Cirrhotic Tissue Research Articles

Comprehensive analysis of genetic aberrations linked to tumorigenesis in regenerative nodules of liver cirrhosis.

Hepatocellular carcinoma (HCC) recurrently develops in cirrhotic liver containing a number of regenerative nodules (RNs). However, the biological tumorigenic potential of RNs is still unclear. To uncover the molecular bases of tumorigenesis in liver cirrhosis, we investigated the genetic aberrations in RNs of cirrhotic tissues using next-generation sequencing. We isolated 205 RNs and 7 HCC tissues from the whole explanted livers of 10 randomly selected patients who had undergone living-donor liver transplantation. Whole-exome sequencing and additional targeted deep sequencing on 30 selected HCC-related genes were conducted to reveal the mutational landscape of RNs and HCCs. Whole-exome sequencing demonstrated that RNs frequently harbored relatively high-abundance genetic alterations, suggesting a clonal structure of each RN in cirrhotic liver. The mutation signature observed in RNs was similar to those determined in HCC, characterized by a predominance of C>T transitions, followed by T>C and C>A mutations. Targeted deep sequencing analyses of RNs identified nonsynonymous low-abundance mutations in various tumor-related genes, including TP53 and ARID1A. In contrast, TERT promoter mutations were not detected in any of the RNs examined. Consistently, TERT expression levels in RNs were comparable to those in normal livers, whereas every HCC tissue demonstrated an elevated level of TERT expression. Analyses of RNs constructing cirrhotic liver indicated that a variety of genetic aberrations accumulate in the cirrhotic liver before the development of clinically and histologically overt HCC. These aberrations in RNs could provide the basis of tumorigenesis in patients with liver cirrhosis.

Identification of key genes and pathways in hepatocellular carcinoma: A preliminary bioinformatics analysis.

Background:Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. However, the precise mechanisms of the development and progression of HCC remain unclear. The present study attempted to identify and functionally analyze the differentially expressed genes between HCC and cirrhotic tissues by using comprehensive bioinformatics analyses.Methods:The GSE63898 gene expression profile was downloaded from the Gene Expression Omnibus (GEO) and analyzed using the online tool GEO2R to identify differentially expressed genes (DEGs). Gene ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the DEGs were performed in DAVID. The STRING database was used to evaluate the interactions of DEGs and to construct a protein-protein interaction (PPI) network using Cytoscape software. Hub genes were selected using the cytoHubba plugin and were validated with the cBioPortal database.Results:A total of 301 DEGs were identified between HCC and cirrhotic tissues. The GO analysis results showed that these DEGs were significantly enriched in certain biological processes including negative regulation of growth and cell chemotaxis. Several significant pathways, including the p53 signaling pathway, were identified as being closely associated with these DEGs. The top 12 hub genes were screened and included TTK, NCAPG, TOP2A, CCNB1, CDK1, PRC1, RRM2, UBE2C, ZWINT, CDKN3, AURKA, and RACGAP1. The cBioPortal analysis found that alterations in hub genes could result in significantly reduced disease-free survival in HCC.Conclusion:The present study identified a series of key genes and pathways that may be involved in the tumorigenicity and progression of HCC, providing a new understanding of the underlying molecular mechanisms of carcinogenesis in HCC.

Hepatocellular carcinomas and their mimics

High resolution cross-sectional imaging techniques means that even small, well-differentiated hepatocellular tumors can also be diagnosed with biopsy. In cirrhotic liver tissue, macroregenerative and dysplastic nodules must be discriminated from hepatocellular cancer (HCC). In non-cirrhotic liver tissue the differential diagnosis includes hepatocellular adenoma, macroregeneratory nodules, fibrolamellar carcinoma, as well as primary tumors and metastases. The diagnostic procedure includes matrix diagnosis of the tumor-bearing liver tissue, cyto- and histomorphologic analysis including capillarization of vascular bed, and adapted immunohistological testing with antibodies which underline possible malignancy or hepatocellular differentiation. Aflow chart for the diagnosis of hepatocellular carcinomas and their mimics on liver biopsies is presented.

Integrating the Epigenome to Identify Drivers of Hepatocellular Carcinoma.

