Phenotype Of Hepatocellular Carcinoma Research Articles

Artificial neural networks and decision tree model analysis of liver cancer proteomes

Hepatocellular carcinoma (HCC) is a heterogeneous cancer and usually diagnosed at late advanced tumor stages of high lethality. The present study attempted to obtain a proteome-wide analysis of HCC in comparison with adjacent non-tumor liver tissues, in order to facilitate biomarkers’ discovery and to investigate the mechanisms of HCC development. A cohort of 66 Chinese patients with HCC was included for proteomic profiling study by two-dimensional gel electrophoresis (2-DE) analysis. Artificial neural network (ANN) and decision tree (CART) data-mining methods were employed to analyze the profiling data and to delineate significant patterns and trends for discriminating HCC from non-malignant liver tissues. Protein markers were identified by tandem MS/MS. A total of 132 proteome datasets were generated by 2-DE expression profiling analysis, and each with 230 consolidated protein expression intensities. Both the data-mining algorithms successfully distinguished the HCC phenotype from other non-malignant liver samples. The detection sensitivity and specificity of ANN were 96.97% and 87.88%, while those of CART were 81.82% and 78.79%, respectively. The three biological classifiers in the CART model were identified as cytochrome b5, heat shock 70 kDa protein 8 isoform 2, and cathepsin B. The 2-DE-based proteomic profiling approach combined with the ANN or CART algorithm yielded satisfactory performance on identifying HCC and revealed potential candidate cancer biomarkers.

Up-regulation of Cyclin D1 by HBx Is Mediated by NF-κB2/BCL3 Complex through κB Site of Cyclin D1 Promoter

Up-regulation of Cyclin D1 by HBx Is Mediated by NF-κB2/BCL3 Complex through κB Site of Cyclin D1 Promoter

Proteomic analysis on metastasis-associated proteins of human hepatocellular carcinoma tissues

A comparative proteomic approach was used to identify and analyze proteins related to metastasis of hepatocellular carcinoma (HCC). Proteins extracted from 12 HCC tissue specimens (six with metastases and six without) were separated by two-dimensional gel electrophoresis (2-DE). The protein spots exhibiting statistical alternations between the two groups through computerized image analysis were then identified by mass spectrometry. In addition immunohistochemistry (IHC), Western blotting and RT-PCR were performed to verify the expression of certain candidate proteins. 16 proteins including HSP27, S100A11, CK18 were annotated by mass spectrometry, relevant to chaperone function, cell mobility, cytoskeletal architecture, respectively. Most were previously unconnected with metastasis of HCC. Of these HSP27 was found overexpressed consistently in 2-DE patterns of all metastatic HCC tissues compared with nonmetastatic ones. IHC and Western blotting of HCC tissues confirmed this difference while RT-PCR did not. There are various proteins joined together in HCC metastasis. The overexpression of HSP27 may serve as a biomarker for early detection and therapeutic targets unique to the metastatic phenotype of HCC.

Microsatellite instability mutator phenotype in hepatocellular carcinoma in non-alcoholic and non-virally infected normal livers.

Microsatellite instability (MSI) seems to be a rare event in hepatocarcinogenesis and might actually be associated with the progression of hepatocellular carcinoma (HCC) in which the liver is often the site of chronic hepatitis or cirrhosis. The aim of this work was to define the MSI phenotype in HCC affecting exclusively normal livers to avoid slippage errors due to cirrhosis. One hundred and sixty-four patients with HCC affecting non-cirrhotic livers were operated on in our hospital between 1984 and 2001. We analyzed 37 patients selected for low alcohol consumption and the absence of HBV or HCV infection. All the livers were histologically normal. MSI was analyzed according to the criteria defined during the conference consensus workshop for colorectal cancer. High MSI (MSI-H > 30%) was found in 6 (16%) and low MSI (MSI-L < 30%) in 10 (27%) of the 37 HCCs. None of the 10 microsatellite markers tested were altered in the remaining 21 tumors (57%). Immunohistochemistry showed that normal amounts of hMLH1 and hMSH2 were present both in MSI-H and in MSI-L HCCs. MSI-H was significantly associated with more aggressive histological tumor features and a shorter median delay before recurrence. Thus, we have found a small subgroup of HCC tumors which can be considered as a new clinical/histological entity.

The prognostic molecular markers in hepatocellular carcinoma.

