Gene In Human Hepatocellular Carcinoma Research Articles

Identification of a Macrophage marker gene signature to evaluate immune infiltration and therapeutic response in hepatocellular carcinoma

BackgroundOnly a minority of hepatocellular carcinoma (HCC) patients can benefit from systemic regimens. Macrophages, which abundantly infiltrate in HCC, could mediate tumour microenvironment remodelling and immune escape, proving to be powerful weapons in combating HCC. Thus, a deeper understanding of macrophages is necessary for improving existing antitumour treatments. MethodsWith a series of bioinformatic approaches, we comprehensively explored the role of macrophage-related genes in human HCCs from multiple single-cell and bulk RNA sequencing datasets. Unsupervised clustering was performed to cluster the macrophage marker genes (MMGs). GSVA and functional enrichment analysis were used to elucidate the functional differences among the MMG-associated clusters. Subsequently, a component analysis algorithm was used to construct a Macrosig score, and the prognosis, biological characteristics, mutation profile, TME cell infiltration status and drug response of patients with different Macrosig scores were further analysed. ResultsWe identified 13 MMGs in 574 HCC samples, based on which three MMG-associated clusters were defined. Overall survival time, clinicopathological features and immune infiltration scores differed among the different clusters. On this basis, 12 hub genes were identified among these clusters; subsequently, a scoring system was constructed to determine the Macrosig score. Importantly, patients with low-Macrosig scores, characterized by increased immune infiltration, increased mutation frequency and increased immune checkpoint expression, including CTLA-4, LAG3, PDCD1 and TIGIT, exhibited enhanced efficacy of immunotherapy when validated in an external database. Moreover, a low-Macrosig score indicates increased sensitivity to AZD.2281, A.443654, ABT.263, ABT.888, AG.014699 and ATRA, while a high Macrosig score indicates increased sensitivity to AZD6482, AKT inhibitor VIII, AS601245, AZ628, AZD.0530 and AZD6244. ConclusionsA novel scoring system was constructed to guide more effective prognostic evaluation and tailoring therapeutic regimens for HCC patients.

Effect of CRISPR Knockout of AXIN1 or ARID1A on Proliferation and Migration of Porcine Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive disease lacking effective treatment. Animal models of HCC are necessary for preclinical evaluation of the safety and efficacy of novel therapeutics. Large animal models of HCC allow testing image-guided locoregional therapies, which are widely used in the management of HCC. Models with precise tumor mutations mimicking human HCC provide valuable tools for testing precision medicine. AXIN1 and ARID1A are two of the most frequently mutated genes in human HCC. Here, we investigated the effects of knockout of AXIN1 and/or ARID1A on proliferation, migration, and chemotherapeutic susceptibility of porcine HCC cells and we developed subcutaneous tumors harboring these mutations in pigs. Gene knockout was achieved by CRISPR/Cas9 and was validated by Next Generation Sequencing. AXIN1 knockout increased the migration of porcine HCC cells but did not alter the cell proliferation. Knockout of ARID1A increased both the proliferation and migration of porcine HCC cells. Simultaneous knockout of AXIN1 and ARID1A increased the migration, but did not alter the proliferation of porcine HCC cells. The effect of gene knockout on the response of porcine HCC cells to two of the most commonly used systemic and locoregional HCC treatments was investigated; sorafenib and doxorubicin, respectively. Knockout of AXIN1 and/or ARID1A did not alter the susceptibility of porcine HCC cells to sorafenib or doxorubicin. Autologous injection of CRISPR edited HCC cells resulted in development of subcutaneous tumors in pigs, which harbored the anticipated edits in AXIN1 and/or ARID1A. This study elucidates the effects of CRISPR-mediated knockout of HCC-associated genes in porcine HCC cells, and lays the foundation for development and utilization of genetically-tailored porcine HCC models for in vivo testing of novel therapeutic approaches in a clinically-relevant large animal model.

Ruthana date extract inhibited proliferation of human hepatocellular carcinoma (HepG2) cells by modulation of BAX gene.

Cancer response to chemotherapeutic agents and its side effects remain a challenge for the development of new anticancer compounds. Dates are consumed worldwide due to their high nutritional value. We investigated the cytotoxicity and expression of the proapoptotic BAX gene in human hepatocellular carcinoma (HepG2) cells treated with Ruthana date ethanolic extract (RDE). The RDE ingredients analyzed by GC/MS and HepG2 cells were treated with different concentrations of RDE for 24, 48, and 72h. Cytotoxicity, cell viability, DNA fragmentation, and BAX expression were determined. The GC/MS analysis of RDE showed its high content of quercetin, myricetin kaempferol, thymine, and catechol as the most active ingredients. HepG2 treated with RDE showed a significant change in morphological characteristics related to cell death. The antiproliferative activity determined by WST-1 demonstrated that RDE significantly reduced cell viability. Cells treated with RDE (10-60mg) showed gradual DNA fragmentation in a dose-dependent manner. Gene expression analysis showed upregulation of BAX at 30mg/ml of RDE (p < 0.001). However, it showed downregulation at (40-60mg/ml) as compared to control. Our findings indicated that RDE exert cytotoxicity against HepG2 cells due to its high content of flavonoids. This effect through DNA fragmentation and activation of the proapoptotic BAX gene.