Disruption of epigenetic mechanisms has been intimately linked to the etiology of human cancer. Understanding how these epigenetic mechanisms (including DNA methylation [5mC], hydroxymethylation [5hmC], and histone post-translational modifications) work in concert to drive cancer initiation and progression remains unknown. Hepatocellular carcinoma (HCC) is increasing in frequency in Western countries but lacks efficacious treatments. The epigenome of HCC remains understudied. To better understand the epigenetic underpinnings of HCC, we performed a genome-wide assessment of 5mC, 5hmC, four histone modifications linked to promoter/enhancer function (H3K4me1, H3K27ac, H3K4me3, and H3K27me3), and transcription across normal, cirrhotic, and HCC liver tissue. Implementation of bioinformatic strategies integrated these epigenetic marks with each other and with transcription to provide a comprehensive epigenetic profile of how and when the liver epigenome is perturbed during progression to HCC. Our data demonstrate significant deregulation of epigenetic regulators combined with disruptions in the epigenome hallmarked by profound loss of 5hmC, locus-specific gains in 5mC and 5hmC, and markedly altered histone modification profiles, particularly remodeling of enhancers. Data integration demonstrates that these marks collaborate to influence transcription (e.g., hyper-5hmC in HCC-gained active enhancers is linked to elevated expression) of genes regulating HCC proliferation. Two such putative epigenetic driver loci identified through our integrative approach, COMT and FMO3, increase apoptosis and decrease cell viability in liver-derived cancer cell lines when ectopically re-expressed. Conclusion: Altogether, integration of multiple epigenetic parameters is a powerful tool for identifying epigenetically regulated drivers of HCC and elucidating how epigenome deregulation contributes to liver disease and HCC.

Mammalian target of rapamycin (mtor) and autophagy in hepatitis c virus-related hepatocellular carcinoma: relation to tumor progression

Introduction: Mammalian target of rapamycin (mTOR) is a key regulator of cell metabolism and a down-regulator of autophagy. The present work was designed to study the role of mTOR and autophagy in the development and progression of hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). Method: Twenty seven patients with HCV-related cirrhosis (13 patients without HCC and 14 patients with HCC who underwent surgical resection) and 15 healthy subjects, were included in the study. Tumor staging was determined according to Barcelona Clinic Liver Cancer system. Measurement of serum mTOR protein was determined using enzyme linked immunosorbant assay. Tumor histological grade was assessed according to Edmonson and Steiner's criteria. Immunohistochemical examination was performed using anti-human antibodies against mTOR and autophagy-related protein 5 (Atg5), a marker of autophagy. Result: Serum mTOR levels were significantly higher in cirrhotic patients with and without HCC than in healthy subjects and in patients with HCC than in patients without HCC (P < 0.001). By plotting ROC curve, the sensitivity and specificity of serum mTOR in discriminating cirrhotic patients with and without HCC were 92.9% and 92.3% respectively (AUC = 0.970, P< 0.001) at a cut-off value of 4.4 ng/ml. mTOR expression showed a significant increase in HCC tissues compared with the surrounding non-neoplastic cirrhotic tissues (P = 0.035) while Atg5 expression was significantly lower in HCCs than in the cirrhotic tissues (P = 0.038). Serum mTOR levels and mTOR expression in HCCs were positively correlated and showed significant positive correlations with serum alpha fetoprotein levels and tumor size, stage and histological grade (P < 0.05) and inverse correlations with intratumoral Atg5 expression (P < 0.001). Conclusion: Activation of mTOR pathway plays an important role in the development and progression of HCV-related HCC, possibly, through inhibition of autophagy. Serum mTOR may represent a potential diagnostic biomarker for HCC in HCV infection.

