Specific gene expression patterns in liver cirrhosis

Liver cirrhosis (LC) is a complex disease that can develop into hepatocellular carcinoma (HCC). In an effort to investigate genetic differences between LC and HCC, we used cDNA microarray analysis to characterize the gene expression profiles in LC and HCC tissues. Consistent differences were observed among the expression patterns in LC, HCC, and normal liver tissues. Interestingly, the expression patterns of LC without tumor association (LCT) were also readily distinguished from those of LC tissues near hepatic tumor tissues (near-tumor tissue, NTT). Moreover, 25 cirrhosis-specific genes could be used to divide the NTT samples into two groups: inflammatory active cirrhosis (NTTa) and inflammatory inactive cirrhosis (NTTi). We found that NTTa samples showed gene expression patterns similar to those of the LCT and HCC groups, whereas the expression patterns of the NTTi group were significantly different from those of the LCT, NTTa, and HCC groups. Finally, we selected two of the 25 LC-specific genes and showed that these markers could be used to successfully discriminate among the different LC subtypes. Collectively, these novel results allow the identification of new genetic subgroups of LC and provide new candidate genes for use as early markers for active cirrhosis and HCC.     

Molecular Signatures Associated with HCV-Induced Hepatocellular Carcinoma and Liver Metastasis

Hepatocellular carcinomas (HCCs) are a heterogeneous group of tumors that differ in risk factors and genetic alterations. In Italy, particularly Southern Italy, chronic hepatitis C virus (HCV) infection represents the main cause of HCC. Using high-density oligoarrays, we identified consistent differences in gene-expression between HCC and normal liver tissue. Expression patterns in HCC were also readily distinguishable from those associated with liver metastases. To characterize molecular events relevant to hepatocarcinogenesis and identify biomarkers for early HCC detection, gene expression profiling of 71 liver biopsies from HCV-related primary HCC and corresponding HCV-positive non-HCC hepatic tissue, as well as gastrointestinal liver metastases paired with the apparently normal peri-tumoral liver tissue, were compared to 6 liver biopsies from healthy individuals. Characteristic gene signatures were identified when normal tissue was compared with HCV-related primary HCC, corresponding HCV-positive non-HCC as well as gastrointestinal liver metastases. Pathway analysis classified the cellular and biological functions of the genes differentially expressed as related to regulation of gene expression and post-translational modification in HCV-related primary HCC; cellular Growth and Proliferation, and Cell-To-Cell Signaling and Interaction in HCV-related non HCC samples; Cellular Growth and Proliferation and Cell Cycle in metastasis. Also characteristic gene signatures were identified of HCV-HCC progression for early HCC diagnosis.

Conclusions

A diagnostic molecular signature complementing conventional pathologic assessment was identified.     

C-myc、p53基因和AFP在家兔诱发性肝癌及癌旁组织中的表达

目的获得家兔二乙基亚硝胺诱发肝脏肿瘤的形态学资料,探讨Cmyc、p53基因和甲胎蛋白的表达情况。方法取5例家兔多结节肝癌的肿瘤性结节25个,瘤旁肝组织每例1块,进行一般病理组织学和超微结构观察。同时,以免疫组化方法检查Cmyc、p53基因和AFP的表达情况。结果和结论25个结节中,良性增生10个,高分化肝细胞癌11个,中分化肝细胞癌4个。家兔肝细胞癌的发生与Cmyc基因、AFP的过表达和p53基因的突变有关。参照肿瘤诱发过程中,增生性肝硬化、腺瘤样增生、高分化癌、中分化癌的变化顺序,在所检测的三种标志物中,Cmyc蛋白为最早出现的肿瘤标志物,p53基因突变相对较晚出现,AFP在恶性肿瘤结节中的表达率最高(933%)。     

Genomic expression patterns distinguish long-term from short-term glioblastoma survivors: a preliminary feasibility study

We used microarray analysis to investigate associations between genotypic expression profiles and survival phenotypes in patients with primary glioblastoma (GBM). Tumor samples from 7 long-term glioblastoma survivors (>24 months) and 13 short-term survivors (<9 months) were analyzed to detect differential patterns of gene expression between these groups and to identify genotypic subclasses of glioblastomas that correlate with survival phenotypes. Five unsupervised and three supervised clustering algorithms consistently and accurately grouped the tumors into genotypic subgroups corresponding to the two clinical survival phenotypes. Three unique prospective mathematical classification algorithms were subsequently trained to use expression data to stratify unknown glioblastomas between survival groups and performed this task with 100% accuracy in validation studies. A set of 1478 genes with significant differential expression (p<0.01) between long-term and short-term survivors was identified, and additional mathematical filtering was used to isolate a 43-gene "fingerprint" that distinguished survival phenotypes. Differential regulation of a subset of these genes was confirmed using RT-PCR. Gene ontology analysis of the fingerprint demonstrated pathophysiologic functions for the gene products that are consistent with current models of tumor biology, suggesting that differential expression of these genes may contribute etiologically to the observed differences in survival. These results demonstrate that unique expression profiles characterize genotypic subsets of primary GBMs associated with differential survival phenotypes, and these profiles can be used in a prospective fashion to assign unknown tumors to survival groups. Future efforts will focus on building more robust classifiers and identifying additional subclasses of gliomas with phenotypic significance.     

