Interactions In Hepatocellular Carcinoma Research Articles

High-fidelity CRISPR/Cas12a dual-crRNA screening reveals novel synergistic interactions in hepatocellular carcinoma.

: CRISPR/Cas12a-based combinational screening has shown remarkable potential for identifying genetic interactions. Here, we describe an innovative method for combinational genetic screening with rapid construction of a dual-CRISPR RNA (crRNA) library using gene splicing through overlap extension PCR (SOE PCR) and the adoption of CeCas12a, which we previously identified with strict PAM recognition and low off-targeting to guarantee fidelity and efficiency. The custom-pooled SOE crRNA array (SOCA) library for double-knockout screening could be conveniently constructed in the laboratory for widespread use, and the CeCas12a-mediated high-fidelity screen displayed good performance even under a negative selection screen. By designing a SOCA dual-crRNA library that covered most of the kinase and metabolism-associated gene targets of FDA-approved drugs implicated in hepatocellular carcinoma (HCC) tumourigenesis, novel cross-talk between the two gene sets was negatively selected to inhibit HCC cell growth in vitro and in vivo and was validated using virtual double-knockdown screening based on TCGA databases. Thus, this rapid, efficient and high-fidelity double-knockout screening system is promising for systemically identifying potential genetic interactions between multiple gene sets or combinations of FDA- approved drugs for clinical translational medicine in the future.

Abstract 3011: The loss of an orphan nuclear receptor NR2E3 augments Wnt/β-Catenin signaling via epigenetic dysregulation that links to the Sp1-β catenin-p300 interactions in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) stands as the fourth leading cause of global cancer-related mortality due to late-stage detection, tumor heterogeneity, and drug resistance. The pivotal role of epigenetic factors in HCC development and progression necessitates the identification of critical epigenetic regulators. The orphan nuclear receptor NR2E3 (Nuclear receptor subfamily 2 group E, Member 3), primarily associated with retinal development, has emerged as a potential tumor suppressor. Earlier studies showed that the loss of NR2E3 leads to epigenetic repression of the estrogen receptor α (ER) through the involvement of Lysine Specific histone Demethylase 1 (LSD1). NR2E3 also forms a transcriptional complex with Sp1, governing the aryl hydrocarbon receptor (AHR) expression, a crucial factor in liver xenobiotic metabolism. In vivo, NR2E3 ablation results in p53 inactivation and severe liver injuries when exposed to liver toxicants, highlighting its role in activating p53. Nonetheless, the precise tumor suppressive and epigenetic role of NR2E3 in HCC remains unclear. HCC patients expressing low NR2E3 exhibit unfavorable clinical outcomes, aligning with heightened activation of the Wnt/β-catenin signaling pathway. The murine HCC models utilizing NR2E3 knockout mice show accelerated liver tumor formation and progression accompanied by enhanced activation of the Wnt/β-catenin signaling pathway and inactivation of the p53 signaling pathway. At a cellular level, losing NR2E3 increases the acquisition of aggressive cancer cell phenotype and tumorigenicity and upregulates vital genes in the Wnt/β-catenin pathway with enhanced chromatin accessibility. This event is mediated through the increased formation of active transcription complex involving Sp1, β-catenin, and p300, a histone acetyltransferase, on the promoters of target genes. Correspondingly, treatment of p300 inhibitor C646 decreased the expression of critical genes in the Wnt/β-catenin signaling pathway. These findings demonstrate that the loss of NR2E3 promotes Wnt/β-catenin signaling activation at cellular, organismal, and clinical levels. In summary, NR2E3 is a novel tumor suppressor that maintains epigenetic homeostasis, thereby preventing activation of Wnt/β-catenin signaling that promotes HCC formation. Citation Format: Yuet-Kin Leung, Sung-Gwon Lee, Jiang Wang, Shuk-Mei Ho, Nancy Rusch, Chungoo Park, Kyounghyun Kim. The loss of an orphan nuclear receptor NR2E3 augments Wnt/β-Catenin signaling via epigenetic dysregulation that links to the Sp1-β catenin-p300 interactions in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3011.