Tetraspanin 5 (TSPAN5), a Novel Gatekeeper of the Tumor Suppressor DLC1 and Myocardin-Related Transcription Factors (MRTFs), Controls HCC Growth and Senescence.

Simple SummaryHepatocellular carcinoma (HCC) ranks second among the leading causes of cancer-related death. Since current therapeutic options are very limited, a deeper understanding of the molecular mechanisms underlying the tumor onset and progression of HCC holds great potential for improved therapeutic options. Although it has been shown that deleted in liver cancer 1 (DLC1) acts as a tumor suppressor whose allele is lost in 50% of liver cancers, alterations in gene expression initiated by DLC1 loss have not yet been the primary focus of liver cancer research. To identify novel gene targets that allow for a personalized medicine approach for HCC therapy, we performed gene expression profiling for HepG2 cells stably expressing DLC1shRNA. We provide evidence that TSPAN5 is required for HCC growth, migration and invasion, and dissected the underlying molecular mechanisms involving myocardin-related transcription factors. Thus, TSPAN5 represents a novel therapeutic target for the treatment of HCC characterized by DLC1 loss.Human hepatocellular carcinoma (HCC) is among the most lethal and common cancers in the human population, and new molecular targets for therapeutic intervention are urgently needed. Deleted in liver cancer 1 (DLC1) was originally identified as a tumor suppressor gene in human HCC. DLC1 is a Rho-GTPase-activating protein (RhoGAP) which accelerates the return of RhoGTPases to an inactive state. We recently described that the restoration of DLC1 expression induces cellular senescence. However, this principle is not amenable to direct therapeutic targeting. We therefore performed gene expression profiling for HepG2 cells depleted of DLC1 to identify druggable gene targets mediating the effects of DLC1 on senescence induction. This approach revealed that versican (VCAN), tetraspanin 5 (TSPAN5) and N-cadherin (CDH2) were strongly upregulated upon DLC1 depletion in HCC cells, but only TSPAN5 affected the proliferation of HCC cells and human HCC. The depletion of TSPAN5 induced oncogene-induced senescence (OIS), mediated by the p16INK4a/pRb pathways. Mechanistically, silencing TSPAN5 reduced actin polymerization and thereby myocardin-related transcription factor A- filamin A (MRTF-A-FLNA) complex formation, resulting in decreased expression of MRTF/SRF-dependent target genes and senescence induction in vitro and in vivo. Our results identify TSPAN5 as a novel druggable target for HCC.

Bromodomain-containing protein BRPF1 is a therapeutic target for liver cancer

Epigenetic deregulation plays an essential role in hepatocellular carcinoma (HCC) progression. Bromodomains are epigenetic “readers” of histone acetylation. Recently, bromodomain inhibitors have exhibited promising therapeutic potential for cancer treatment. Using transcriptome sequencing, we identified BRPF1 (bromodomain and PHD finger containing 1) as the most significantly upregulated gene among the 43 bromodomain-containing genes in human HCC. BRPF1 upregulation was significantly associated with poor patient survival. Gene ablation or pharmacological inactivation of BRPF1 significantly attenuated HCC cell growth in vitro and in vivo. BRPF1 was involved in cell cycle progression, senescence and cancer stemness. Transcriptome sequencing revealed that BRPF1 is a master regulator controlling the expression of multiple key oncogenes, including E2F2 and EZH2. We demonstrated that BRPF1 activated E2F2 and EZH2 expression by facilitating promoter H3K14 acetylation through MOZ/MORF complex. In conclusion, BRPF1 is frequently upregulated in human HCCs. Targeting BRPF1 may be an approach for HCC treatment.

Prognostic value of aberrantly expressed methylation genes in human hepatocellular carcinoma.

