Liver Tumor Samples Research Articles

Molecular Investigation of Occult Hepatitis B Virus Infection Among Patients with Liver Cancers

Background: Hepatitis B virus (HBV) is still the leading cause of hepatocellular carcinoma (HCC). HBV could persist in the low replicate state that is defines as occult hepatitis B virus infection (OBI). Recently, OBI has been defined important in the development and/or exacerbation of HCC and other liver diseases. Objectives: This study tried to determine the frequency of OBI among liver tumor samples. Methods: This cross-sectional study was performed among patients with HCC and intrahepatic cholangiocarcinoma (iCCA) in hospitals affiliated with Iran University of Medical Sciences. Liver tumor samples (Fresh frozen and formalin fixed paraffin embedded) were processed for isolation of DNA and then subjected to molecular assays, including PCR examinations of HBV genes (S, X, and C), determination of viral loads, detection of HBV-cccDNA, phylogenetic analysis, and assessment of mutations in HBsAg and RT regions. Results: In total, there were 93 participants, including HCC (n = 60), iCCA (n = 33); among which, 15% were detected positive for OBI. The OBI among HCC and iCCA were 18.3% and 9.1%, respectively. The mean intrahepatic viral load was 1.3 × 105 ± 1.4 × 102 copies/µl, and 5 had detectable cccDNA. The OBI subjects belonged to the HBV genotype D and subgenotype D1. In the HBsAg protein, T45N (33.3%) and P105A (25%) were the most prevalent mutations. N53K (33.3%), Y54H (25%), L80I (33.3%), and A113G (25%) were missense mutations that were observed in the RT domain. Conclusions: HBV-DNA was detected among liver tumor samples with negative HBsAg status. The results of viral load and cccDNA tests are important in identification and prognosis of liver diseases. It’s needed more precise molecular and cohort studies to clarify the functions of OBI in liver diseases.

Re-analysis of hepatitis B virus integration sites reveals potential new loci associated with oncogenesis in hepatocellular carcinoma.

Hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC). HBV DNA can get integrated into the hepatocyte genome to promote carcinogenesis. However, the precise mechanism by which the integrated HBV genome promotes HCC has not been elucidated. To analyze the features of HBV integration in HCC using a new reference database and integration detection method. Published data, consisting of 426 Liver tumor samples and 426 paired adjacent non-tumor samples, were re-analyzed to identify the integration sites. Genome Reference Consortium Human Build 38 (GRCh38) and Telomere-to-Telomere Consortium CHM13 (T2T-CHM13 (v2.0)) were used as the human reference genomes. In contrast, human genome 19 (hg19) was used in the original study. In addition, GRIDSS VIRUSBreakend was used to detect HBV integration sites, whereas high-throughput viral integration detection (HIVID) was applied in the original study (HIVID-hg19). A total of 5361 integration sites were detected using T2T-CHM13. In the tumor samples, integration hotspots in the cancer driver genes, such as TERT and KMT2B, were consistent with those in the original study. GRIDSS VIRUSBreakend detected integrations in more samples than by HIVID-hg19. Enrichment of integration was observed at chromosome 11q13.3, including the CCND1 pro-moter, in tumor samples. Recurrent integration sites were observed in mitochondrial genes. GRIDSS VIRUSBreakend using T2T-CHM13 is accurate and sensitive in detecting HBV integration. Re-analysis provides new insights into the regions of HBV integration and their potential roles in HCC development.