Single-Cell RNA Sequencing: A Deep Dive into The Cellular Landscape and Interactions in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide, with its progression highly influenced by the cellular interplay within the tumor microenvironment that is underexplored. Aiming to bridge this gap, our study utilizes single-cell RNA sequencing (scRNA-seq) to examine the cellular heterogeneity of HCC and investigate the roles of distinct cell populations. scRNA-seq was performed on eight DEN mice HCC samples, followed by bioinformatic analysis with Seurat package. Nine distinct cell populations were identified, with three unique macrophage populations suggestive of their role as tumor-associated macrophages (TAMs). The detected endothelial cells and pericytes hint at ongoing neoangiogenesis, with implications that endothelial cells might function as tumor-associated endothelial cells (TECs) and pericytes as carcinoma-associated fibroblasts (CAFs). Our findings provide insights into the potential roles of various cell populations in the HCC tumor microenvironment, which paves the way for developing novel therapies. These postulations, while offering a deeper understanding of HCC's cellular landscape, necessitate experimental validation for confirmation.

Six immune-related promising biomarkers may promote hepatocellular carcinoma prognosis: a bioinformatics analysis and experimental validation

BackgroundAbnormal miRNA and mRNA expression and dysregulated immune microenvironment have been found to frequently induce the progression of hepatocellular carcinoma (HCC) in recent reports. In particular, the immune-related competing endogenous RNAs (ceRNA) mechanism plays a crucial role in HCC progression. However, the underlying mechanisms remain unclear.MethodsDifferentially expressed immune-related genes were obtained from the Immport, GEO, and TCGA databases. The mRNA and protein expression levels in HCC tissues and adjacent normal tissues were confirmed, and we further investigated the methylation levels of these biomarkers to explore their function. Then, the TIMER and TISCH databases were used to assess the relationship between immune infiltration and hub genes. Survival analysis and univariate and multivariate Cox models were used to evaluate the association between hub genes and HCC diagnosis. Hub gene expression was experimentally validated in six HCC cell lines and 15 HCC samples using qRT-PCR and immunohistochemistry. The hub genes were uploaded to DSigDB for drug prediction enrichment analysis.ResultsWe identified that patients with abnormal miRNAs (hsa-miR-125b-5p and hsa-miR-21-5p) and their targeted genes (NTF3, PSMD14, CD320, and SORT1) had a worse prognosis. Methylation analysis of miRNA-targeted genes suggested that alteration of methylation levels is also a factor in the induction of tumorigenesis. We also found that the development of HCC progression caused by miRNA-mRNA interactions may be closely correlated with the infiltration of immunocytes. Moreover, the GSEA, GO, and KEGG analysis suggested that several common immune-related biological processes and pathways were related to miRNA-targeted genes. The results of qRT-PCR, immunohistochemistry, and western blotting were consistent with our bioinformatics results, suggesting that abnormal miRNAs and their targeted genes may affect HCC progression.ConclusionsBriefly, our study systematically describes the mechanisms of miRNA-mRNA interactions in HCC and predicts promising biomarkers that are associated with immune filtration for HCC progression.

Comprehensive analysis of prognostic value and immune infiltration of IAPs family in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality rates. The inhibitors of apoptosis (IAP) family act as oncogenes in various tumor types; however, their functions in HCC remain unclear. Here, we used integrated bioinformatics analysis and experimental verification to assess the expression and the prognostic and clinical value of the IAP family in HCC. Using the University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) and the Tumor Immune Estimation Resource (TIMER), we analyzed the expression profiles of IAP family members in HCC tissue, normal tissues, and in patients with different stages and grades of HCC. We further verified the expression level of BIRC2 in 25 HCC samples and matched adjacent normal tissues using quantitative real-time PCR (qRT-PCR), and analyzed its correlation with the marker gene of T-helper type 1 cells (Th1)-STAT1. Meanwhile, the association between BIRC2 and the immunotherapeutic response or immunomodulators was confirmed using the Biomarker Exploration of Solid Tumors (BEST) database. The results showed that NAIP, BIRC2, BIRC3, XIAP, BIRC5, and BIRC6 mRNAs were overexpressed in HCC. The clinical stages, pathological grades, and other clinicopathological features of HCC were closely related to the expression levels of the IAP family members, especially the BIRC2 and BIRC5, which were found to be potential prognostic biomarkers for HCC. Expression of the IAPs was strongly associated with immune cell infiltration. Based on the infiltrative status of various immune cells, HCC patients with high BIRC2 and BIRC5 expression demonstrated poor overall survival (OS) rates. In patients with HCC, BIRC2 expression was noticeably elevated. Concurrently, the expression levels of BIRC2 and STAT1 showed a favorable correlation. BEST database analysis revealed that BIRC2 was a negative predictor of responsiveness to anti-programmed cell death ligand 1 (PD-L1)/cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) inhibitor treatment in HCC, and BIRC2 mRNA expression levels were positively correlated with the expression levels of the immune checkpoint genes programmed cell death protein 1 (PD-1), PD-L1, and CTLA-4 in HCC. Consequently, the IAP family may play a role in carcinogenesis and cancer-immune system interactions in HCC. Our results demonstrate that IAP family members may be viable predictive biomarkers and therapeutic targets for HCC.