Objectives: To identify the prognostic value of aberrantly methylated differentially expressed genes (DEGs) in hepatocellular carcinoma (HCC) and to explore the underlying mechanisms of tumorigenesis.Methods: Gene expression profiles (GSE65372 and GSE37988) were analyzed using GEO2R to obtain aberrantly methylated DEGs. Functional enrichment analysis of screened genes was performed by the Database for Annotation, Visualization, and Integrated Discovery (DAVID). Cytoscape software was used to analyze the PPI network and to select hub genes. Transcriptional and proteinic expression data of hub genes were obtained through UALCAN and the Human Protein Reference Database. Finally, we analyzed the prognostic value of hub genes with the Kaplan–Meier Plotter and MethSurv database.Results: In total, 24 up-hypomethylated oncogenes and 37 down-hypermethylated tumor suppressor genes (TSGs) were identified, and 8 hub genes, including 4 up-hypomethylated oncogenes (CDC5L, MERTK, RHOA and YBX1) and 4 down-hypermethylated TSGs (BCR, DFFA, SCUBE2 and TP63), were selected by PPI. Higher expression of methylated CDC5L-cg05671347, MERTK-cg08279316, RHOA-cg05657651 and YBX1-cg16306148, and lower expression of methylated BCR-cg25410636, DFFA-cg20696875, SCUBE2-cg19000089 and TP63-cg06520450, were associated with better overall survival (OS) in HCC patients. Multivariate analysis also showed they were independent prognostic factors for OS of HCC patients.Conclusions: In summary, different expression of methylated genes above mentioned were associated with better prognosis in HCC patients. Altering the methylation status of these genes may be a therapeutic target for HCC, but it should be further evaluated in clinical studies.

MicroRNA-1305 Inhibits the Stemness of LCSCs and Tumorigenesis by Repressing the UBE2T-Dependent Akt-Signaling Pathway

MicroRNAs (miRNAs) are involved in the maintenance of the cancer stem cell (CSC) phenotype by binding to genes and proteins that modulate cell proliferation and/or cell apoptosis. In our study, we aimed to investigate the role of miR-1305 in the proliferation and self-renewal of liver CSCs (LCSCs) via the ubiquitin-conjugating enzyme E2T (UBE2T)-mediated Akt-signaling pathway. Differentially expressed genes in human hepatocellular carcinoma (HCC) were obtained by in silico analysis. The relationship between miR-1305 and UBE2T was verified by dual luciferase reporter gene assay. qRT-PCR and western blot analysis were performed to determine the expression of UBE2T, the Akt-signaling pathway, and stemness-related factors in LCSCs. In addition, miR-1305 disrupted the activation of the Akt-signaling pathway by targeting UBE2T, and, ultimately, it repressed the sphere formation, colony formation, and proliferation, as well as tumorigenicity of LCSCs. In summary, miR-1305 targeted UBE2T to inhibit the Akt-signaling pathway, thereby suppressing the self-renewal and tumorigenicity of LCSCs. Those findings may provide an enhanced understanding of miR-1305 as a therapeutic target to limit the progression of LCSCs.

HELLS Regulates Chromatin Remodeling and Epigenetic Silencing of Multiple Tumor Suppressor Genes in Human Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the third most lethal cancer worldwide. Increasing evidence shows that epigenetic alterations play an important role in human carcinogenesis. Deregulation of DNA methylation and histone modifications have recently been characterized in HCC, but the significance of chromatin remodeling in liver carcinogenesis remains to be explored. In this study, by systematically analyzing the expression of chromatin remodeling genes in human HCCs, we found that helicase, lymphoid-specific (HELLS), an SWI2/SNF2 chromatin remodeling enzyme, was remarkably overexpressed in HCC. Overexpression of HELLS correlated with more aggressive clinicopathological features and poorer patient prognosis compared to patients with lower HELLS expression. We further showed that up-regulation of HELLS in HCC was conferred by hyperactivation of transcription factor specificity protein 1 (SP1). To investigate the functions of HELLS in HCC, we generated both gain-of-function and loss-of-function models by the CRISPR activation system, lentiviral short hairpin RNA, and the CRISPR/Cas9 genome editing system. We demonstrated that overexpression of HELLS augmented HCC cell proliferation and migration. In contrast, depletion of HELLS reduced HCC growth and metastasis both in vitro and in vivo. Moreover, inactivation of HELLS led to metabolic reprogramming and reversed the Warburg effect in HCC cells. Mechanistically, by integrating analysis of RNA sequencing and micrococcal nuclease sequencing, we revealed that overexpression of HELLS increased nucleosome occupancy, which obstructed the accessibility of enhancers and hindered formation of the nucleosome-free region (NFR) at the transcription start site. Though this mechanism, up-regulation of HELLS mediated epigenetic silencing of multiple tumor suppressor genes including E-cadherin, FBP1, IGFBP3, XAF1 and CREB3L3 in HCC. Conclusion: Our data reveal that HELLS is a key epigenetic driver of HCC; by altering the nucleosome occupancy at the NFR and enhancer, HELLS epigenetically suppresses multiple tumor suppressor genes to promote HCC progression.

