Early Event In Hepatocarcinogenesis Research Articles

Pathology of Hepatocellular Carcinoma and Tumor-Bearing Liver Tissue in Association with hTERT Promoter Mutation.

The hTERT promoter mutation represents a common and early event in hepatocarcinogenesis, but its linkage to the morphological status of the underlying liver tissue is poorly understood. We analyzed the connection between the histopathological changes in tumor-bearing liver tissue and the occurrence of the hTERT promoter mutation in hepatocellular carcinoma (HCC), correlated with clinical data. The study cohort comprised 160 histologically confirmed HCC in patients with or without cirrhosis that were investigated for the hTERT promoter mutation. We evaluated the frequency of the hTERT promoter mutation in patients with HCC with or without cirrhosis and correlated it with potential clinical and histopathological drivers. In particular, we examined tumor-bearing noncirrhotic liver tissue regarding inflammation; the modified histological activity index (mHAI), fibrosis, and steatosis; and its correlation with the frequency of the hTERT promoter mutation in HCC. We evaluated overall survival with multivariate Cox regression. Furthermore, we compared hTERT antibody immunohistochemistry and molecular hTERT promoter mutation analysis of both HCC and background liver tissue. The hTERT promoter mutation was especially related to HCC in cirrhotic compared with noncirrhotic liver (p < 0.001) and independently of cirrhosis in patients ≥ 60 years (p = 0.005). Furthermore, the hTERT promoter mutation was associated with cirrhosis caused by alcohol toxicity and hepatitis C virus infection. In noncirrhotic liver tissue, the frequency of hTERT-promoter-mutated HCC increased with the degree of inflammation and fibrosis. Nevertheless, 25% of the hTERT-promoter-mutated HCC developed in normal liver tissue without HCC risk factors. Multivariate Cox regression analysis did not reveal an influence of the hTERT promoter mutation in HCC on overall survival at 3, 5, and 16 years. Immunohistochemical analysis with the hTERT antibodies LS-B95 and 2D8 in hTERT-promoter-mutated HCC and hTERT-wildtype HCC showed a mildly stronger immunoreaction compared with the tumor-bearing liver tissue (LS-B95: p < 0.01, 2D8: p < 0.01). Our study reveals a connection between pathological changes in tumor-bearing liver tissue and the hTERT promoter mutation in most HCC, even in noncirrhotic liver tissue. Immunohistochemical hTERT antibodies do not discriminate between hTERT-promoter-mutated and wildtype HCC.

Vitamin D receptor gene methylation in hepatocellular carcinoma

Worldwide, hepatocellular carcinoma (HCC) is the major subtype of primary liver cancers. HCC is typically diagnosed late in its course. With respect to cancer, the genomic actions of vitamin D are mediated through binding to the Vitamin D Receptor (VDR), which allows it to modulate the expression of genes in a cell-and tissue-specific manner. Epigenetics is a rapidly evolving field of genetic study applicable to HCC. Changes in DNA methylation patterns are thought to be early events in hepatocarcinogenesis. Curcumin has great potential as an epigenetic agent. Accordingly, the current study has been designed to study the methylation status of VDR gene promoter for the first time in HCC aiming to find its clinical significance and potential screening role in chronic Liver Disease (CLD). Additionally, we aimed to investigate, the effect of Curcumin on HCC cell line, aiming to discover new therapeutic targets through epigenetics. This study was conducted on 45 formalin-fixed, paraffin-embedded liver tissue blocks including 15 HCC samples (group A), 15 CLD samples (group B) and 15 apparently normal tissue taken from around benign lesions (group C). Methylation Specific Restriction Digestion and qPCR were done on all samples after DNA extraction. The percentage of VDR gene promoter methylation was significantly higher in the HCC group compared to both CLD and control groups (p < 0.01). VDR promoter methylation by (MS-qPCR) was decreased and the relative expression of VDR by (qRT-PCR) was markedly increased in a dose-dependent fashion in cells grown in Curcumin-adequate medium. In conclusion, this study may open a new gate for the use of VDR promoter methylation as a potential biomarker in HCC.