The prognosis of hepatocellular carcinoma (HCC) still remains dismal, although many advances in its clinical study have been made. It is important for tumor control to identify the factors that predispose patients to death. With new discoveries in cancer biology, the pathological and biological prognostic factors of HCC have been studied quite extensively. Analyzing molecular markers (biomarkers) with prognostic significance is a complementary method. A large number of molecular factors have been shown to associate with the invasiveness of HCC, and have potential prognostic significance. One important aspect is the analysis of molecular markers for the cellular malignancy phenotype. These include alterations in DNA ploidy, cellular proliferation markers (PCNA, Ki-67, Mcm2, MIB1, MIA, and CSE1L/CAS protein), nuclear morphology, the p53 gene and its related molecule MD M2, other cell cycle regulators (cyclin A, cyclin D, cyclin E, cdc2, p27, p73), oncogenes and their receptors (such as ras, c-myc, c-fms, HGF, c-met, and erb-B receptor family members), apoptosis related factors (Fas and FasL), as well as telomerase activity. Another important aspect is the analysis of molecular markers involved in the process of cancer invasion and metastasis. Adhesion molecules (E-cadherin, catenins, serum intercellular adhesion molecule-1, CD44 variants), proteinases involved in the degradation of extracellular matrix (MMP-2, MMP-9, uPA, uPAR, PAI), as well as other molecules have been regarded as biomarkers for the malignant phenotype of HCC, and are related to prognosis and therapeutic outcomes. Tumor angiogenesis is critical to both the growth and metastasis of cancers including HCC, and has drawn much attention in recent years. Many angiogenesis-related markers, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PD-ECGF), thrombospondin (TSP), angiogenin, pleiotrophin, and endostatin (ES) levels, as well as intratumor microvessel density (MVD) have been evaluated and found to be of prognostic significance. Body fluid (particularly blood and urinary) testing for biomarkers is easily accessible and useful in clinical patients. The prognostic significance of circulating DNA in plasma or serum, and its genetic alterations in HCC are other important trends. More attention should be paid to these two areas in future. As the progress of the human genome project advances, so does a clearer understanding of tumor biology, and more and more new prognostic markers with high sensitivity and specificity will be found and used in clinical assays. However, the combination of some items, i.e., the pathological features and some biomarkers mentioned above, seems to be more practical for now.

Expression of the multidrug resistance proteins MRP2 and MRP3 in human hepatocellular carcinoma.

Treatment of hepatocellular carcinoma (HCC) by chemotherapy is often impeded by the intrinsic multidrug resistance (MDR) of this frequent primary cancer of the liver. The MDR phenotype can be caused by ATP-dependent export of chemotherapeutic drugs across the plasma membrane being mediated by transporters of the MDR P-glycoprotein family or of the multidrug resistance protein (MRP) family. To elucidate the role of MRP family members in HCC, we analyzed the expression and subcellular localization of MRP1 (ABCC1), MRP2 (ABCC2) and MRP3 (ABCC3); all 3 isoforms have been shown to confer resistance to chemotherapeutic drugs. Semiquantitative RT-PCR demonstrated that MRP2 and MRP3 mRNA expression in HCC was at least 10-fold higher than MRP1 mRNA expression. MRP2 immunostaining was observed in 87% (33/38) of HCC samples. MRP2 was localized in the plasma membrane in a polarized fashion, either in trabecular structures resembling the canalicular membrane or in the luminal membrane when cells had a pseudoglandular arrangement. MRP3 was detected in all samples examined (9/9) by RT-PCR and by immunofluorescence microscopy. MRP3 was localized to the basolateral membrane of carcinoma cells. Double-label immunofluorescence microscopy with antibodies specific for MRP2 or MRP3 indicated that carcinoma cells expressed both MRP isoforms simultaneously. When MRP1 was detected by immunofluorescence microscopy, it was localized on the intracellular membranes of carcinoma cells. Thus, plasma membrane expression of MRP2 and MRP3, but not of MRP1, can contribute to the MDR phenotype of 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.

Inhibitory effect of IGF- II antisense RNA on malignant phenotype of hepatocellular carcinoma.

Inhibitory effect of IGF-II antisense RNA on malignant phenotype of hepatocellular carcinoma

Immunohistochemical studies on endothelial cell phenotype in hepatocellular carcinoma.