Genetic susceptibility to hepatocellular carcinoma in chromosome 22q13.31, findings of a genome-wide association study.

Background and AimChronic hepatitis C virus (HCV) infection, long‐term alcohol use, cigarette smoking, and obesity are the major risk factors for hepatocellular carcinoma (HCC) in the United States, but the disease risk varies substantially among individuals with these factors, suggesting host susceptibility to and gene–environment interactions in HCC. To address genetic susceptibility to HCC, we conducted a genome‐wide association study (GWAS).MethodsTwo case‐control studies on HCC were conducted in the United States. DNA samples were genotyped using the Illumian microarray chip with over 710 000 single nucleotide polymorphisms (SNPs). We compared these SNPs between 705 HCC cases and 1455 population controls for their associations with HCC and verified our findings in additional studies.ResultsIn this GWAS, we found that two SNPs were associated with HCC at P < 5E‐8 and six SNPs at P < 5E‐6 after adjusting for age, sex, and the top three principal components (PCs). Five of the SNPs in chromosome 22q13.31, three in PNPLA3 (rs2281135, rs2896019, and rs4823173) and two in SAMM50 (rs3761472, rs3827385), were replicated in a small US case‐control study and a cohort study in Singapore. The associations remained significant after adjusting for body mass index and HCV infection. Meta‐analysis of multiple datasets indicated that these SNPs were significantly associated with HCC.ConclusionsSNPs in PNPLA3 and SAMM50 are known risk loci for nonalcoholic fatty liver disease (NAFLD) and are suspected to be associated with HCC. Our GWAS demonstrated the associations of these SNPs with HCC in a US population. Biological mechanisms underlying the relationship remain to be elucidated.

Genetic Polymorphisms in CD35 Gene Contribute to the Susceptibility and Prognosis of Hepatocellular Carcinoma.

CD35, an important molecule implicated in inflammation and immunity, is reportedly associated with several cancers. However, very few studies have investigated the relationship between CD35 polymorphisms and hepatocellular carcinoma (HCC). The current study was conducted to investigate the association between tag SNPs in CD35 and HCC susceptibility and postoperative recurrence, in an attempt to elucidate the gene-environment interactions in HCC. A total of 1233 Chinese Han people, including 647 healthy controls and 586 HCC cases, were sampled in this study. Six Tag SNPs (rs10494885, rs2296160, rs3737002, rs3849266, rs669117, and rs7525160) of CD35 were selected using the HaploView 4.2 program and genotyped by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Overall, the mutation genotypes CC/CG of CD35 rs7525160 significantly increased the risk of HCC. Stratification analysis indicated that CD35 rs7525160 CC/CG genotypes increased HCC risk in patients younger than 65 years and were closely related to the pathological type of poor prognosis of HCC. Cox proportional hazard ratio model analysis revealed that the rs7525160 CC/CG genotype remains a significant independent risk factor for postoperative recurrence of HCC. In conclusion, CD35 rs7525160 polymorphism may contribute to the susceptibility and prognosis of HCC in the Chinese Han population.