Expression of c-MET Protein in Various Subtypes of Hepatocellular Adenoma Compared to Hepatocellular Carcinoma and Non-Neoplastic Liver in Human Tissue

Hepatocellular adenoma (HA) is a benign neoplasm of the liver, whose aetiopathogenesis is little known. Newest research allowed dividing all cases into three types based on molecular characteristics: inflammatory HA, HA with HNF1A mutation, β-catenin- mutated HA. The clinical significance of HA is chiefly due to the possibility of malignant transformation into hepatocellular carcinoma (HCC). The aim of the present study was to immunohistochemically assess the expression pattern and level of c-MET protein in hepatocellular adenoma (taking into account its status of Wnt/β-catenin pathway functioning) and intertwining the results into a wider pattern of expression in non-neoplastic liver and hepatocellular carcinoma of various histological grades. It was found that expression of c-MET in poorly-differentiated HCC was significantly higher than in non-neoplastic liver and well- to moderately-differentiated HCC. The expression in HA was variable and differed between molecular subtypes of this neoplasm: inflammatory and HNF1A mutation-associated type are characterized by overexpression of c-MET to an extent comparable with poorly-differentiated HCC, whereas Wnt/β-catenin dysfunction-associated type lacks overexpression, and the amount of c-MET protein accumulated in its cells is similar to the levels in non-neoplastic tissue and well- to moderately-differentiated HCC. These findings suggest that c-MET overexpression in HA is not an early event in hepatocarcinogenesis, but constitutes a divergent molecular pathway leading to neoplastic change compared to overexpression observed in the late stages of tumour progression.

Induction of autophagy promotes the growth of early preneoplastic rat liver nodules.

Although inhibition of autophagy has been implicated in the onset and progression of cancer cells, it is still unclear whether its dysregulation at early stages of tumorigenesis plays an oncogenic or a tumor suppressor role. To address this question, we employed the Resistant-Hepatocyte rat model to study the very early stages of hepatocellular carcinoma (HCC) development. We detected a different autophagy-related gene expression and changes in the ultrastructural profile comparing the most aggressive preneoplastic lesions, namely those positive for the putative progenitor cell marker cytokeratin-19 (KRT-19) with the negative ones. The ultrastructural and immunohistochemical analyses of KRT-19-positive preneoplastic hepatocytes showed the presence of autophagic vacuoles which was associated with p62, Ambra1 and Beclin1 protein accumulation suggesting that a differential modulation of autophagy occurs at early stages of the oncogenesis in KRT-19-positive vs negative lesions. We observed an overall decrease of the autophagy-related genes transcripts and a strong up-regulation of miR-224 in the KRT-19-positive nodules. Interestingly, the treatment with the autophagy inducer, Amiodarone, caused a marked increase in the proliferation of KRT-19 positive preneoplastic lesions associated with a strong increase of their size; by contrast, Chloroquine, an inhibitor of the autophagic process, led to their reduction. These results show that autophagy modulation is a very early event in hepatocarcinogenesis and is restricted to a hepatocytes subset in the most aggressive preneoplastic lesions. Our findings highlight the induction of autophagy as a permissive condition favouring cancer progression indicating in its inhibition a therapeutic goal to interfere with the development of HCC.

Stem cell expression profile in hepatocellular carcinoma, small cell dysplasia, and cirrhosis

