Upregulation In Hepatocellular Carcinoma Research Articles

Hepatoma cell-intrinsic TLR9 activation induces immune escape through PD-L1 upregulation in hepatocellular carcinoma.

A TLR9 agonist in combination with a PD-1 inhibitor produced powerful antitumor responses in a clinical trial despite TLR9 agonists as monotherapies failing to generate systemic antitumor immune responses due to immunosuppressive effects. However, the mechanism involved in the improved response induced by their combination remains unknown.Methods: Subcutaneous and orthotopic Hepa1-6 tumor model was used for single-drug and combined-drug treatment. We used TLR9 agonist stimulation or lentiviral vectors to overexpress TLR9 and activate TLR9 signaling. We next investigated the crosstalk between PARP1 autoPARylation and ubiquitination and between STAT3 PARylation and phosphorylation mediated by TLR9. Tissue chips were used to analyze the relationships among TLR9, PARP1, p-STAT3 and PD-L1 expression.Results: In this study, we found that the TLR9 agonist in combination with anti-PD-1 therapy or anti-PD-L1 therapy yielded an additive effect that inhibited HCC growth in mice. Mechanistically, we found that TLR9 promoted PD-L1 transcription by enhancing STAT3 Tyr705 phosphorylation. Then, we observed that TLR9 negatively regulated PARP1 expression, which mediated a decrease in STAT3 PARylation and an increase in STAT3 Tyr705 phosphorylation. Moreover, we found that TLR9 enhanced PARP1 autoPARylation by inhibiting PARG expression, which then promoted the RNF146-mediated ubiquitination and subsequent degradation of PARP1. Finally, we observed positive associations between TLR9 and p-STAT3 (Tyr705) or PD-L1 expression and negative associations between TLR9 and PARP1 in HCC patient samples.Conclusions: We showed that hepatoma cell-intrinsic TLR9 activation regulated the crosstalk between PARP1 autoPARylation and ubiquitination and between STAT3 PARylation and phosphorylation, which together upregulated PD-L1 expression and finally induces immune escape. Therefore, combination therapy with a TLR9 agonist and an anti-PD-1 antibody or anti-PD-L1 had much better antitumor efficacy than either monotherapy in HCC.

RP11-81H3.2 Acts as an Oncogene via microRNA-490-3p Inhibition and Consequential Tankyrase 2 Up-Regulation in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a serious threat to human lives and is usually diagnosed at the late stages. Recently, there has been a rapid advancement in the treatment options for HCC, but novel therapeutic targets are still needed, especially for precision medicine. We aimed to investigate the involvement of non-coding RNA RP11-81H3.2 in HCC. The expression of RP11-81H3.2 was examined in the blood samples of HCC patients, and in the human HCC cell lines, including HepG2, Smmc-7721, and Huh7. Cell proliferation was determined using the CCK-8 and EdU assay, and cell invasion and migration were determined using the transwell/wound healing assay. The effects of RP11-81H3.2 knockdown on in vivo tumor growth were evaluated utilizing the nude mice HepG2 tumor xenograft model. Here, we have identified a long non-coding RNA, RP11-81H3.2, which is enriched in HCC and can promote its proliferation, migration, and invasion both in vitro and in vivo. In addition, our results showed that RP11-81H3.2 binds to and regulate miR-490-3p expression in the HCC cells. Moreover, we found that RP11-81H3.2 regulates the expression of TNKS2 via miR-490-3p. Further, we found that RP11-81H3.2 and miR-490-3p form a regulatory loop; the release of RP11-81H3.2 leads to the suppression of miR-490-3p expression, thus, further enhancing the expression of RP11-81H3.2. Our data have provided a novel target for the diagnosis and treatment of HCC, and sheds light on the lncRNA-miRNA regulatory nexus that can control the HCC related pathogenesis.

