Methylation of the CpG sites only on the sense strand of the APC gene is specific for hepatocellular carcinoma

Hypermethylation of the promoter of the tumor suppressor gene, adenomatous polyposis coli (APC), occurs in various malignancies, including hepatocellular carcinoma (HCC). However, reports on the specificity of the methylation of the APC gene for HCC have varied. To gain insight into how these variations occur, bisulfite PCR sequencing was performed to analyze the methylation status of both sense and antisense strands of the APC gene in samples of HCC tissue, matched adjacent non-HCC liver tissue, hepatitis, cirrhosis, and normal liver tissues. DNA derived from fetal liver and 12 nonhepatic normal tissue was also examined. These experiments revealed liver-specific, antisense strand-biased CpG methylation of the APC gene and suggested that, although methylation of the antisense strand of the APC gene exists in normal liver and other non-HCC disease liver tissue, methylation of the sense strand of the APC gene occurs predominantly in HCC. To determine the effect of the DNA strand on the specificity of the methylated APC gene as a biomarker for HCC detection, quantitative methylation-specific PCR assays for sense and antisense strand DNA were developed and performed on DNA isolated from HCC (n = 58), matched adjacent non-HCC (n = 58), cirrhosis (n = 41), and hepatitis (n = 39). Receiver operating characteristic curves were constructed. With the cutoff value set at the limit of detection, the specificity of sense and antisense strand methylation was 84% and 43%, respectively, and sensitivity was 67.2% and 72.4%, respectively. This result demonstrated that the identity of the methylated DNA strand impacted the specificity of APC for HCC detection. Interestingly, methylation of the sense strand of APC occurred in 40% of HCCs from patients with serum AFP levels less than 20 ng/mL, suggesting a potential role for APC as a biomarker to complement AFP in HCC screening.     

Monitoring liver alterations during hepatic tumorigenesis by NMR profiling and pattern recognition

Human hepatocellular carcinoma (HCC) is the most recurrent malignancy of the liver and represents one of the main causes of cancer death worldwide. Furthermore, the liver is the most frequent site of metastatic colonization, and hepatic metastases are far more common than primary cancers in Western countries. A possible way of investigating liver diseases is to study the tissue metabolic profiles. High-resolution nuclear magnetic resonance (NMR) spectroscopy of hepatic tissue extracts was combined with pattern-recognition and visualization techniques to uncover metabolic differences among analyzed tissue types. Extracts were from primary HCC, chronic hepatitis-C-virus related cirrhotic tissues, hepatic metastases from colorectal carcinomas, and non-cirrhotic normal liver tissues adjacent to metastases as controls. We identified all metabolites present in the NMR spectra, and after statistical evaluation of all spectral classes, we were able to define the metabolic changes underlying the different liver conditions and diseases. In particular, the lactate and the glucose tissue signals were found to primarily discriminate the different histological samples. We followed the biochemical changes of human hepatic lesions through primary (HCC) and secondary (metastases from colorectal carcinoma) liver tumors, cirrhotic tissues, and non-cirrhotic histologically-confirmed normal liver tissues adjacent to metastases, achieving a metabolic differentiation of the various pathological states based upon the variation of the intracellular lactate/glucose ratio. It is suggested that such a signal pattern may act as a potential marker for assessing pathological hepatic lesions.     

Comparison of pathways associated with hepatitis B- and C-infected hepatocellular carcinoma using pathway-based class discrimination method

Molecular signatures causing hepatocellular carcinoma (HCC) from chronic infection of hepatitis B virus (HBV) or hepatitis C virus (HCV) are not clearly known. Using microarray datasets composed of HCV-positive HCC or HBV-positive HCC, pathways that could discriminate tumor tissue from adjacent non-tumor liver tissue were selected by implementing nearest shrunken centroid algorithm. Cancer-related signaling pathways and lipid metabolism-related pathways were predominantly enriched in HCV-positive HCC, whereas functionally diverse pathways including immune-related pathways, cell cycle pathways, and RNA metabolism pathways were mainly enriched in HBV-positive HCC. In addition to differentially involved pathways, signaling pathways such as TGF-β, MAPK, and p53 pathways were commonly significant in both HCCs, suggesting the presence of common hepatocarcinogenesis process. The pathway clustering also verified segregation of pathways into the functional subgroups in both HCCs. This study indicates the functional distinction and similarity on the pathways implicated in the development of HCV- and/or HBV-positive HCC.     