To examine the phenotype of the sinusoidal endothelial cells (SECs) surrounding tumor cells and the process of capillarization in hepatocellular carcinoma (HCC), 51 primary HCCs, 4 adrenal metastases, and 3 portal tumor thrombi were immunohistochemically stained with monoclonal antibodies (MAbs) for CD4, CD14 (lipopolysaccharide-binding protein complex receptors), and CD32 (Fc gamma receptor II), which are specifically found on the SECs in normal liver, but not on ordinary vascular endothelial cells (ECs). Immunostaining was also performed for CD36 (thrombospondin receptors), EN4 antigen (Ag) (a pan-vascular endothelial cell Ag), PAL-E Ag (a venous and capillary EC Ag), factor VIII-related Ag (FVIIIRAg), and laminin. MAb 25F9, which identifies macrophages, was simultaneously used with the other MAbs to distinguish macrophages from SECs in HCCs (HCC SECs). CD4, CD14, and/or CD32 were found on HCC SECs only in 12 well-differentiated primary HCCs showing a thin trabecular or pseudoglandular tumor cell arrangement. These 12 tumors were smaller than those without CD4-, CD14-, and/or CD32-positive SECs (P < .05). Among them, 7, 5, and 11 tumors were negative or only partially positive for laminin, PAL-E Ag, and FVIIIRAg, respectively. Staining for laminin and PAL-E Ag showed an inverse relationship to the expression of CD4, CD14, and CD32 on HCC SECs and the tumor differentiation. In conclusion, the phenotypes of the SECs in early and well-differentiated HCC are thought to be similar to those of the SECs in normal liver. With progressing tumor dedifferentiation the HCC SECs lose the phenotypes peculiar to liver SECs and acquire the characteristics of capillary ECs, though both types of phenotypical change occur independently of each other.

Expression of the integrin? 5 subunit and its mediated cell adhesion in hepatocellular carcinoma

Tumor invasion and metastasis are complex processes, requiring the ability of tumor cells to interact with proteins of the extracellular matrix through cell-adhesion molecules on the cell surface. Integrins are heterodimeric membrane glycoproteins, consisting of alpha and beta subunits, which enable cells to recognize adhesive substrates in the extracellular matrix. The roles of the integrin alpha5beta1 in tumor invasion are highlighted by finding that some tumor cells have lost or reduced alpha5beta1 expression. It therefore functions as a negative signaling regulator. Expression of alpha5beta1 and its mediation of cell adhesion in hepatocellular carcinoma (HCC) have not been elucidated. In surgical specimens of HCC we found, by immunohistochemistry and Northern blot analysis, that the alpha5-positive rates in cancerous tissues were lower than the corresponding rates in non-cancerous tissues. Reduced expression of the integrin alpha5 occurred more frequently in HCC with more malignant phenotypes, such as poor differentiation, large size (more than 10-cm in diameter), absence of capsule and high invasion. Reverse transcription/polymerase chain reaction, a more sensitive assay, was used to detect the alpha5 mRNA level in LCID20, a highly metastatic model of human HCC, and LCID35, a low-metastasis model. The results showed that integrin alpha5 was negative in the former and positive in the latter. Cell adhesion assays showed the maximal percentage inhibition of anti-alpha5 mAb on SMMC 7721 cell adhesion to fibronectin to be 68.9 +/- 4.9% at the saturation concentrations of each antibody (200 microg/ml). If anti-alpha5 mAb was combined with anti-beta1 mAb, the inhibition was 74.1 +/- 11.1%. It is concluded that reduced expression of the integrin alpha5 subunit is correlated with more malignant phenotypes of human HCC. Any change in the adhesion of hepatocellular carcinoma cells to fibronectin is mainly dependent upon the function of the integrin alpha5beta1.

Molecular characterization of S-adenosylmethionine synthetase in human liver cancer

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.

The expression of C-myc and N-ras oncogenes in human hepatocellular carcinoma—Anin situ hybridization study on paraffin embedded tissue sections

In this research, we investigated the expression of C-myc and N-ras mRNAs on 21 cases paraffin-embedded tissue sections of hepatocellular carcinoma (HCC) usingin situ hybridization technique with biotinylated labelled cDNA probes. Of 21 cases of hepatoma, C-myc mRNA was positive-expressed in 9 cases (42.9%) and N-ras positive in 4 cases (19%) in hepatoma cells, and C-myc and N-ras positive in 4 and 1 cases respectively in peritumor hepatocytes. C-myc mRNAs were localized within cytoplasms of both hepatoma cells and peritumor hepatocytes. However, the positive intensities of C-myc and N-ras mRNAs in hepatoma cells were much greater than those in peritumor hepatocytes. The results indicated that C-myc and N-ras oncogenes were overexpressed in HCC, and may play an important role in coordinatively maintaince of the malignant phenotypes in HCC.

