Molecular Mechanisms Of Hepatocarcinogenesis Research Articles

Screening Prognosis-Related lncRNAs Based on WGCNA to Establish a New Risk Score for Predicting Prognosis in Patients with Hepatocellular Carcinoma.

Background Hepatocellular carcinoma (HCC) remains an important cause of cancer death. The molecular mechanism of hepatocarcinogenesis and prognostic factors of HCC have not been completely uncovered. Methods In this study, we screened out differentially expressed lncRNAs (DE lncRNAs), miRNAs (DE miRNAs), and mRNAs (DE mRNAs) by comparing the gene expression of HCC and normal tissue in The Cancer Genome Atlas (TCGA) database. DE mRNAs were used to perform Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Then, the miRNA and lncRNA/mRNA modules that were most closely related to the survival time of patients with HCC were screened to construct a competitive endogenous RNA (ceRNA) network by weighted gene coexpression network analysis (WGCNA). Moreover, univariable Cox regression and Kaplan-Meier curve analyses of DE lncRNAs and DE mRNAs were conducted. Finally, the lasso-penalized Cox regression analysis and nomogram model were used to establish a new risk scoring system and predict the prognosis of patients with liver cancer. The expression of survival-related DE lncRNAs was verified by qRT-PCR. Results A total of 1896 DEmRNAs, 330 DElncRNAs, and 76 DEmiRNAs were identified in HCC and normal tissue samples. Then, the turquoise miRNA and turquoise lncRNA/mRNA modules that were most closely related to the survival time of patients with HCC were screened to construct a ceRNA network by WGCNA. In this ceRNA network, there were 566 lncRNA-miRNA-mRNA regulatory pairs, including 30 upregulated lncRNAs, 16 downregulated miRNAs, and 75 upregulated mRNAs. Moreover, we screened out 19 lncRNAs and 14 hub mRNAs related to prognosis from this ceRNA network by univariable Cox regression and Kaplan-Meier curve analyses. Finally, a new risk scoring system was established by selecting the optimal risk lncRNAs from the 19 prognosis-related lncRNAs through lasso-penalized Cox regression analysis. In addition, we established a nomogram model consisting of independent prognostic factors to predict the survival rate of HCC patients. Finally, the correlation between the risk score and immune cell infiltration and gene set enrichment analysis were determined. Conclusions In conclusion, the results may provide potential biomarkers or therapeutic targets for HCC and the establishment of the new risk scoring system and nomogram model provides the new perspective for predicting the prognosis of HCC.

Transgenic expression of tgfb1a induces hepatic inflammation, fibrosis and metastasis in zebrafish

TGFB signaling pathway plays a key role on liver disease progression. In our previous study, we have demonstrated the oncogenic ability of Tgfb signaling pathway as a chronic induction of tgfb1a specifically in hepatocytes led to both hepatocellular carcinoma (HCC) and cholangiocarcinoma in zebrafish. Here we would like to examine the potential mechanisms of Tgfb1a induced tumorigenesis. As majority of HCC developed from the background of liver inflammation and fibrosis, by immune-fluorescent staining on markers of liver inflammation, we indeed observed a progressively increased liver inflammation during tumorigenesis. Examination of liver fibrosis also revealed marked increase of liver fibrosis during early liver tumorigenesis and it was dramatically dropped in late liver tumorigenesis. Hence, induction of tgfb1a drives HCC through association of liver inflammation and fibrosis. Furthermore, we found high expression of EMT markers in late liver tumorigenesis, indicating a tumor metastasis potential. These observations are generally consistent with the molecular mechanisms of hepatocarcinogenesis in human.

Molecular Mechanisms of Hepatocarcinogenesis Following Sustained Virological Response in Patients with Chronic Hepatitis C Virus Infection.

