellular carcinoma and their relationship

Objective To explore the expression of Glut1, HIF-1α and Ki-67 in hepatocellular carcinoma (HCC) and their relationship between their expression and clinicopathological features.Methods Immunohistochemical study (EnVision method) for Glut1, HIF-1a and Ki-67 were performed on tissue microarray which consisted of 171 cases of HCC, 55 cases of adjacent non-neoplastic liver tissues, and 22cases of normal liver tissues.Results The expression rate of Glut1 ,HIF-1α and ki-67 in 171 cases of HCC was 15.2％, 19.9％ and 66.1％, respectively, which was much higher than that in the adjacent non-neoplastic liver tissues (1.8％ ,1.8％and 5.5％) and normal liver tissues(all negative).The expressions of Glut1 and HIF-1α were positively correlated with the differentiation degree of HCC and TNM stage(P ＜0.01, P ＜0.05).The expression of ki-67 was positively correlated with the differentiation degree of HCC.There was a significant positive correlation between the expressions of Glut1 and HIF-1α in HCC (r1 =0.553, P ＜0.05), the expressions of Glut1 and HIF-1α were positively correlated with ki-67(r2 =0.560,r3 =0.613, P ＜0.05).Conclusions Glut1, HIF-1α and ki-67 may play a role in the tumorigenesis and progression of HCC in some degree.Combined detection of Glut1, HIF-1α and Ki-67 may be helpful to judge the degree of malignancy and potential metastasis and evaluate the prognosis. Key words: Glucose transport proteins,facilitative/ME,Hypoxia-inducible factor 1/ME; Ki-67antigen/ME; Carcinoma, hepatocellular/ME

MAGE-1 and MAGE-3 mRNA expressions as molecular biomarkers in patients with hepatitis C virus-related hepatocellular carcinoma

IntroductionThe melanoma antigen (MAGE) family members are tumour-specific antigens exclusively expressed in neoplastic cells. Therefore, the present work was designed to study the expression of MAGE-1 and MAGE-3 mRNAs in...

Deregulated expression of microRNA-221 with the potential for prognostic biomarkers in surgically resected hepatocellular carcinoma

Aberrant expression of specific microRNAs in hepatocellular carcinomas has recently been reported. We examined expression patterns of 4 microRNAs (microRNA-221, microRNA-222, microRNA-21, and microRNA-155) to evaluate their potential as relevant biomarkers by quantitative real-time reverse transcriptase-polymerase chain reaction using formalin-fixed, paraffin-embedded tissues of 115 surgically resected hepatocellular carcinoma and paired nonneoplastic liver cases as well as 21 normal liver samples from cancer-free individuals. MicroRNA-221, microRNA-222, and microRNA-21 were differentially overexpressed in hepatocellular carcinoma compared with nonneoplastic and normal livers (P < .001). The mean fold changes in microRNA-221, microRNA-222, and microRNA-21(hepatocellular carcinoma to matched nonneoplastic liver) were 4.00, 4.44, and, 3.67, respectively. In addition, nonneoplastic liver tissues displayed higher levels of microRNA-221, microRNA-222, microRNA-21, and microRNA-155 than normal livers (P < .001, respectively). However, the overexpression of the 4 microRNAs showed no consistent relevance to the known prognostic clinicopathologic parameters. High expression of microRNA-221 in hepatocellular carcinomas was significantly related to shorter time to local recurrence (P < .001) and determined as an independent predictor for local recurrence (P = .001). The fold changes in microRNA-221 (hepatocellular carcinoma to matched nonneoplastic liver) less than 1 were more commonly detected in cases of distant metastases than those of disease-free and local recurrence (P = .009). The fold changes less than 1 were related to reduced metastasis-free survival (P = .006) and thus can be used as an independent predictor of distant metastasis after surgical resection (P = .027). Based on these results, we propose the possible role of microRNA-221, microRNA-222, microRNA-21, and microRNA-155 dysregulation in hepatocarcinogenesis and the potential of microRNA-221 dysregulation for predicting local recurrence and distant metastasis after curative surgery.

