Pathogenesis Of HCC Research Articles

Construction of a long non-coding RNA-associated ceRNA network reveals potential prognostic lncRNA biomarkers in hepatocellular carcinoma

ObjectivesHepatocellular carcinoma (HCC) is the sixth leading cause of cancer-related mortality in the world. Accumulating evidence has highlighted the regulatory roles of long non-coding RNAs (lncRNAs) acting as competing endogenous RNAs (ceRNAs) in HCC. MethodsThe lncRNA expression data and corresponding patient information were obtained from The Cancer Genome Atlas (TCGA) database. Competing lncRNA-mRNA interactions were identified using the hypergeometric test. Co-expression analysis was implemented using the Spearman correlation coefficient. Multivariate Cox regression survival analysis was utilized to extract prognostic lncRNAs in the network. ResultsBased on the “ceRNA hypothesis”, a global lncRNA-associated ceRNA network (LCeNET) in HCC was constructed. Nine lncRNAs were identified as hubs and found to be enriched in various cancer-related biological processes. In addition, ceRNA pairs associated with survival were screened to construct a lncRNA-miRNA-mRNA sub network. Finally, we developed a sixteen-lncRNA model that could classify patients into high- and low-risk subgroups with different survival outcomes, and MCM3AP-AS1 functioned as a hub in both LCeNET and prognostic model. ConclusionsOur work will improve the understanding of lncRNA-mediated ceRNA regulatory mechanisms in HCC pathogenesis and facilitate the identification of candidate prognostic biomarkers for HCC.

PO-137 Liver specific NEMO ablation induces mixed hepatocellular-cholangiocarcinoma in MYC-overexpressing mice

IntroductionThe MYC oncogene appears to be critically involved in the pathogenesis of HCC. The role of NF- κB in liver cancer is complex, either acts as a tumor-promoter or tumor-suppressor in liver carcinogenesis. However, the role of NF- κB in MYC-induced liver carcinogenesis has not been reported. In the present study, we have generated a mouse model of MYC-induced HCC with or without liver specific NEMO deletion. Our aim is to study the effect of inhibiting the IKK/NF-κB signalling in MYC-induced liver carcinogenesis.Material and methodsLAP-tTA mice were first crossed to Alb-cre transgenic mice. Double transgenic offspring were crossed to NEMOfl mice to obtain LAP-tTA+/Alb-cre+/NEMOfl/wt mice, which later were crossed with tetO-Myc +mice. MYC expression was conditionally regulated by doxycycline (DOX) treatment. The combination of all these genetically modified mouse lines resulted in mice that expressed MYC in a liver-specific fashion (MYCLAP-tTA) and had deleted NEMO in liver parenchymal cells (NEMOΔLPC). Mice were analysed at 35 days and 45 days old. Survival curve was plotted and compared. Serum liver enzymes were measured, and analysis was based mainly on histology, protein and gene expression levels.Results and discussionsMacroscopic analyses of the livers of mice with MYC overexpression revealed substantially enlarged livers with multiple visible tumour nodules. MYCLAP-tTA/NEMOΔLPC mice showed significantly reduced survival time than MYCLAP-tTA mice. A significant increase of AST, ALP, GGT and bilirubin levels were observed in MYCLAP-tTA/NEMOΔLPC mice compared to MYCLAP-tTA mice. Unlike the solid tumour nodules present in the MYCLAP-tTA mice, the tumour nodules in the MYCLAP-tTA/NEMOΔLPC mice contained multiple cyst-like/glandular structures, positively stained for CK19, which have been described in human intrahepatic cholangiocarcinoma (ICC). MYCLAP-tTA/NEMOΔLPC mice showed stronger proliferation, apoptosis and fibrosis and an upregulation of progenitor genes expression compared to MYCLAP-tTA mice. We found an activation of RAF/MEK/ERK pathway in MYCLAP-tTA/NEMOΔLPC mice. 10 days DOX treatment could reverse tumorigenesis in MYC transgenic mice.ConclusionWe showed that liver specific NEMO deletion together with MYC overexpression induce a mixed tumour type of HCC-ICC. Deregulation of the IKK/NF-κB signalling may be one of the possible triggers for the development of mixed HCC-ICC and therefore may act as a tumour suppressor in liver carcinogenesis. MYC-driven liver tumorigenesis is reversible by DOX treatment.