Expression and significance of new tumor suppressor gene PTEN in primary liver cancer

Objective : To investigate expression and significance of PTEN gene in primary hepatocellular carcinoma (HCC). Methods: Immunohistochemical peroxidase-conjugated streptavidin (SP) method was used to detect expression of PTEN gene in 120 cases of primary HCC and its adjacent tissue 10 cases of normal liver tissue. The relationship between expression of tumor suppressor gene of PTEN and the percentage of lymph node metastasis of HCC was analyzed. Results: It was shown that PTEN gene was expressed in all 10 cases of normal liver tissues and paracancerous liver tissues. The staining was localized mainly in cytoplasm. Expression of PTEN in 120 cases of HCC were as follows: 12.5% were negative, 17.5% were weak positive, and 70% were strong positive. At time of diagnosis, 33/120 (27.5%) presented lymph node metastasis. Lymph node metastases were present in 80% (12 out of 15) PTEN negative HCC, 57.14% (12 out of 21) PTEN weak positive HCC and only 10.71% (9 out of 84) PTEN intense positive HCC, ( P <0.05). Therefore, PTEN tumor suppresor gene malfunction seems to be involed in mtastasing capacity of HCC. Conclusion: This study suggests that PTEN gene was deleted or weakly expressed in primary hepatocellar carcinoma, which is probably related to its tumorigenesis.     

Overexpression of alpha enolase in hepatitis C virus-related hepatocellular carcinoma: association with tumor progression as determined by proteomic analysis

To identify proteins that could be molecular targets for diagnosis and treatment of hepatitis C virus-related hepatocellular carcinoma (HCV-related HCC), we used a proteomic approach to analyze protein expression in samples of human liver. Twenty-six pairs of tumorous and corresponding nontumorous liver samples from patients with HCV-related HCC and six normal liver samples were analyzed by two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry. One of the numerous spots that showed stronger intensity in tumorous than in nontumorous samples was identified as alpha enolase, a key enzyme in the glycolytic pathway. Expression of this protein increased with tumor dedifferentiation and was significantly higher in poorly differentiated HCC than in well-differentiated HCC. This pattern was reproduced by immunoblot analysis and immunohistochemistry. Expression of alpha enolase also correlated positively with tumor size and venous invasion. These results suggest that alpha enolase is one of the candidates for biomarkers for tumor progression that deserves further investigation in HCV-related HCC.     

Global Gene Expression Profiling Reveals SPINK1 as a Potential Hepatocellular Carcinoma Marker

Background

Liver cirrhosis is the most important risk factor for hepatocellular carcinoma (HCC) but the role of liver disease aetiology in cancer development remains under-explored. We investigated global gene expression profiles from HCC arising in different liver diseases to test whether HCC development is driven by expression of common or different genes, which could provide new diagnostic markers or therapeutic targets.

Methodology and Principal Findings

Global gene expression profiling was performed for 4 normal (control) livers as well as 8 background liver and 7 HCC from 3 patients with hereditary haemochromatosis (HH) undergoing surgery. In order to investigate different disease phenotypes causing HCC, the data were compared with public microarray repositories for gene expression in normal liver, hepatitis C virus (HCV) cirrhosis, HCV-related HCC (HCV-HCC), hepatitis B virus (HBV) cirrhosis and HBV-related HCC (HBV-HCC). Principal component analysis and differential gene expression analysis were carried out using R Bioconductor. Liver disease-specific and shared gene lists were created and genes identified as highly expressed in hereditary haemochromatosis HCC (HH-HCC) were validated using quantitative RT-PCR. Selected genes were investigated further using immunohistochemistry in 86 HCC arising in liver disorders with varied aetiology. Using a 2-fold cut-off, 9 genes were highly expressed in all HCC, 11 in HH-HCC, 270 in HBV-HCC and 9 in HCV-HCC. Six genes identified by microarray as highly expressed in HH-HCC were confirmed by RT qPCR. Serine peptidase inhibitor, Kazal type 1 (SPINK1) mRNA was very highly expressed in HH-HCC (median fold change 2291, p = 0.0072) and was detected by immunohistochemistry in 91% of HH-HCC, 0% of HH-related cirrhotic or dysplastic nodules and 79% of mixed-aetiology HCC.