Correlation of KiSS-1, KiSS-1R and MMP-9 expression to invasion and metastasis of hepatocellular carcinoma and its significance

Objective To detect the expression of KiSS-1, KiSS-1R and MMP-9 in hepatocellular carcinoma (HCC). To study the correlation of KiSS-1, KiSS-1R and MMP-9 expression with invasion and metastasis of HCC, and to explore the underlying mechanisms. Methods The expression of KiSS-1 , KiSS-1R mRNA in 33 HCC samples, 26 non-neoplastic adjacent liver tissue samples and 13 non-neoplastic distant liver tissue samples were detected by RT-PCR. Tissue chips were constructed by modified manual tools, which contained HCC, non-neoplastic adjacent liver tissues, non-neoplastic distant liver tissues, normal liver tissues and intrahepatic metastasis lesions. The expression of KiSS-1 and MMP-9 protein was determined by tissue chips, immunohistochemistry and semi-quantitative image analysis in 150 HCC, 137 non-neoplastic adjacent liver tissue, 98 non-neoplastic distant liver tissues, 16 normal liver tissues and 37 intrahepatic metastasis lesion samples. Results The results of RT-PCR showed that compared with the non-neoplastic adjacent liver tissues and the non-neoplastic distant liver tissues, the expression of KiSS-1 mRNA in HCC was significantly lower (P＜0.01). The expression of KiSS-1R mRNA did not changed in HCC and non-neoplastic liver tissues (P＞0.05). The expression of KiSS-1 protein was lower in HCC with metastasis and in clinical stage Ⅲ than that in those with non-metastasis, and in clinical stages Ⅰ and Ⅱ . It was also higher in the primary than in the metastasis lesions (P＜0.01, respectively). The expression of MMP-9 was higher in tumors having peplos invasion and metastasis than in those with negative peplos invasion and non-metastasis. It was lower in the primary than the metastasis lesions (P＜0. 01, respectively).Negative correlation between KiSS-1 and MMP-9 expression was found in HCC(r=- 0.340,P＜0.01). Conclusions The imbalance between KiSS-1 and MMP-9 expression might play an important role in enhancing the invasive and metastatic capacity of HCC. Loss of KiSS-1 expression might predict an aggressive clinical behavior and was associated with metastatic potential in HCC. Key words: Liver neoplasm; KiSS-1; KiSS-1R; MMP-9; Tissue chip

Gene expression profiling of human hepatoblastoma using archived formalin-fixed and paraffin-embedded tissues

We elucidated the genetic profile of hepatoblastomas (HBLs) to identify diagnostic and prognostic markers. RNA was extracted from 32 formalin-fixed, paraffin-embedded HBLs and corresponding nonneoplastic liver (NNL) tissues, and cDNA-mediated annealing, selection, extension, and ligation (DASL) chip assays were performed. Immunohistochemistry was performed to confirm the expression of Yin Yang 1 (YY1) protein in HBL. Twenty-four genes that were associated with signal transduction, cell-cell adhesion, cell cycle regulation, and apoptosis were differentially expressed in HBL and NNL tissues. Two apoptosis-associated genes, MYCN and BIRC5, were highly upregulated in HBL. Eight genes, including YY1 and IGF1, were upregulated in HBL cases that had a poor prognosis. Thirty-eight genes, including YY1, were differentially expressed according to histologic differentiation of HBL, and the immunohistochemical expression of YY1 was correlated with poor HBL differentiation. Thus, using DASL chip assays, we report the gene expression profiles of HBL, which suggest new candidate prognostic and diagnostic genetic markers and putative therapeutic targets for HBL.

CpG island methylator phenotype in plasma is associated with hepatocellular carcinoma prognosis