Liver cancer cell lines distinctly mimic the metabolic gene expression pattern of the corresponding human tumours

BackgroundAlthough metabolism is profoundly altered in human liver cancer, the extent to which experimental models, e.g. cell lines, mimic those alterations is unresolved. Here, we aimed to determine the resemblance of hepatocellular carcinoma (HCC) cell lines to human liver tumours, specifically in the expression of deregulated metabolic targets in clinical tissue samples.MethodsWe compared the overall gene expression profile of poorly-differentiated (HLE, HLF, SNU-449) to well-differentiated (HUH7, HEPG2, HEP3B) HCC cell lines in three publicly available microarray datasets. Three thousand and eighty-five differentially expressed genes in ≥2 datasets (P < 0.05) were used for pathway enrichment and gene ontology (GO) analyses. Further, we compared the topmost gene expression, pathways, and GO from poorly differentiated cell lines to the pattern from four human HCC datasets (623 tumour tissues). In well- versus poorly differentiated cell lines, and in representative models HLE and HUH7 cells, we specifically assessed the expression pattern of 634 consistently deregulated metabolic genes in human HCC. These data were complemented by quantitative PCR, proteomics, metabolomics and assessment of response to thirteen metabolism-targeting compounds in HLE versus HUH7 cells.ResultsWe found that poorly-differentiated HCC cells display upregulated MAPK/RAS/NFkB signaling, focal adhesion, and downregulated complement/coagulation cascade, PPAR-signaling, among pathway alterations seen in clinical tumour datasets. In HLE cells, 148 downregulated metabolic genes in liver tumours also showed low gene/protein expression – notably in fatty acid β-oxidation (e.g. ACAA1/2, ACADSB, HADH), urea cycle (e.g. CPS1, ARG1, ASL), molecule transport (e.g. SLC2A2, SLC7A1, SLC25A15/20), and amino acid metabolism (e.g. PHGDH, PSAT1, GOT1, GLUD1). In contrast, HUH7 cells showed a higher expression of 98 metabolic targets upregulated in tumours (e.g. HK2, PKM, PSPH, GLUL, ASNS, and fatty acid synthesis enzymes ACLY, FASN). Metabolomics revealed that the genomic portrait of HLE cells co-exist with profound reliance on glutamine to fuel tricarboxylic acid cycle, whereas HUH7 cells use both glucose and glutamine. Targeting glutamine pathway selectively suppressed the proliferation of HLE cells.ConclusionsWe report a yet unappreciated distinct expression pattern of clinically-relevant metabolic genes in HCC cell lines, which could enable the identification and therapeutic targeting of metabolic vulnerabilities at various liver cancer stages.

Bioinformatical identification of key pathways and genes in human hepatocellular carcinoma after CSN5 depletion

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. It has been previously reported that CSN5 depletion is an effective method in human HCC. In the current study, we aimed to uncover gene signatures and key pathways during HCC. Gene expression profiles of GSE26485 were downloaded from GEO database. Totally, 101 differentially expressed genes (DEGs) were up-regulated and 146 ones were down-regulated. Biological processes (BP) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) analysis showed that the DEGs were mainly enriched in regulation of cell growth, oxidation-reduction process, mitotic cytokinesis, negative regulation of macroautophagy, endosome organization, lysosome, biosynthesis of antibiotics, small cell lung cancer and glutathione metabolism and so on (P < 0.05). Protein–protein interaction (PPI) network, Kaplan–Meier, log-rank method, western blot, immunohistochemistry and encyclopedia of DNA elements (ENCODE) analysis showed that CSN5 depletion took effects through down-regulation of SMAD5-related pathways which include EXO1, CENPA and NCAPG, resulting in the inactivation of H3K4me3 and H3K36me3. Those genes represent the promising targets for therapeutic intervention in HCC patients.

Abstract B40: Model liver cancer metastasis using 3D spheroids derived from primary tumors in liver cancer genetic mouse models