Living on the edge: how stromal cells restrict liver cancer invasion

The global incidence of primary liver cancer is increasing and currently ranks amongst the top-five causes of premature death from cancer annually. Like in many solid tumours, liver cancer cells are closely associated with stromal cells, known as non-parenchymal cells (NPCs), in the liver, including fibroblasts and endothelial cells, which normally support liver function and structure. Although single-cell RNA sequencing has begun to unpick the interactions between tumour cells and this non-epithelial microenvironment, these -omic approaches cannot mechanistically resolve how the non-parenchymal stroma interacts with tumour cells, nor can they resolve whether a fibroblast exerts differential outcomes on mutant versus healthy epithelial cells.In this issue of Disease Models & Mechanisms, Liqin Zhu and colleagues describe a novel in vitro microwell culture system to co-culture NPCs with either healthy hepatocytes or tumour cells of intrahepatic cholangiocarcinoma (iCCA), an aggressive type of primary liver cancer. When the authors co-cultured NPCs with primary hepatocytes, the NPCs supported hepatocyte spheroid growth, maintaining the phenotype of the healthy hepatocytes in culture. Interestingly, when cultured with mutant iCCA cells, NPCs supressed the growth of cancer spheroids, suggesting a divergent role of NPCs in tumour growth.To understand the role of these stromal cells in tumour growth further, the authors then co-cultured healthy hepatocytes and tumour cells in their microwell system and found that tumour cells invade and kill healthy hepatocytes. The addition of NPCs to this system significantly limited this process, and in vitro cancer cells were constrained, no longer killing healthy hepatocytes. Following on from this, the authors used an orthotopic transplantation mouse model of liver cancer and liver tumour samples from patients to show that activated myofibroblasts localised at the interface between tumour and normal tissue. Together, the data suggest that, in liver cancer, NPCs, in particular myofibroblasts, form a physical barrier that limits the ability of cancer cells to invade and damage normal tissue.Targeting the tumour microenvironment to limit disease progression is an attractive therapeutic prospect that could be agnostic to the underlying genetic and molecular heterogeneity seen in cancer. However, as NPCs have previously been shown to provide pro-proliferative signals in cancer and modify the intratumoral immune landscape, more research is required to understand the nuanced NPC-epithelial cell relationship in cancer. We should carefully consider the diverse and potentially conflicting roles of NPCs in tumour progression, and advanced in vitro systems, such as those developed by Zhu and colleagues, will help us decipher this.

Glutamine Transporter SLC1A5 Regulates Ionizing Radiation-Derived Oxidative Damage and Ferroptosis.

Ionizing radiation-derived oxidative stress and ferroptosis are one of the most important biological effects on destroying the liver tumor, whereas radioresistance of liver tumor remains a leading cause of radiotherapy (RT) failure mainly because of the protective antiferroptosis, in which oxidative stress and subsequent lipid peroxidation are the key initiators. Thus, it is of great importance to overcome ferroptosis resistance to improve the therapeutic efficacy of RT in liver tumor patients. Irradiation-resistant HepG2 cells (HepG2-IRR) were established by long-term exposure to X-ray (2 to 8 Gy), and targeted metabolomics analysis revealed an obvious increase in intracellular amino acids in HepG2-IRR cells upon ferroptosis stress. Among these amino acids with obvious changes, N-acetylglutamine, a derivative of glutamine, is essential for the redox homeostasis and progression of tumor cells. Interestingly, the treatment of glutamine starvation could promote the ferroptosis effect significantly, whereas glutamine supplementation reversed the ferroptosis effect completely. Consistent with the changes in amino acids pattern, the glutamine transporter SLC1A5 was verified in liver tumor samples from TCGA training and validation cohorts as an independent prognostic amino acid-ferroptosis gene (AFG). A risk score for screening prognosis based on the SLC1A5, SLC7A11, ASNS, and TXNRD1 demonstrated that a high-risk score was correlated with poor survival. In vitro studies had shown that the knockdown of SLC1A5 resulted in a significant decrease in cell viability and promoted lipid peroxidation and oxidative damage introduced by irradiation (10 Gy). Collectively, our findings indicated that SLC1A5 may act as a suppressor gene against ferroptosis and can be a potential target for ionizing radiation mediated effects.

Ketamine Induces Ferroptosis of Liver Cancer Cells by Targeting lncRNA PVT1/miR-214-3p/GPX4.

BackgroundLiver cancer ranks the top four malignant cancer type worldwide, which needs effective and safe treatment. Ferroptosis is a novel form of regulated cell death driven by iron-dependent lipid peroxidation and has been regarded as a promising therapeutic target for cancers. In this work, we aimed to study the effects of anesthetic ketamine on proliferation and ferroptosis of liver cancer.MethodsCell viability and proliferation were detected by cell counting kit 8 (CCK-8), colony formation, and 5-ethynyl-2′-deoxyuridine (EdU) assay. Ferroptosis was determined by levels of Fe2+, lipid reactive oxygen species (ROS), and malondialdehyde (MDA). RNA levels of lncPVT1, miR-214-3p, and glutathione peroxidase 4 (GPX4) were checked by real-time PCR assay. Clinical liver tumor samples were collected to detect the levels of long noncoding RNA lncPVT1, miR-214-3p, and GPX4, and their correlation was evaluated by Pearson comparison test. Luciferase reporter gene assay and RNA pulldown were conducted to determine the binding between lncPVT1, miR-214-3p, and GPX4 3ʹUTR.ResultsKetamine significantly suppressed viability and proliferation of liver cancer cells both in vitro and in vivo, as well as stimulated ferroptosis, along with decreased expression of lncPVT1 and GPX4. LncPVT1 directly interacted with miR-214-3p to impede its role as a sponge of GPX4. Depletion of lncPVT1 accelerated the ferroptosis of live cancer cells, whereas miR-214-3p inhibition and GPX4 overexpression reversed this effect. Ketamine-induced cell growth suppression and ferroptosis were also suppressed by miR-214-3p inhibition and GPX4 overexpression.ConclusionIn this work, we determined that ketamine suppressed viability of liver cancer cells and induced ferroptosis and identified the possible regulatory mechanism of lncPVT1/miR-214-3p/GPX4 axis.