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.     

BRE over-expression promotes growth of hepatocellular carcinoma

BRE, also known as TNFRSF1A modulator and BRCC45, is an evolutionarily highly conserved protein. It is a death receptor-associated protein in cytoplasm and a component of BRCA1/2-containing DNA repair complex in nucleus. BRE was found to have anti-apoptotic activity. Over-expression of BRE by transfection promoted survival of cell lines against apoptotic induction; whereas depletion of the protein by siRNA resulted in the opposite. In vivo anti-apoptotic activity of BRE was demonstrated by significant attenuation of Fas-induced acute fulminant hepatitis in transgenic mice expressing the human protein specifically in the liver. BRE was also implicated in tumor promotion by the accelerated tumor growth of Lewis Lung carcinoma transfected with human BRE; and by high expression of BRE specifically in the tumoral regions of human hepatocellular carcinoma (HCC). The present study was to test directly if transgenic expression of BRE in livers could promote HCC development in neonatal diethylnitrosamine model. By 8 months after tumor induction, the maximal sizes of tumor nodules of transgenic mice were significantly larger than those of the non-transgenic controls, although the numbers of tumor nodules between the two groups did not significantly differ. Importantly, as in human HCC, the mouse endogenous BRE level was up-regulated in mouse HCC nodules. These results show that BRE over-expression can indeed promote growth, though not initiation, of liver tumors. Furthermore, the common occurrence of BRE over-expression in human and mouse HCC suggests that up-regulation of BRE is functionally important in liver tumor development.     

Deficiency of the Promyelocytic Leukemia Protein Fosters Hepatitis C-Associated Hepatocarcinogenesis in Mice

Overwhelming lines of epidemiological evidence have indicated that persistent infection with hepatitis C virus (HCV) is a major risk for the development of hepatocellular carcinoma (HCC). We have recently shown that HCV core protein mediates functional inactivation of the promyelocytic leukemia (PML) tumor suppressor pathway. However, the role of PML in HCC development yet remains unclear. To clarify the function of PML in liver carcinogenesis and HCV-associated pathogenesis we crossed PML-deficient mice with HCV transgene (HCV-Tg) expressing mice and treated the resulting animals with DEN/Phenobarbital, an established protocol for liver carcinogenesis. Seven months after treatment, livers were examined macroscopically and histologically. Genetic depletion of the tumor suppressor PML coincided with an increase in hepatocyte proliferation, resulting in development of multiple dysplastic nodules in 100% of the PML-deficient livers and of HCCs in 53%, establishing a tumor suppressive function of PML in the liver. In animals expressing the HCV-transgene in PML-deficient background, HCC development occurred even in 73%, while only 7% of their wildtype littermates developed HCC. The neoplastic nature of the tumors was confirmed by histology and expression of the HCC marker glutamine synthetase. Several pro- and antiapoptotic factors were tested for differential expression and liver carcinogenesis was associated with impaired expression of the proapoptotic molecule TRAIL in PML-deficient mice. In conclusion, this study provides first in vivo evidence that the tumor suppressor PML acts as an important barrier in liver carcinogenesis and HCV-dependent liver pathology.     

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.     

Methylation Profiles Reveal Distinct Subgroup of Hepatocellular Carcinoma Patients with Poor Prognosis

Hepatocellular Carcinoma (HCC) is one of the leading causes of cancer-associated mortality worldwide. However, the role of epigenetic changes such as aberrant DNA methylation in hepatocarcinogenesis remains largely unclear. In this study, we examined the methylation profiles of 59 HCC patients. Using consensus hierarchical clustering with feature selection, we identified three tumor subgroups based on their methylation profiles and correlated these subgroups with clinicopathological parameters. Interestingly, one tumor subgroup is different from the other 2 subgroups and the methylation profile of this subgroup is the most distinctly different from the non-tumorous liver tissues. Significantly, this subgroup of patients was found to be associated with poor overall as well as disease-free survival. To further understand the pathways modulated by the deregulation of methylation in HCC patients, we integrated data from both the methylation as well as the gene expression profiles of these 59 HCC patients. In these patients, while 4416 CpG sites were differentially methylated between the tumors compared to the adjacent non-tumorous tissues, only 536 of these CpG sites were associated with differences in the expression of their associated genes. Pathway analysis revealed that forty-four percent of the most significant upstream regulators of these 536 genes were involved in inflammation-related NFκB pathway. These data suggest that inflammation via the NFκB pathway play an important role in modulating gene expression of HCC patients through methylation. Overall, our analysis provides an understanding on aberrant methylation profile in HCC patients.     