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with various etiologies and its incidence still continues to increase. Tumors with progenitor/stem cell (P/SC) immunophenotype are currently the main focus of interest. Human hepatic P/SCs are located in ductal plates in fetal and neonatal livers and they remain relatively constant throughout life. Enhanced self-renewal level of normal hepatic P/SCs serves as an early event in hepatocarcinogenesis. Approximately 40% of HCCs arise from P/SCs and these cells have a critical role in development and progression of HCC. Thus, CD133 expression was found to be upregulated during early liver restoration. In spite of the increasing evidence of the impact of cancer stem cells (CSCs) in hepatocarcinogenesis, the role of CSCs in the sequence of cirrhosis (Crh)-dysplastic nodules (DN)-HCC is still not clear. In our recent research, we investigated the expression rate and staining patterns of CD133 and CD90 in Crh, small cell dysplasia, and HCC. We also evaluated the relationship between the expression rates of CSC markers and the etiology of liver diseases. Among the P/SC markers, we like to attract more attention to CD133 and CD90. Both CD133 and CD90 expressions were higher in poorly differentiated HCC cases than well differentiated ones. According to the etiology, we found that the highest staining rate for CD133 in HCC cases developed in a cirrhotic background with chronic hepatitis B and D co-infection. The highest rate for CD90 was determined in HCC cases with chronic hepatitis C. Since Crh is the end-stage of chronic injury with continuing regeneration, detection of accompanying P/SC activation by CSC markers in suspicious nodules may suggest the initiation of early phases of hepatocarcinogenesis. Some other immunophenotypical features such as Glypican 3, heat shock protein-70, and glutamine synthetase stainings are also being used to diagnose precursor lesions. Facing the emerging concept of personalized treatment strategy, it is obvious that the studies in hepatocarcinogenesis related to the theory of CSCs will provide new ideas on genesis, development, and metastasis of HCC and will bring new insights for the diagnosis and treatment of these tumors.

Wnt-/-β-catenin pathway signaling in human hepatocellular carcinoma.

The molecular basis of the carcinogenesis of hepatocellular carcinoma (HCC) has not been adequately clarified, which negatively impacts the development of targeted therapy protocols for this overwhelming neoplasia. The aberrant activation of signaling in the HCC is primarily due to the deregulated expression of the components of the Wnt-/-β-catenin. This leads to the activation of β-catenin/T-cell factor-dependent target genes that control cell proliferation, cell cycle, apoptosis, and cell motility. The deregulation of the Wnt pathway is an early event in hepatocarcinogenesis. An aggressive phenotype was associated with HCC, since this pathway is implicated in the proliferation, migration, and invasiveness of cancer cells, regarding the cell's own survival. The disruption of the signaling cascade Wnt-/-β-catenin has shown anticancer properties in HCC's clinical evaluations of therapeutic molecules targeted for blocking the Wnt signaling pathway for the treatment of HCC, and it represents a promising perspective. The key to bringing this strategy in to clinical practice is to identify new molecules that would be effective only in tumor cells with aberrant signaling β-catenin.

Disease Progression from Chronic Hepatitis C to Cirrhosis and Hepatocellular Carcinoma is Associated with Increasing DNA Promoter Methylation

Changes in DNA methylation patterns are believed to be early events in hepatocarcinogenesis. A better understanding of methylation states and how they correlate with disease progression will aid in finding potential strategies for early detection of HCC. The aim of our study was to analyze the methylation frequency of tumor suppressor genes, P14, P15, and P73, and a mismatch repair gene (O6MGMT) in HCV related chronic liver disease and HCC to identify candidate epigenetic biomarkers for HCC prediction. 516 Egyptian patients with HCV-related liver disease were recruited from Kasr Alaini multidisciplinary HCC clinic from April 2010 to January 2012. Subjects were divided into 4 different clinically defined groups - HCC group (n=208), liver cirrhosis group (n=108), chronic hepatitis C group (n=100), and control group (n=100) - to analyze the methylation status of the target genes in patient plasma using EpiTect Methyl qPCR Array technology. Methylation was considered to be hypermethylated if >10% and/or intermediately methylated if >60%. In our series, a significant difference in the hypermethylation status of all studied genes was noted within the different stages of chronic liver disease and ultimately HCC. Hypermethylation of the P14 gene was detected in 100/208 (48.1%), 52/108 (48.1%), 16/100 (16%) and 8/100 (8%) among HCC, liver cirrhosis, chronic hepatitis and control groups, respectively, with a statistically significant difference between the studied groups (p-value 0.008). We also detected P15 hypermethylation in 92/208 (44.2%), 36/108 (33.3%), 20/100 (20%) and 4/100 (4%) , respectively (p-value 0.006). In addition, hypermethylation of P73 was detected in 136/208 (65.4%), 72/108 (66.7%), 32/100 (32%) and 4/100 (4%) (p-value <0.001). Also, we detected O6MGMT hypermethylation in 84/208 (40.4%), 60/108 (55.3%), 20/100 (20%) and 4/100 (4%), respectively (p value <0.001. The epigenetic changes observed in this study indicate that HCC tumors exhibit specific DNA methylation signatures with potential clinical applications in diagnosis and prognosis. In addition, methylation frequency could be used to monitor whether a patient with chronic hepatitis C is likely to progress to liver cirrhosis or even HCC. We can conclude that methylation processes are not just early events in hepatocarcinogenesis but accumulate with progression to cancer.