C- erbB-2 oncogene expression in hepatocellular carcinoma and cholangiocarcinoma

The c- erbB-2 proto-oncogene encodes a transmembrane protein which is homologous to the epidermal growth factor receptor. This protein can be localized immunohistochemically in formalin-fixed paraffin-embedded material using a monoclonal antibody NCL-CB11; positive membrane staining correlates with gene amplification and protein overexpression in breast cancer. Using this technique we have shown that only 2 26 (8%) of hepatocellular carcinomas, 0 10 (0%) of cholangiocarcinomas and 0 2 (0%) hepatoblastomas overexpressed c- erbB-2 as evidenced by membrane staining. Moreover c- erbB-2 mRNA was not detected in seven hepatocellular carcinomas examined by Northern blot analysis. c- erbB-2 overexpression is, therefore, unlikely to be contributing to the malignant phenotype in hepatocellular carcinoma and cholangiocarcinoma.

Glycolysis module activated by hypoxia-inducible factor 1α is related to the aggressive phenotype of hepatocellular carcinoma

An increased level of glycolysis, an intracellular hallmark of neoplasms, enables cancer cells to survive under various conditions. To elucidate the role of increased glycolysis in the progression of hepatocellular carcinoma (HCC), we investigated the associations between the expression patterns of 14 glycolysis-related genes and clinicopathologic factors in 60 HCCs by using pooled transcriptome data. We then evaluated the therapeutic efficacy of the knockdown of ENO1, which is encoded by a glycolysis-related gene, in HCC cells. Among the 14 genes, levels of 8 genes (GPI, ALDOA, TPI1, GAPD, PGK, PGAM, ENO1 and PKM), all of which can be transcriptionally activated by hypoxia-inducible factor 1alpha (HIF-1alpha), were significantly higher in HCC with venous invasion (VI) than in HCC without VI. Our cluster analysis showed that HCC patients with activation of the 8 HIF-1alpha-regulated genes had significantly shorter overall survival (P=0.023) than did HCC patients without increased expression levels of these genes. The association between the levels of ENO1 and VI was confirmed in an independent sample set of 49 HCCs by real-time reverse-transcription PCR. The knockdown of ENO1 by small-interfering RNA significantly inhibited the proliferation of an HCC cell line (HLE cells) in both the glucose-rich and glucose-free conditions, accompanied by a decreased S phase and increased G2/M phase of the cell cycle. Collectively, these data suggest that activation of an HIF-1alpha-regulated glycolysis module is closely related to the aggressive phenotype of HCC, and that ENO1, a glycolysis module gene, might serve as a new target to circumvent HCC metastasis.

Alpha-1-Antitrypsin phenotypes in hepatocellular carcinoma.

alpha-1-Antitrypsin deficiency due to homozygous Pi ZZ state is reported to be associated with cirrhosis and hepatocellular carcinoma (HCC); however, the role of heterozygous Pi Z state is not definitely known. In order to investigate the possible association, we studied the phenotypic distribution of alpha-1-antitrypsin variants (Pi) in 124 cases of HCC. Two thousand ten normal American Red Cross blood donors were studied as controls. Twelve patients with HCC had aberrant phenotypes, an incidence of 9.67% as compared to 8.36% among normal controls. Nine of 12 patients with HCC with aberrant Pi type had cirrhosis; 5 of the 9 had cirrhosis due to hepatitis B virus; 2 of the 9 had alcoholic liver disease with cirrhosis, and 2 had cryptogenic cirrhosis. The three patients with HCC arising in noncirrhotic livers who also had aberrant Pi phenotypes, had a relatively rare variety of HCC called fibrolamellar type. Z gene was found in five patients: all five were MZ. Incidence of MZ phenotype in HCC was similar to that of the normal control population (4.0% in HCC and 2.9% in the controls). However, 3 of 5 MZ were associated with fibrolamellar HCC. Another aberrant phenotype found among the patients with HCC was MF (fast moving) which occurred with an incidence of 2.41% as compared to none in the control group. In conclusion, we found no significant increase in the incidence of Z gene among 124 patients with HCC as compared to the normal population.