PO-140 Cellular localization of survivin influences the response of colorectal cancer cells to topoisomerase I inhibitors

IntroductionThe MYC oncogene appears to be critically involved in the pathogenesis of HCC. The role of NF- κB in liver cancer is complex, either acts as a tumor-promoter or tumor-suppressor in liver carcinogenesis. However, the role of NF- κB in MYC-induced liver carcinogenesis has not been reported. In the present study, we have generated a mouse model of MYC-induced HCC with or without liver specific NEMO deletion. Our aim is to study the effect of inhibiting the IKK/NF-κB signalling in MYC-induced liver carcinogenesis.Material and methodsLAP-tTA mice were first crossed to Alb-cre transgenic mice. Double transgenic offspring were crossed to NEMOfl mice to obtain LAP-tTA+/Alb-cre+/NEMOfl/wt mice, which later were crossed with tetO-Myc +mice. MYC expression was conditionally regulated by doxycycline (DOX) treatment. The combination of all these genetically modified mouse lines resulted in mice that expressed MYC in a liver-specific fashion (MYCLAP-tTA) and had deleted NEMO in liver parenchymal cells (NEMOΔLPC). Mice were analysed at 35 days and 45 days old. Survival curve was plotted and compared. Serum liver enzymes were measured, and analysis was based mainly on histology, protein and gene expression levels.Results and discussionsMacroscopic analyses of the livers of mice with MYC overexpression revealed substantially enlarged livers with multiple visible tumour nodules. MYCLAP-tTA/NEMOΔLPC mice showed significantly reduced survival time than MYCLAP-tTA mice. A significant increase of AST, ALP, GGT and bilirubin levels were observed in MYCLAP-tTA/NEMOΔLPC mice compared to MYCLAP-tTA mice. Unlike the solid tumour nodules present in the MYCLAP-tTA mice, the tumour nodules in the MYCLAP-tTA/NEMOΔLPC mice contained multiple cyst-like/glandular structures, positively stained for CK19, which have been described in human intrahepatic cholangiocarcinoma (ICC). MYCLAP-tTA/NEMOΔLPC mice showed stronger proliferation, apoptosis and fibrosis and an upregulation of progenitor genes expression compared to MYCLAP-tTA mice. We found an activation of RAF/MEK/ERK pathway in MYCLAP-tTA/NEMOΔLPC mice. 10 days DOX treatment could reverse tumorigenesis in MYC transgenic mice.ConclusionWe showed that liver specific NEMO deletion together with MYC overexpression induce a mixed tumour type of HCC-ICC. Deregulation of the IKK/NF-κB signalling may be one of the possible triggers for the development of mixed HCC-ICC and therefore may act as a tumour suppressor in liver carcinogenesis. MYC-driven liver tumorigenesis is reversible by DOX treatment.

PO-114 Histone deacetylase 6 regulates NOTCH3 endosomal trafficking and degradation in T-cell acute lymphoblastic leukaemia cells

IntroductionHepatocellular carcinoma (HCC) is developed by multiple steps accompanying progressive alterations of gene expression, which leads to increased cell proliferation and malignancy. Although environmental factors and intracellular signalling pathways that are critical for HCC progression have been identified, gene expression changes and the related genetic factors contributing to HCC pathogenesis are still insufficiently understood. In this study, we identify a transcriptional repressor Capicua/CIC as a suppressor of HCC progression and a potential therapeutic target.Material and methodsWe used human HCC patients samples, tissue microarray, and TCGA database to check CIC levels between normal and HCC patients. We used various HCC cell lines to check cell proliferation, migration, and invasion activity by using CIC knockdown, CIC overexpression, or ETV4 knockdown cells and so on. Also we used two different mouse models, Xenograft and liver specific CIC knockout mice to evaluate tumour progression, metastasis, or survival.Results and discussionsExpression of CIC is posttranscriptionally reduced in HCC cells. CIC levels are correlated with survival rates in patients with HCC. CIC overexpression suppresses HCC cell proliferation and invasion, whereas loss of CIC exerts opposite effects in vivo as well as in vitro. Levels of polyoma enhancer activator 3 (PEA3) group genes, the best-known CIC target genes, are correlated with lethality in patients with HCC. Among the PEA3 group genes, ETS translocation variant 4 (ETV4) is the most significantly up-regulated in CIC-deficient HCC cells, consequently promoting HCC progression. Furthermore, it induces expression of matrix metalloproteinase 1 (MMP1), the MMP gene highly relevant to HCC progression, in HCC cells; and knockdown of MMP1 completely blocks the CIC deficiency–induced HCC cell proliferation and invasion.ConclusionOur study demonstrates that the CIC–ETV4–MMP1 axis is a novel regulatory module controlling HCC progression.