Prognostic Value of PLXND1 and TGF-β1 Coexpression and Its Correlation With Immune Infiltrates in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy with no curative treatments. Plexin D1 (PLXND1) is a cellular receptor whose functions have been explored in several human cancers; however, the critical roles of PLXND1 in HCC have rarely been probed. Therefore, the present study attempted to elucidate the expression pattern, prognostic significance, and potential roles of PLXND1 in HCC. We found that PLXND1 expression in HCC tissues was significantly higher compared with normal liver tissue from Gene Expression Profiling Interactive Analysis (GEPIA) and Integrative Molecular Database of Hepatocellular Carcinoma (HCCDB) databases. This result was further validated by immunohistochemistry staining (IHC) using tissue microarrays, which contained 216 HCC cases collected from our hospital. Additionally, PLXND1 expression showed a significant correlation with several clinical characteristics, including tumor grade and tumor hemorrhage (TH). Moreover, TISIDB and GEPIA databases were used to investigate the roles of PLXND1 in tumor-immune system interactions in HCC. As an immunoinhibitor, transforming growth factor-beta (TGF-β1) displayed the greatest correlations with PLXND1 in HCC. Finally, Kaplan-Meier curves and Cox analysis were conducted to further examine the potential clinical value of PLXND1 in HCC. We described a subclassification of HCC based on PLXND1 and TGF-β1 expression, which could be used to predict clinical outcomes and patient prognosis. Taken together, the results of this study indicate that PLXND1 might be a promising prognostic biomarker and potential therapeutic target in HCC.

Simulations of Combination Therapy in Hepatocellular Carcinoma Using a Quantitative Systems Pharmacology Model

Hepatocellular carcinoma (HCC) is a major health concern and despite efforts to screen at‐risk individuals, diagnosis often occurs at late stages when patients are no longer candidates for potentially curative therapies (i.e. surgical resection, ablation, or transplantation). For patients with advanced HCC, tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors may provide clinical benefit, but overall survival remains low. One strategy that has shown promise in on‐going clinical trials is to administer a combination of TKIs and immune checkpoint inhibitors. Despite the increased number of patients that respond to combination therapy compared to individual therapies, there is still a large fraction of patients who remain unresponsive. Therefore, to improve therapy outcomes for patients with HCC, there are two broad questions which need to be addressed. First, through which mechanisms are the therapies interacting in HCC, and secondly, why are they effective in only a subset of patients?To address these questions, we developed a mechanistic quantitative systems pharmacology (QSP) modeling platform for immuno‐oncology (IO) that can be used to predict the progression of HCC in response to molecular and immune therapies. This model integrates cancer growth, immune response, and intracellular signaling in four compartments (tumor, tumor‐draining lymph node, central and peripheral), and considers the interactions and transport of various immune cells (e.g. effector T cells, T regulatory cells and antigen presenting cells) as well as the growth of tumor cells which are partially dictated by interactions with immune cells in the tumor compartment. Additionally, the model includes the binding kinetics of immune checkpoint proteins. Further, pharmacokinetic models are used to model the uptake, transport and clearance of immune checkpoint inhibitors, and the antibodies in the model by bind the immune checkpoint proteins. In this work, the model was applied to an on‐going clinical trial (ClinicalTrials.gov Identifier: NCT03299946) of the combination of TKI, cabozantinib and immune checkpoint inhibitor, nivolumab.Overall, this work presents a modular platform for the development of QSP models in IO that integrate features at the molecular, cellular, tumor and whole patient scales. This framework can then be used to predict therapeutic response to the treatment of HCC, and is the first to predict the outcome of combination therapy for liver cancer. Additionally, it enables the analysis and interpretation of the complex interactions in HCC that may ultimately lead to the understanding of the drug interactions in cancer and enable the prediction of patient response to therapies, facilitating planning for patient cohorts and even individual patients.Support or Funding InformationSupported by NIH grant U01CA212007.