Use of X-Ray Fluorescence Microscopy for Studies on Research Models of Hepatocellular Carcinoma.

Introduction: TheraSphere® microspheres containing yttrium 90Y are among many radioembolization agents used clinically to reduce liver tumor burden, and their effects on cancer volume reduction are well-established. At the same time, concerns about off target tissue injury often limit their use. Deeper investigation into tissue distribution and long-term impact of these microspheres could inform us about additional ways to use them in practice.Methods: Healthy rat liver and rabbit liver tumor samples from animals treated with TheraSpheres were sectioned and their elemental maps were generated by X-ray fluorescence microscopy (XFM) at the Advanced Photon Source (APS) synchrotron at Argonne National Laboratory (ANL).Results: Elemental imaging allowed us to identify the presence and distribution of TheraSpheres in animal tissues without the need for additional sample manipulation or staining. Ionizing radiation produced by 90Y radioactive contaminants present in these microspheres makes processing TheraSphere treated samples complex. Accumulation of microspheres in macrophages was observed.Conclusions: This is the first study that used XFM to evaluate the location of microspheres and radionuclides in animal liver and tumor samples introduced through radioembolization. XFM has shown promise in expanding our understanding of radioembolization and could be used for investigation of human patient samples in the future.

Occurrence and distribution of per- and polyfluoroalkyl substances (PFASs) in human livers with liver cancer

Per- and polyfluoroalkyl substances (PFASs) are anthropogenic compounds that are widely accumulated in human tissues, and the liver is considered a primary target organ for PFASs exposure. The occurrence and distribution of 21 PFASs in liver tissues with tumors (n = 55) and without tumors (n = 55) are investigated in this study. Eleven perfluorinated carboxylic acids (PFCAs) and five perfluorinated sulfonic acids (PFSAs) were detected at high frequencies (45.5%–100 %), while the detection frequencies of five perfluoroalkyl phosphate (PFPAs) were relatively lower (≤29.1 %). PFSAs and PFCAs accounted for up to 82.5%–92.7 % of the total PFASs. Although it was not found to be statistically significant, the concentrations of the total PFASs were slightly higher in the tumor liver samples (mean 64.3, range 5.70–303 ng/g) than those in the non-tumor liver samples (mean 62.7, range 4.08–240 ng/g).The perfluorooctanoic acid (PFOA), perfluorotridecanoic acid (PFTrDA), and perfluorobutanesulphonate (PFBS) showed significant differences (p < 0.05) between the tumor and non-tumor liver samples, and the different distribution levels of these three PFASs may have been a consequence of oxidative stress. The total concentrations of PFASs in the three age groups were in the decreasing order of middle-aged people (45–60) > old people (>60) > young people (<45). The PFASs in females were generally lower than in males, which may have been related to women's special excretion methods (such as childbirth and breastfeeding). The results should be valuable for further mechanistic studies regarding the toxic effects of PFASs in human livers.

Evaluation of Ultrasonic Attenuation in Primary and Secondary Human Liver Tumors and Its Potential Effect on High-Intensity Focused Ultrasound Treatment

Primary and secondary liver tumors are completely different diseases but are usually treated similarly using high-intensity focused ultrasound (HIFU). However, the acoustic parameters of these tissues are not well documented. In this study, attenuation coefficients were evaluated in fresh primary (N = 8) and secondary (N = 13) human liver tumor samples recovered by hepatectomy. The average attenuation coefficients of the primary and secondary liver tumors were 0.10 ± 0.03 and 0.20 ± 0.04 Np/cm/MHz, respectively. The average attenuation coefficients of the liver tissue surrounding the primary and secondary tumors were 0.16 ± 0.07 and 0.07 ± 0.02 Np/cm/MHz, respectively. Numerical simulations performed using these values revealed that completely different HIFU ablation patterns were created in primary and secondary liver tumors using the same exposure parameters. The dimensions of a typical HIFU lesion were two times larger in secondary liver tumors than in primary tumors. HIFU treatment parameters should be set properly according to the acoustic properties of the diseased liver tissue.