Kinesin family member 15 promotes cancer stem cell phenotype and malignancy via reactive oxygen species imbalance in hepatocellular carcinoma

The development and progression of hepatocellular carcinoma (HCC) is associated with the presence of cancer stem cells (CSCs). In the present study, kinesin family member 15 (KIF15) expression was shown to be overexpressed in HCC tissues, cell lines, and CSCs. Patients with HCC with high KIF15 expression had shortened overall survival (OS) and high recurrence probability. Downregulation of KIF15 in vitro as well as in HCC organoids resulted in a significant reduction in sphere formation and expression of stemness-related genes. KIF15 downregulation in human HCC xenograft models delayed tumor initiation, growth, and metastasis. KIF15 was also demonstrated to interact with phosphoglycerate dehydrogenase (PHGDH) and inhibit proteasomal degradation of PHGDH, thus promoting CSC phenotype and malignancy via PHGDH-mediated intracellular reactive oxygen species (ROS) imbalance in HCC. Moreover, AAA nuclear coregulator cancer-associated protein (ANCCA) upregulation acts as a key mediator in KIF15 expression upregulation in HCC. Conclusion: In this study, we found that KIF15 promotes the CSC phenotype and malignancy via PHGDH-mediated ROS imbalance in HCC. These findings highlight potential therapeutic targets for HCC.

APOBEC3B interaction with PRC2 modulates microenvironment to promote HCC progression

ObjectiveAPOBEC3B (A3B), a cytidine deaminase acting as a contributor to the APOBEC mutation pattern in many kinds of tumours, is upregulated in patients with hepatocellular carcinoma (HCC). However, APOBEC mutation...

Lnc-UCID Promotes G1/S Transition and Hepatoma Growth by Preventing DHX9-Mediated CDK6 Down-regulation.

Although thousands of long noncoding RNAs (lncRNAs) have been annotated, only a limited number of them have been functionally characterized. Here, we identified an oncogenic lncRNA, named lnc‐UCID (lncRNA up‐regulating CDK6 by interacting with DHX9). Lnc‐UCID was up‐regulated in hepatocellular carcinoma (HCC), and a higher lnc‐UCID level was correlated with shorter recurrence‐free survival of HCC patients. Both gain‐of‐function and loss‐of function studies revealed that lnc‐UCID enhanced cyclin‐dependent kinase 6 (CDK6) expression and thereby promoted G1/S transition and cell proliferation. Studies from mouse xenograft models revealed that tumors derived from lnc‐UCID‐silenced HCC cells had a much smaller size than those from control cells, and intratumoral injection of lnc‐UCID small interfering RNA suppressed xenograft growth. Mechanistically, the 850‐1030‐nt domain of lnc‐UCID interacted physically with DEAH (Asp‐Glu‐Ala‐His) box helicase 9 (DHX9), an RNA helicase. On the other hand, DHX9 post‐transcriptionally suppressed CDK6 expression by binding to the 3′‐untranslated region (3′UTR) of CDK6 mRNA. Further investigation disclosed that lnc‐UCID enhanced CDK6 expression by competitively binding to DHX9 and sequestering DHX9 from CDK6‐3′UTR. In an attempt to explore the mechanisms responsible for lnc‐UCID up‐regulation in HCC, we found that the lnc‐UCID gene was frequently amplified in HCC. Furthermore, miR‐148a, whose down‐regulation was associated with an increase of lnc‐UCID in HCC, could bind lnc‐UCID and inhibit its expression. Conclusion: Up‐regulation of lnc‐UCID, which may result from amplification of its gene locus and down‐regulation of miR‐148a, can promote HCC growth by preventing the interaction of DHX9 with CDK6 and subsequently enhancing CDK6 expression. These findings provide insights into the biological functions of lncRNAs, the regulatory network of cell cycle control, and the mechanisms of HCC development, which may be exploited for anticancer therapy.

Overexpression of PARPBP Correlates with Tumor Progression and Poor Prognosis in Hepatocellular Carcinoma.