Abstract Introduction: Liver cancer, predominantly hepatocellular carcinoma (HCC) in adults, has become the fastest-growing cause of cancer death in the United States. Most cases of HCC are diagnosed at the metastatic stage and have a 5-year survival rate below 3%. Liver cancer in children, most commonly hepatoblastoma (HB), has a better prognosis than in adults, but the 5-year survival falls from 70% to 30% if the tumor has disseminated. Without basic research into the biology of liver cancer metastasis, cure rates for this disease are unlikely to improve. Preclinical mouse models have played a central role in linking basic and translational cancer research. However, current mouse models of liver cancer have mostly addressed the early stages of the disease and have rarely focused on its metastatic forms. Accurate model systems of liver cancer metastasis are urgently needed to elucidate the associated cellular and molecular mechanisms. Methods: Metastasis-initiating cells often hijack normal stem cell pathways to gain survival and growth advantages. Recent advances in 3D stem cell spheroid culture have provided a means to rapidly select and propagate cancer cells with stemness potential for in-depth biologic analysis. We have previously generated two liver cancer genetic mouse models (GMMs) of HCC and HB, both of which developed frequent primary tumors in the liver but only rare metastases. Our laboratory recently developed a robust protocol for growing liver cancer spheroids (LCSs) from primary tumors in the two GMM using 3D stem cell culture. To evaluate the in vivo tumorigenicity of the LCSs, we developed a novel ultrasound-guided, noninvasive intrahepatic injection method for transplanting cells into the liver with high accuracy and minimal animal manipulation. Results: We found that a subset of LCSs from each model exhibited potent tumorigenicity, driving widespread metastases in vivo upon orthotopic transplant. Gene set enrichment analysis demonstrated substantial enrichment of a number of metastatic and recurrent human HCC gene signatures in the LCS-driven HCC tumors and the enrichment of a human HB gene signature in the LCS-driven HB tumors. A large RNA-seq transcriptomic analysis revealed a significant enrichment of inflammatory and extracellular processes in the metastatic LCSs and tumors they generated compared to their nonmetastatic counterparts in both models, suggesting a critical role of tumor microenvironment (TME) in liver cancer metastasis. Conclusion: Our LCS-based approach to model liver cancer metastasis represents a unique strategy to model liver cancer metastasis by using primary tumors from non- or low-metastatic GMMs, an approach that can potentially be extended to other cancer types, considering the rapid advances in cancer spheroid studies across a wide range of solid malignant tumors. It also permits rapid mechanistic studies of liver cancer metastasis in the future by allowing gene manipulation in vitro within a uniform population of tumor-derived, metastasis-initiating LCSs, with subsequent tracking of metastasis development in transplantation models to be built in disease-relevant TMEs. Citation Format: Liyuan Li, Maoxiang Qian, David Finkelstein, Melissa Johnson, Dolores H. López-Terrada, Liqin Zhu. Model liver cancer metastasis using 3D spheroids derived from primary tumors in liver cancer genetic mouse models [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B40.

Ganodermanontriol inhibits expression of special AT rich sequence binding protein 1 gene in human hepatocellular carcinoma.

The metastasis of liver cancer is a major cause of clinical treatment failure, restrain, and control the cancer metastasis is the major strategy of the treatment and prevention of the disease. Special AT-rich sequence-binding protein 1 (SATB1) gene was overexpressed in many malignant tumors and considered as a potential target of anticancer drug. This study investigated the mechanism how ganodermanontriol effect the expression of SATB1 and thus inhibits the growth and metastasis in hepatocellular carcinoma (HCC). This study explored mainly on the mechanism how ganodermanontriol affects the expression of SATB1 and inhibits proliferation of tumor on human hepatoma cell line HepG2. The cancer cells were treated with ganodermanontriol. The status of the cells was detected by different methods. The mechanism was checked by various methods. In HepG2 cancer cells treated with various concentrations of ganodermanontriol, the cell proliferation of was detected by MTT assay, cell apoptosis was analyzed by flow cytometry; the mRNA of SATB1, Bcl-2, Bax were detected by reverse transcription-polymerase chain reaction (RT-PCR) and the protein level of SATB1, Bcl-2, Bax, and caspase 3 were analyzed by Western blot. Data are presented as the mean ± standard deviation. The data were analyzed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). A one-way analysis of variance test was used to compare the differences among groups. This study showed that ganodermanontriol could significantly reduce the expression level of SATB1. Therefore, downregulate the cascade effect caused by the expression level of Bcl-2 in HCC HepG2 cells.

Abstract LB-156: Delineating the pathological roles and molecular functions of histone methyltransferase G9a in hepatocellular carcinoma

Abstract Liver cancer, primarily hepatocellular carcinoma (HCC), is the third leading cause of cancer mortality worldwide. In HCC, epigenetic changes including aberrant DNA methylations and histone modifications occur far more frequently than genetic alternations. However, the underlying mechanism and specific gene targets are still unknown. Using RNA-Sequencing, we identified Euchromatic histone-lysine N-methyltransferase 2 (EHMT2/G9a) as one the most significantly deregulated epigenetic regulatory genes in human HCC. G9a is a SET domain containing histone methyltransferase specific for histone H3 lysine 9 methylation. However, little is known about the functional roles of G9a in human HCC. Herein, we showed that G9a expression was frequently up-regulated in primary HCC. Up-regulation of G9a was significantly associated with multiple clinicopathological features related to HCC metastasis. We demonstrated that the frequent G9a up-regulation in HCC was attributed to the G9a gene copy number gain. In addition, we identified miR-1 as a novel post-transcriptional regulator of G9a. Loss of miR-1 significantly contributed to G9a up-regulation in human HCC. Functionally, stable knockdown (by lenti-viral based shRNA) and genetic knock-out of G9a (by CRISPR/Cas9 gene editing system) significantly suppressed HCC cell proliferation, migration and colony formation. We also demonstrated that G9a specific inhibitors, UNC0638 and BIX01294, could effectively inhibit HCC cell growth. By utilizing nude mice model, we showed that depletion of G9a drastically inhibited in vivo tumorigenicity of HCC cells. Mechanistically, we found that up-regulation of G9a epigenetically silence the expression of tumor suppressive gene RARRES3 to promote HCC tumorigenicity and metastasis. In summary, our finding suggested that up-regulation of G9a contributed to the liver carcinogenesis and could be a therapeutic target for HCC treatment. Citation Format: Lei Wei, Felice Ho-Ching Tsang, Sandy Leung-Kuen Au, Joyce Lee, Carmen Wong, Irene Oi-Lin Ng, Chun-Ming Wong. Delineating the pathological roles and molecular functions of histone methyltransferase G9a in 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 LB-156.