Loss of heterozygosity and methylation of multiple tumor suppressor genes on chromosome 3 in hepatocellular carcinoma

Genetic and epigenetic alterations are the two key mechanisms in the development of hepatocellular carcinoma (HCC). However, how they contribute to hepatocarcinogenesis and the correlation between them has not been fully elucidated. A total of 48 paired HCCs and noncancerous tissues were used to detect loss of heterozygosity (LOH) and the methylation profiles of five tumor suppressor genes (RASSF1A, BLU, FHIT, CRBP1, and HLTF) on chromosome 3 by using polymerase chain reaction (PCR) and methylation-specific PCR. Gene expression was analyzed by immunohistochemistry and reverse transcription (RT)-PCR. Sixteen of 48 (33.3 %) HCCs had LOH on at least one locus on chromosome 3, and two smallest common deleted regions (3p22.3-24.3 and 3p12.3-14.2) were identified. RASSF1A, BLU, and FHIT showed very high frequencies of methylation in HCCs (100, 81.3, and 64.6 %, respectively) and noncancerous tissues, but not in liver tissues from control patients. Well-differentiated HCCs showed high methylation frequencies of these genes but very low frequencies of LOH. Furthermore, BLU methylation was associated with an increased level of alpha-fetoprotein, and FHIT methylation was inversely correlated with HCC recurrence. In comparison, CRBP1 showed moderate frequencies of methylation, while HLTF showed low frequencies of methylation, and CRBP1 methylation occurred mainly in elderly patients. Treatment with 5-aza-2'-deoxycytidine demethylated at least one of these genes and restored their expression in a DNA methylation-dependent or -independent manner. Hypermethylation of RASSF1A, BLU, and FHIT is a common and very early event in hepatocarcinogenesis; CRBP1 methylation may also be involved in the later stage. Although LOH was not too frequent on chromosome 3, it may play a role as another mechanism in hepatocarcinogenesis.

Impact of Drug-Resistance Polymerase Mutations on the Replication of HBeAg-Positive and HBeAg-Negative Hepatitis B Virus Strains in Vitro

Impact of Drug-Resistance Polymerase Mutations on the Replication of HBeAg-Positive and HBeAg-Negative Hepatitis B Virus Strains in Vitro

Upregulation of the Insulin Receptor and Type I Insulin-Like Growth Factor Receptor Are Early Events in Hepatocarcinogenesis

The molecular mechanisms underlying the development of hepatocellular carcinoma (HCC) are not yet fully understood. Preneoplastic foci of altered hepatocytes regularly precede HCC in various species. The predominant earliest type of foci of altered hepatocytes, the glycogen storage focus (GSF), shows an excess of glycogen (glycogenosis) in the cytoplasm. During progression from GSF to HCC, the stored glycogen is gradually reduced, resulting in complete loss in basophilic HCC. We have previously shown that in N-nitrosomorpholine-induced hepatocarcinogenesis, insulin receptor substrate (IRS-1) is strongly expressed in GSF and reduced during progression to HCC, thus correlating with the glycogen content. In the present study, we observed increased levels of insulin receptor, IGF-I receptor (IGF-IR), IRS-2, and mitogen-activated kinase/extracellular regulated kinase-1 in GSF, following the same pattern of expression as IRS-1. We conclude that the abundance of IRS-1, IRS-2, and mitogen-activated kinase/extracellular regulated kinase-1 coincides with a concerted upregulation of both IR and IGF-IR induced by the hepatocarcinogen. Our data suggest that in early hepatocellular preneoplasia, the upregulation of IR elicits glycogenosis through IRS-1 and/or IRS-2, whereas the increased level of the IGF-IR may lead to the increased cell proliferation previously reported in GSF. Therefore, the concerted upregulation of both IR and IGF-IR may represent initial events in hepatocarcinogenesis.