Prostate-specific membrane antigen expression in hepatocellular carcinoma, cholangiocarcinoma, and liver cirrhosis.

BACKGROUNDPrimary liver cancer includes three subtypes: Hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (CCA), and combined hepatocellular carcinoma. Patients with primary liver cancer experienced poor prognosis and high mortality, so early detection of liver cancer and improved management of metastases are both key strategies to reduce the death toll from liver cancer. Prostate-specific membrane antigen (PSMA) expression in the tumor-associated neovasculature of nonprostate malignancies including liver cancer has been reported recently, but conclusive evidence of PSMA expression based on the pathological type of liver cancer remains limited.AIMTo study the expression of PSMA in HCC, CCA, and liver cirrhosis.METHODSA total of 446 formalin-fixed paraffin-embedded (FFPE) liver tumor and liver cirrhosis tissue samples were obtained retrospectively from the Pathology Department of Tongji Hospital. Immunohistochemistry was used to detect PSMA expression in these 446 FFPE liver biopsy specimens (213 HCC, 203 CCA, and 30 liver cirrhosis). The tumor compartment and the associated neovascular endothelium were separately analyzed. PSMA expression was examined by two certified pathologists, and the final results were presented in a 4-point scoring system (0-3 points). Correlation between PSMA expression and clinicopathological information was also assessed.RESULTSPSMA was expressed primarily in the neovascular endothelium associated with tumors. The positive rate of PSMA staining in HCC was significantly higher than that in CCA (86.8% vs 79.3%; P = 0.001) but was only 6.6% in liver cirrhosis (P = 0.000). HCC cases had more 3-score PSMA staining than CCA had (89/213, 41.8% vs 35/203, 17.2%; P = 0.001). PSMA expression correlated positively with the stage and grade of HCC and CCA. In both liver cancer subtypes, there were more PSMA+ cases in stages III–V diseases than in stages I and II. High staining intensity of PSMA was more frequently observed in liver cancers at high grade and advanced stage. There was no significant association of PSMA expression with sex, age, region, α-fetoprotein, hepatitis B surface antigen, or tumor size in both tumor subtypes.CONCLUSIONNeovascular PSMA may be a promising marker to differentiate HCC from liver cirrhosis and a prognostic marker for anti-tumor angiogenesis therapy for HCC.

Comparison of the Hepatic Effects of Phenobarbital in Chimeric Mice Containing Either Rat or Human Hepatocytes With Humanized Constitutive Androstane Receptor and Pregnane X Receptor Mice.

Using a chimeric mouse humanized liver model, we provided evidence that human hepatocytes are refractory to the mitogenic effects of rodent constitutive androstane receptor (CAR) activators. To evaluate the functional reliability of this model, the present study examined mitogenic responses to phenobarbital (PB) in chimeric mice transplanted with rat hepatocytes, because rats are responsive to CAR activators. Treatment with 1000 ppm PB for 7 days significantly increased replicative DNA synthesis (RDS) in rat hepatocytes of the chimeric mice, demonstrating that the transplanted hepatocyte model is functionally reliable for cell proliferation analysis. Treatment of humanized CAR and pregnane X receptor (PXR) mice (hCAR/hPXR mice) with 1000 ppm PB for 7 days significantly increased hepatocyte RDS together with increases in several mitogenic genes. Global gene expression analysis was performed with liver samples from this and from previous studies focusing on PB-induced Wnt/β-catenin signaling and showed that altered genes in hCAR/hPXR mice clustered most closely with liver tumor samples from a diethylnitrosamine/PB initiation/promotion study than with wild-type mice. However, different gene clusters were observed for chimeric mice with human hepatocytes for Wnt/β-catenin signaling when compared with those of hCAR/hPXR mice, wild-type mice, and liver tumor samples. The results of this study demonstrate clear differences in the effects of PB on hepatocyte RDS and global gene expression between human hepatocytes of chimeric mice and hCAR/hPXR mice, suggesting that the chimeric mouse model is relevant to humans for studies on the hepatic effects of rodent CAR activators whereas the hCAR/hPXR mouse is not.

SDC1 promotes cisplatin resistance in hepatic carcinoma cells via PI3K-AKT pathway.