Despite the success of direct-acting antiviral (DAA) agents in treating chronic hepatitis C virus (HCV) infection, the number of cases of HCV-related hepatocellular carcinoma (HCC) is expected to increase over the next five years. HCC develops over the span of decades and is closely associated with fibrosis stage. HCV both directly and indirectly establishes a pro-inflammatory environment favorable for viral replication. Repeated cycles of cell death and regeneration lead to genomic instability and loss of cell cycle control. DAA therapy offers >90% sustained virological response (SVR) rates with fewer side effects and restrictions than interferon. While elimination of HCV helps to restore liver function and reverse mild fibrosis, post-SVR patients remain at elevated risk of HCC. A series of studies reporting higher than expected rates of HCC development among DAA-treated patients ignited debate over whether use of DAAs elevates HCC risk compared to interferon. However, recent prospective and retrospective studies based on larger patient cohorts have found no significant difference in risk between DAA and interferon therapy once other factors are taken into account. Although many mechanisms and pathways involved in hepatocarcinogenesis have been elucidated, our understanding of drivers specific to post-SVR hepatocarcinogenesis is still limited, and lack of suitable in vivo and in vitro experimental systems has hampered efforts to examine etiology-specific mechanisms that might serve to answer this question more thoroughly. Further research is needed to identify risk factors and biomarkers for post-SVR HCC and to develop targeted therapies based on more complete understanding of the molecules and pathways implicated in hepatocarcinogenesis.

비만과 간세포암

Obesity is closely associated with hepatocellular carcinoma (HCC) as well as other malignancies. Obesity is an important risk factor for cancer development and overall mortality in HCC. Molecular mechanisms of hepatocarcinogenesis in obesity are adipose tissue remodeling, dysregulation of adipokines, increased reactive oxygen species, insulin resistance or hyperinsulinemia, alteration of gut microbiota, and dysregulation of microRNA. Obesity is the most common cause of non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). NAFLD or NASH leads to HCC as well as liver cirrhosis. Hepatitis C virus regulates lipid homeostasis in liver. Obesity and its’ related factors (metabolic syndrome and diabetes mellitus) are significantly related to the risk of HCC development in chronic hepatitis C. However, it is not clear whether obesity is a risk factor for HCC in chronic hepatitis B. The relationship between obesity and HCC seems to be different according to etiology of background liver disease. Further studies are needed to clarify the effect of obesity on HCC in different etiologies of chronic liver disease.

Abstract 5346: Identification of essential genes for the development of hepatitis B virus-associated hepatocellular carcinoma

Abstract Background and Aim: Hepatocellular carcinoma (HCC) is a highly lethal and prevalent cancer, posing a grave threat to human health globally. Hepatitis B virus (HBV) infection is considered as a major risk factor for this cancer, especially in the Asia-Pacific region. Unfortunately, the molecular mechanisms of hepatocarcinogenesis remain obscure, which hinders the development of effective therapies for the disease. In the present study, we attempted to elucidate the molecular details of HBV-induced hepatocarcinogenesis by investigating differentially regulated genes at multiple developmental stages of HCC in a HBV transgenic mouse model. Materials and Methods: The transgenic mice which overproduced HBV large envelope polypeptide in hepatocytes and developed liver tumors spontaneously were used in this study. To unravel transcriptomics dynamics underlying hepatocarcinogenesis, RNA prepared from livers of both transgenic and wild type mice of different ages (at months 2, 12, 18 and 19) were subjected to RNA sequencing. Selected target genes were first validated by quantitative PCR (qPCR) using a larger set of mouse liver tissues (n=96) collected from 8 time points. Clinical implications of the selected genes were then explored in a set of human liver samples comprising 18 normal, 29 cirrhosis and 96 pairs of HCC. RNA and protein expression levels were determined by qPCR, immunohistochemical staining and Western blotting, respectively. Results: Upon analysis of 20,209 gene transcripts, 2574 and 1035 transcripts were found to be up-regulated (≥2 folds) and down-regulated (≤2 folds) in tumors, respectively, when compared with the wild type controls. Among these, 133 most prominent genes that exhibited concordant differential expression throughout the stages of tumor progression were chosen for validation in mouse liver tissues. Correlation analysis showed a high correlation between RNA sequencing and qPCR data (r=0.7495; P<0.0001), indicating a high validity of the data. Forty-six biologically informative genes were further validated in human liver samples. By Gene Ontology analysis, the target genes were revealed to play roles in a variety of biological processes including stress and inflammation responses, metabolic and apoptotic processes. Immunohistochemical staining and Western blotting demonstrated significant differential expression of these genes between HCC and non-tumorous livers. Statistical analyses revealed their significant correlation with clinicopathological parameters including venous infiltration, tumor size and overall survival, implicating their roles in hepatocarcinogenesis. Conclusion: This study has demonstrated a systematic strategy for identifying crucial genes for HBV-associated HCC, which may have profound implications in combating this deadly cancer. Citation Format: CT Lam, ZF Yang, JC Lau, MN Ng, WC Yu, DW Ho, ST Fan. Identification of essential genes for the development of hepatitis B virus-associated hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5346. doi:10.1158/1538-7445.AM2014-5346