中国启东肝癌高发区肝癌中AKR1C2基因异常表达及其在肝癌发生中作用(英文)

已在许多肿瘤中发现AKR1C2基因的异常表达 .为研究启东肝癌中AKR1C2基因异常表达的意义及其在肝癌发生中作用 .通过制备兔抗人AKR1C2多克隆抗体、免疫组化、蛋白质印迹、RT PCR、RNA印迹、原位杂交、cDNA表达芯片、免疫共沉淀、体内外致瘤试验等方法 ,对 68例启东肝癌标本、 8例正常肝组织、QGY 770 3启东肝癌细胞株中AKR1C2表达及作用进行分析 .并研究了AKR 1C2蛋白、mRNA表达与肝癌临床病理特征 ,侵袭性间关系 .研究表明正常及癌旁肝组织中AKR1C2蛋白为膜染色 ,偶见弱的细胞浆染色 .95 3 %肝癌显示胞浆或核染的累积型 .癌及癌旁肝组织中标记指数(LI)分别为 61 4± 2 7 8,10 2± 8 7(P <0 . 0 1) .较高的LI与HCC侵袭性密切相关 .蛋白质印迹显示癌组织中AKR1C2表达升高 .RT PCR显示 ,肝癌中AKR1C2表达指数 (EI)高于癌旁及正常组织 ,而且存在序列差异 .RNA印迹显示 91 2 %为上调表达 .原位杂交显示肝癌细胞胞浆中染色强于癌旁及正常肝 .AKR1C2过表达与肝癌转移潜能有关 .AKR1C2过表达刺激QGY770 3细胞中DNA合成与阻止细胞凋亡 .转染AKR1C2基因的QGY770 3细胞在软琼脂上集落形成能力增强 ,并能促进QGY770 3在裸鼠体内肿瘤形成能力 .cDNA表达芯片显示转染AKR1C2后导致QGY770 3细胞中一些基因表达改变 .AKR     

Variation in gene expression patterns in human gastric cancers

Gastric cancer is the world's second most common cause of cancer death. We analyzed gene expression patterns in 90 primary gastric cancers, 14 metastatic gastric cancers, and 22 nonneoplastic gastric tissues, using cDNA microarrays representing approximately 30,300 genes. Gastric cancers were distinguished from nonneoplastic gastric tissues by characteristic differences in their gene expression patterns. We found a diversity of gene expression patterns in gastric cancer, reflecting variation in intrinsic properties of tumor and normal cells and variation in the cellular composition of these complex tissues. We identified several genes whose expression levels were significantly correlated with patient survival. The variations in gene expression patterns among cancers in different patients suggest differences in pathogenetic pathways and potential therapeutic strategies.     

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.     

MicroRNA Profiling of Laser-Microdissected Hepatocellular Carcinoma Reveals an Oncogenic Phenotype of the Tumor Capsule

Several microRNAs (miRNAs) are associated with the molecular pathogenesis of hepatocellular carcinoma (HCC). However, previous studies analyzing the dysregulation of miRNAs in HCC show heterogeneous results. We hypothesized that part of this heterogeneity might be attributable to variations of miRNA expression deriving from the HCC capsule or the fibrotic septa within the peritumoral tissue used as controls. Tissue from surgically resected hepatitis C–associated HCC from six well-matched patients was microdissected using laser microdissection and pressure catapulting technique. Four distinct histologic compartments were isolated: tumor parenchyma (TP), fibrous capsule of the tumor (TC), tumor-adjacent liver parenchyma (LP), and cirrhotic septa of the tumor-adjacent liver (LC). MiRNA expression profiling analysis of 1105 mature miRNAs and precursors was performed using miRNA microarray. Principal component analysis and consecutive pairwise supervised comparisons demonstrated distinct patterns of expressed miRNAs not only for TP versus LP (e.g., intratumoral down-regulation of miR-214, miR-199a, miR-146a, and miR-125a; P< .05) but also for TC versus LC (including down-regulation within TC of miR-126, miR-99a/100, miR-26a, and miR-125b; P< .05). The tumor capsule therefore demonstrates a tumor-like phenotype with down-regulation of well-known tumor-suppressive miRNAs. Variations of co-analyzed fibrotic tissue within the tumor or in controls may have profound influence on miRNA expression analyses in HCC. Several miRNAs, which are proposed to be HCC specific, may indeed be rather associated to the tumor capsule. As miRNAs evolve to be important biomarkers in liver tumors, the presented data have important translational implications on diagnostics and treatment in patients with HCC.     