Conclusion

HCC, arising from diverse backgrounds, uniformly over-express a small set of genes. SPINK1, a secretory trypsin inhibitor, demonstrated potential as a diagnostic HCC marker and should be evaluated in future studies.     

Comparative proteomic and transcriptomic profiling of the human hepatocellular carcinoma

Proteome analysis of human hepatocellular carcinoma (HCC) was done using two-dimensional difference gel electrophoresis. To gain an understanding of the molecular events accompanying HCC development, we compared the protein expression profiles of HCC and non-HCC tissue from 14 patients to the mRNA expression profiles of the same samples made from a cDNA microarray. A total of 125 proteins were identified, and the expression profiles of 93 proteins (149 spots) were compared to the mRNA expression profiles. The overall protein expression ratios correlated well with the mRNA ratios between HCC and non-HCC (Pearson’s correlation coefficient: r = 0.73). Particularly, the HCC/non-HCC expression ratios of proteins involved in metabolic processes showed significant correlation to those of mRNA (r = 0.9). A considerable number of proteins were expressed as multiple spots. Among them, several proteins showed spot-to-spot differences in expression level and their expression ratios between HCC and non-HCC poorly correlated to mRNA ratios. Such multi-spotted proteins might arise as a consequence of post-translational modifications.     

Colon-Derived Liver Metastasis,Colorectal Carcinoma,and Hepatocellular Carcinoma Can Be Discriminated by the Ca2+-Binding Proteins S100A6 and S100A11

Background

It is unknown, on the proteomic level, whether the protein patterns of tumors change during metastasis or whether markers are present that allow metastases to be allocated to a specific tumor entity. The latter is of clinical interest if the primary tumor is not known.

Methodology/Principal Findings

In this study, tissue from colon-derived liver metastases (n = 17) were classified, laser-microdissected, and analysed by ProteinChip arrays (SELDI). The resulting spectra were compared with data for primary colorectal (CRC) and hepatocellular carcinomas (HCC) from our former studies. Of 49 signals differentially expressed in primary HCC, primary CRC, and liver metastases, two were identified by immunodepletion as S100A6 and S100A11. Both proteins were precisely localized immunohistochemically in cells. S100A6 and S100A11 can discriminate significantly between the two primary tumor entities, CRC and HCC, whereas S100A6 allows the discrimination of metastases and HCC.

Conclusions

Both identified proteins can be used to discriminate different tumor entities. Specific markers or proteomic patterns for the metastases of different primary cancers will allow us to determine the biological characteristics of metastasis in general. It is unknown how the protein patterns of tumors change during metastasis or whether markers are present that allow metastases to be allocated to a specific tumor entity. The latter is of clinical interest if the primary tumor is not known.     

Role of cadherin-17 in oncogenesis and potential therapeutic implications in hepatocellular carcinoma

Cadherin is an important cell adhesion molecule that plays paramount roles in organ development and the maintenance of tissue integrity. Dysregulation of cadherin expression is often associated with disease pathology including tissue dysplasia, tumor formation, and metastasis. Cadherin-17 (CDH17), belonging to a subclass of 7D-cadherin superfamily, is present in fetal liver and gastrointestinal tract during embryogenesis, but the gene becomes silenced in healthy adult liver and stomach tissues. It functions as a peptide transporter and a cell adhesion molecule to maintain tissue integrity in epithelia. However, recent findings from our group and others have reported aberrant expression of CDH17 in major gastrointestinal malignancies including hepatocellular carcinoma (HCC), stomach and colorectal cancers, and its clinical association with tumor metastasis and advanced tumor stages. Furthermore, alternative splice isoforms and genetic polymorphisms of CDH17 gene have been identified in HCC and linked to an increased risk of HCC. CDH17 is an attractive target for HCC therapy. Targeting CDH17 in HCC can inhibit tumor growth and inactivate Wnt signaling pathway in concomitance with activation of tumor suppressor genes. Further investigation on CDH17-mediated oncogenic signaling and cognate molecular mechanisms would shed light on new targeting therapy on HCC and potentially other gastrointestinal malignancies.     