To evaluate the clinical significance of CpG island methylator phenotype (CIMP) in plasma and its association with hepatocellular carcinoma (HCC) progress. CIMP status of 108 HCC patients was analyzed using a methylation marker panel in tumor tissues and plasma with methylation-specific polymerase chain reaction. Fifteen samples of non-neoplastic liver tissues and 60 of plasma from healthy persons were examined simultaneously. Examined genes included APC, WIF-1, RUNX-3, DLC-1, SFRP-1, DKK and E-cad. The frequencies of high-level methylation in HCC tissue and plasma were at least 15% for the seven genes: APC, 48/108, 44.44% in tissue and 26/108, 24.07% in plasma; WIF-1, 53/108, 49.07% in tissue and 35/108, 32.41% in plasma; RUNX-3, 52/108, 48.14% in tissue and 42/108, 38.89% in plasma; DLC-1, 38/108, 35.18% in tissue and 23/108, 21.30% in plasma; SFRP-1, 40/108, 37.04% in tissue and 31/108, 28.7% in plasma; DKK, 39/108, 36.1% in tissue and 25/108, 23.14% in plasma; and E-cad, 37/108, 34.3% in tissue and 18/108, 16.67% in plasma. CIMP+ (≥ 3 methylated genes) was detected in 68 (60.2%) tumor tissue samples and 62 (57.4%) plasma samples. CIMP was not detected in non-neoplastic liver tissues or plasma of healthy persons. CIMP status in tumor tissues differed significantly in gender, hepatitis B surface antigen, alpha-fetoprotein, and tumor-node-metastasis stage (P < 0.05). Similar results were obtained with plasma samples (P < 0.05). There was no difference in CIMP status in age, presence of hepatitis C virus antibody, cirrhosis, number of nodes, number of tumors, tumor size, or Edmondson-Steiner stage. A one-year follow-up found that the metastatic rate and recurrence rate in the CIMP+ group were significantly higher than in the CIMP- group as assessed with plasma samples (P < 0.05). Plasma DNA can be a reliable sample source for CIMP analysis. CIMP in plasma may serve as a molecular marker of late-stage and poor-prognosis HCC.

Overexpression of SUMO-1 in hepatocellular carcinoma: a latent target for diagnosis and therapy of hepatoma

To investigate the expression of SUMO-1 in human hepatocellular carcinoma (HCC) cell lines and clinical HCC samples. RT-PCR and Western blot were used to detect the expressions of SUMO-1 in HCC cell lines, clinical HCC samples,and the non-neoplastic liver tissues adjacent to HCC. After transfection of SUMO-1 siRNA into HCC cell line SMMC-7721, the expression levels of Bcl-2, c-Myc and α-tubulin were examined, and MTT assay and cell cycle analysis were carried out as well. Overexpressions of SUMO-1 were detected in HCC cell lines and clinical HCC samples, while the expression level of SUMO-1 in the non-neoplastic liver tissues was significantly lower (P < 0.001). Transfection of SUMO-1 siRNA resulted in 73.43% of maximal silencing efficiency of SUMO-1 in 48 h. The expressions of Bcl-2 and c-Myc were down-regulated coincidentally. SUMO-1 siRNA notably inhibited SMMC-7721 cells proliferation in vitro and increased the ratios of G2 phase and S phase in the cells. Owing to overexpression of SUMO-1 in HCC and its important role in the development of HCC, SUMO-1 could be a latent target in diagnosis and therapy of HCC.

Genomic and immunophenotypical differences between hepatocellular carcinoma with and without cirrhosis

To compare the expression of genes involved in p53, Wnt/beta-catenin, and retinoblastoma (Rb) 1 pathways between cirrhosis-associated hepatocellular carcinoma (HCC-C) and hepatocellular carcinoma arising in non-cirrhotic liver (HCC-NC). The gene expression profile was analysed using oligo-DNA arrays, and then validated at protein level in a tissue microarray using immunohistochemistry. Compared with their background non-neoplastic liver tissue, HCC-C showed a significantly higher rate of p53, beta-catenin (protein only) and cyclin D1 expression, whereas HCC-NC showed a significantly higher rate of p21(Waf1/cip1) and p27(Kip1) expression. HCC-C had a significantly higher rate of p53 expression and a significantly lower rate of p21(waf1/cip1) expression than HCC-NC. There was no statistically significant association between the expression of genetic markers and tumour histological grade, underlying aetiology, or lymphovascular invasion. Aberrant beta-catenin expression was more commonly seen in single tumours in comparison with multiple tumours. Increased p16(INK4) and p21(waf1/cip1) expression was more commonly observed in large-sized tumours (>50 mm) than small-sized tumours. Alteration of the p53 pathway plays a more important role in the pathogenesis of HCC-C, whereas alterations in cell cycle regulators p21(waf1/cip1) and p27(Kip1) play a more important role in the pathogenesis of HCC-NC.