A classification based on tumor budding and immune score for patients with hepatocellular carcinoma

ABSTRACT Background: The role of immune profiling and tumor budding in hepatocellular carcinoma (HCC) remains largely unknown. This study evaluated the association between tumor budding and lymphocytic infiltration in HCC. Meanwhile, HCC patients were stratified based on tumor budding grade and immune score. Patients and methods: A total of 423 HCC patients were divided into training (n = 212) and validation (n = 211) cohort. Tumor slides from resected HCC samples were used for tumor budding assessment. A prognosis-relevant immune score was developed based on five types of immune cells out of eleven immune markers. A classification based on tumor budding grade and immune type was established (IS-TB type). To explore the association of IS-TB type and molecular alterations of HCC, 100 HCC samples and adjacent non-tumor tissues from 100 patients were investigated by whole-exome sequencing. Results: Tumor budding was an independent adverse prognostic factor for OS and DFS in both of the training and validation cohorts (all P values <.05). The rate of high-grade tumor budding was significantly higher in HCC with immature stroma (P < .001), strong α-SMA expression (P = .005), non-steatotic tumors and non-fibrolamellar-HCC (P < .001). Additionally, tumor budding was related to both anti- and pro-tumor immune responses. Patients were classified into immune type A and immune type B according to the immune score. Based on tumor budding grade and immunotype, patients were classified into four subgroups: ISA-TBhigh (type I), ISB-TBhigh (type II), ISA-TBlow (type III) and ISB-TBlow (type IV). Patients with type III tumor had the best OS and DFS, whereas OS and DFS were the worst for cases with type II tumor. TP53 mutation was more frequent in IS-TB type I (ISATBhigh) patients, while IS-TB type IV (ISBTBlow) harbored high number of CTNNB1 mutation. Conclusion: Tumor-immune cell interactions in HCC is heterogeneous. HCC classification based on tumor budding and immune score correlates with patient survival and molecular alterations. The defined subtypes may have significance for utilizing individualized treatment in patients with HCC.

A Classification Based on Tumor Budding and Immune Score for Patients with Hepatocellular Carcinoma

Background: The role of immune profiling and tumor budding in hepatocellular carcinoma (HCC) remains largely unknown. This study evaluated the association between tumor budding and lymphocytic infiltration in HCC. Meanwhile, HCC patients were stratified based on tumor budding grade and immune score. Patients and Methods: A total of 472 HCC patients were divided into training (n = 236) and validation (n = 236) cohort. Tumor slides from resected HCC samples were used for tumor budding assessment. A prognosis-relevant immune score was developed based on five types of immune cells out of eleven immune markers. A classification based on tumor budding grade and immune type was established (IS-TB type). To explore the association of IS-TB type and molecular alterations of HCC, 100 HCC samples and adjacent non-tumor tissules from 100 patients were investigated by whole exome sequencing. Results: Tumor budding was an independent adverse prognostic factor for OS and DFS in both of the training and validation cohorts (all P values <0.05). The rate of high grade tumor budding was significantly higher in HCC with immature stroma (P< 0.001), strong α-SMA expression (P = 0.005), non-steatotic tumors and non-fibrolamellar-HCC (P < 0.001). Additionally, tumor budding was related to both anti- and pro-tumor immune responses. Patients were classified into immune type A and immune type B according to the immune score. Based on tumor budding grade and immunotype, patients were classified into four subgroups: ISA-TBhigh (type I), ISBTBhigh (type II), ISA-TBlow (type III) and ISB-TBlow (type IV). Patients with type III tumor had the best OS and DFS, whereas OS and DFS were the worst for cases with type II tumor. TP53 mutation was more frequent in IS-TB type I (ISATBhigh) patients, while IS-TB type IV (ISBTBlow) harbored high number of CTNNB1 mutation. Conclusion: Tumor-immune cell interactions in HCC is heterogeneous. HCC classification based on tumor budding and immune score correlates with patient survival and molecular alterations. The defined subtypes may have significance for utilizing individualized treatment in patients with HCC. Funding Statement: This study was funded by grants from the Science and Technology Support Program of Sichuan Province (2017SZ0003) and the Natural Science Foundation of China (81770615, 81700555, 81672882 and 81502441). Declaration of Interests: The authors stated: none. Ethics Approval Statement: Informed consent was obtained in all cases. The study was approved by review board committees in the West China Hospital.

Hepatitis B virus genotypes, expression quantitative trait loci for ZNRD1-AS1 and their interactions in hepatocellular carcinoma.