PARP1-binding protein (PARPBP/PARI/C12orf48), a negative regulator of homologous recombination (HR), has been suggested to function as an oncogene in cervical, lung, and pancreatic cancer. To investigate the expression profile of PARPBP and its role in hepatocellular carcinoma (HCC). Using data from the Cancer Genome Atlas and Human Protein Atlas databases, PARPBP expression level and its clinical implication in HCC were identified by t test and Chi-square test. The prognostic value of PARPBP in HCC was evaluated by Kaplan-Meier method, Cox regression analysis, and nomogram. Gene set enrichment analysis (GSEA) was used to screen biological pathways correlated with PARPBP expression in HCC. PARPBP was significantly upregulated in HCC tissues compared with normal liver tissues (P < 0.05). High PARPBP expression was significantly associated with elevated serum AFP level, vascular invasion, poor tumor differentiation, and advanced TNM stage (all P < 0.05). Kaplan-Meier analyses suggested that upregulation of PARPBP was correlated with worse overall survival (OS) and recurrence-free survival (RFS) in HCC. Multivariate analyses further confirmed that PARPBP upregulation was an independent indicator of poor OS and RFS (all P < 0.05). The prognostic nomograms based on PARPBP mRNA expression and TNM stage were superior to those based on the TNM staging system alone (all P < 0.05). Besides, PARPBP DNA copy gain and miR-139-5p downregulation were associated with PARPBP upregulation in HCC. GSEA revealed that "cell cycle," "HR," "DNA replication," and "p53 signaling" pathways were enriched in high PARPBP expression group. PARPBP may be a promising prognostic biomarker and candidate therapeutic target in HCC.

Overexpression of TONSL might be an independent unfavorable prognostic indicator in hepatocellular carcinoma

BackgroundTONSL has been suggested to function as an oncogene in lung, esophageal and cervical cancer. This study was aimed to identify the expression of TONSL and its role in hepatocellular carcinoma (HCC). MethodsBy data mining in the Cancer Genome Atlas (TCGA) and Human Protein Atlas (HPA) databases, the expression profile of TONSL, its clinical significance, the potential mechanisms of its dysregulation and its underlying biological function in HCC were investigated. ResultsTONSL was significantly upregulated in HCC tissues relative to normal liver tissues (P < 0.05). High TONSL expression was significantly correlated with advanced TNM stage, poorly differentiated tumors, vascular invasion, elevated serum alpha-fetoprotein expression and a worse prognosis (all P < 0.05). Multivariate analysis further confirmed that TONSL overexpression was an independent risk factor for poor overall survival (OS) and recurrence-free survival (RFS) in HCC (all P < 0.05). Additionally, 16% of HCC cases (n = 370) had TONSL DNA amplification. The total methylation level of TONSL was moderately and negatively correlated with its mRNA expression (P < 0.05). TONSL was predictively targeted by miR-133b, which was downregulated in HCC and negatively related to TONSL mRNA expression (all P < 0.05). Kaplan-Meier analyses demonstrated that low miR-133b expression was significantly associated with poor OS and RFS (all P < 0.05). Moreover, gene set enrichment analysis revealed that cases with TONSL overexpression were enriched in cell cycle regulation pathways (all P < 0.05). ConclusionsTONSL holds promise for serving as a prognostic biomarker for HCC. DNA amplification, hypomethylation and miR-133b downregulation could be the mechanisms associated with TONSL upregulation in HCC. TONSL might function as an oncogene via cell cycle regulation pathways in HCC.

The Long Noncoding RNA Cancer Susceptibility 9 and RNA Binding Protein Heterogeneous Nuclear Ribonucleoprotein L Form a Complex and Coregulate Genes Linked to AKT Signaling.