Transposon mutagenesis identifies genes and cellular processes driving epithelial-mesenchymal transition in hepatocellular carcinoma

Epithelial-mesenchymal transition (EMT) is thought to contribute to metastasis and chemoresistance in patients with hepatocellular carcinoma (HCC), leading to their poor prognosis. The genes driving EMT in HCC are not yet fully understood, however. Here, we show that mobilization of Sleeping Beauty (SB) transposons in immortalized mouse hepatoblasts induces mesenchymal liver tumors on transplantation to nude mice. These tumors show significant down-regulation of epithelial markers, along with up-regulation of mesenchymal markers and EMT-related transcription factors (EMT-TFs). Sequencing of transposon insertion sites from tumors identified 233 candidate cancer genes (CCGs) that were enriched for genes and cellular processes driving EMT. Subsequent trunk driver analysis identified 23 CCGs that are predicted to function early in tumorigenesis and whose mutation or alteration in patients with HCC is correlated with poor patient survival. Validation of the top trunk drivers identified in the screen, including MET (MET proto-oncogene, receptor tyrosine kinase), GRB2-associated binding protein 1 (GAB1), HECT, UBA, and WWE domain containing 1 (HUWE1), lysine-specific demethylase 6A (KDM6A), and protein-tyrosine phosphatase, nonreceptor-type 12 (PTPN12), showed that deregulation of these genes activates an EMT program in human HCC cells that enhances tumor cell migration. Finally, deregulation of these genes in human HCC was found to confer sorafenib resistance through apoptotic tolerance and reduced proliferation, consistent with recent studies showing that EMT contributes to the chemoresistance of tumor cells. Our unique cell-based transposon mutagenesis screen appears to be an excellent resource for discovering genes involved in EMT in human HCC and potentially for identifying new drug targets.

Identification of allelic expression imbalance genes in human hepatocellular carcinoma through massively parallel DNA and RNA sequencing.

Hepatocellular carcinoma (HCC) is a common malignant tumor worldwide. The prognosis and treatment of this disease have changed little in recent decades because the mechanisms underlying most events of this disease remain obscure. Allelic variation of gene expression is associated with many important biological processes, which provide a new perspective to understand HCC pathogenesis at the molecular level. To identify allelic expression imbalance (AEI) genes in HCCs, we developed a computational method that considered accurate mapping and vigorous AEI detection using paired DNA-seq and RNA-seq data. We analyzed the DNA-seq and RNA-seq data derived from two HCC samples and two cell lines. By applying a strict criterion, a total of 203 tumor-specific AEI genes were identified with high confidence, and several genes have been reported to be associated with the migration or proliferation of cancer cells, such as the genes RELN and DHRS3. In addition, we also found some novel AEI genes in HCCs, such as HNRNPR and PTAFR. Our study provides new insight into AEI events that may contribute to understanding gene expression regulation, cell proliferation and migration, and tumorigenesis.

Global analysis of DNA methylation in hepatocellular carcinoma by a liquid hybridization capture-based bisulfite sequencing approach.

BackgroundEpigenetic alterations, such as aberrant DNA methylation of promoter and enhancer regions, which lead to atypical gene expression, have been associated with carcinogenesis. In hepatocellular carcinoma (HCC), genome-wide analysis of methylation has only recently been used. For a better understanding of hepatocarcinogenesis, we applied an even higher resolution analysis of the promoter methylome to identify previously unknown regions and genes differentially methylated in HCC.ResultsOptimized liquid hybridization capture-based bisulfite sequencing (LHC-BS) was developed to quantitatively analyze 1.86 million CpG sites in individual samples from eight pairs of HCC and adjacent tissues. By linking the differentially methylated regions (DMRs) in promoters to the differentially expressed genes (DEGs), we identified 12 DMR-associated genes. We further utilized Illumina MiSeq combining the bisulfite sequencing PCR approach to validate the 12 candidate genes. Analysis of an additional 78 HCC pairs on the Illumina MiSeq platform confirmed that 7 genes showed either promoter hyper-methylation (SMAD6, IFITM1, LRRC4, CHST4, and TBX15) or hypo-methylation (CCL20 and NQO1) in HCC.ConclusionsNovel methylome profiling provides a cost-efficient approach to identifying candidate genes in human HCC that may contribute to hepatocarcinogenesis. Our work provides further information critical for understanding the epigenetic processes underlying tumorigenesis and development of HCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0121-1) contains supplementary material, which is available to authorized users.