Hsa-let-7b inhibits cell proliferation by targeting PLK1 in HCC

Previous studies have shown that high levels of PLK1 are expressed in HCC, and PLK1 inhibitors are being tested in clinical trials. However, the mechanisms, which regulate PLK1 expression in HCC, have not been clarified. Here, we show that induction of let-7b over-expression inhibits the PLK1-regulated luciferase activity in HEK-293T cells, and decreases the levels of PLK1 expression in HCC cells. Furthermore, the levels of let-7b expression were negatively correlated with PLK1 expression in HCC tissues. Let-7b over-expression inhibited the proliferation of HCC cells and promoted their apoptosis, which were partially rescued by increased PLK1 expression. Let-7b over-expression decreased the levels of PLK1, CDC25C and Survivin phosphorylation and CDC2, β-catenin, TCF-4 expression, which were mitigated by increased PLK1 expression in MHCC-97H cells. Let-7b over-expression inhibited the development and growth of implanted HCC tumors in mice by decreasing PLK1 and Survivin expression in the tumors. Together, our data indicated that let-7b targeted PLK1 to inhibit HCC growth and induce their apoptosis by attenuating the PLK1-mediated Survivin phosphorylation. Our findings may provide new insights into the pathogenesis of HCC.

Patient-derived multicellular tumor spheroids towards optimized treatment for patients with hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide and has poor prognosis. Specially, patients with HCC usually have poor tolerance of systemic chemotherapy, because HCCs develop from chronically damaged tissue that contains considerable inflammation, fibrosis, and cirrhosis. Since HCC exhibits highly heterogeneous molecular characteristics, a proper in vitro system is required for the study of HCC pathogenesis. To this end, we have established two new hepatitis B virus (HBV) DNA-secreting HCC cell lines from infected patients.MethodsBased on these two new HCC cell lines, we have developed chemosensitivity assays for patient-derived multicellular tumor spheroids (MCTSs) in order to select optimized anti-cancer drugs to provide more informative data for clinical drug application. To monitor the effect of the interaction of cancer cells and stromal cells in MCTS, we used a 3D co-culture model with patient-derived HCC cells and stromal cells from human hepatic stellate cells, human fibroblasts, and human umbilical vein endothelial cells to facilitate screening for optimized cancer therapy.ResultsTo validate our system, we performed a comparison of chemosensitivity of the three culture systems, which are monolayer culture system, tumor spheroids, and MCTSs of patient-derived cells, to sorafenib, 5-fluorouracil, and cisplatin, as these compounds are typically standard therapy for advanced HCC in South Korea.ConclusionIn summary, these findings suggest that the MCTS culture system is the best methodology for screening for optimized treatment for each patients with HCC, because tumor spheroids not only mirror the 3D cellular context of the tumors but also exhibit therapeutically relevant pathophysiological gradients and heterogeneity of in vivo tumors.

Tonicity-responsive enhancer-binding protein promotes hepatocellular carcinogenesis, recurrence and metastasis

ObjectivesHepatocellular carcinoma (HCC) is a common cancer with high rate of recurrence and mortality. Diverse aetiological agents and wide heterogeneity in individual tumours impede effective and personalised treatment. Tonicity-responsive enhancer-binding...

Concomitant use of heat-shock protein 70, glutamine synthetase and glypican-3 is useful in diagnosis of HBV-related hepatocellular carcinoma with higher specificity and sensitivity.