This study is to analyze the potential contribution of Syndecan 1 (SDC1) to cisplatin resistance in hepatic carcinoma. Cell proliferation and viability were determined by direct counting and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. The protein levels of SDC1, p-AKT, AKT and β-actin were quantified by western blotting. The SDC1 transcript abundance was measured by real-time polymerase chain reaction. The relative expression of SDC1 in clinical liver tumor samples was analyzed with immunohistochemistry. SDC1 was up-regulated in cisplatin-resistant HepG2 cells (denoted as HepG2 CR hereafter). SDC1-knockdown re-sensitized HepG2 CR cells to cisplatin treatment. Ectopic over-expression of SDC1 conferred drug resistance to naïve HepG2 cells. PI3K/AKT pathway was over-activated in HepG2 CR cells, and simultaneous administration with PI3K inhibitor greatly surmounted the resistance. We also demonstrated that SDC1 was aberrantly up-regulated in clinical hepatocellular carcinoma samples. Our study highlighted the importance of SDC1-PI3K/AKT signaling in the cisplatin resistance in hepatocellular carcinoma.

Analysis of β-catenin gene mutations and gene expression in liver tumours of C57BL/10J mice produced by chronic administration of sodium phenobarbital

In this study liver tumours produced in male and female mice of the low spontaneous liver tumour incidence C57BL/10J strain treated for 99 weeks with 1000 ppm in the diet with the model constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were analysed for β-catenin mutations by Western immunoblotting and DNA/RNA analysis. Some gene array analysis was also performed to identify genes involved in CAR activation and in β-catenin and Hras gene mutations. Analysis of 8 male and 2 female NaPB-induced liver tumour samples (comprising 2 adenomas, 6 carcinomas and 2 samples containing separate adenomas and carcinomas) revealed truncated β-catenin forms in just 4 male liver tumour samples, with the presence of the truncated β-catenin forms being confirmed by β-catenin exon 1–3 mutation analysis. Microarray gene expression analysis was performed with three of the NaPB-induced male mouse liver tumour samples where β-catenin mutations had not been identified by Western immunoblotting and DNA/RNA analysis and with three liver samples from both NaPB-induced non-tumour tissue and control animals. Treatment with NaPB resulted in induction of Cyp2b subfamily gene expression in both NaPB-induced mouse liver tumours and in NaPB-treated non-tumour tissue. In addition, the gene expression analysis demonstrated that the β-catenin and Hras pathways were not modified in NaPB-induced mouse liver tumours not exhibiting truncated β-catenin forms. Overall, while chronic administration of the model CAR activator NaPB results in both hepatocellular adenoma and carcinoma in the low spontaneous liver tumour incidence C57BL/10J mouse strain, only 40 % of the liver tumours evaluated in this study had β-catenin mutations. These results are in agreement with previous studies with the CAR activator oxazepam and demonstrate that mouse liver tumours induced by nongenotoxic CAR activators in the absence of initiation with a genotoxic agent are due to a number of mechanisms, including those largely independent of either the Wnt/β-catenin signalling pathway or Hras oncogene mutations.

Monozygotic twins with neuroblastoma MS have a similar molecular profile: a case of twin-to-twin metastasis

Fetoplacental neuroblastoma metastasis has been postulated as a mechanism accounting for concordant cases where one twin develops a primary tumour and the second twin manifests the disease without an identifiable primary site. These tumours may originate and spread concomitantly due to the same genetic background shared by monozygotic twins. This study investigated the molecular profile of stage MS neuroblastoma presenting concomitantly in monozygotic twins. Comparative genomic hybridisation (aCGH) was done for each of the twin liver tumour and peripheral blood samples at diagnosis. Comparison of copy-number variation (CNV) regions revealed a set of CNVs that were common to both tumour specimens and not apparent in the blood. The CNV signature in both twins’ tumours was highly similar, suggesting a common clonal origin. Additional findings included large deletion of chromosome 10 and amplification of chromosome 17. Notably, both liver samples had amplification of a short region involving DEIN (chromosome 4q34.1). Similar CNVs strongly support a common clonal origin and metastatic spread from one twin to the other. DEIN is a long-coding RNA (IncRNA) that has been found highly expressed in stage MS neuroblastoma and is likely involved in biological processes such as cell migration and metastasis.