Targeting HSP90 with the small molecule inhibitor AUY922 (luminespib) as a treatment strategy against hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly malignant tumor that responds very poorly to existing therapies, most probably due to its extraordinary inter- and intra-tumor molecular heterogeneity. The modest therapeutic response to molecular targeted agents underlines the need for new therapeutic approaches for HCC. In our study, we took advantage of well-characterized human HCC cell lines, differing in transcriptomic subtypes, DNA mutation and amplification alterations, reflecting the heterogeneity of primary HCCs, to provide a preclinical evaluation of the specific heat shock protein 90 (HSP90) inhibitor AUY922 (luminespib). Indeed, HSP90 is highly expressed in different tumor types, but its role in hepatocarcinogenesis remains unclear. Here, we analyzed HSP90 expression in primary human HCC tissues and evaluated the antitumor effects of AUY922 in vitro as well as in vivo. HSP90 expression was significantly higher in HCC tissues than in cirrhotic peritumoral liver tissues. AUY922 treatment reduced the cell proliferation and viability of HCC cells in a dose-dependent manner, but did not do so for normal human primary hepatocytes. AUY922 treatment led to the upregulation of HSP70 and the simultaneous depletion of HSP90 client proteins. In addition, in a cell type-dependent manner, treatment induced either both caspase-dependent β-catenin cleavage and the upregulation of p53, or Mcl-1 expression, or NUPR1 expression, which contributed to the increased efficacy of, or resistance to, treatment. Finally, in vivo AUY922 inhibited tumor growth in a xenograft model. In conclusion, HSP90 is a promising therapeutic target in HCC, and AUY922 could be a drug candidate for its treatment.

Retinoic Acid Receptor-β Is Downregulated in Hepatocellular Carcinoma and Cirrhosis and Its Expression Inhibits Myosin-Driven Activation and Durotaxis in Hepatic Stellate Cells.

Hepatic stellate cells (HSCs) are essential perisinusoidal cells in both healthy and diseased liver. HSCs modulate extracellular matrix (ECM) homeostasis when quiescent, but in liver fibrosis, HSCs become activated and promote excess deposition of ECM molecules and tissue stiffening via force generation and mechanosensing. In hepatocellular carcinoma (HCC), activated HSCs infiltrate the stroma and migrate to the tumor core to facilitate paracrine signaling with cancer cells. Because the function of HSCs is known to be modulated by retinoids, we investigated the expression profile of retinoic acid receptor beta (RAR-β) in patients with cirrhosis and HCC, as well as the effects of RAR-β activation in HSCs. We found that RAR-β expression is significantly reduced in cirrhotic and HCC tissues. Using a comprehensive set of biophysical methods combined with cellular and molecular biology, we have elucidated the biomechanical mechanism by which all trans-retinoic acid promotes HSC deactivation via RAR-β-dependent transcriptional downregulation of myosin light chain 2 expression. Furthermore, this also abrogated mechanically driven migration toward stiffer substrates. Conclusion: Targeting mechanotransduction in HSCs at the transcriptional level may offer therapeutic options for a range of liver diseases.

Thioredoxin interacting protein promotes invasion in hepatocellular carcinoma.

BackgroundConsiderable evidence suggests that oxidative stress plays an essential role in the progression of hepatocellular carcinoma (HCC). While acquired resistance to oxidative stress is the main driver of aggressive cell phenotype, the underlying mechanisms remain unknown. Here, we tested the hypothesis that elevated expression of Thioredoxin-interacting protein (TXNIP) is a main regulator of the aggressive phenotype in HCC.Materials and MethodsTo test this hypothesis, we measured TXNIP expression levels in 11 HCC cell lines by qPCR and western blotting. In addition, 80 pairs of HCC tissues and matched liver tissues of 73 cases, as well as 11 normal liver tissue samples were examined by immunohistochemistry. Besides, TXNIP expression levels were analyzed by Oncomine Platform in seven independent microarray datasets. Finally, the functional role of TXNIP in HCC was investigated in vitro and in vivo by silencing and overexpression studies.ResultsOur results show that TXNIP expression is significantly increased in HCC compared to non-tumor counterparts (p < 0.0001) as well as to normal (p < 0.0001) and cirrhotic (p < 0.0001) liver tissues. Moreover, stable overexpression of TXNIP in HCC cells (i) significantly increases ROS levels, (ii) induces EMT phenotype, (iii) increases motility, invasion and 3D branching tubulogenesis, (iv) decreases apoptosis, and (v) elevates in vivo metastasis in zebrafish embryos. Finally, we identify sinusoidal/stromal and cytoplasmic TXNIP staining patterns as risk factors for intrahepatic vascular invasion (p:0.0400).ConclusionOur results strongly suggest that overexpression of TXNIP has a pivotal role in HCC progression by inducing cell survival, invasion, and metastasis.

Immunohistochemical Expression of Epidermal Growth Factor Receptor in Hepatocellular Carcinoma.