OC-029 Expression of heat shock protein 27 and caspase-3 in hepatitis C virus-related hepatocellular carcinoma: relation to tumour progression

Introduction Hepatitis C virus (HCV) is a major risk factor for development of hepatocellular carcinoma (HCC), however, the mechanism of hepatocarcinogenesis in HCV infection is still undefined. Heat shock protein (HSP) 27 is a ubiquitous chaperone molecule induced in cells exposed to different stress conditions, including carcinogenesis. It also has potent anti-apoptotic properties through inhibition of caspase-3 activation. Therefore, the aim of the present work was to study the expression of HSP27 and caspase-3 in HCV-related HCCs in relation to tumour progression. Methods Twenty cirrhotic patients with HCV-related HCC were enrolled in the study. The severity of liver disease was assessed according to the Model for End Stage Liver Disease (MELD) score. Serum levels of α-fetoprotein (AFP) were measured by enzyme immunoassay kit. The tumour stage was classified using the scoring system proposed by the Cancer of the Liver Italian Program (CLIP). Histological grading of tumour s was performed according to the Edmondson and Steiner9s criteria and the surrounding liver tissue was examined for the presence of cirrhosis and steatosis. Expression of HSP27 and caspase-3 was studied in HCC and adjacent non-neoplastic liver tissues by immunohistochemistry and the staining intensity of the tumour was designated as “negative/low expression” or “high expression” if 25% of cells were positively-stained, respectively. Results HCV-related HCCs showed a significant increase in HSP27 expression and a significant decrease in caspase-3 expression as compared with adjacent non-neoplastic liver tissues (p=0.029 and p=0.040, respectively). The expression of HSP27 showed an inverse correlation with caspase-3 expression in HCC tissues (r=−0.691, p=0.001). High HSP27 expression and negative/low caspase-3 expression in HCCs were associated with significant increases in serum levels of aminotransferases, HCV RNA and AFP, MELD score, tumour size, tumour stage, histological tumour grade, body mass index and the presence of steatosis in the surrounding liver tissue (P 0.05). Conclusion HSP27 plays an important role in the pathogenesis and progression of HCC in HCV infection through inhibition of caspase-3 mediated cell apoptosis and may serve as a potentially useful therapeutic target.

Reduced Expression of Fibroblast Growth Factor Receptor 2IIIb in Hepatocellular Carcinoma Induces a More Aggressive Growth

Fibroblast growth factor receptor 2 isoform b (FGFR2-IIIb) is highly expressed in hepatocytes and plays an important role in liver homeostasis and regeneration. Here, we analyzed the expression and function of FGFR2-IIIb in hepatocellular carcinoma (HCC). FGFR2-IIIb expression in HCC tissues and cell lines was lower than in primary human hepatocytes and nontumorous tissue. FGFR2-IIIb-negative HCCs showed a significantly higher Ki-67 labeling index, and loss of FGFR2-IIIb expression correlated significantly with vascular invasion and more advanced tumor stages. A decrease in FGFR-2IIIb expression in HCC cell lines was not related to promoter hypermethylation. However, PCR analysis indicated that chromosomal deletion at 10q accounted for the loss of FGFR2 expression in a subset of HCC cells. FGFR2-IIIb re-expression in stable transfected HCC cell lines induced a higher basal apoptosis rate and a significantly reduced proliferation and migratory potential in vitro. In nude mice, FGFR2-IIIb re-expressing HCC cells grew significantly slower, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay revealed higher apoptosis rates. The antitumorigenic effects of FGFR2-IIIb expression in HCC cells were not affected by keratinocyte growth factor or an inhibitor of FGFR-phosphorylation, indicating that they are independent of tyrosine kinase activation. In conclusion, our data indicate that FGFR2-IIIb inhibits tumorigenicity of HCC cells. Identification of the molecular mechanisms promoting regeneration in normal tissue while suppressing malignancy may lead to novel therapeutic targets of this highly aggressive tumor.