Hepatocellular carcinoma is the third leading cause of cancer-related death worldwide and late diagnosis is the main cause of death in HCC patients. In this study expression patterns of HSP70, GPC3 and GS and their relationships with pathogenesis of HCC in Iranian patients were investigated. The expression of HSP70, GPC3 and GS were determined by immunohistochemistry and quantitative real-time PCR (q-PCR) methods, using 121 cases from patients with HBV alone, HCC without HBV, HBV+HCC and 30 normal tissues as control group. HSP70, GPC3 and GS were expressed in higher levels in HBV-related HCC samples compared to HBV alone group. The results showed that the labeling index of HSP70, GPC3 and GS are correlated with immunohistochemical and molecular expressions of HSP70, GPC3 and GS. The sensitivity and specificity for HCC diagnosis were 43.4% and 89.7% for HSP70, 64.3% and 90.4% for GPC3, and 60.7% and 94.3% for GS, respectively. The sensitivity and specificity of the panels with 3, 2 and 1 positive markers, regardless of which one, were 21.6% and 100%, 51.3% and 100% and 93.4% and 80.5% respectively. The current study demonstrated an association between HSP70, GPC3 and GS expressions and HBV-related HCC in our population. It was concluded that HSP70, GPC3 and GS expressions could be useful biomarkers for increasing the specificity and sensitivity of HCC diagnosis to acceptable level. Also, proper combinations of these 3 markers could improve diagnostic accuracy.

Inhibition of the Cell Death Pathway in Nonalcoholic Steatohepatitis (NASH)-Related Hepatocarcinogenesis Is Associated with Histone H4 lysine 16 Deacetylation

Hepatocellular carcinoma (HCC) is one of the most aggressive human cancers, and its incidence is steadily increasing worldwide. Recent epidemiologic findings have suggested that the increased incidence of HCC is associated with obesity, type II diabetes mellitus, and nonalcoholic steatohepatitis (NASH); however, the mechanisms and the molecular pathogenesis of NASH-related HCC are not fully understood. To elucidate the underlying mechanisms of the development of NASH-related HCC, we investigated the hepatic transcriptomic and histone modification profiles in Stelic Animal Model mice, the first animal model of NASH-related HCC to resemble the disease pathogenesis in humans. The results demonstrate that the development of NASH-related HCC is characterized by progressive transcriptomic alterations, global loss of histone H4 lysine 20 trimethylation (H4K20me3), and global and gene-specific deacetylation of histone H4 lysine 16 (H4K16). Pathway analysis of the entire set of differentially expressed genes indicated that the inhibition of cell death pathway was the most prominent alteration, and this was facilitated by persistent gene-specific histone H4K16 deacetylation. Mechanistically, deacetylation of histone H4K16 was associated with downregulation of lysine acetyltransferase KAT8, which was driven by overexpression of its inhibitor nuclear protein 1 (Nupr1). The results of this study identified a reduction of global and gene-specific histone H4K16 acetylation as a key pathophysiologic mechanism contributing to the development of NASH-derived HCC and emphasized the importance of epigenetic alterations as diagnostic and therapeutic targets for HCC.Implications: Histone H4K16 deacetylation induces silencing of genes related to the cell death that occurred during the development of NASH-related HCC. Mol Cancer Res; 15(9); 1163-72. ©2017 AACR.

Abstract 5059: Preclinical study on the efficacy of Panobinostat in hepatocellular carcinoma