Modelling hepatocellular carcinoma (HCC) initiation in mice with biliary fibrosis reveals strain- and gender-specific apoptosis rates

Introduction: In most patients, hepatocellular carcinoma (HCC) develops as a consequence of advanced fibrosis but the mechanisms are still incompletely understood. Recently it has been shown that initiation of HCC can be modelled by a single dose of the hepatocarcinogen diethylnitrosamine (DEN) in mice (Naugler et al. Science 2007). ABCB4-deficient mice develop biliary liver fibrosis and can be used as a spontaneous model to dissect profibrotic pathways. To investigate the early events in hepatocarcinogenesis, our aim now was to examine tumour initiation in ABCB4-deficient mice.

Successful antiviral therapy determines a significant decrease in squamous cell carcinoma antigen‐associated (SCCA) variants’ serum levels in anti‐HCV positive cirrhotic patients*

Aberrant squamous cell carcinoma antigen (SCCA) expression is an early event in hepatocarcinogenesis, and increasing serum levels of SCCA variants IgM immune complexes (SCCA-IgM IC) have been found in cirrhotic patients developing hepatocellular carcinoma (HCC). We longitudinally evaluated a cohort of cirrhotic patients with hepatitis C virus infection (HCV) who underwent pegylated interferon (PEG-IFN) and ribavirin treatment. SCCA-IgM IC levels were assessed in the sera of 33 cirrhotic patients with HCV (21 males, median age 57 years) before, at the end and at 6-month and 1-year follow-up after treatment with PEG-IFN and ribavirin. SCCA-IgM IC serum levels (arbitrary units/mL, AU/mL) were evaluated according to treatment outcome: sustained virological response (SVR) vs nonresponse (NR). Overall, 15 patients obtained a SVR to antiviral therapy (45%). There was no significant difference in baseline SCCA-IgM IC serum levels between SVR and NR patients. When compared to baseline (451.2 AU/mL), SVR patients showed a significant decrease in median SCCA-IgM IC serum levels at the end of treatment (186.8 AU/mL, P = 0.013) and at both 6-month (96.8 AU/mL, P < 0.001) and 1-year follow-up (52.4 AU/mL, P < 0.001), while no significant modification was observed in NR patients. In patients with HCV-related liver cirrhosis, successful antiviral therapy is associated with a dramatic and significant decrease in SCCA-IC serum levels. Because of the pathophysiological correlation between SCCA and liver carcinogenesis, it is hypothesized that in patients with liver cirrhosis, SVR may be accompanied by a decreased proliferative stimulation.

Clinicopathologic significance of mitotic arrest defective protein 2 overexpression in hepatocellular carcinoma

Mitotic arrest defective protein 2 (MAD2) gene plays a central role in the mitotic checkpoint. Elevated MAD2 expression was observed in a number of human malignancies; its role in the development of hepatocellular carcinoma is still not understood and is controversial. The purpose of this study was to investigate the clinicopathologic significance of MAD2 expression in hepatocellular carcinoma. The MAD2 protein and its messenger RNA levels were measured in hepatocellular carcinomas, high-grade dysplastic nodules, and their paired nontumorous liver tissues by quantitative real-time polymerase chain reaction, Western blot, and immunohistochemistry. The results showed that MAD2 at both messenger RNA and protein levels was overexpressed in 8 of 9 high-grade dysplastic nodules and in 51 of 58 hepatocellular carcinomas, including 12 of 14 unifocal small hepatocellular carcinomas. There was a tendency for MAD2 expression to increase in the process of this multistep carcinogenesis. A significantly high tumor MAD2 immunostaining was associated with the progression of histologic grade and the overall low survival. In conclusion, MAD2 is overexpressed frequently in hepatocellular carcinoma, including high-grade dysplastic nodules and early-stage small hepatocellular carcinoma, indicating that overexpression of MAD2 plays a role in the development and progression of hepatocellular carcinoma. It may be an early event in hepatocarcinogenesis and could be used as a potential prognostic indicator.