CpG Island Methylator Phenotype Associated with Tumor Recurrence in Tumor–Node–Metastasis Stage I Hepatocellular Carcinoma

CpG island methylator phenotype (CIMP), characterized by simultaneous methylation of multiple tumor suppressor genes (TSGs), has been reported to be associated with biological malignancy in many cancers. Whether CIMP is potentially predictive of clinical outcome in hepatocellular carcinoma (HCC) remains unknown. We investigated the methylation status of ten TSGs and CIMP in 115 samples of HCC and 48 samples of corresponding nonneoplastic liver tissues using a methylation-specific polymerase chain reaction. The methylation frequencies of the ten genes examined in HCC were 40.0% for p14 ( ARF ), 60.9% for p15 ( INK4b ), 70.4% for p16 ( INK4a ), 34.8% for p73, 70.4% for GSTP1, 64.3% for MGMT, 13.0% for hMLH1, 59.1% for RARbeta, 82.6% for SOCS-1, and 80.9% for OPCML. CIMP+ (with six or more methylated genes) was detected in 68 (59.1%) of 115 HCCs and none of 48 nonneoplastic liver tissues. On stratified univariate analysis, patients with tumor-node-metastasis (TNM) stage I HCC with CIMP+ had significantly shorter overall survival (OS) (P = 0.002) and recurrence-free survival (RFS) (P = 0.042) than those with CIMP-. Furthermore, multivariate analysis revealed CIMP+ as an independent prognostic factor for both OS [hazard ratio (HR), 12.266; P = 0.015] and RFS (HR, 2.275; P = 0.032) in TNM stage I patients. CIMP+ may specifically define a subgroup of patients with unfavorable outcome in TNM stage I HCC. Examination of CIMP status may be useful for stratifying prognosis of patients with early-stage HCC and identifying patients who are at higher risk for recurrence.

OCT4 expression in hepatocellular carcinoma and its clinical significance

Recently, many studies have focused on stem cells in hepatocellular carcinoma (HCC) and found some stem cell markers in HCC, which are associated with the prognosis. OCT4, as a member of the POU transcription factor family, is a key factor to maintain self-renewal and pluripotency of embryonic stem cells (ESCs). This study was to explore the expression of the ESCs marker OCT4A in HCC, and its correlations with clinicopathologic features and prognosis of HCC. OCT4A mRNA expression was detected in five liver cancer cell lines (SMMC-7721, BEL-7402, Hep-G2, MHCC97-L, and MHCC97-H), one immortalized liver cell line L-O2, tumor tissues with matched non-neoplastic liver tissues in 107 HCC patients, and normal liver tissues of 20 cases using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The correlations between OCT4A mRNA and clinicopathologic features and prognosis of HCC were analyzed. OCT4A mRNA was detected in SMMC-7721, BEL-7402, Hep-G2, MHCC-97L, and MHCC-97H cells, but not in L-O2 cells. The positive rate of OCT4A mRNA expression was significantly higher in the HCC tissues than in the non-neoplastic liver tissues (72.0% vs. 30.8%, P<0.001). No OCT4A mRNA expression was found in the normal liver tissues. OCT4A mRNA expression was correlated with the tumor size, vascular invasion, and TNM stage (P<0.05). Kaplan-Meier survival curves showed that patients with positive expression of OCT4A mRNA had lower overall survival and disease-free survival rates. OCT4A mRNA, which is highly expressed in a subset of liver cancer cell lines and HCC tissues, may be involved in the carcinogenesis of HCC. OCT4A mRNA may be a valuable biomarker for assessing the prognosis of HCC.

Histological evaluation of intracapsular venous invasion for discrimination between portal and hepatic venous invasion in hepatocellular carcinoma

Histological criteria for intracapsular venous invasion (IVI) that would allow its discrimination between portal and hepatic venous invasion in hepatocellular carcinoma (HCC) have not been established. We evaluated IVI immunohistochemically to discriminate between portal and hepatic venous invasion in 89 resected specimens from patients with HCC. IVI was defined as the microscopic involvement of the vessels within the fibrous capsule of HCC. The hepatic venous system was subdivided into the central vein and the sublobular/hepatic vein. Immunohistochemical analysis with the D2-40 monoclonal antibody revealed lymphatic vessels. In non-neoplastic liver tissues, the portal veins (n = 4355) were accompanied by lymphatic vessels (99.7%), bile ductules (100%) and arteries (96%), whereas the central veins (n = 3932) and sublobular/hepatic veins (n = 662) were rarely accompanied by lymphatic vessels (0% and 17%, respectively) and bile ductules (12% and 33%, respectively). In total, 29 IVI foci were detected; three foci were clearly visible within vessels that contained a distinct layer of connective tissue fibers, signifying sublobular/hepatic venous invasion. As the remaining 26 foci were accompanied by lymphatic vessels (26/26 [100%]), bile ductules (21/26 [81%]) and arteries (10/26 [38%]), these foci were considered to reflect intracapsular portal venous invasion rather than venous invasion of the central vein. Intracapsular portal venous invasion was significantly associated with extratumoral portal venous invasion (P < 0.001). D2-40 immunoreactivity for the histological evaluation of IVI in HCC allows discrimination between portal and hepatic venous invasion for cases in which portal venous invasion predominates.