The identification of viability-associated long noncoding RNAs (lncRNAs) might be a promising rationale for new therapeutic approaches in liver cancer. Here, we applied an RNA interference screening approach in hepatocellular carcinoma (HCC) cell lines to find viability-associated lncRNAs. Among the multiple identified lncRNAs with a significant impact on HCC cell viability, we selected cancer susceptibility 9 (CASC9) due to the strength of its phenotype, expression, and up-regulation in HCC versus normal liver. CASC9 regulated viability across multiple HCC cell lines as shown by clustered regularly interspaced short palindromic repeats interference and single small interfering RNA (siRNA)-mediated and siRNA pool-mediated depletion of CASC9. Further, CASC9 depletion caused an increase in apoptosis and a decrease of proliferation. We identified the RNA binding protein heterogeneous nuclear ribonucleoprotein L (HNRNPL) as a CASC9 interacting protein by RNA affinity purification and validated it by native RNA immunoprecipitation. Knockdown of HNRNPL mimicked the loss-of-viability phenotype observed upon CASC9 depletion. Analysis of the proteome (stable isotope labeling with amino acids in cell culture) of CASC9-depleted and HNRNPL-depleted cells revealed a set of coregulated genes which implied a role of the CASC9:HNRNPL complex in AKT signaling and DNA damage sensing. CASC9 expression levels were elevated in patient-derived tumor samples compared to normal control tissue and had a significant association with overall survival of HCC patients. In a xenograft chicken chorioallantoic membrane model, we measured decreased tumor size after knockdown of CASC9. Conclusion: Taken together, we provide a comprehensive list of viability-associated lncRNAs in HCC; we identified the CASC9:HNRNPL complex as a clinically relevant viability-associated lncRNA/protein complex which affects AKT signaling and DNA damage sensing in HCC.

The E3 ligase for metastasis associated 1 protein, TRIM25, is targeted by microRNA-873 in hepatocellular carcinoma

Tumor metastasis accounts for 90% of all cancer-related deaths. Epithelial to mesenchymal transition (EMT) considered to be centrally important in acquired resistance to chemotherapy and in progression of tumors to secondary organs. One of the important mediators of metastatic progression in hepatocellular carcinoma (HCC) is the metastasis associated protein 1 (MTA-1). We have earlier shown that in the context of HCC and normal liver cell lines, MTA-1 protein is actively stabilized in HCC cell lines and actively degraded in normal liver cells. We have also shown that TRIM25 is the E3 ligase that interacts with and degrades MTA-1 protein. The identity of the factor regulating expression of TRIM25 in normal liver cells and HCC is unknown. In the current work we elucidate that microRNA (miR)− 873 targets TRIM25 in HCC cells. Both metagenomic analysis and quantification of miR-873 and TRIM25 in 25 HCC patients revealed an inverse correlation between the two in HCC patients with high miR-873 and low TRIM25 expression, respectively. The expression pattern was mimicked in the normal liver cells THLE-2 and the HCC cell line, HuH6. In vitro luciferase reporter assays confirmed TRIM25 as the target of miR-873. Transient transfection of HuH6 cells with an anti-miR-873 antagomir significantly decreased both transwell motility in these cells. Furthermore, in in vivo xenograft assays treatment with anti-miR-873 antagomir significantly decreased hepatic nodules formation. Cumulatively, our data indicate that suppression of TRIM25 expression by high levels of miR-873 dictates MTA1 protein upregulation in HCC.

Pharmacological or transcriptional inhibition of both HDAC1 and 2 leads to cell cycle blockage and apoptosis via p21Waf1/Cip1 and p19INK4d upregulation in hepatocellular carcinoma.