Genetic variants in zinc ribbon domain-containing 1 antisense RNA 1 (ZNRD1-AS1) have been reported to be associated with development of hepatocellular carcinoma (HCC). We sought to determine the influences of ZNRD1-AS1 polymorphisms and their interactions with Hepatitis B virus (HBV) genotypes on the risk of HCC. In this study, we conducted a large population case-control study with 1,507 HBV-related HCC cases and 1,560 HBV persistent carriers. Three single-nucleotide polymorphisms (SNPs) in ZNRD1-AS1 (rs3757328, rs6940552 and rs9261204) were genotyped using a TaqMan allelic discrimination assay, and the HBV genotypes were identified by multiplex PCR. We found consistently significant associations between the ZNRD1-AS1 rs6940552 and rs9261204 SNPs with an increased risk of HCC (additive genetic model: adjusted OR = 1.16, 95% CI = 1.03-1.32 for rs6940552; adjusted OR =1.20, 95% CI = 1.06-1.35 for rs9261204) and found a borderline association between rs3757328 and HCC risk. Besides, we observed a dose-dependent relationship between increasing numbers of variant alleles of the SNPs and HCC risk (P for trend <0.001). Moreover, we observed a stronger combined effect of the three SNPs on HCC risk among the subjects infected with non-B genotype HBV (adjusted OR = 1.26, 95% CI = 1.05-1.50) compared with HBV B-related genotypes (adjusted OR = 0.89, 95% CI = 0.69-1.15; P= 0.029 for heterogeneity test). We also found that a multiplicative interaction between the variant alleles and the HBV genotype significantly affected HCC susceptibility (P = 0.030). Together, these results indicate that ZNRD1-AS1 may influence HCC risk accompanied by HBV genotypes.

Hepatitis B virus mutations, expression quantitative trait loci for PTPN12, and their interactions in hepatocellular carcinoma.

Previously we identified that HBV(Hepatitis B virus) sequence variation, which may interact with host human leukocyte antigen (HLA) genetic variation, could influence host risk of hepatocellular carcinoma (HCC). More HBV‐host interactions need to be identified. Protein tyrosine phosphatase nonreceptor type 12 (PTPN12), serves as an antagonist to tyrosine kinase signaling, may play integral roles in immune response against HBV infection and the development of HCC. Rs11485985 was an expression quantitative trait loci (eQTL) for PTPN12 by bioinformatics analyses. In this study, we genotyped the PTPN12 eQTL and sequenced the HBV region EnhII/BCP/PC in a case–control cohort including 1507 HBV‐related HCC cases and 1560 HBV persistent carriers as controls. The variant genotype GG of rs11489585 increased HCC risk compared to the HBV persistent carriers (adjusted OR = 2.03, 95% confidence interval [CIs] = 1.30–3.18). We also detected borderline significant associations of PTPN12 eQTL rs11489585 with HBV mutations (P = 0.05 for G1799C). Taken together, PTPN12 may influence HCC risk accompanied by HBV mutations.

Hepatitis B virus genotype, mutations, human leukocyte antigen polymorphisms and their interactions in hepatocellular carcinoma: a multi-centre case-control study.

Three genome-wide association studies (GWAS) have been conducted on the genetic susceptibility of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), two of which consistently identified tagging single nucleotide polymorphisms (SNPs) around HLA-DQ/DR. In contrast, large multi-centre association studies between HBV genotype, mutations and the risk of HCC are relatively rare, and their interactions with host variants are even less. We performed a multi-centre study of 1,507 HBV-related HCC cases and 1,560 HBV persistent carriers as controls to evaluate the effects of HBV genotype, mutations, GWAS-identified HLA-DQ/DR SNPs (rs9272105 and rs9275319) and their interactions on HCC risk. We found HBV genotype C was more frequent in HBV-related HCC. And 11 HBV hotspot mutations were independently and significantly associated with HCC risk. We also detected significant interactions of rs9272105 with both the HBV genotype and mutations. Through stepwise regression analysis, HBV genotype, the 11 mutations, HLA-DQ/DR SNPs, and the interaction of rs9272105 with mutation A1752G were all entered into the HCC prediction model, and the area under the curve for the panel including the HLA-DQ/DR SNPs, HBV genotype and mutations was 0.840. The HBV genotype, the mutations and the HLA-DQ/DR SNPs may serve as biomarkers for the surveillance of HBV persistent carriers.