Epidermal growth factor receptor (EGFR) signaling plays an important role in various cancers, including hepatocellular carcinoma (HCC). We aimed to evaluate immunoexpression of EGFR in HCC and surrounding non-tumor liver tissue and to correlate it to multiple clinicopathologic data. We analyzed 60 patients with HCC for multiple clinicopathologic characteristics and survival. Presence of the immunosignal and the percentage of positive tumor cells at the whole tumor tissue sample and adjacent cirrhotic liver tissue were semi-quantitatively determined. Nineteen patients (31.67%) were female and 41 (68.33%) were male ranging in age from 31 to 85 years, median 61.88±10.51. Mean survival time for female patients was 8.86±1.76 months, for male 13.03±1.50 months and overall survival was 11.6051±1.19 months. The most patients had: T2 status (41.67%), no enlarged lymph nodes (90%), vascular invasion (63.33%) and well differentiated (43.33%) tumors. EGFR immunoexpression was determined in range from 0% to 100% in both tumor and non-tumor tissue with mean value of 39.58% in tumor and 86.86% in cirrhotic tissue (p<0.00). Higher percent of tumor EGFR positive cells were found in cases with higher T status, higher levels of AFP and poorly differentiated carcinoma, but not significantly. Lower percent of tumor EGFR positive cells were found in patients with vascular invasion and enlarged lymph nodes, but also not significantly. EGFR expression in tumor tissue significantly influenced survival of the patients (p<0.05). The study showed that expression of EGFR in lower percentage of tumor cells was associated to favorable prognosis, making it a potential prognostic marker and therapeutic target.

Transcriptomic analysis of hepatocellular carcinoma reveals molecular features of disease progression and tumor immune biology

Hepatocellular carcinoma (HCC) develops in the context of chronic inflammatory liver disease and has an extremely poor prognosis. An immunosuppressive tumor microenvironment may contribute to therapeutic failure in metastatic HCC. Here, we identified unique molecular signatures pertaining to HCC disease progression and tumor immunity by analyzing genome-wide RNA-Seq data derived from HCC patient tumors and non-tumor cirrhotic tissues. Unsupervised clustering of gene expression data revealed a gradual suppression of local tumor immunity that coincided with disease progression, indicating an increasingly immunosuppressive tumor environment during HCC disease advancement. IHC examination of the spatial distribution of CD8+ T cells in tumors revealed distinct intra- and peri-tumoral subsets. Differential gene expression analysis revealed an 85-gene signature that was significantly upregulated in the peri-tumoral CD8+ T cell-excluded tumors. Notably, this signature was highly enriched with components of underlying extracellular matrix, fibrosis, and epithelial–mesenchymal transition (EMT). Further analysis condensed this signature to a core set of 23 genes that are associated with CD8+ T cell localization, and were prospectively validated in an independent cohort of HCC specimens. These findings suggest a potential association between elevated fibrosis, possibly modulated by TGF-β, PDGFR, SHH or Notch pathway, and the T cell-excluded immune phenotype. Indeed, targeting fibrosis using a TGF-β neutralizing antibody in the STAM™ model of murine HCC, we found that ameliorating the fibrotic environment could facilitate redistribution of CD8+ lymphocytes into tumors. Our results provide a strong rationale for utilizing immunotherapies in HCC earlier during treatment, potentially in combination with anti-fibrotic therapies.

Caveolin-1 is related to lipid droplet formation in hepatic stellate cells in human liver

Caveolins (CAVs) regulate intracellular cholesterol transport by a complex process involving caveolae, endoplasmic reticulum (ER), and the Golgi network. Hepatic stellate cells (HSCs) are the central site for retinoid storage in the liver and indeed the entire body. Herein, we attempted to elucidate the ultrastructural localization and expression of caveolin-1 (CAV-1) in human HSCs during the progression of liver cirrhosis (LC). Normal and hepatitis C-related cirrhotic liver samples were prepared using a modified perfusion-fixation method to fix organelle structures and molecules in their in vivo positions, and examined using immunoelectron microscopy. In control liver specimens, CAV-1 was minimally associated with low electron density lipid droplets (LDs) segregated around zones 1―2, and specifically associated with membranes surrounding LDs. CAV-1 was segregated in high-density LDs, consistent with the formation of membrane-enclosed lipid-rich vesicular structures, as well as caveolae on plasma membranes around zones 2―3. In cirrhotic liver specimens, CAV-1 molecules were inserted into the cytoplasmic leaflets of ER membranes for transportation to LDs. Thus, CAV-1 transport to LDs might represent an intracellular pathway from the ER in cirrhotic liver tissue.