MicroRNA-101 inhibits human hepatocellular carcinoma progression through EZH2 downregulation and increased cytostatic drug sensitivity

Oncogene polycomb group protein enhancer of zeste homolog 2 (EZH2) has been proposed to be a target gene of putative tumor suppressor microRNA-101 (miR-101). The aim of our study was to investigate the functional role of both miR-101 and EZH2 in human hepatocellular carcinoma (HCC). MiR-101 and EZH2 expressions were evaluated in tumor tissues of 99 HCC patients and 7 liver cancer cell lines by real-time PCR. Luciferase reporter assay was employed to validate whether EZH2 represents a target gene of miR-101. The effect of miR-101 on HCC growth as well as programmed cell death was studied in vitro and in vivo. MiR-101 expression was significantly downregulated in most of HCC tissues and all cell lines, whereas EZH2 was significantly overexpressed in most of HCC tissues and all cell lines. There was a negative correlation between expression levels of miR-101 and EZH2. Luciferase assay results confirmed EZH2 as a direct target gene of miR-101, which negatively regulates EZH2 expression in HCC. Ectopic overexpression of miR-101 dramatically repressed proliferation, invasion, colony formation as well as cell cycle progression in vitro and suppressed tumorigenicity in vivo. Furthermore, miR-101 inhibited autophagy and synergized with either doxorubicin or fluorouracil to induce apoptosis in tumor cells. Tumor suppressor miR-101 represses HCC progression through directly targeting EZH2 oncogene and sensitizes liver cancer cells to chemotherapeutic treatment. Our findings provide significant insights into molecular mechanisms of hepatocarcinogenesis and may have clinical relevance for the development of novel targeted therapies for HCC.

Molecular mechanisms of hepatocarcinogenesis in chronic hepatitis C virus infection

Hepatitis C virus (HCV) infection is a major cause of hepatocellular carcinoma (HCC) and chronic liver disease worldwide. Recent developments and advances in HCV replication systems in vitro and in vivo, transgenic animal models, and gene expression profiling approaches have provided novel insights into the mechanisms of HCV replication. They have also helped elucidate host cellular responses, including activated/inactivated signaling pathways, and the relationship between innate immune responses by HCV infection and host genetic traits. However, the mechanisms of hepatocyte malignant transformation induced by HCV infection are still largely unclear, most likely due to the heterogeneity of molecular paths leading to HCC development in each individual. In this review, we summarize recent advances in knowledge about the mechanisms of hepatocarcinogenesis induced by HCV infection.

Abstract 234: Transcription factor LSF is a novel oncogene for hepatocellular carcinoma (HCC)

Abstract While the incidence of overall cancers is decreasing, Hepatocellular Carcinoma (HCC) is one of the few cancers the incidence of which is on an increase. The mortality rate of HCC parallels that of incidence since HCC is a highly aggressive cancer with extremely limited currently available therapeutic strategies. Understanding the molecular mechanism of hepatocarcinogenesis will help develop novel, targeted and more effective therapeutic regimen to counteract this fatal disease. Here, we demonstrate that the cellular transcriptional factor Late SV40 Factor (LSF) functions as an oncogene in HCC. The level of LSF expression is significantly increased in human HCC patients compared to matched non-neoplastic controls and normal liver and LSF expression significantly correlates with the stages and grades of HCC. Ectopic overexpression of LSF in nontumorigenic human HCC cells leads to highly aggressive, multi-organ metastatic tumors while inhibition of LSF in aggressive human HCC cells profoundly inhibits tumorigenic and metastatic properties in athymic nude mice. Affymetrix microarray studies identified novel LSF-downstream genes that are associated with cell growth, angiogenesis, invasion, metastasis, chemoresistance, and senescence. Osteopontin, a key molecule mediating tumor progression, especially metastasis, was identified as a downstream target gene of LSF. Promoter-reporter and chromatin immunoprecipitation analyses confirmed that LSF transcriptionally upregulates OPN by binding to its promoter. Knockdown of OPN gene in LSF-overexpressing HCC cells markedly inhibits LSF-mediated tumor progression including metastasis. This is the first report on the novel function of LSF as an oncogene in any cancer indication. Since LSF expression correlates with the advanced stages of the disease, it might be employed as a diagnostic/prognostic marker for HCC. The DNA binding domain of LSF might be targeted with small molecule inhibitors that might be developed as potential HCC therapeutic. Our findings thus have significant translational implications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 234.