Abstract Background Aberrant regulation of histone deacetylases (HDACs) is known to play a pivotal role in HCC pathogenesis as well as other human malignancies. Panobinostat (LBH589) is a pan-HDAC inhibitor covering a wide range of HDACs (Class I, II and IV) with high inhibitory activity at nanomolar concentration. It has been approved by FDA for treating multiple myeloma and has demonstrated promising anti-proliferative and cytotoxic activity in breast, prostate, colon and pancreatic cancer cell lines. This study investigated in vitro and in vivo effect of Panobinostat in HCC cell lines. Methods Basal expressions of HR23B and HDACs of 7 HCC cell lines (HepG2, PLC/PRF/5, Huh-7, Hep3B, SNU-182, SNU-398 and SNU-449) were determined by western blotting. Their corresponding IC50 for 24, 48 and 72 hours towards Panobinostat were determined by cell viability assay. Huh-7, Hep3B and SNU-449 were selected for further in vitro experiments. Their cell cycle distribution after Panobinostat treatment was evaluated by flow cytometry. Apoptosis was detected by Cell Death Detection ELISA. Huh-7 and Hep3B xenograft model were used for in vivo investigation. Cells were inoculated subcutaneously into the flanks of 3-4 week old male athymic nude mice. When tumors were established, Panobinostat was administrated intraperitoneally at 7.5mg/kg and 15mg/kg five days per week for 2 weeks. Results All cell lines were able to achieve nearly 100% growth inhibition and had displayed a dose- and time-dependent manner towards Panobinostat. Maximum growth inhibition was 20-70% at 24hr compared to over 90% at 72hr. There was significant reduction in cell viability at low nanomolar concentrations (IC50 at 48hr: HepG2=8.81±0.72nM, PLC/PRF/5=18.9±0.74nM, Huh-7=14.01±1.12nM, Hep3B=25.00±3.69nM, SNU-182=73.33±15.52nM, SNU-398=12.86±3.25nM, SNU-449=73.01±9.09nM). Flow cytometry analysis showed Panobinostat induced accumulation of cells at G0/G1 phase in Huh-7 and SNU-449. Meanwhile, an increase in sub G1 population was detected in Hep3B after exposure to 25nM Panobinostat for 48h. Apoptotic induction was further confirmed by cell death detection ELISA and western blotting. Panobinostat promoted apoptosis more remarkable in Hep3B than other 2 cell lines as evidenced by a stronger cleaved PARP expression level. Panobinostat treatment delayed tumor growth in Hep3B (p<0.0005) and Huh-7 (p<0.0005) xenografts compared to vehicle control. The overall weight loss was less than 20% despite of greater drop during middle of the treatment. Conclusion Panobinostat has been demonstrated to inhibit in vitro and in vivo HCC cell growth. Further study on the mechanism behind Panobinostat sensitivity is warranted. The study was supported by Novartis. Citation Format: Chi Tung Choy, Wing Yu Man, Chi Hang Wong, Stephen Lam Chan. Preclinical study on the efficacy of Panobinostat in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5059. doi:10.1158/1538-7445.AM2017-5059

Abstract 2677: Role of CLEC4D in inflammation-driven liver carcinogenesis

Abstract Hepatocellular carcinoma (HCC) accounts for approximately 90% of all primary liver tumors and is the second most deadly cancer in the world. HCC commonly arises in a chronically damaged liver that contains large amounts of inflammation and fibrosis. Indeed, interactions among hepatocytes, stromal, and inflammatory cells create a complex microenvironment permissive to tumor development. Myeloid cells are crucial players in cancer-related inflammation. From transcriptional profiling of liver myeloid cells, we identified the gene encoding for the C-type lectin receptor CLEC4D as one of the genes upregulated during the inflammatory response that precedes cancer development in a spontaneous model of murine HCC. We hypothesized that CLEC4D may be involved in the molecular mechanisms that drive HCC pathogenesis. We used the murine model of diethylnitrosamine (DEN)-induced liver carcinogenesis and evaluated tumor growth in genetically modified mice lacking CLEC4D (CLEC4D KO mice), as compared to C57BL/6 wild type (WT) mice. We observed that tumor burden (number of tumors per mouse and tumor dimensions) was reduced in CLEC4D KO mice compared to WT mice. Gene expression analysis of tumor lesions showed that the tumor microenvironment of CLEC4D KO mice was less inflammatory. CLEC4D expression was then evaluated in surgical specimens and tissue microarrays from HCV+ HCC patients. CLEC4D-positive inflammatory cells (macrophages and granulocytes) were present both in the peritumor and in the tumor areas. The percentage of intratumor CLEC4D-positive cells inversely correlated with the degree of the lesion: CLEC4D expression was higher in dysplastic nodules compared to high grade HCCs. Our results indicate that the CLEC4D-dependent pathway contributes to the initiation and the progression of HCC. By targeting CLEC4D, we could modulate immune responses and provide an environment less favorable to tumor growth. An intervention at this level could represent a chemopreventive strategy to arrest the development of HCC in a cirrhotic liver.This work has been supported by Fondazione Cariplo, grant n° 2014-0962. Citation Format: Elena Riboldi, Luca Di Tommaso, Nausicaa Clemente, Chiara Raggi, Elisa Forti, Simone Merlin, Fabio Pasqualini, Antonia Follenzi, Antonio Sica. Role of CLEC4D in inflammation-driven liver carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2677. doi:10.1158/1538-7445.AM2017-2677

E167K polymorphism of TM6SF2 gene affects cell cycle of hepatocellular carcinoma cell HEPA 1-6