Phosphorylated Heat Shock Protein 27 Represses Growth of Hepatocellular Carcinoma via Inhibition of Extracellular Signal-regulated Kinase

Heat shock protein 27, one of the low molecular weight stress proteins, is recognized as a molecular chaperone; however, other functions have not yet been well established. Phosphorylated heat shock protein 27 levels inversely correlate with the progression of human hepatocellular carcinoma. This study shows that phosphorylated heat shock protein 27 interferes with cell growth of the hepatocellular carcinoma-derived HuH7 cells in the presence of the proinflammatory cytokine, tumor necrosis factor-alpha, via inhibition of the sustained activation of the extracellular signal-regulated kinase signal pathway. The activities of Raf/extracellular signal-regulated kinase and subsequent activator protein-1 transactivation and the induction levels of cyclin D1 were lower in HuH7 cells transfected with phosphorylated heat shock protein 27 than those with unphosphorylated heat shock protein 27. Moreover, phosphorylated heat shock protein 27 up-regulated the levels of p38 mitogen-activated protein kinase and mitogen-activated protein kinase phosphatase-1, an inhibitory protein of extracellular signal-regulated kinase. These results indicate that phosphorylated heat shock protein 27 might suppress the extracellular signal-regulated kinase activity in the hepatocellular carcinoma cells via two separate pathways in an inflammatory state. The extracellular signal-regulated kinase activity is inversely correlated with phosphorylated heat shock protein 27 at serine 15 and also in human hepatocellular carcinoma tissues in vivo. Because the extracellular signal-regulated kinase signal pathway is a major proliferation signal of hepatocellular carcinoma, activator protein-1 activation is an early event in hepatocarcinogenesis. These findings strongly suggest that the control of the phosphorylated heat shock protein 27 levels could be a new therapeutic strategy especially to counter the recurrence of hepatocellular carcinoma.

DNA methyltransferase expression and DNA methylation in human hepatocellular carcinoma and their clinicopathological correlation

Aberrant DNA methylation on CpG islands is one of the most consistent epigenetic changes in human cancers, and the methylation process is catalyzed by DNA methyltransferase (DNMT). We evaluated i) the mRNA levels of three DNMTs; DNMT1, DNMT3a and DNMT3b, in 25 hepatocellular carcinomas (HCCs), in their corresponding non-cancerous liver tissues and in 7 normal livers by using real-time reverse transcriptase-polymerase chain reaction; ii) nuclear expression of DNMT1 and DNMT3a proteins in the HCCs by immunohistochemistry, iii) the methylation status of 5 genes; p16, p15, E-cadherin, HIC-1 and RASSF1A in the same tissues, and iv) the relationships between the above results and the clinicopathological characteristics, including prognosis. The differences in mRNA expression levels for DNMT1, DNMT3a and DNMT3b were statistically significant between HCC and normal livers (p<0.001), HCC and chronic hepatitis (p<0.001) and HCC and cirrhosis (p<0.001). An increase in mRNA expression levels of >4-fold for DNMT3b in HCCs was significantly associated with a poorer overall survival (p=0.027) and shorter metastasis-free survival (p=0.0299). A poorer recurrence-free survival was noted in HCCs with a >4-fold increase in DNMT3a mRNA (p=0.0120). The average numbers of methylated genes were 0, 1.27, 1.38 and 2.72 for normal livers, chronic hepatitis, cirrhosis and HCCs, respectively, and this progressive increase from normal livers to chronic hepatitis/cirrhosis through HCC may suggest that tumor suppressor gene methylation is an early event in hepatocarcinogenesis. These results first suggest that hepatocarcinogenesis involves an increased expression of DNMT1, DNMT3a and DNMT3b mRNA and a progressive increase in the number of methylated genes from normal liver, chronic hepatitis/cirrhosis to HCC and secondly that an increase in the DNMT3a and DNMT3b mRNA levels in HCCs relative to their non-cancerous tissues may be a predictor of poor survival.