The survivin gene expression in neoplastic hepatocytes from chickens infected with Marek’s virus

P53 protein is one of the main proteins in apoptosis pathway. The role of survivin protein in inhibition of P53 in different human cancers has been proved. The expression of survivin can be a main marker in diagnosis and prognosis of different human cancer. Until now, the survivin gene expression in birds infected with Marek’s disease was not investigated. In this study, liver tissue samples of chickens infected with Marek’s disease virus (MDV) was collected. The identification of MDV was carried out with PCR and histopathologic examination. After identification of MDV, the pathogenicity of infected virus was investigated with specific primers targeted on 132 bp tandem repeat. After this, the survivin gene expression was examined in neoplastic liver samples by Real-Time PCR. The PCR amplification of tumor liver samples showed that all samples were infected with MDV. The result of histopathology and amplification of 132 bp tandem repeat in tumor samples showed that the chickens were infected to pathogenic MDV. Results showed that the survivin gene expression in neoplastic hepatocytes was significantly higher than normal hepatocytes. In conclusion, survivin gene expression can be utilized as a suitable biomarker in diagnosis of Marek’s disease in birds. It seems that viral meq oncogene in Marek’s disease can play role in induction of survivin expression in this disease that it is necessary to be proved.

Repressing of NHERF1 inhibits liver cancer progression by promoting the production of ROS

NHERF1/EBP50 is a PDZ-scaffold protein initially identified as an organizer and modulator of transporters and channels at the apical side of epithelia via actin-binding ezrin-moesin-radixin proteins. Presently, hepatocellular carcinoma (HCC) is one of the most deadly cancers in the world and has no effective therapeutic strategies. In the present study, we attempted to explore the role of NHERF1 in regulating liver cancer progression. The results indicated that NHERF1 was significantly expressed in liver tumor samples compared to the corresponding adjacent normal tissues. HCC patients with low NHERF1 exhibited better survival rate. Additionally, repressing NHERF1 expression markedly down-regulated the cell proliferation. G0/G1 transition was highly induced by NHERF1 knockdown, accompanied with reduced expressions of Cyclin D1 and cyclin-dependent kinase 4 (CDK4), as well as the enhanced expression of p27, phosphatase and tensin homolog (PTEN) and p53. Moreover, NHERF1 suppression significantly induced apoptosis in liver cancer cells by promoting the activation of Caspase-3 and poly (ADP-ribose) polymerase (PARP). We also observed a remarkable increase of reactive oxygen species (ROS) production in NHERF1-knockdown cells, along with c-Jun-N-terminal kinase (JNK) phosphorylation. Importantly, suppressing ROS production abolished NHERF1 knockdown-induced JNK activation. Moreover, cell cycle-regulatory proteins meditated by NHERF1 knockdown in liver cancer cells were abrogated by the pre-treatment of ROS scavenger. Further, restraining ROS generation also diminished NHERF1 knockdown-induced apoptosis. In vivo, we also found that NHERF1 knockdown markedly reduced the tumor growth. In conclusion, the results suggested that NHERF1 played an essential role in regulating liver cancer progression, and repressing NHERF1 expression exhibited significant anticancer effects via the induction of G0/G1 phase arrest, apoptosis and ROS generation.

Key signaling pathways, genes and transcription factors associated with hepatocellular carcinoma.

The purpose of the present study was to investigate the underlying molecular mechanism of hepatocellular carcinoma (HCC) using bioinformatics approaches. The microarray dataset GSE64041 was downloaded from the Gene Expression Omnibus database, which included 60 tumor liver samples and 60 matched control samples. Differentially expressed genes (DEGs) between HCC and control groups were identified. Then functional enrichment analyses, protein-protein interaction (PPI) network, sub-network and integrated transcription factor (TF)-microRNA (miRNA)-target network analyses were performed for these DEGs. A total of 378 DEGs were obtained, including 101 upregulated and 277 downregulated DEGs. In addition, functional enrichment analysis for DEGs in the sub-network revealed ‘cell division’ and ‘cell cycle’ as key Gene Ontology (GO) terms and pathways. Topoisomerase (DNA) IIα (TOP2A) and integrin subunit α2 (ITGA2) were hub nodes in the PPI network. TOP2A, cyclin dependent kinase 1 (CDK1) and polo like kinase 1 (PLK1) were revealed to be hub nodes in the sub-network. Finally, 4 TFs including forkhead box M1 (FOXM1), E2F transcription factor 4 (E2F4), SIN3 transcription regulator family member A (SIN3A) and transcription factor 7 like 1 (TCF7L1) were obtained through integrated network analysis. TOP2A, ITGA2, PLK1 and CDK1 may be key genes involved in HCC development. ‘Cell division’ and ‘cell cycle’ were indicated to act as key GO terms and Kyoto Encyclopedia of Genes and Genomes pathways in HCC. In addition, FOXM1, TCF7L1, E2F4 and SIN3A were revealed to be key TFs associated with HCC.