BackgroundSome studties reported that the polymorphism of TM6SF2 gene E167K affects the occurrence and the progression of hepatocytes carcinoma (hepatocellular, HCC). In oeder to investigate the effects of the polymorphism of TM6SF2 gene E167K in the pathogenesis of HCC, we explored its influence on the cell cycle in hepatocellular carcinoma cell HEPA1-6.MethodsHEPA 1–6 cells which could respectively overexpress TM6SF2 wild type and E167K variant were cultured and HEPA 1–6 cells with zero load plasmids were used as matched control. Flow cytometry was used to detect the cell cycles of these 3 type of HEPA 1–6 cells. Realtime fluores-cence quantitative PCR and western blot were used to analyzed the expression of regulatory factors (Cyclin D1、p53、P16、P27、P21 and Rb) of cell cycle. T-test was used in statistical analysis.ResultsCell cycle phase distribution was presented by the proportion of cells in each phases (%). Compared with the control group, the cell cycle phase distribution (G1 phase 57.36 ± 0.21%, G2/M phase 25.61 ± 0.36%,S phases 19.31 ± 0.25%) had no differences in wild type group (G1 phase 57.63 ± 0.28%, G2/M phase 25.77 ± 0.51%, S phases 19.54 ± 0.25%; P < 0.05). Between variant type group and wild type group,G1 phase was significantly decreased (variant type group G1 phase 36.26 ± 0.31%, P < 0.05),S phase and G2/M phase were increased(variant type group S phase 28.41 ± 0.31%, P < 0.05;G2/M phase 35.23 ± 0.14%, P < 0.05), respectively. Compared with control group,the relative expression of CyclinD1、P53 and Rb mRNA in variant type group was significantly upregulated (2.03 ± 0.01 VS 1.04 ± 0.06, 1.88 ± 0.05 VS 1.37 ± 0.03, 1.29 ± 0.06 VS 1.15 ± 0.03, P < 0.05) and P27 mRNA in variant type group was significantly downregulated (0.56 ± 0.02 VS 0.85 ± 0.05, P < 0.05).Compared with wild type group, the relative expression of CyclinD1、P53 and Rb mRNA in variant type group was significantly upregulated (wild type group 1.00 ± 0.00, 1.48 ± 0.09, 1.18 ± 0.01, P < 0.05) and P27 mRNA in variant type group was significantly downregulated (variant type group 0.82 ± 0.05,P < 0.05). There was no statistical significance between wild type group and control group (P > 0.05). P16 and P21 expression showed no statistical sigtfificance in any of these three groups (P > 0.05).ConclusionE167K polymorphism of TM6SF2 gene affects cell cycles of HEPA1–6 cells via up-regulating CyclinD1、P53 and Rb and down-regulating P27.

Hepatocarcinoma: genetic and epigenetic features.

HCC is the third leading cause of cancer-related deaths worldwide, accounting for about 1 million deaths annually. The incidence of HCC is highest in Asia and Africa, where the endemic high prevalence of hepatitis B and hepatitis C strongly predisposes to the development of chronic liver disease and subsequent development of HCC. Patients with HCC generally present at an advanced stage due to compensated cirrhosis defined by the absence of pathognomonic symptoms, resulting in death within 6 to 20 months, suggesting an urgent need in treatment modalities that will dramatically decrease the mortality rate of HCC. The molecular hepatocarcinogenesis is, however, a gradual process during which genetic alterations progressively accumulate and lead to HCC through intermediate preneoplastic stages. With the advent of whole genome sequencing tools, various mutations associated with HCC have been identified, which have advanced our molecular understanding of HCC. However, the frequency of these mutations is rare, and these genetic mutations only partly explain the etiology of the disease. Better understanding and characterization of novel genetic and epigenetic alterations, which are important to hepatocarcinogenesis, may help understand the molecular pathogenesis of HCC, as well as providing novel therapeutic targets for HCC treatment. Further consideration should be given to developing more effective molecular diagnostic markers and targeted drug therapy.

Innovative molecular targeted agents in hepatocellular carcinoma: new gladiators on the arena.

Treatment of advanced hepatocellular carcinoma remains a challenge, with discouraging results in terms of survival. Following the approval of the multikinase inhibitor sorafenib, a large number of molecular targeted agents have been tested, but many have failed to demonstrate significant efficacy in clinical trials. However, the deeper knowledge in HCC pathogenesis achieved through the years has enabled us to explore new targetable pathways as well as biomarkers that could lead to treatment personalization. In this review, we provide a comprehensive update of the most recent data regarding new drugs under investigation ‒ some like regorafenib, very close to its approval ‒ and new possible targets for future treatments.