Enhanced Self-Renewal Capability in Hepatic Stem/Progenitor Cells Drives Cancer Initiation

Transformed hematopoietic stem/progenitor cells with an enhanced or acquired self-renewal capability function as leukemic stem cells. In a variety of solid cancers, stem/progenitor cells could be also targets of carcinogenesis. However, it remains unclear whether disruption of stem cell function directly contributes to cancer initiation. We sought to elucidate the mechanisms of self-renewal in hepatic stem/progenitor cells and the relation between stem cell function and hepatocarcinogenesis. Functional analyses of polycomb-group protein Bmi1 and Wnt/beta-catenin, the molecules that are responsible for the self-renewal capability of many types of stem cells, were conducted in c-Kit(-)CD29(+)CD49f(+/low)CD45(-)Ter-119(-) hepatic stem/progenitor cells using retrovirus- or lentivirus-mediated gene transfer. The tumorigenicity of these cells transduced with the indicated retroviruses was also assessed by transplantation into nonobese diabetic/severe combined immunodeficient mice. Forced expression of Bmi1 and constitutively active beta-catenin mutant similarly promoted the self-renewal of hepatic stem/progenitor cells. The transplantation of Bmi1- or beta-catenin-transduced cells clonally expanded from single hepatic stem/progenitor cells produced tumors, which exhibited the histologic features of combined hepatocellular and cholangiocarcinoma. These observations imply that the dysregulated self-renewal of hepatic stem/progenitor cells serves as an early event in hepatocarcinogenesis, and they highlight the important roles of Bmi1 and the Wnt/beta-catenin pathway in regulating the self-renewal of normal or cancer stem cells in liver.

Telomerase Activation in Liver Regeneration and Hepatocarcinogenesis: Dr. Jekyll or Mr. Hyde?

The liver has a remarkable capability to restore its functional capacity following liver injury. According to the current paradigm, differentiated and usually quiescent hepatocytes are the primary cell type responsible for liver repair. As reserve compartment, bipotent hepatic progenitor cells are activated, especially if extensive loss or damage of hepatocytes with impaired replication occurs, e.g. in cirrhotic liver tissue. Recently, animal studies have suggested that liver regeneration following partial hepatectomy is associated with telomerase activation. Telomerase, a ribonucleoprotein with reverse transcriptase activity, plays a pivotal role in maintaining telomere length and chromosomal stability in proliferating cells. In cells lacking telomerase activity, replication-associated telomere shortening limits the replicative lifespan. Therefore, in the context of liver regeneration, telomerase activation might be a cellular mechanism to confer an extended lifespan to replicating hepatocytes and hepatic progenitor cells. On the other hand, high levels of telomerase activity are a hallmark of cancer, including hepatocellular carcinoma. Moreover, recent data indicate that telomerase activation may be an early event in hepatocarcinogenesis. At present, it is unclear, whether telomerase activation preserves the non-malignant phenotype and replicative longevity of liver cells or constitutes an early alteration obligatory for an unlimited proliferation and malignant transformation.

Downregulation of KLF6 is an early event in hepatocarcinogenesis, and stimulates proliferation while reducing differentiation

Hepatocellular carcinoma (HCC) has the most rapidly rising cancer incidence in the US and Europe. The KLF6 tumor suppressor is frequently inactivated in HCC by loss-of-heterozygosity (LOH) and/or mutation. Here we have analyzed 33 HBV- and 40 HCV-related HCCs for mRNA expression of wildtype KLF6 (wtKLF6) as well as the KLF6 variant 1 (SV1), a truncated, growth-promoting variant that antagonizes wtKLF6 function. The HCV-related tumors analyzed represented the full histologic spectrum from cirrhosis and dysplasia to metastatic cancer. Expression of KLF6 mRNA is decreased in 73% of HBV-associated HCCs compared to matched surrounding tissue (ST), with reductions of approximately 80% in one-third of the patients. KLF6 mRNA expression is also reduced in dysplastic nodules from patients with HCV compared to cirrhotic livers (p<0.005), with an additional, marked decrease in the very advanced, metastatic stage (p<0.05). An increased ratio of KLF6SV1/wt KLF6 is present in a subset (6/33, 18%) of the HBV-related HCCs compared to matched ST. Reconstituting KLF6 in HepG2 cells by retroviral infection decreased proliferation and related markers including cyclin D1 and beta-catenin, increased cellular differentiation based on induction of albumin, E-cadherin, and decreased alpha fetoprotein. We conclude that reduced KLF6 expression is common in both HBV- and HCV-related HCCs and occurs at critical stages during cancer progression. Effects of KLF6 are attributable to regulation of genes controlling hepatocyte growth and differentiation.