Evaluation of the role of Notch1 expression in hepatic carcinogenesis with clinico-pathological correlation

The role of Notch pathway in hepatocarcinogenesis is unclear with conflicting results reported from different researchers. This study aimed to investigate the exact role of Notch1 in hepatocarcinogenesis and its influence on survival and to determine the possibility of it being a target therapy. Differential immunohistochemical expression of Notch1 in 100 cases of hepatocellular carcinoma (HCC) and adjacent non-neoplastic liver tissue was performed. The results showed that expression of Notch1 was significantly higher in the non-neoplastic hepatic tissues than in HCC tissues (p<0.001), but there was no significant difference in Notch1 expression between cirrhotic and non-cirrhotic liver tissue (p=0.197). Notch1 expression was higher in low grade than in high grade HCC (p=0.036). Notch1 expression showed reverse correlation with mitotic count (p=0.008), and necrosis (p=0.005). The disease free survival was shorter in patients displaying low levels of Notch1 expression (p=0.045). The overall survival showed no significant difference between high and low levels of Notch1 expression; however, it was somewhat longer in patients with high Notch1 expression (p=0.220). In conclusion, the tumour suppressor role of Notch1 was supported and the use of Notch1 agonists may have a role in improving the prognosis of HCC.

LncRNAs as novel players in hepatocellular carcinoma recurrence.

Long non-coding RNAs (lncRNAs) are ncRNAs more than 200 nucleotides long that participate to a wide range of biological functions. However, their role in cancer is poorly known. By using an NGS-based approach we analyzed the intragenic and poliA-lncRNAs in hepatocellular carcinoma (HCC) and we assayed the relationships between their deregulated expression and clinical-pathological characteristics. The expression profile of lncRNAs was studied in a discovery series of 28 HCC and matched cirrhosis and was validated in an independent cohort of 32 HCC patients both in tissue and serum. The correlation between lncRNA expression and clinical-pathological variables, EMT markers and putative sponged microRNAs level were investigated. Functional experiments were performed in HCC-derived cell lines to clarify the role of selected lncRNAs in HCC. A panel of deregulated lncRNAs differentiated HCC from cirrhotic tissue. CASC9 and LUCAT1 were up-regulated in a subset of HCC-derived cell lines and in half of HCCs which displayed a lower recurrence after surgery. LUCAT1 and CASC9 silencing increased cell motility and invasion capability in HCC cells and influenced the EMT phenotype. LUCAT1 was demonstrated to directly sponge the onco-miR-181d-5p. Both LUCAT1 and CASC9 were secreted in exosomes, and higher circulating CASC9 levels were associated with tumor size and HCC recurrence after surgery, suggesting its potential usage as putative non-invasive prognostic biomarker of recurrence.

Rapid 3D bioprinting of decellularized extracellular matrix with regionally varied mechanical properties and biomimetic microarchitecture

Hepatocellular carcinoma (HCC), as the fifth most common malignant cancer, develops and progresses mostly in a cirrhotic liver where stiff nodules are separated by fibrous bands. Scaffolds that can provide a 3D cirrhotic mechanical environment with complex native composition and biomimetic architecture are necessary for the development of better predictive tissue models. Here, we developed photocrosslinkable liver decellularized extracellular matrix (dECM) and a rapid light-based 3D bioprinting process to pattern liver dECM with tailorable mechanical properties to serve as a platform for HCC progression study. 3D bioprinted liver dECM scaffolds were able to stably recapitulate the clinically relevant mechanical properties of cirrhotic liver tissue. When encapsulated in dECM scaffolds with cirrhotic stiffness, HepG2 cells demonstrated reduced growth along with an upregulation of invasion markers compared to healthy controls. Moreover, an engineered cancer tissue platform possessing tissue-scale organization and distinct regional stiffness enabled the visualization of HepG2 stromal invasion from the nodule with cirrhotic stiffness. This work demonstrates a significant advancement in rapid 3D patterning of complex ECM biomaterials with biomimetic architecture and tunable mechanical properties for in vitro disease modeling.