Abstract 1948: In silico screening for novel Wnt/β-catenin pathway target and regulator genes in human hepatocellular carcinoma

Abstract Introduction Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the third leading cause of all cancer deaths worldwide. A fraction of HCC has increased activity of Wnt/β-catenin (Wnt) pathway, which reportedly enhances malignant phenotypes like epithelial-mesenchymal transition (EMT) in various kinds of cancer. Identification of Wnt pathway target and regulator genes gives us better understanding of molecular biological behavior of HCC. In this study, we performed in silico screening for a novel Wnt pathway target and regulator genes in human HCC using public databases. Method First, we obtained TCGA data sets of human HCC including mRNA expression (268 samples) and mutational status (198 samples) from the Broad GDAC Firehose web site (gdac.broadinstitute.org/). Next, for β-catenin transcriptional target genes prepared by literature curation, we performed EEM analysis (Niida et al. 2009) on the HCC expression data set to obtain Wnt pathway target genes in HCC. Assuming the sum of mRNA expression of the Wnt pathway target genes as Wnt pathway activity, we then measured association between the pathway alteration and the activity by linear multiple regression analysis ([Wnt pathway activity] = a0 + a1[CTNNB1 mutation] + a2[AXIN1 mutation] + a3[AXIN2 mutation]+a4[APC mutation]+ a5[CTNNB1 mRNA expression] : a0∼a5; partial regression coefficient). By focusing on known epithelial markers (CDH1, KRT18, MUC1, OCLN, TJP1) and mesenchymal markers (ACTA2, CDH2, FN1, VIM, VTN), we also examined association between the Wnt pathway activity and EMT-manifesting genotype. Finally, we performed multiple regression analysis to search for novel Wnt pathway regulator genes ([Wnt pathway activity] = b0 + b1[CTNNB1 mutation] + b2[APC mutation]+ b3[CTNNB1 mRNA expression] + b4[mRNA of a regulator] : b0∼b4; partial regression coefficient). Result (1)EEM identified AASS, ASPSCR1, BMP4, C6orf97, DUT, GGH, GNPAT, GREB1, HGD, IKBKAP, ITPR2, LAMA3, LGR5, NEK3, RELN, RHBG, RHOBTB1, SLC13A3, SMYD2, TBX3, TDGF1, TRIB2, VSNL1, ZBTB38 as the Wnt pathway target genes in HCC. (2)Multiple regression analysis revealed the Wnt pathway activity had strong correlations with CTNNB1 mutation (p-value &amp;lt; 2.00×10-16), APC mutation (p-value = 2.73×10-3), and CTNNB1 expression (p-value = 2.19×10-07) (3)The Wnt pathway activity had a strong correlation with the EMT genotype (p-value = 3.58×10-4). (4)We identified 100 statistically significant Wnt pathway regulator candidates (q-values &amp;lt; 1.00×10-10). Conclusion Our in silico screening identified novel Wnt pathway target genes in HCC, whose expression profiles were correlated with Wnt pathway alterations and the EMT-manifesting gene signature. We also have predicted Wnt pathway regulator genes, and are now conducting in vitro validation. Citation Format: Hisateru Komatsu, Atsushi Niida, Masami Ueda, Hidenari Hirata, Ryutaro Uchi, Sho Nambara, Tomoko Saito, Shotaro Sakimura, Yuki Takano, Yoshiaki Shinden, Tomohiro Iguchi, Hidetoshi Eguchi, Keishi Sugimachi, Yuichiro Doki, Masaki Mori, Koshi Mimori. In silico screening for novel Wnt/β-catenin pathway target and regulator genes in human hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1948. doi:10.1158/1538-7445.AM2015-1948

Integrated analyses of DNA methylation and hydroxymethylation reveal tumor suppressive roles of ECM1, ATF5, and EOMES in human hepatocellular carcinoma.