Histone deacetylases (HDACs) are commonly dysregulated in cancer and represent promising therapeutic targets. However, global HDAC inhibitors have shown limited efficacy in the treatment of solid tumours, including hepatocellular carcinoma (HCC). In this study, we investigated the therapeutic effect of selectively inhibiting HDAC1 and 2 in HCC. HDAC1 inhibitor Tacedinaline (CI994), HDAC2 inhibitor Santacruzamate A (CAY10683), HDAC1/2 common inhibitor Romidepsin (FK228) and global HDAC inhibitor Vorinostat (SAHA) were used to treat HCC cells. Cell cycle, apoptosis and the protein levels of CDKs and CDKNs were performed to evaluate HCC cell growth. Inhibition of HDAC1/2 by RNAi was further investigated. Combined inhibition of HDAC1/2 led to HCC cell morphology changes, growth inhibition, cell cycle blockage and apoptosis in vitro and suppressed the growth of subcutaneous HCC xenograft tumours in vivo. p21Waf1/Cip1 and p19INK4d , which play roles in cell cycle blockage and apoptosis induction, were upregulated. Inhibition of HDAC1/2 by siRNA further demonstrated that HDAC1 and 2 cooperate in blocking the cell cycle and inducing apoptosis via p19INK4d and p21Waf1/Cip1 upregulation. Finally, H3K18, H3K56 and H4K12 in the p19INK4d and p21Waf1/Cip1 promoter regions were found to be targets of HDAC1/2. Pharmacological or transcriptional inhibition of HDAC1/2 increases p19INK4d and p21Waf1/Cip1 expression, decreases CDK expression and arrests HCC growth. These results indicated a potential pharmacological mechanism of selective HDAC1/2 inhibitors in HCC therapy.

MicroRNA-500a Promotes the Progression of Hepatocellular Carcinoma by Post-Transcriptionally Targeting BID

Background/Aims: Hepatocellular carcinoma (HCC) is one of the most common human malignant diseases in the world, and the mechanisms underlying HCC carcinogenesis and progression need further investigation. MicroRNAs play important roles in the development of cancer, and miR-500a is suggested to be deregulated in some types of cancer. However, the underlying molecular mechanisms of miR-500a in HCC remain unknown. Methods: The expression of miR-500a in HCC was analyzed in The Cancer Genome Atlas (TCGA) database and examined in 33 pairs of HCC tissues and matched nontumor tissues. The correlation between miR-500a expression and prognosis of HCC patients was analyzed from the survival data in TCGA. The mechanism of miR-500a upregulation in HCC was detected using chromatin immunoprecipitation-quantitative real-time PCR. The roles of miR-500a in HCC development were examined using a cell counting kit-8 assay in vitro and growth of transplanted tumors in nude mice in vivo. Apoptosis of HCC was detected using Annexin V/propidium iodide staining. The expression of BH3-interacting death agonist (BID) protein was examined using western blot analysis. Results: miR-500a expression was upregulated in HCC tissues, and high miR-500a expression was significantly correlated with the poor prognosis of HCC patients. Histone modifications in the promoter region of miR-500a may be responsible for its increased expression. Inhibition of miR-500a in HCC cell lines significantly promoted apoptosis, as well as inhibiting the proliferation of HCC cells and growth of transplanted tumors in nude mice. miR-500a directly targeted the 3′ untranslated region of BID mRNA, and inhibition of miR-500a-promoted apoptosis was almost completely abolished by the administration of ABT-199 via the BID-mitochondria pathway. Conclusion: Our results suggest that histone modifications in the promoter region of miR-500a may be responsible for the increased expression of miR-500a in HCC, which promotes cancer progression by targeting BID, indicating that miR-500a may be a potential prognostic predictor and therapeutic target for HCC patients.

Indoleamine 2,3-dioxygenase: As a potential prognostic marker and immunotherapeutic target for hepatocellular carcinoma.

Tumor cells induce an immunosuppressive microenvironment which leads towards tumor immune escape. Understanding the intricacy of immunomodulation by tumor cells is essential for immunotherapy. Indoleamine 2,3-dioxygenase (IDO) is an immunosuppressive enzyme which mediates tumor immune escape in various cancers including hepatocellular carcinoma (HCC). IDO up-regulation in HCC may lead to recruitment of regulatory T-cells into tumor microenvironment and therefore inhibit local immune responses and promote metastasis. HCC associated fibroblasts stimulate natural killer cells dysfunction through prostaglandin E2 and subsequently IDO promotes favorable condition for tumor metastasis. IDO up-regulation induces immunosuppression and may enhance the risk of hepatitis C virus and hepatitis B virus induced HCC. Therefore, IDO inhibitors as adjuvant therapeutic agents may have clinical implications in HCC. This review proposes future prospects of IDO not only as a therapeutic target but also as a prognostic marker for HCC.