Linc-SCRG1 accelerates liver fibrosis by decreasing RNA-binding protein tristetraprolin.

The biologic roles of long noncoding RNAs (lncRNAs) in liver fibrosis remained unknown. Through microarray analysis, linc-SCRG1 (a lncRNA with transcript length 3118 bp) was found up-regulated 13.62-fold in human cirrhotic tissues. Quantitative PCR verified that linc-SCRG1 increased along with liver fibrosis progression in human tissues and in activated LX2 cells induced by TGF-β1. Knockdown of linc-SCRG1 significantly reversed the effects of TGF-β1 on LX2, including inhibiting activation, promoting apoptosis, reducing proliferation, lessening invasion, and down-regulating genes [fibrosis-related mRNA: α-smooth muscle actin ( α-SMA), type I collagen, and B-cell lymphoma-2; invasion-related mRNA: matrix metallopeptidase-2 ( MMP-2), MMP-9, and MMP-13; inflammation-related mRNA: TNF-α, IL-6, and IL-10]. linc-SCRG1 had binding sites with tristetraprolin (TTP), a kind of RNA-binding protein, and specifically combined to TTP proteins. Overexpression of linc-SCRG1 would cause TTP mRNA unstably and proteins decreasing. TTP mRNA was proved having negative relevance with linc-SCRG1 and was gradually reduced during human liver fibrosis progression. Overexpressing TTP resulted in knockdown of lincSCRG1 and degraded downstream target genes ( MMP-2 and TNF-α) in activated LX2. Overexpressing TTP had the same effects as small interfering RNA-lincSCRG1 (si- lincSCRG1), whereas knockdown of TTP had reversal effects on si- lincSCRG1 in activated LX2. In summary, linc-SCRG1 reduced TTP and restricted its degradation of target genes TNF-α and MMP-2. Therefore, linc-SCRG1 had a repressing TTP-elicited inactivation effect on hepatic stellate cell (HSC) phenotypes. Inhibition of linc-SCRG1 may be a novel therapeutic approach to inactivate HSCs and extenuate human liver fibrosis.-Wu, J.-C., Luo, S.-Z., Liu, T., Lu, L.-G., Xu, M.-Y. linc-SCRG1 accelerates liver fibrosis by decreasing RNA-binding protein tristetraprolin.

Liver Angiopoietin-2 Is a Key Predictor of De Novo or Recurrent Hepatocellular Cancer After Hepatitis C Virus Direct-Acting Antivirals.

Recent reports suggested that direct acting antivirals (DAAs) might favor hepatocellular carcinoma (HCC). In study 1, we studied the proangiogenic liver microenvironment in 242 DAA‐treated chronic hepatitis C patients with advanced fibrosis. Angiopoietin‐2 (ANGPT2) expression was studied in tissue (cirrhotic and/or neoplastic) from recurrent, de novo, nonrecurrent HCC, or patients never developing HCC. Circulating ANGPT2,vascular endothelial growth factor (VEGF), and C‐reactive protein (CRP) were also measured. In study 2, we searched for factors associated with de novo HCC in 257 patients with cirrhosis of different etiologies enrolled in a dedicated prospective study. Thorough biochemical, clinical, hemodynamic, endoscopic, elastographic, and echo‐Doppler work‐up was performed in both studies. In study 1, no patients without cirrhosis developed HCC. Of 183 patients with cirrhosis, 14 of 28 (50.0%) with previous HCC recurred whereas 21 of 155 (13.5%) developed de novo HCC. Patients with recurrent and de novo HCCs had significantly higher liver fibrosis (LF) scores, portal pressure, and systemic inflammation than nonrecurrent HCC or patients never developing HCC. In recurrent/de novo HCC patients, tumor and nontumor ANGPT2 showed an inverse relationship with portal vein velocity (PVv; r = –0.412, P = 0.037 and r = –0.409, P = 0.047 respectively) and a positive relationship with liver stiffness (r = 0.526, P = 0.007; r = 0.525, P = 0.003 respectively). Baseline circulating VEGF and cirrhotic liver ANGPT2 were significantly related (r = 0.414, P = 0.044). VEGF increased during DAAs, remaining stably elevated at 3‐month follow‐up, when it significantly related with serum ANGPT2 (r = 0.531, P = 0.005). ANGPT2 expression in the primary tumor or in cirrhotic tissue before DAAs was independently related with risk of HCC recurrence (odds ratio [OR], 1.137; 95% confidence interval [CI], 1.044‐1.137; P = 0.003) or occurrence (OR, 1.604; 95% CI, 1.080‐2.382; P = 0.019). In study 2, DAA treatment (OR, 4.770; 95% CI, 1.395‐16.316; P = 0.013) and large varices (OR, 3.857; 95% CI, 1.127‐13.203; P = 0.032) were independent predictors of de novo HCC. Conclusion: Our study indicates that DAA‐mediated increase of VEGF favors HCC recurrence/occurrence in susceptible patients, that is, those with more severe fibrosis and splanchnic collateralization, who already have abnormal activation in liver tissues of neo‐angiogenetic pathways, as shown by increased ANGPT2. (Hepatology 2018; 00:000‐000).