BackgroundDifferences in 5-hydroxymethylcytosine, 5hmC, distributions may complicate previous observations of abnormal cytosine methylation statuses that are used for the identification of new tumor suppressor gene candidates that are relevant to human hepatocarcinogenesis. The simultaneous detection of 5-methylcytosine and 5-hydroxymethylcytosine is likely to stimulate the discovery of aberrantly methylated genes with increased accuracy in human hepatocellular carcinoma.ResultsHere, we performed ultra-performance liquid chromatography/tandem mass spectrometry and single-base high-throughput sequencing, Hydroxymethylation and Methylation Sensitive Tag sequencing, HMST-seq, to synchronously measure these two modifications in human hepatocellular carcinoma samples. After identification of differentially methylated and hydroxymethylated genes in human hepatocellular carcinoma, we integrate DNA copy-number alterations, as determined using array-based comparative genomic hybridization data, with gene expression to identify genes that are potentially silenced by promoter hypermethylation.ConclusionsWe report a high enrichment of genes with epigenetic aberrations in cancer signaling pathways. Six genes were selected as tumor suppressor gene candidates, among which, ECM1, ATF5 and EOMES are confirmed via siRNA experiments to have potential anti-cancer functions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0533-9) contains supplementary material, which is available to authorized users.

Identification of Epigenetically Inactivated Genes in Human Hepatocellular Carcinoma by Integrative Analyses of Methylation Profiling and Pharmacological Unmasking

Objectives: DNA methylation-dependent transcriptional inactivation of tumor suppressor genes (TSGs) is critical for the pathogenesis of hepatocellular carcinoma (HCC). This study identifies potential TSGs in HCCs using methylation profiling and pharmacological unmasking of methylated TSGs. Methods: Methylation profiling was performed on 22 pairs of HCCs and their corresponding noncancerous liver tissues using the Infinium HumanMethylation27 BeadChip. We also determined the gene reexpression after treatment with 5-aza-2′-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) in 5 HCC cell lines. Results: We selected CpGs that exhibited a significant increase in methylation in HCC tissues compared with that of the noncancerous control group. Two hundred and thirteen CpGs on different gene promoters with a mean difference in the β value ≥0.15 and a value of p < 0.05 were selected. Of the 213 genes, 45 genes were upregulated in 3 or more HCC cell lines with multiplier value of differences ≥2.0 after 5-Aza-dC and TSA treatment. Conclusions: We identified several potential TSGs that participate in transcription inactivation through epigenetic interactions in HCC. The results of this study are important for the understanding of functionally important epigenetic alterations in HCC.

Abstract 4717: Detection of the Cyclin J (CCNJ) as a new cancer-related gene in human hepatocellular carcinoma by using a method of triple combination array analysis

Abstract Background: Hepatocellular carcinoma (HCC) has a high rate of recurrence and a poor prognosis. To detect genes correlated with HCC, we have developed a new technique, triple combination array analysis, consisting of methylation array, gene expression array and single nucleotide polymorphism (SNP) array analysis. Methods: A surgical specimen obtained from a 68-year-old female HCC patient was analyzed using triple combination array analysis, which identified a candidate cancer-related gene of HCC. Subsequently, samples from nine cell lines and 48 HCC patients were evaluated for their methylation and expression status of the identified gene using methylation-specific polymerase chain reaction (MSP) and quantitative real-time reverse transcriptase PCR, respectively. Results: Using the triple combination array analysis, cyclin J (CCNJ) was detected as a candidate cancer-related gene. CCNJ was located on chromosome 10q24.1 and the copy number, using SNP chip array, revealed no loss of heterozygosity. According to expression array results, The expression of CCNJ in tumor tissue decreased at two points of the expression array chip, and the decreased values of the chip were −1.3 and −2.3. CCNJ was found to be hypermethylated (methylation value 0.906, range 0-1.0) in cancer tissue compared with adjacent normal tissue (0.112) using the methylation array. Using MSP, hypermethylation of the promoter region of CCNJ was shown to occur in 37 (77.1%) tumor samples. CCNJ expression was significantly decreased in cases with methylation (p&amp;lt;0.0001). Furthermore, HCC patients with methylated CCNJ had a significantly worse prognosis for recurrence-free survival (p=0.0353) and tended to have a worse prognosis for overall survival than those with unmethylated CCNJ (p=0.0838). Conclusion: The present study indicates that triple combination array analysis is an effective method to detect novel genes related to HCC. We suggest that CCNJ acts as a cancer-related gene in HCC. Citation Format: Nao Takano, Shuji Nomoto, Mitsuhiro Hishida, Yoshikuni Inokawa, Masamichi Hayashi, Mitsuro Kanda, Yukiyasu Okamura, Yoko Nishikawa, Chie Tanaka, Daisuke Kobayashi, Suguru Yamada, Goro Nakayama, Tsutomu Fujii, Hiroyuki Sugimoto, Masahiko Koike, Michitaka Fujii, Shin Takeda, Yasuhiro Kodera. Detection of the Cyclin J (CCNJ) as a new cancer-related gene in human hepatocellular carcinoma by using a method of triple combination array analysis. [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 4717. doi:10.1158/1538-7445.AM2014-4717