Characteristic gene expression profiles in the progression from liver cirrhosis to carcinoma induced by diethylnitrosamine in a rat model

BackgroundLiver cancr is a heterogeneous disease in terms of etiology, biologic and clinical behavior. Very little is known about how many genes concur at the molecular level of tumor development, progression and aggressiveness. To explore the key genes involved in the development of liver cancer, we established a rat model induced by diethylnitrosamine to investigate the gene expression profiles of liver tissues during the transition to cirrhosis and carcinoma.MethodsA rat model of liver cancer induced by diethylnitrosamine was established. The cirrhotic tissue, the dysplasia nodules, the early cancerous nodules and the cancerous nodules from the rats with lung metastasis were chosen to compare with liver tissue of normal rats to investigate the differential expression genes between them. Affymetrix GeneChip Rat 230 2.0 arrays were used throughout. The real-time quantity PCR was used to verify the expression of some differential expression genes in tissues.ResultsThe pathological changes that occurred in the livers of diethylnitrosamine-treated rats included non-specific injury, fibrosis and cirrhosis, dysplastic nodules, early cancerous nodules and metastasis. There are 349 upregulated and 345 downregulated genes sharing among the above chosen tissues when compared with liver tissue of normal rats. The deregulated genes play various roles in diverse processes such as metabolism, transport, cell proliferation, apoptosis, cell adhesion, angiogenesis and so on. Among which, 41 upregulated and 27 downregulated genes are associated with inflammatory response, immune response and oxidative stress. Twenty-four genes associated with glutathione metabolism majorly participating oxidative stress were deregulated in the development of liver cancer. There were 19 members belong to CYP450 family downregulated, except CYP2C40 upregulated.ConclusionIn this study, we provide the global gene expression profiles during the development and progression of liver cancer in rats. The data obtained from the gene expression profiles will allow us to acquire insights into the molecular mechanisms of hepatocarcinogenesis and identify specific genes (or gene products) that can be used for early molecular diagnosis, risk analysis, prognosis prediction, and development of new therapies.

Deletion mapping of chromosome 16q24 in hepatocellular carcinoma in Taiwan and mutational analysis of the 17-beta-HSD gene localized to the region.

Human chromosome band 16q24 commonly undergoes loss of heterozygosity (LOH) in human hepatocellular carcinoma (HCC). To further localize the region of deletion on 16q24 and to evaluate the genetic role of 17-beta-HSD, which is near 16q24, in HCC, we examined the pattern of loss of heterozygosity in 88 HCC patients. DNAs from 88 pairs of HCCs and corresponding non-tumor parts were prepared. Loss of heterozygosity on chromosomes 16q24 was investigated by 11 sets of microsatellite markers. Mutation analysis of type II 17-beta-HSD was performed by automatic sequencing. LOH on 16q24 for at least 1 locus was found in 43 of the 88 tumor DNAs (49%). Three non-overlapping regions of frequent LOH were defined in these 43 tumors with partial deletions. The first region was between D16S516 loci and D16S507, encompassed by a 1-cM region, defined by the D16S504. The second region was defined by the 17HSDB2 locus between D16S505 and D16S422, encompassed approximately by a 1-cM region. The third region was between D16S520 and D16S413, defined by D16S3048, encompassed approximately by a 4-cM region. Homozygous deletions of any exons in 17HSDB2 gene were identified in 7 of 27 cases (26%). Automated sequencing analysis of 17HSDB2 failed to demonstrate mutations in any of these specimens. Our data suggest that the 17HSDB2 locus is a frequent target of deletion in HCC but the inactivation of 17HSDB2 may not involve sequence mutations. Furthermore, the presence of the other 2 frequent LOH regions suggest that the putative tumor suppressor genes at these locations might be involved in the development of HCC.

Germ-line mutations of the p16INK4(MTS1) gene occur in a subset of patients with hepatocellular carcinoma.