Distinct Chromosomal Bias of Gene Expression Signatures in the Progression of Hepatocellular Carcinoma

To identify the chromosomal aberrations associated with the progression of liver cancer, we applied expression imbalance map analysis to gene expression data from 31 hepatocellular carcinomas and 19 noncancerous tissues. Expression imbalance map analysis, which detects mRNA expression imbalance correlated with chromosomal regions, showed that expression gains of 1q21-23 (74%), 8q13-21 (48%), 12q23-24 (41%), 17q12-21(48%), 17q25 (25%), and 20q11 (22%) and losses of 4q13 (48%), 8p12-21 (32%), 13q14 (32%), and 17p13 (29%) were significantly associated with hepatocellular carcinoma. Most regions with altered expression identified by expression imbalance map were also identified in previous reports using comparative genomic hybridization. We demonstrated chromosomal copy number gain in 1q21-23 and loss in 17p13 by genomic quantitative PCR, suggesting that gene expression profiles reflect chromosomal alterations. Furthermore, expression imbalance map analysis revealed that more poorly differentiated hepatocellular carcinoma contain more chromosomal alterations, which are accumulated in a stepwise manner in the course of hepatocellular carcinoma progression: expression imbalance of 1q, 8p, 8q, and 17p occur as early events in hepatocarcinogenesis, and 12q, 17q25 and 20q occur as later events. In particular, expression gain of 17q12-21 and loss of 4q were seen to accumulate constantly through the dedifferentiation process. Our data suggest that gene expression profiles are subject to chromosomal bias and that expression imbalance map can correlate gene expression to gene loci with high resolution and sensitivity.

Association of low p16INK4a and p15INK4b mRNAs expression with their CpG islands methylation with human hepatocellular carcinogenesis.

To study the significance of p16 and p15 transcription suppression with hypermethylation of their genes' 5'CpG islands during human hepatocellular carcinogenesis. The mRNA expression levels of p16 and p15 genes were evaluated in cancerous, para-cancerous and non-cancerous tissues of 20 HCC, 3 normal liver tissues from 3 accidentally died healthy adults using semi-quantitatively Northern blot. The methylation status was also assessed with methylation specific PCR. p16 mRNA expression level was decreased in the cancerous tissues in 60% (12/20) of HCC patients, of which 2 cases had no p16 mRNA detected, 5 cases (25%) displayed variation in the order of cancerous<para-cancerous<non-cancerous liver tissues. p15 mRNA expression level was decreased in the cancerous tissues in 50% (10/20) HCC patients, of which one case had no p15 mRNA detected, 4 cases (20%) displayed variation in the order of cancerous<para-cancerous<non-cancerous liver tissues. In cancerous, para-cancerous and non-cancerous tissues, p16 promoter CpG islands hypermethylation occurred 65%, 60% and 35%, while p15 promoter CpG islands hypermethylation occurred 50%, 40% and 25%. Of 12 HCCs with lower p16 mRNA expression level, 11 cases showed p16 promoter CpG islands methylation (91.6%). Hundred percent (10/10) HCCs with lower p15 mRNA expression level showed p15 promoter CpG islands methylation. Significant correlation between 5'CpG islands methylation and p16/p15 mRNA expression suppression was found. The decreased expression of p16/p15 mRNA or methylation of p16/p15 promoters 5'CpG island was significantly correlate with poor differentiation of HCC (P=0.0083, 0.0102, 0.00271, 0.0218, respectively, P<0.05). p16 and p15 genes transcriptional inactivation might play an important role in hepatocarcinogenesis. 5'CpG islands methylation might be the major mechanism of p16 and p15 genes inactivation in primary HCC in the studied population. 5'CpG islands methylation of p16 and p15 genes might be an early event in hepatocarcinogenesis.