Cirrhotic stiffness affects the migration of hepatocellular carcinoma cells and induces sorafenib resistance through YAP.

A majority of hepatocellular carcinomas (HCCs) combine with liver cirrhosis. The cirrhotic liver has been implicated in interfering with the effects of HCC-targeted drugs, including sorafenib. Alterations in the tumor microenvironment of the cirrhotic liver include both biochemical and biomechanical factors. In this study, we induced sorafenib resistance in HCC cells. We observed changes in cell morphology, cytoskeletal architecture, and cellular stiffness in these sorafenib-resistant cells, resembling those adapted to stiffer substrates. To examine the contribution of mechanical factors in HCC cell growth and drug resistance, we used an in vitro cell culture system with adjustable stiffness mimicking the normal or cirrhotic liver tissues. We identified that mechanical adaptation conferred HCC cells with increased motility and sorafenib resistance. We further reported the mechanism underlying the involvement of the transcription coactivator YAP. Our results underline the important role of mechanical factors in the interaction between tumor cells and their microenvironment.

N-Linked Glycan Branching and Fucosylation Are Increased Directly in Hcc Tissue As Determined through in Situ Glycan Imaging.

Hepatocellular carcinoma (HCC) remains as the fifth most common cancer in the world and accounts for more than 700,000 deaths annually. Changes in serum glycosylation have long been associated with this cancer but the source of that material is unknown and direct glycan analysis of HCC tissues has been limited. Our laboratory previously developed a method of in situ tissue based N-linked glycan imaging that bypasses the need for microdissection and solubilization of tissue prior to analysis. We used this methodology in the analysis of 138 HCC tissue samples and compared the N-linked glycans in cancer tissue with either adjacent untransformed or tissue from patients with liver cirrhosis but no cancer. Ten glycans were found significantly elevated in HCC tissues as compared to cirrhotic or adjacent tissue. These glycans fell into two major classes, those with increased levels of fucosylation and those with increased levels of branching with or without any fucose modifications. In addition, increased levels of fucosylated glycoforms were associated with a reduction in survival time. This work supports the hypothesis that the increased levels of fucosylated N-linked glycans in HCC serum are produced directly from the cancer tissue.

Abstract 3349: TEAD4 expression in human hepatocellular carcinoma correlates with poor prognosis and promotes tumor growth and migration