Abstract 850: Hepatitis B virus genotype, mutations, human leukocyte antigen polymorphisms and their interactions in hepatocellular carcinoma: a multi-centre case-control study

Abstract Objective:Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. Our aim was to evaluate the effects of the hepatitis B virus (HBV) genotype, mutations, HLA single nucleotide polymorphisms (SNPs) and their interactions on HCC risk. Methods: A multi-centre case-control study of 1,507 HBV-related HCC cases and 1,560 HBV persistent carriers as controls was performed. HBV genotype, mutations and genome-wide association studies (GWAS)-identified HLA-DQ/DR SNPs (rs9272105 and rs9275319) were detected. Results: We found that the frequencies of the HBV genotypes B and BC (coinfection) were varied among the studied areas, but genotype C was consistently more frequent in HBV-related HCC (80.6% among HCC vs. 34.4% among the controls). Moreover, 11 HBV hotspot mutations in the EnhII/BCP/PC region were independently and significantly associated with HCC risk. We also detected significant interactions of HLA-DQ/DR rs9272105 with both the HBV genotype and mutations (P &amp;lt; 0.05 for each), but there were not significant interactions for rs9275319. Through a stepwise regression analysis, the HBV genotype, the 11 mutations, HLA-DQ/DR rs9272105 and rs9275319, and the interaction of the mutation A1752G with rs9272105 were all entered into the prediction model of HCC, and the area under the curve (AUC) for the panel including the HLA-DQ/DR SNPs, HBV genotype and mutations was 0.840 (sensitivity = 81.3%, specificity = 74.8%). Conclusions: The HBV genotype, the mutations and the HLA-DQ/DR SNPs may serve as biomarkers for the surveillance of HBV persistent carriers. Large studies with prospective designs are warranted to further evaluate our results. Note: This abstract was not presented at the meeting. Citation Format: Juan Wen, Ci Song, Deke Jiang, Tianbo Jin, Xiangjun Zhai, Jianfeng Xu, Hongbing Shen, Zhibin Hu. Hepatitis B virus genotype, mutations, human leukocyte antigen polymorphisms and their interactions in hepatocellular carcinoma: a multi-centre case-control study. [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 850. doi:10.1158/1538-7445.AM2015-850

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.

Hepatic stellate cells promote tumor progression by enhancement of immunosuppressive cells in an orthotopic liver tumor mouse model

The immunosuppressive properties of hepatic stellate cells (HSCs) contribute to the occurrence and development of hepatocellular carcinoma (HCC). The accumulation of cells with immune suppressive activities, such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) is a key mechanism for tumor immune evasion. However, the impact of HSCs on immune cell populations in tumor-bearing hosts is unclear. In this study, we established an orthotopic liver tumor mouse model for studying the complex tumor–host interactions in HCC. The activated HSCs promoted HCC growth not only induced tumor angiogenesis and lymphangiogenesis, but also significantly increased the suppressive immune cell population of Tregs and MDSCs in the spleen, bone marrow, and tumor tissues of the tumor-bearing mice. Murine HCC cell line H22-activated HSCs also expanded the expression of Tregs and MDSCs in vitro. In conclusion, our study suggests a novel role for HSCs in the HCC microenvironment. HSCs can promote HCC progression by enhancement of the immunosuppressive cell population. Targeting HSCs, which is a new concept in adjuvant immunotherapy, may be introduced in the near future to improve the outcome of patients with HCC.

Down-Regulation of Connective Tissue Growth Factor by Inhibition of Transforming Growth Factor β Blocks the Tumor–Stroma Cross-Talk and Tumor Progression in Hepatocellular Carcinoma