−509C>T polymorphism in the TGF-β1 gene promoter,impact on the hepatocellular carcinoma risk in Chinese patients with chronic hepatitis B virus infection

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. The risk for developing HCC increases with severity of inflammation and fibrosis. Transforming growth factor-β1 (TGF-β1) is most frequently upregulated in tumor cells. The most studied −509C>T polymorphism of TGF-β1 gene has been associated with colorectal, gynecologic, and lung cancers. To assess whether this polymorphism in TGF-β1 gene is associated with susceptibility to and/or clinicopathologic characteristics of HBV-related HCC, a total of 575 patients with chronic HBV infection and 299 healthy volunteers with no evidence of recent or remote HBV infection were prospectively enrolled. The patients were divided into two groups: those without (n = 196) and those with HCC (n = 379). These 379 HCC patients with chronic HBV infection were designated as cases, the remaining 196 patients without HCC and 299 healthy volunteers served as disease and healthy controls, respectively. −509C>T polymorphism in the TGF-β1 gene promoter was studied using restriction fragment-length polymorphism. In addition, tumor tissues of liver (n = 60) were obtained from the studied HCC patients for measurement of TGF-β1 mRNA expression levels. We also assessed the plasma TGF-β1 levels of HBV patients without (n = 94) or with HCC (n = 136) and healthy subjects (n = 120). In our study group, the risk of HCC in Chinese patients with HBV infection was significantly lower with the TT genotypes than in those with the CC genotypes at position −509 of TGF-β1 gene (P = 0.01). In addition, in the case group, patients with the CC genotype had a statistically significant higher median plasma TGF-β1 or liver tumor tissue TGF-β1 mRNA level compared with the individuals with the TT genotype. However, in a subsequent analysis of the association between this polymorphism and clinicopathological characteristics including tumor number, size, grade, stage, and invasiveness, there was no significant difference in both the distribution of genotype or allelic frequency within HCC patients, indicating that −509C>T exchange in TGF-β1 gene may play an important role in the occurrence, not the progression of HBV-related HCC through influencing plasma concentrations of TGF-β1 or TGF-β1 mRNA expression of liver tumor tissue.     

Epithelial–mesenchymal transition markers expressed in circulating tumor cells in hepatocellular carcinoma patients with different stages of disease

The presence of circulating tumor cells (CTCs) in peripheral blood is associated with metastasis and prognosis in hepatocellular carcinoma (HCC) patients. The epithelial–mesenchymal transition (EMT) has a pivotal role in tumor invasion and dissemination. To identify more sensitive biomarkers for evaluating metastasis and prognosis, we investigated the expression of EMT markers, including vimentin, twist, ZEB1, ZEB2, snail, slug and E-cadherin in CTCs, primary HCC tumors and adjacent non-tumoral liver tissues. After isolating viable CTCs from the peripheral blood of HCC patients using asialoglycoprotein receptors (ASGPRs), the CTCs were identified with immunofluorescence staining. CTCs were detected in the peripheral blood obtained from 46 of 60 (76.7%) HCC patients. Triple-immunofluorescence staining showed that twist and vimentin expression could be detected in CTCs obtained from 39 (84.8%) and 37 (80.4%) of the 46 patients, respectively. The expression of both twist and vimentin in CTCs was significantly correlated with portal vein tumor thrombus. Coexpression of twist and vimentin in CTCs could be detected in 32 (69.6%) of the 46 patients and was highly correlated with portal vein tumor thrombus, TNM classification and tumor size. Quantitative fluorescence western blot analysis revealed that the expression levels of E-cadherin, vimentin and twist in HCC tumors were significantly associated with the positivity of isolated CTCs (P=0.013, P=0.012, P=0.009, respectively). However, there was no significant difference in ZEB1, ZEB2, snail and slug expression levels in CTCs, primary HCC tumors and adjacent non-tumoral liver tissues across samples with regard to the clinicopathological parameters. Our results demonstrate that the EMT has a role in promoting the blood-borne dissemination of primary HCC cells, and the twist and vimentin expression levels in CTCs could serve as promising biomarkers for evaluating metastasis and prognosis in HCC patients.