The molecular mechanisms of hepatocarcinogenesis are poorly understood. Only very recently has there been a suggestion of familial hepatocellular carcinoma (HCC). We have analyzed the status of the p16INK4(MTS1) gene, a cyclin-dependent kinase inhibitor, in 26 patients with HCC of different etiologies. Four patients carried hemizygous germ-line point mutations of the p16INK4(MTS1) gene, suggesting the existence of familial HCC involving this gene. The wild-type allele was lost in the tumor in 2 of these 4 patients. Three of the patients carrying a germ-line mutation had non-cirrhosis-associated HCC. No somatic mutations of p16INK4(MTS1) were observed in the 26 cases of HCC. The most common somatic alteration of the p16INK4(MTS1) gene in HCC was de novo methylation, which was detected in 48% of the cases. Low levels (21%) of p16INK4(MTS1) gene allele loss were observed. Altogether, these results indicate that alteration of the p16INK4(MTS1) gene plays an important role in the genesis of HCC.

Cellular and molecular mechanisms of hepatocarcinogenesis.

For colorectal carcinomas, the rate of tumor development is proportional to the fourth to sixth power of elapsed time, suggesting that four to six independent events are necessary. Although similar calculations have not been made for HBV-associated HCCs, it is likely that this is also the case for HCCs, since individuals with persistent HBV infection do not usually develop HCC until they are 45 or greater years old. As evidence for specific genetic and epigenetic changes in HCCs accumulate, the important players in multistep hepatocarcinogenesis are becoming clearer. However, even though Myc family oncogenes are clearly implicated in woodchuck HCC, similar integrations have not been found in human HCCs. Therefore, although rodent and human systems have many similarities, we must realize that important differences may also exist. Regarding tumor suppressor genes, the evidence for p53 alterations in HCC is strong. A growing body of evidence suggests further that alterations in the retinoblastoma gene and one or more tumor suppressor genes on chromosome 11 are also involved in HCC. HBV integrations may certainly play a role in the generation of chromosome aberrations leading to loss of tumor suppressor alleles, since chromosomes 11 and 17 are the most common integration sites. Finally, the role of X proteins as participants in malignant transformation has been demonstrated for certain immortalized, nontransformed hepatocytes. Altered autocrine mechanisms of cell growth control, possibly involving IGF-II, are clearly implicated in HCC. Paracrine mechanisms for the control of hepatocyte growth and differentiated functions may also be altered as a result of the synthesis and secretion of a complex array of interleukins, HGF, and basic and acidic FGFs by cells in the inflammatory and cirrhotic lesions of precancerous livers. Whether the order of molecular changes in the hepatocyte is important for malignant progression is presently not clear. What is clear, however, is that hepatocarcinogenesis involves alterations in the concerted action of protooncogenes, growth factor, and tumor suppressor genes. How these factors interact will provide a more complete understanding of the mechanism or mechanisms of hepatic oncogenesis.

Abnormal structure and expression of p53 gene in human hepatocellular carcinoma.

There is little information regarding the molecular mechanisms of hepatocarcinogenesis. We studied the p53 gene at the DNA, RNA, and protein level in seven human hepatocellular carcinoma (HCC)-derived cell lines; six of seven showed p53 abnormalities. By Southern blotting, the p53 gene was found to be partially deleted in Hep 3B and rearranged in SK-HEP-1 cells. Transcripts of the p53 gene were undetectable in Hep 3B as well as in FOCUS cells that had no apparent deletion or rearrangement of the p53 gene. Immunoprecipitation after [35S]methionine labeling of HCC cells demonstrated that p53 protein was absent in Hep 3B and FOCUS and reduced in concentration in PLC/PRF/5 cells. p53 synthesized by Mahlavu cells showed a slower migration on SDS/polyacrylamide gels suggesting it was an abnormal protein. In Huh7 cells, p53 protein had a prolonged half-life leading to its accumulation in the nuclei; increased levels of p53 protein were also found by immunoblotting. The p53 gene and its expression appeared to be unaltered in the hepatoblastoma-derived Hep G2 cell line. We found that the loss of p53 expression did not occur as a late in vitro event in the FOCUS cell line because p53 protein was also nondetectable at an early passage. We conclude that the loss of p53 expression or the presence of abnormal forms of the protein are frequently associated with HCC cell lines. These observations suggest that alterations in p53 may be important events in the transformation of hepatocytes to the malignant phenotype.