Identification and expression analysis of the aldo–ketoreductase1-B10 gene in primary malignant liver tumours

The aim of our study was to search for highly up-regulated genes in primary malignant liver tumours and to analyse their expression at the mRNA- and protein level. Using a random-based gene fishing approach (representational difference analysis coupled to array hybridisation) we identified 7 genes high abundantly expressed in hepatocellular carcinoma (HCC) as compared to non-neoplastic liver tissue, among them a gene fragment of the aldo-ketoreductase (AKR) superfamily. Full length cloning and sequencing of the gene fragment identified it as B10 gene of the AKR-family 1 (AKR1B10). For expression analysis on transcriptional level quantitative real-time RT-PCR was performed in 22 HCC and 22 non-neoplastic liver cirrhotic tissues. Our data demonstrate significantly higher expression levels of AKR1B10-mRNA in HCC compared to non-tumourous cirrhotic liver tissue (p<0.0001). To evaluate its protein expression in primary malignant liver tumours, we investigated tissue arrays of 210 HCC and 51 cholangiocarcinomas (CC) by immunohistochemistry, using a monoclonal antibody against AKR1B10. Protein staining of AKR1B10 was significantly increased in well and moderately differentiated tumours compared to corresponding non-neoplastic liver tissue (p=0.023). However, AKR1B10-staining decreased in advanced, low differentiated tumours with a significant inverse correlation between AKR1B10-staining and tumour proliferation, indicated by Ki67 (MIB-1) staining (r=-0.89, p=0.02). The over-expression of AKR1B10 in early stages of well and moderately differentiated tumours and its down-regulation in advanced tumour-stages with low grade of differentiation demonstrated that AKR1B10 may be a helpful marker for differentiation and proliferation of HCC and CC.

Expression of xenobiotic and steroid hormone metabolizing enzymes in hepatocellular tumors of the non-cirrhotic liver

Hepatocellular adenomas (HCA) and some hepatocellular carcinomas (HCC) arise in the non-cirrhotic liver. Although the liver is involved in the metabolism of a huge number of exogenous and endogenous substances, little is known about the role of metabolic enzymes in the development of liver tumors in the absence of cirrhosis. We analyzed the expression of glutathione S-transferases (GST) and cytochrome P450 enzymes (CYP) in 23 HCA, 20 HCC, and 22 focal nodular hyperplasias (FNH) using immunohistochemistry. The liver tissue revealed consistent specific staining for GST alpha, CYP1A1, 1A2, 2E1, and 3A4. In HCA and HCC, GST alpha expression was significantly reduced ( p<0.001 and 0.043). Reduced GST alpha expression was significantly associated with steatosis in HCA and HCC ( n=12, p=0.006), but not in non-neoplastic liver tissue. CYP3A4 expression was also reduced in HCA and HCC ( p=0.03 and 0.02), and this was correlated with diabetes mellitus type 2 ( p=0.02). In conclusion, HCA and HCC revealed changes in the expression of certain metabolic enzymes as compared with the non-neoplastic liver tissue or FNH. Therefore, reduced expression of GST alpha and CYP3A4 may indicate specific metabolic defects in the tumor tissue characterizing subgroups of HCA and HCC.