Tumor-stroma interactions in hepatocellular carcinoma (HCC) are of key importance to tumor progression. In this study, we show that HCC invasive cells produce high levels of connective tissue growth factor (CTGF) and generate tumors with a high stromal component in a xenograft model. A transforming growth factor beta (TGF-beta) receptor inhibitor, LY2109761, inhibited the synthesis and release of CTGF, as well as reducing the stromal component of the tumors. In addition, the TGF-beta-dependent down-regulation of CTGF diminished tumor growth, intravasation, and metastatic dissemination of HCC cells by inhibiting cancer-associated fibroblast proliferation. By contrast, noninvasive HCC cells were found to produce low levels of CTGF. Upon TGF-beta1 stimulation, noninvasive HCC cells form tumors with a high stromal content and CTGF expression, which is inhibited by treatment with LY2109761. In addition, the acquired intravasation and metastatic spread of noninvasive HCC cells after TGF-beta1 stimulation was blocked by LY2109761. LY2109761 interrupts the cross-talk between cancer cells and cancer-associated fibroblasts, leading to a significant reduction of HCC growth and dissemination. Interestingly, patients with high CTGF expression had poor prognosis, suggesting that treatment aimed at reducing TGF-beta-dependent CTGF expression may offer clinical benefits. Taken together, our preclinical results indicate that LY2109761 targets the cross-talk between HCC and the stroma and provide a rationale for future clinical trials.

Down-regulation of c-myc and Cyclin D1 genes by antisense oligodeoxy nucleotides inhibits the expression of E2F1 and in vitro growth of HepG2 and Morris 5123 liver cancer cells.

A number of genetic interactions are involved in the control of cell cycle, but their role and nature have not been completely clarified. The knowledge of the behavior of these interactions in hepatocellular carcinoma, could optimize preventive and therapeutic strategies based on cell cycle restraint. We studied downstream events following c-MYC and CYCLIN D1 gene inhibition, by lipoplex-delivered MYC and CYCLIN D1 antisense oligodeoxy nucleotides (aODNM, aODND1), in in vitro cultured human HepG2 and rat Morris 5123 hepatoma cells. 0.5-20 micro M aODN(M) and aODND1 inhibited in vitro growth of both cell types. Scramble oligomer (SCR) and sense ODNs had no or relatively poor effect. Ten micromolar aODNM and aODND1, but not SCR, also induced a significant increase in the apoptotic index of HepG2 and 5123 cells, and inhibited colony formation in soft agar by HepG2 cells. Treatment of the cells with aODNM plus aODND1 had no additive effect on growth and apoptosis. aODNM and aODND1 induced >50% decrease in c-MYC and CYCLIN D1 gene expression, respectively, at both mRNA and protein level. The inhibition of gene expression by aODNs was highly specific, and SCR was without effect. The reduction in c-MYC and CYCLIN D1 expression by aODNs, was associated with a >50% decrease in E2F1 mRNA and protein production, without changes in CYCLIN A and CYCLIN E expression. These results suggest the involvement of both c-MYC and CYCLIN D1 on E2F1 gene function, and indicate that aODNM and aODND1 may inhibit hepatoma cell growth through down-regulation of the E2F1 gene. The inhibition of E2F1 gene expression by E2F1 aODN, was associated with strong growth restraint of HepG2 cells. Thus, interactions of c-MYC and CYCLIN D1 with E2F1 gene are essential for cell cycle activity in hepatoma cells, and their inhibition may have a therapeutic effect.

Coordinated expression of integrin α6β1 and laminin in hepatocellular carcinoma

The interaction between tumor cells and laminin mediated by laminin-binding integrins is critical for tumor invasion and metastasis. The aim of this study was to clarify the altered expression of lamininbinding integrins with the change of laniinin deposition in hepatocellular carcinoma (HCC) in comparison with cirrhotic or normal liver by immunohistochemistry. In HCC, hepatoma cells and sinusoidal endothelial cells expressed integrins α1β1, α2β1, α3β1, and α6β1. Integrins α1β1 and α6β1 were detected in a continuous pattern along the sinusoids in accordance with laminin assembly. Integrins α2β1 and α3β1 were detected in a discontinuous pattern at these sites. Integrin α6β4 was not detected. In cirrhotic liver, although integrins α1β1 and α6β1 as well as laminin were detected in a continuous pattern along the sinusoids, integrins α2β1, α3β1, and α6β4 were not detected. In normal liver, although integrin α1β1 was detected in a continuous pattern along the sinusoids, neither integrins α2β1, α3β1, α6β1, α6β4, nor laminin were detected. We have clarified that, of laminin-binding integrins, the localization of integrin α6β1 shows the best correspondence with the localization of laminin. These results suggest that of laminin-binding integrins, integrin α6β1 is very important for cell-laminin interactions in HCC.