TMED3 promotes hepatocellular carcinoma progression via IL-11/STAT3 signaling.

Transmembrane p24 trafficking protein 3(TMED3) is a metastatic suppressor in colon cancer, but its function in the progression of hepatocellular carcinoma (HCC) is unknown. Here, we report that TMED3 was up-regulated in HCC and portal vein tumor thrombus. TMED3 up-regulation in HCC was significantly correlated with aggressive characteristics and predicted poor prognosis in HCC patients. TMED3 overexpression in HCC cell lines promoted cell migration and invasion. In contrast, TMED3 knockdown suppressed HCC metastasis both in vitro and in vivo. Gene microarray analysis revealed decreased IL-11 expression in TMED3-knockdown cells. We propose that TMED3 promotes HCC metastasis through IL-11/STAT3 signaling. Taken together, these findings demonstrate that TMED3 promotes HCC metastasis and is a potential prognostic biomarker in HCC.

Hepatitis B virus X protein mediates yes-associated protein 1 upregulation in hepatocellular carcinoma.

Hepatitis B virus (HBV) X protein (HBx) is implicated in the development of hepatocellular carcinoma (HCC). Yes-associated protein 1 (YAP) is an important proto-oncogene, which is a downstream effector molecule in the Hippo signaling pathway. The aim of the present study was to investigate the association between HBx expression in HCC samples and YAP expression in the Hippo pathway. A total of 20 pathologically confirmed HCC samples, 20 corresponding adjacent non-tumor liver tissues and 5 normal liver tissue samples were collected. The expression of HBx and YAP in the tissues was analyzed by quantitative reverse transcription-polymerase chain reaction and western blot analysis. The intensity and location of YAP expression were analyzed by immunohistochemistry. YAP mRNA and protein expression levels in HCC samples infected with HBV were significantly higher than those of normal liver tissues. Furthermore, YAP expression was positively correlated with HBx expression in HBV-positive HCC samples. Immunohistochemical staining revealed that YAP was predominantly expressed in the nuclei in HBV-positive HCC tissues. YAP expression was significantly decreased in the normal liver tissue and corresponding adjacent liver tissue when compared with the HCC tissues and by contrast to HCC tissues, YAP was predominantly located in the cytoplasm. In conclusion, these results indicate that the YAP gene is a key driver of HBx-induced liver cancer. Therefore, YAP may present a novel target in the treatment of HBV-associated HCC.

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.

Abstract 3492: Differential sensitivity of normal liver and hepatocarcinoma tissues to tBid induced apoptosis is determined by the abundance of VDAC2

Abstract A conserved step in extrinsic apoptosis is the outer mitochondrial membrane (OMM) permeabilization by truncated Bid (tBid) that is generated from Bid upon cleavage by Caspase-8 activated at death receptors. tBid directly or indirectly activate proapoptotic proteins i.e. Bak in OMM and/ or Bax in cytosol which is followed by a change in their conformation and oligomerization, leading to a pore formation in the OMM. To better understand the mitochondrial phase of this pathway we applied tBid to semi-permeabilized cells and isolated mitochondria. We monitored cytochrome c release, and the ensuing mitochondrial membrane potential collapse, using immunoblotting and fluorimetry as the markers for mitochondrial cell death. We found that normal liver tissue and normal hepatocytes (Rat, Mouse or Human) are unexpectedly resistant to tBid, whereas hepatocellular carcinoma (HCC) tissues and cell lines (e.g. PLC, Huh7) are greatly sensitized. Considering that the effect of tBid is most effectively mediated through Bak, its abundance in the mitochondria was tested. Bak level is significantly higher in tumor tissue/cell lines, whereas OMM-bound tBid and Bax levels are similar in both cancerous and normal tissue. Since we have recently found that voltage dependent anion channel 2 (V2), a pore forming OMM protein, is specifically required for Bak targeting to OMM, the abundance of V2 was also measured. V2 showed significant upregulation in HCC tissue/cell lines. Rescue studies using V2 sensitized normal hepatocytes and liver mitochondria to tBid. We propose that V2 might be the main regulator for the differential tBid sensitivity in normal liver and hepatocellular carcinoma. Citation Format: Shamim Naghdi, Soumya Sinha Roy, Ludivine Walter, Gyorgy Hajnoczky. Differential sensitivity of normal liver and hepatocarcinoma tissues to tBid induced apoptosis is determined by the abundance of VDAC2. [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 3492.