Predicting hepatitis B virus-positive metastatic hepatocellular carcinomas using gene expression profiling and supervised machine learning

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Its high mortality rate is mainly a result of intra-hepatic metastases. We analyzed the expression profiles of HCC samples without or with intra-hepatic metastases. Using a supervised machine-learning algorithm, we generated for the first time a molecular signature that can classify metastatic HCC patients and identified genes that were relevant to metastasis and patient survival. We found that the gene expression signature of primary HCCs with accompanying metastasis was very similar to that of their corresponding metastases, implying that genes favoring metastasis progression were initiated in the primary tumors. Osteopontin, which was identified as a lead gene in the signature, was over-expressed in metastatic HCC; an osteopontin-specific antibody effectively blocked HCC cell invasion in vitro and inhibited pulmonary metastasis of HCC cells in nude mice. Thus, osteopontin acts as both a diagnostic marker and a potential therapeutic target for metastatic HCC.     

Prevalence and characteristics of hepatitis C virus-related hepatocellular carcinoma in a Spanish university hospital

BackgroundHepatocellular Carcinoma (HCC) arises in chronic liver diseases, particularly caused by hepatitis C virus (HCV) and alcohol in Europe. We aimed at evaluating the characteristics and mortality of patients with HCV-related HCC as compared to other HCC etiologies.MethodsWe retrospectively evaluated data from 887 patients with HCC identified through the Hospital del Mar Cancer Registry (Barcelona, Spain), during the 2001–2020 period. We estimated crude and adjusted hazard ratios (aHR) of dying and its 95% confidence interval (95%CI).ResultsAmong 887 patients with HCC, 617 (69.6%) were HCV-infected. Underlying cirrhosis was more frequent in HCV-related HCC compared to other etiologies (97% vs. 89%, p < 0.001). The prevalence of HCV-related HCC decreased from 79% in 2001–2005 to 55% in 2015–2020 (p < 0.001). HCV infection did not increase the hazard of death [aHR 0.95 (CI95% 0.81–1.13)]. Mortality was independently related to age > 75 years, advanced BCLC stage at diagnosis, and diagnosis before 2010.ConclusionIn our cohort, HCV-related HCC frequently occurred in a cirrhotic background, but showed similar clinical characteristics and mortality as compared to other HCC etiologies.     

Epigenetic mechanisms in hepatocellular carcinoma: how environmental factors influence the epigenome

Epigenetic mechanisms maintain heritable changes in gene expression and chromatin organization over many cell generations. Importantly, deregulated epigenetic mechanisms play a key role in a wide range of human malignancies, including liver cancer. Hepatocellular carcinoma (HCC), which originates from the hepatocytes, is by far the most common liver cancer, with rates and aetiology that show considerable geographic variation. Various environmental agents and lifestyles known to be risk factors for HCC (such as infection by hepatitis B virus (HBV) and hepatitis C virus (HCV), chronic alcohol intake, and aflatoxins) are suspected to promote its development by eliciting epigenetic changes, however the precise gene targets and underlying mechanisms have not been elucidated. Many recent studies have exploited conceptual and technological advances in epigenetics and epigenomics to investigate the role of epigenetic events induced by environmental factors in HCC tumors and non-tumor precancerous (cirrhotic) lesions. These studies have identified a large number of genes and pathways that are targeted by epigenetic deregulation (changes in DNA methylation, histone modifications and RNA-mediated gene silencing) during the development and progression of HCC. Frequent identification of aberrant epigenetic changes in specific genes in cirrhotic tissue is consistent with the notion that epigenetic deregulation of selected genes in pre-malignant lesions precedes and promotes the development of HCC. In addition, several lines of evidence argue that some environmental factors (such as HBV virus) may abrogate cellular defense systems, induce silencing of host genes and promote HCC development via an "epigenetic strategy". Finally, profiling studies reveal that HCC tumors and pre-cancerous lesions may exhibit epigenetic signatures associated with specific risk factors and tumor progression stage. Together, recent evidence underscores the importance of aberrant epigenetic events induced by environmental factors in liver cancer and highlights potential targets for biomarker discovery and future preventive and therapeutic strategies.     