Elucidating genetic events in human hepatocellular carcinoma

e15590 Background: Hepatocellular carcinoma (HCC) is prevalent world wide with increasing incidence in North America. 4q is a commonly deleted region in HCC and is lost in 32% of premalignant cirrhotic nodules but the precise molecular lesion involved in tumor initiation remains unknown. Human Plk4, a member of the polo family of cell cycle regulating serine /threonine kinases is located on 4q28. Plk4 is a haploinsufficient tumor suppressor in mice, with 50% of animals developing spontaneous tumors, most commonly multifocal primary HCC. We hypothesize that Plk4 plays a role in the initiation of human HCC. Our purpose was to determine the levels of Plk4 in human HCC and investigate possible genetic interactions with the tumor suppressor p53. Methods: Formalin-fixed paraffin-embedded sections of human HCCs, adjacent non-neoplastic liver tissue and gallbladder tissue were microdissected. DNA was extracted and manual LOH analysis was performed. To asses Plk4 expression, RNA from the hepatoma cases was used for real time RT-PCR. To elucidate a genetic interaction, Plk4 p53 compound mutant mice were generated and murine embryonic fibroblasts (MEFs) were derived and cultured. Results: In HCC Plk4 LOH rates varied from 57% for the marker closest to Plk4 to 8% for the marker furthest away (n=32). Using an intragenic marker the rate of LOH was 45% (n=24). Studies in CRC (n=46) and pancreas cancer specimens (n=40) showed only background levels of LOH. Expression of Plk4 in HCC tumor samples that displayed LOH at the Plk4 locus was reduced compared to non-neoplastic liver. Plk4±p53−/− mice showed acceleration of tumor formation compared to Plk4+/+p53−/− mice. Plk4±p53−/− MEFs had increased levels multinucleation, centrosome amplification and aneuploidy compared to Plk4+/+p53−/− cells. In addition these cells were tumorigenic in vivo whereas Plk4+/+p53−/− cells were not. Conclusions: Our results demonstrate significant rates of LOH at the Plk4 locus specifically in human HCC and indicate that this is associated with decreased Plk4 expression. In addition, our studies of compound mutant mice suggest a possible genetic interaction of Plk4 and p53 in tumor development. No significant financial relationships to disclose.

Methylation Regulates Hepatitis B Viral Protein Expression

Hepatitis B virus (HBV) DNA has been shown to contain CpG islands that are methylated in human tissue, which suggests a role for methylation in regulating viral protein production. However, data are lacking about whether methylation regulates viral gene expression. To investigate the hypothesis that methylation of viral DNA regulates viral gene expression, unmethylated, partially methylated, and fully methylated viral DNA was transfected into HepG2 cells. In addition, a new assay was designed that specifically identifies methylated covalently closed circular DNA (cccDNA) in human liver tissue. Transfection of methylated HBV DNA led to reduced HBV mRNA levels in HepG2 cells, decreased surface and core protein expression in these cells, and decreased secretion of HBV viral proteins into the cell supernatant. These data provide direct evidence that CpG islands regulate gene transcription of HBV. Furthermore, methylated cccDNA was found in tumor and nonneoplastic human liver tissues. Finally, an in vitro equivalent of cccDNA showed decreased viral protein production in HepG2 cells after DNA methylation. Taken together, these data demonstrate that methylation of viral CpG islands can regulate viral protein production.

GLUT1 Expression Is Increased in Hepatocellular Carcinoma and Promotes Tumorigenesis

Accelerated glycolysis is one of the biochemical characteristics of cancer cells. The glucose transporter isoform 1 (GLUT1) gene encodes a key rate-limiting factor in glucose transport into cancer cells. However, its expression level and functional significance in hepatocellular cancer (HCC) are still disputed. Therefore, we aimed to analyze the expression and function of the GLUT1 gene in cases of HCC. We found significantly higher GLUT1 mRNA expression levels in HCC tissues and cell lines compared with primary human hepatocytes and matched nontumor tissue. Immunohistochemical analysis of a tissue microarray of 152 HCC cases revealed a significant correlation between Glut1 protein expression levels and a higher Ki-67 labeling index, advanced tumor stages, and poor differentiation. Accordingly, suppression of GLUT1 expression by siRNA significantly impaired both the growth and migratory potential of HCC cells. Furthermore, inhibition of GLUT1 expression reduced both glucose uptake and lactate secretion. Hypoxic conditions further increased GLUT1 expression levels in HCC cells, and this induction was dependent on the activation of the transcription factor hypoxia-inducible factor-1alpha. In summary, our findings suggest that increased GLUT1 expression levels in HCC cells functionally affect tumorigenicity, and thus, we propose GLUT1 as an innovative therapeutic target for this highly aggressive tumor.