HDAC inhibition activates the apoptosome via Apaf1 upregulation in hepatocellular carcinoma.

BackgroundHistone deacetylation, a common hallmark in malignant tumors, strongly alters the transcription of genes involved in the control of proliferation, cell survival, differentiation and genetic stability. We have previously shown that HDAC1, HDAC2, and HDAC3 (HDAC1–3) genes encoding histone deacetylases 1–3 are upregulated in primary human hepatocellular carcinoma (HCC). The aim of this study was to characterize the functional effects of HDAC1–3 downregulation and to identify functionally important target genes of histone deacetylation in HCC.MethodsTherefore, HCC cell lines were treated with the histone deacetylase inhibitor (HDACi) trichostatin A and by siRNA-knockdown of HDAC1–3. Differentially expressed mRNAs were identified after siRNA-knockdown of HDAC1–3 using mRNA expression profiling. Findings were validated after siRNA-mediated silencing of HDAC1–3 using qRTPCR and Western blotting assays.ResultsmRNA profiling identified apoptotic protease-activating factor 1 (Apaf1) to be significantly upregulated after HDAC inhibition (HLE siRNA#1/siRNA#2 p < 0.05, HLF siRNA#1/siRNA#2 p < 0.05). As a component of the apoptosome, a caspase-activating complex, Apaf1 plays a central role in the mitochondrial caspase activation pathway of apoptosis. Using annexin V, a significant increase in apoptosis could also be shown in HLE (siRNA #1 p = 0.0034) and HLF after siRNA against HDAC1–3 (Fig. 3a, b). In parallel, caspase-9 activity was increased after siRNA-knockdown of HDAC1–3 leading to enhanced apoptosis after HDAC inhibition (Fig. 3c, d).ConclusionsThe present data show that siRNA-knockdown of HDAC1–3 plays a major role in mediating apoptotic response to HDAC inhibitors through regulation of Apaf1.

Abstract 5147: Deregulation of G9a and its functional roles in liver cancer

Abstract Liver cancer, primarily hepatocellular carcinoma (HCC), is the fifth most common cancers and the third leading cause of cancer mortality worldwide. In recent years, deregulated epigenetic mechanisms were found important in a wide range of human carcinogenesis, including HCC. The epigenetic changes elicited by various environmental factors and lifestyles (such as hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, chronic alcohol intake) were suspected to promote HCC development. However, the underlying mechanism and specific gene targets are still unknown. Our previous study suggested that deregulation of epigenetic regulators was a common event in human HCC. Among 90 epigenetic modifiers analyzed in human HCC, Euchromatic histone-lysine N-methyltransferase 2 (EHMT2/G9a) was the most significantly up-regulated one. G9a is a SET domain containing histone methyltransferase specific for histone H3 lysine 9 mono- and di-methylation. Previous work had revealed that G9a is important for embryonic development and adipocyte differentiation. However, little is known about functional roles of G9a in human HCC. We hypothesize that deregulation of G9a may led to aberrant epigenetic changes and contribute to initiation and progression of human HCC. In this study, we found that G9a mRNA expression in primary HCC was 3 folder than their corresponding non-tumor liver and 10 fold higher than normal livers. The up-regulation of G9a was detected in 63% (24/38) of primary HCC samples and was significantly associated with the presence of venous invasion (P = 0.006). The frequent G9a up-regulation in HCC was attributed to gene amplification and microRNA deregulation. We showed that 53% of HCC samples have 3 or more copies of G9a gene. In addition, we identified that miR-1 was a post-transcriptional regulator of G9a. Ectopic expression of miR-1 suppressed G9a 3′-untranslated-region-coupled luciferase activity. Functionally, knockdown of G9a significantly suppressed HCC cell proliferation, migration and colony formation. We also demonstrated that UNC0638, as a G9a specific inhibitor, could effectively inhibit HCC cell growth and cause cell morphology changes. By utilizing nude mice model, we showed that depletion of G9a drastically inhibited in vivo tumorigenicity. Our finding suggested that up-regulation of G9a contributed to the liver carcinogenesis and could be a therapeutic target for HCC treatment. Citation Format: Lai Wei, Felice Ho-Ching Tsang, Jeffery Chi-Fai Lau, Sandy Leung-Kuen Au, Joyce Man-Fong Lee, Carmen Chak-Lui Wong, Irene Oi-Lin Ng, Chun-Ming Wong. Deregulation of G9a and its functional roles in liver cancer. [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 5147. doi:10.1158/1538-7445.AM2014-5147