Gene signatures derived from a c-MET-driven liver cancer mouse model predict survival of patients with hepatocellular carcinoma

Biomarkers derived from gene expression profiling data may have a high false-positive rate and must be rigorously validated using independent clinical data sets, which are not always available. Although animal model systems could provide alternative data sets to formulate hypotheses and limit the number of signatures to be tested in clinical samples, the predictive power of such an approach is not yet proven. The present study aims to analyze the molecular signatures of liver cancer in a c-MET-transgenic mouse model and investigate its prognostic relevance to human hepatocellular carcinoma (HCC). Tissue samples were obtained from tumor (TU), adjacent non-tumor (AN) and distant normal (DN) liver in Tet-operator regulated (TRE) human c-MET transgenic mice (n = 21) as well as from a Chinese cohort of 272 HBV- and 9 HCV-associated HCC patients. Whole genome microarray expression profiling was conducted in Affymetrix gene expression chips, and prognostic significances of gene expression signatures were evaluated across the two species. Our data revealed parallels between mouse and human liver tumors, including down-regulation of metabolic pathways and up-regulation of cell cycle processes. The mouse tumors were most similar to a subset of patient samples characterized by activation of the Wnt pathway, but distinctive in the p53 pathway signals. Of potential clinical utility, we identified a set of genes that were down regulated in both mouse tumors and human HCC having significant predictive power on overall and disease-free survival, which were highly enriched for metabolic functions. In conclusions, this study provides evidence that a disease model can serve as a possible platform for generating hypotheses to be tested in human tissues and highlights an efficient method for generating biomarker signatures before extensive clinical trials have been initiated.     

Expression of cyclooxygenase-2 and transforming growth factor-beta1 in HCV-induced chronic liver disease and hepatocellular carcinoma

Cyclooxygenase-2 (COX-2) and transforming growth factor-beta1 (TGF-beta1) were modulated in a variety of viral infections, but there is a paucity of data about their role in the pathologic process of cirrhosis and/or hepatocellular carcinoma (HCC) following chronic hepatitis C virus (HCV) infection. The material of the current study included 50 cases of chronic hepatitis C (CHC) without cirrhosis, 30 cases of CHC with cirrhosis, and 30 cases of HCC with HCV admitted to the Gastroenterology and Hepatology Department of Theodor Bilharz Research Institute, Giza, Egypt. Fifteen wedge liver biopsies, taken during laparoscopic cholecystectomy, were included in the study as normal controls. Laboratory investigations, serologic markers for viral hepatitis, and serum alpha fetoprotein levels (alpha-FP) were done for all cases of the study. Immunohistochemistry using primary antibodies against both factors revealed weak to faint immunoreactivity to COX-2 and TGF-beta1 in normal hepatic tissue (< 30% and < 50% of the cells, respectively). COX-2 expression was upregulated in patients with CHC with and without cirrhosis, yet 80% of positively stained cirrhotic cases showed marked staining intensity. Higher COX-2 expression was observed in well-differentiated HCC cases (80%) with marked staining intensity (75%) compared with advanced HCC tumors (P < .001). TGF-beta1 was expressed in the hepatocytes of all cases of CHC with and without cirrhosis as well as in 67% of HCC cases. Extensive cytoplasmic expression was detected in 52%, 93.3%, and 46.6% of CHC patients without cirrhosis, patients with cirrhosis, and patients with HCC, respectively. A positive correlation was observed between hepatic expression of COX-2 and TGF-beta1 (r = 0.67, P < .05); however, no correlation was detected between the latter and grade of HCC differentiation (r = 0.33, P > .05). CONCLUSION: These findings may suggest that TGF-beta1 plays a role in hepatic cell damage following HCV infection thus stressing the usefulness of this cytokine as a prognostic marker for liver cell injury. However, COX-2 is a predictive marker for malignant transformation and has a role in the early stages of hepatocarcinogenesis, but not in the advanced stages. The combined expression of both factors in HCV-related HCC suggests their synergistic action in the pathophysiology of hepatocarcinogenesis.     