WITHDRAWN: Identification of aberrantly expressed circRNAs in subgroup J avian leucosis virus induced tumor livers by RNA sequencing

// Xinheng Zhang 1, 4, 5 , Yiming Yan 1, 4 , Xiaoya Lei 1, 4 , Aijun Li 2 , Huanmin Zhang 3 , Zhenkai Dai 1, 4 , Xinjian Li 1, 4 , Weiguo Chen 1, 4, 5 , Wencheng Lin 1, 4, 5 , Feng Chen 1, 4, 5 , Jingyun Ma 1, 4, 5 and Qingmei Xie 1, 4, 5 1 College of Animal Science, South China Agricultural University and Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding and Key Laboratory of Chicken Genetics, Breeding and Reproduction, Ministry of Agriculture, Guangzhou, 510642, P. R. China 2 College of Science and Engineering, Jinan University, Guangzhou, 510632, P. R. China 3 USDA, Agriculture Research Service, Avian Disease and Oncology Laboratory, East Lansing, MI, 48823, USA 4 Key Laboratory of Animal Health Aquaculture and Environmental Control, Guangdong, Guangzhou, 510642, P. R. China 5 South China Collaborative Innovation Center for Poultry Disease Control and Product Safety, Guangzhou, 510642, P. R. China Correspondence to: Qingmei Xie, email: qmx@scau.edu.cn Keywords: RNA sequencing; ALV-J; tumor; circular RNA; bioinformatics analysis Received: October 16, 2017&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Accepted: December 05, 2017&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Published: January 12, 2018 ABSTRACT ALV-J (subgroup J avian leucosis virus) is a kind of oncogenic exogenous retrovirus and diseases associated with ALV-J have caused severe reproduction problems in the poultry industry worldwide. However, the pathogenesis of ALV-J-induced tumor formation is still unclear. In recent years, circRNAs are validated to be related to cancer, which prompts us to explore whether aberrant expression of circRNAs exists in ALV-J-infected tumor samples. In this study, a total of 2822 differentially expressed circRNAs were detected by RNA sequencing in the tumor livers of ALV-J-susceptible chickens ( n = 3) and the normal liver samples of ALV-J-resistant chickens ( n = 3), among which 2808 circRNAs in the tumor liver samples were detected as unregulated compared to those in the normal liver samples. As circRNAs are considered as miRNA sponge, we predicted top 5 miRNA targets for 24 selected upregulated circRNAs in the ALV-J-induced tumor livers. CircRNA-miRNA-mRNA regulatory axes were also constructed to provide clear interaction relationship among them. Furthermore, Gene Ontology and KEGG pathway analyses were performed for miRNA target genes, the result of which implied that metabolism plays a key role in circRNAs alterations in ALV-J-induced tumor livers. In summary, we identified that circRNAs are involved in ALV-J-induced tumor formation in chickens. However, the pathogenicity mechanism still needs to be further explored.

Abstract 5425: The role of six transmembrane epithelial antigen of the prostate 2 in hepatocellular carcinoma