Abstract Background: TEAD family is a group of four transcription factor members, including TEAD1, TEAD2, TEAD3 and TEAD4. These genes are known to be involved in the regulation of different genes associated with cell proliferation, differentiation and apoptosis. TEAD proteins represent the major downstream effectors of the conserved Hippo signalling pathway, known to be deregulated in different cancer entities. In the present study, we sought to explore the expression of TEAD4 in hepatocellular carcinoma (HCC) and its effect in in vitro models. Methods: We evaluated TEAD4 expression at mRNA level using data from The Cancer Genome Atlas (liver dataset) and at protein level using a tissue microarray containing 192 HCCs, 108 cirrhotic tissues and 79 normal liver samples. For in vitro experiments, two liver cancer cell lines were stably overexpressed and downregulated for TEAD4 to characterize its role in in vitro models, in terms of proliferation, migration and anchorage-independent growth. Results: TEAD4 was found overexpressed in a subset of HCCs in the TCGA dataset and its overexpression correlated with worst overall survival (p=0.03). The tissue microarray analysis showed a similar trend not only in the HCCs but also in cirrhotic tissues. The in vitro assays demonstrated that the overexpression of TEAD4 promoted tumour growth and migration as well as increased foci formation capacity. On the other hand, TEAD4 knockdown was able to revert the phenotype, suggesting a putative oncogenic role of TEAD4 in hepatocarcinogenesis. Conclusions: Finally, TEAD4 is frequently overexpressed in HCCs and in cirrhotic tissues and its expression is associated with poor prognosis. Furthermore, in vitro evidence suggests that TEAD4 may act as oncogene and may have a pivotal role in hepatocarcinogenesis. Citation Format: Nadia Tosti, Manuela Lanzafame, Viola Paradiso, Charlotte K. Ng, Salvatore Piscuoglio, Cristina Quintavalle, Luigi Maria Terracciano. TEAD4 expression in human hepatocellular carcinoma correlates with poor prognosis and promotes tumor growth and migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3349.

Abstract LB-235: DNA methylation of individual repetitive elements in HCV infection-induced HCC

Abstract Hepatocellular carcinoma (HCC) is the second most common cause of cancer deaths worldwide. Hepatitis C virus (HCV) infection is a major risk factor for HCC in the US. HCV infection may cause cancer by suppressing host defense that controls the propagation of repetitive element (RE) sequences, including LINE-1 and Alu, the most representative REs in the human genome. DNA methylation of RE helps stabilize the genome, while reduced RE methylation, also referred as global hypomethylation, leads to genomic instability and is known as a hallmark of cancer. However, current available high-throughput technology for DNA methylation profiling only enables genomewide RE methylation study on a global scale. The role of individual RE methylation in cancer is largely understudied. In this study, using our recently developed and published machine learning algorithm to enhance the RE coverage of the Illumina methylation array platform, we examined the role of individual RE methylation in HCV-induced HCC across the entire genome. We conducted an epigenome-wide association study on 22 HCC, 20 cirrhotic, and 44 normal liver tissues. We included equal numbers of HCV-induced and alcohol-induced HCC/cirrhosis cases. The Illumina 450k array profiled 1939 REs and we computationally predicted 33184 REs; correlations between the overlapping 1541 profiled and predicted RE methylation were &amp;gt; 0.91. Sex, tumor differentiation, and tumor stage were similarly distributed across the HCV and alcohol groups. We adjusted for batch effects and used Benjamini-Hochberg adjusted false discovery rate (FDR) to account for multiple testing. Relative to normal liver tissue, we observed over twice as many differentially methylated (FDR &amp;lt; 0.05) REs in HCV-HCC compared to alcohol-HCC tissue (2911 vs 1304, 749 in common). We validated our results in The Cancer Genome Atlas (TCGA) (325 vs 129, 74 in common) with high correlation (r = 0.90). We also observed that 169 REs were significantly altered in HCV-cirrhosis tissue (compared to none in alcohol-cirrhosis tissue), of which, 17 REs in 13 genes, including PIK3CD, LY9, GCK, NLRC3, and PRKCZ, were consistently hypomethylated in both HCC and cirrhotic liver tissue induced by HCV (r = 0.87). Using TCGA RNA sequencing data, we found that these REs were located in gene body and their hypomethylation was associated with lower expression of their host genes (r &amp;gt; 0.3, p &amp;lt; 0.05). Gene Ontology Enrichment analysis showed that these genes were enriched in T-cell activation &amp; differentiation and cytokine production (FDR &amp;lt; 0.05). Our findings indicate that HCV infection has a more extensive impact on the DNA methylation landscape in RE than alcohol consumption, and that HCV infection may also induce similar hypomethylation in cirrhotic liver. Hypomethylated RE may alter local chromatin environment and disrupt expression of genes involved in immune and inflammatory responses, both of which may promote HCC development. RE hypomethylation in specific genomic locus may serve as early detection biomarker of HCC in patients with HCV-induced cirrhosis. Citation Format: Yinan Zheng, Jimmy Ren, Ryan A. Hlady, Keith D. Robertson, Robert L. Murphy, Lewis R. Roberts, Lifang Hou. DNA methylation of individual repetitive elements in HCV infection-induced HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-235.