Novel tumor-associated antigen of hepatocellular carcinoma defined by monoclonal antibody E4-65

A monoclonal antibody, E4-65, produced by immunizing mice with SMMC-7721 cells, a human hepatocellular carcinoma (HCC) cell line, was used to identify and characterize an unreported HCC-associated antigen. Indirect immunofluorescence studies showed that E4-65 antibody reacted with five out of eight HCC cell lines, but not with 10 non-HCC tumor cell lines or a normal liver cell line. Using immunohistochemical examination, E4-65 antigen was detected on the cell membranes and in the cytoplasm of human liver tumor tissues, but was not found in most other tumors, or normal adult or fetal tissues, except for a weakly positive reaction in tissues of the digestive system. Western blot analysis showed that E4-65 antibody bound to a 45 kDa protein in the human HCC cell line and tissue lysates. Enzyme treatment and lectin blotting did not detect the carbohydrate chain in E4-65 antigen. This HCC-associated protein represents a potentially useful target for diagnoses and immunotherapy of human HCC.     

Integrative genomics: liver regeneration and hepatocellular carcinoma

Numerous genome wide profiles of gene expression changes in human hepatocellular carcinoma (HCC), compared to normal liver tissue, have been reported. Hierarchical clustering of these data reveal distinct patterns, which underscore conservation between human disease and mouse models of HCC, as well as suggest specific classification of subtypes within the heterogeneous disease of HCC. Global profiling of gene expression in mouse liver, challenged by partial hepatectomy to regenerate, reveals alterations in gene expression that occur in response to acute injury, inflammation, and re-entry into cell cycle. When we integrated datasets of gene expression changes in mouse models of HCC and those that are altered at specific times of liver regeneration, we saw shared, conserved alterations in gene expression within specific biological pathways, both up-regulated, for example, cell cycle, cell death, and cellular development, or down-regulated, for example, vitamin and mineral metabolism, lipid metabolism, and molecular transport. Additional molecular mechanisms shared by liver regeneration and HCC, as yet undiscovered, may have important implications in tumor development and recurrence. These comparisons may offer a way to judge how liver resection, in the treatment of HCC, introduces challenges to care of the disease. Further, uncovering the pathways conserved in inflammatory response, hypertrophy, proliferation, and architectural remodeling of the liver, which are shared in liver regeneration and HCC, versus those specific to tumor development and progression in HCC, may reveal new biomarkers or potential therapeutic targets in HCC.     

Enhanced expression of mRNAs of antisecretory factor-1, gp96, DAD1 and CDC34 in human hepatocellular carcinomas

To identify differentially expressed genes in hepatocarcinogenesis, we performed differential display analysis using surgically resected hepatocellular carcinoma (HCC) and adjacent non-tumorous liver tissues. We identified four cDNA fragments upregulated in HCC samples, encoding antisecretory factor-1 (AF), gp96, DAD1 and CDC34. Northern blot analysis demonstrated that these mRNAs were expressed preferentially in HCCs compared with adjacent non-tumorous liver tissues or normal liver tissues from non-HCC patients. The expression of these mRNAs was increased along with the histological grading of HCC tissues. These mRNA levels were also high in three human HCC cell lines (HuH-7, HepG2 and HLF), irrespective of the growth state. We also demonstrate that sodium butyrate, an inducer of differentiation, downregulated the expression of AF and gp96 mRNAs, supporting in part our pathological observation. Immunohistochemical analysis revealed that gp96 and CDC34 proteins were preferentially accumulated in cytoplasm and nuclei of HCC cells, respectively. Overexpression of these genes could be an important manifestation of HCC phenotypes and should provide clues to understand the molecular basis of hepatocellular carcinogenesis.