Rpb3 promotes hepatocellular carcinoma through its N-terminus.

The expression of RNA polymerase II subunit 3 (Rpb3) was found frequent up-regulation in Hepatocellular carcinoma (HCC) tumors. Significant associations could also be drawn between increased expressions of Rpb3 and advance HCC staging and shorter disease-free survival of patients. Overexpression of Rpb3 increased HCC cell proliferation, migratory rate and tumor growth in nude mice, whereas suppression of Rpb3 using shRNA inhibited these effects. For mechanism study, we found that Rpb3 bound directly to Snail, downregulated E-cadherin, induced HCC cells epithelial-mesenchymal transition (EMT). In particular, N-terminus of Rpb3 blocked Rpb3 binding to Snail, inhibited Rpb3-high-expression HCC cells proliferation, migration, tumor growth in nude mice, and also inhibited DEN-induced liver tumorigenesis. Furthermore, N-terminus of Rpb3 did not inhibit normal liver cells or Rpb3-low-expression HCC cells proliferation. These findings suggest that N-terminus of Rpb3 selectively inhibits Rpb3-high-expression HCC cells proliferation. N-terminus of Rpb3 may be useful in treating patients diagnosed with Rpb3-high-expression HCC.

New insights on the role of epigenetic alterations in hepatocellular carcinoma.

Emerging evidence assigns to epigenetic mechanisms heritable differences in gene function that come into being during cell development or via the effect of environmental factors. Epigenetic deregulation is strongly involved in the development of hepatocellular carcinoma (HCC). It includes changes in methionine metabolism, promoter hypermethylation, or increased proteasomal degradation of oncosuppressors, as well as posttranscriptional deregulation by microRNA or messenger RNA (mRNA) binding proteins. Alterations in the methylation of the promoter of methyl adenosyltransferase MAT1A and MAT2A genes in HCC result in decreased S-adenosylmethionine levels, global DNA hypomethylation, and deregulation of signal transduction pathways linked to methionine metabolism and methyl adenosyltransferases activity. Changes in S-adenosylmethionine levels may also depend on MAT1A mRNA destabilization associated with MAT2A mRNA stabilization by specific proteins. Decrease in MAT1A expression has also been attributed to miRNA upregulation in HCC. A complex deregulation of miRNAs is also strongly involved in hepatocarcinogenesis, with up-regulation of different miRNAs targeting oncosuppressor genes and down-regulation of miRNAs targeting genes involved in cell-cycle and signal transduction control. Oncosuppressor gene down-regulation in HCC is also induced by promoter hypermethylation or posttranslational deregulation, leading to proteasomal degradation. The role of epigenetic changes in hepatocarcinogenesis has recently suggested new promising therapeutic approaches for HCC on the basis of the administration of methylating agents, inhibition of methyl adenosyltransferases, and restoration of the expression of tumor-suppressor miRNAs.