Abstract Introduction: The incidence of hepatocellular carcinoma (HCC) in Hispanics is three times higher than non-Hispanic whites, and even higher in South Texas (STX) Hispanics. This is attributed to a higher prevalence of hepatitis C, diabetes, obesity and perhaps genetic and epigenetic alterations. Knowledge regarding genetic alterations in Hispanics is sparse as demonstrated by the lack of Hispanics with HCC in The Cancer Genome Atlas (TCGA). Therefore, our group sequenced paired adjacent liver and HCC tumor samples from STX Hispanics, which highlighted a gene over-expressed in tumors of Hispanics, called the Six Transmembrane Epithelial Antigen of the Prostate 2 (STEAP2). STEAP2 is a metalloreductase of iron and copper and is implicated in increased iron and reactive oxygen species in the liver which can lead to the progression of inflammation and cirrhosis. STEAP2 may play an important oncogenic role in HCC, especially in the setting of obesity. We propose to test the hypothesis that overexpression of STEAP2 will lead to malignant property in HCC cells resulting in enhanced proliferation, survival, invasiveness, and eventually development of HCC, especially in obese hosts. Methods: Hispanic paired tissue continues to be collected from our institution for RNA sequencing and establishment of Hispanic HCC cell lines. STEAP2 RNA and protein expression levels in Hispanic paired samples versus Caucasian paired samples were evaluated by RT-PCR, Western blot, and immunohistochemistry. Knockdown and overexpression of STEAP2 were established in HCC cell lines and in primary Hispanic HCC cell lines to examine the effects on iron levels, oxidative stress, proliferation, invasiveness, apoptosis and cell cycle in vitro. Results: Hispanic HCC RNA sequencing data compared to TCGA HCC RNA sequencing data (no Hispanics) demonstrated the overexpression of STEAP2 in HCC tumors in Hispanic patients, which were validated by RT-PCR data and Western blot data. Lipid peroxidation product, 4-hydroxynonenal, and copper levels were higher in HCC tumor versus adjacent tissue. Iron levels were higher in adjacent tissue versus tumor tissue in Hispanics. Knockdown of STEAP2 in SNU398 cells decreased proliferation and migration, while in HUH7 cells STEAP2 knockdown only decreased migration. Conclusions: STEAP2 is specifically overexpressed in HCC tumors in Hispanics in comparison to HCC tumors in non-Hispanic whites and appears to play a malignant-promoting role in HCC cells. Further studies to establish the role of STEAP2 as a tumor promoter in HCC and the mechanisms by which it promotes carcinogenesis are underway. The proposed studies will likely yield mechanistic insights into the molecular mechanisms that drive HCC development and progression in South Texas Hispanics and potential therapeutic targets involving STEAP2-mediated metal ion metabolism and oxidative stress. Citation Format: Carla R. Zeballos, Hakim Bouamar, Xiang Gu, Yidong Chen, Francisco G. Cigarroa, LuZhe Sun. The role of six transmembrane epithelial antigen of the prostate 2 in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5425. doi:10.1158/1538-7445.AM2017-5425

The cell polarity protein Scrib functions as a tumor suppressor in liver cancer.

Scrib is a membrane protein that is involved in the maintenance of apical-basal cell polarity of the epithelial tissues. However, Scrib has also been shown to be mislocalized to the cytoplasm in breast and prostate cancer. Here, for the first time, we report that Scrib not only translocates to the cytoplasm but also to the nucleus in hepatocellular carcinoma (HCC) cells, and in mouse and human liver tumor samples. We demonstrate that Scrib overexpression suppresses the growth of HCC cells in vitro, and Scrib deficiency enhances liver tumor growth in vivo. At the molecular level, we have identified the existence of a positive feed-back loop between Yap1 and c-Myc in HCC cells, which Scrib disrupts by simultaneously regulating the MAPK/ERK and Hippo signaling pathways. Overall, Scrib inhibits liver cancer cell proliferation by suppressing the expression of three oncogenes, Yap1, c-Myc and cyclin D1, thereby functioning as a tumor suppressor in liver cancer.

Abstract 4936: The function role of Brf1 in alcohol-induced human and mouse hepatocellular carcinoma*

Abstract Brf1 (TFIIB-related factor 1) specifically regulates the transcription of Pol III genes (RNA polymerase III-dependent genes), including tRNAs and 5S rRNAs, which are elevated in both transformed and tumor cells, suggesting that they play a crucial role in tumorigenesis. Our recent studies have demonstrated that alcohol consumption increases Brf1 expression and Pol III gene transcription to promote tumor development. However, it remains to be investigated that Brf1 is expressed in human cancers. At the present studies, we have determined the significance of Brf1 overexpression in human hepatocellular carcinoma (HCC) and the impaction of repressing Brf1 expression in alcohol-promoted cell transformation. Biopsies of human HCC, liver tumor samples of mice and cell lines of normal and tumor liver were utilized to determine alteration of Brf1 expression using cytological and molecular biological approaches. The result indicates that Brf1 expression is increased in human cases of HCC. High expression of Brf1 displays shorter overall survival period. Levels of Brf1 and Pol III gene transcription in HCC patients with alcohol consumption show an additional increase, compared with the cases of non-HCC with or without alcohol intake. Induction of Brf1 and Pol III genes by ethanol in hepatoma cells is higher than in non-tumor cells. Ethanol increases the rate of cell transformation. Repression of Brf1 inhibits alcohol-promoted cell transformation. Together, these studies demonstrate that Brf1 is overexpressed in HCC cases and liver tumor cell lines. High level of Brf1 in human HCC is correlated with short survival time. Alcohol consumption enhances Brf1 expression to promote cell transformation. Brf1 is a new biomarker of diagnosis and prognosis of HCC. *: The project is supported by NIH grants: AA017288, AA021114 and AA02324 to Shuping Zhong Citation Format: Shuping Zhong. The function role of Brf1 in alcohol-induced human and mouse hepatocellular carcinoma*. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4936.