Icaritin inhibits the expression of alpha-fetoprotein in hepatitis B virus-infected hepatoma cell lines through post-transcriptional regulation.

Although it has showed that icaritin can apparently suppress growth of HCC by reducing the level of AFP, the intrinsic mechanism remains unclear. In this study, we explored the possible mechanism of miRNAs on post-transcriptional regulation of AFP gene, as well as the effects of HBV infection and icaritin in hepatoma cells. The results showed that miR-620, miR-1236 and miR-1270 could bind target sites in the range of 9–18 nt and 131–151 nt downstream of the stop codon in the AFP mRNA 3′-UTR to suppress the expression of AFP. Mutation of these target sites could reverse the effects of these miRNAs. Icaritin (10 μM) might reduce the stability and translational activity of AFP mRNA by increasing the expression levels of these mentioned miRNAs. HBV infection resulted in apparent decreases of these miRNAs and, consequently, increased AFP expression. The results indicated that miR-620, miR-1236 and miR-1270 are critical factors in the post-transcriptional regulation of AFP. Icaritin can counteract the effect of HBV. These findings will contribute to full understanding of the regulatory mechanism of AFP expression in hepatoma cells. And also it revealed a synergistic mechanism of HBV infection and elevation of AFP in the pathogenesis of HCC, as well as the potential clinical significance of icaritin on the therapy of HCC induced by HBV.

Abstract 1099: Up-regulated miR-96-5p inhibits apoptosis by targeting FOXO1 in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is one of the most common cancers and the third leading cause of cancer death worldwide, especially in Asia and sub-Saharan Africa. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and can act as tumor suppressors or oncogenes. To identify miRNA involved in HCC, we performed a genome-wide miRNA gene expression analysis using the human miRNA microarray (Agilent) in paired tumor and non-tumor tissues from patients with primary HCC. The array-based miRNA expression profiles were validated by quantitative PCR. We identified miR-96-5p as the most significantly upregulated miRNA in HCC tumors. Although miR-96-5p has been recognized as an oncogenic miRNA, its role in the pathogenesis of HCC remains unknown. Our study showed that the inhibition of miR-96-5p reduced proliferation of HCC cells and induced apoptosis. Furthermore, we found that the forkhead box O1 (FOXO1) gene was a target of miR-96-5p. FOXO1, a transcription factor, plays an essential role in cell fate decisions. FOXO1 can induce apoptosis through mitochondria-dependent and mitochondria-independent pathways. Collectively, our results suggested that miR-96-5p may function as an oncogenic miRNA through inhibiting apoptosis by targeting FOXO1 in HCC. Citation Format: Naoto Iwai, Kohichiroh Yasui, Akira Tomie, Tomoko Kitaichi, Osamu Dohi, Yasuyuki Gen, Yoshito Itoh. Up-regulated miR-96-5p inhibits apoptosis by targeting FOXO1 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 1099.

Abstract 3820: Epigenetic re-expression of fetal IGF2 as therapeutic target in hepatocellular carcinoma

Abstract Background and aims Hepatocellular carcinoma (HCC) is a major health problem. Most patients are diagnosed at advanced stages when the only approved therapy is the multi-kinase inhibitor sorafenib. Consequently, there is a great need for the development of new effective treatments. IGF signaling pathway is aberrantly activated in HCC; however, its contribution to HCC pathogenesis is still unclear. Since IGF2 is overexpressed in HCC, we aimed to elucidate the oncogenic potential and mechanism of dis-regulation of this protein and determine the antitumoral efficacy of molecular abrogation of this ligand by targeted therapies. Methods Transcriptomic profiling, miRNAs expression, RNA- and whole exome- sequencing and methylation were analyzed in 228 HCCs with a focus on IGF-pathway. Gene Set Enrichment Analysis and Ingenuity Pathway Analysis were carried out in IGF2-overexpressing tumors. Stable HCC cell lines with knock-down and ectopic overexpression of IGF2 were generated. A chemically-induced mouse model of HCC, and two genetically-engineered mosaic mouse models (GEMM) overexpressing IGF2 specifically in the liver were generated to assess IGF2 oncogenicity in hepatocarcinogenesis. The therapeutic potential of a monoclonal-antibody against IGF-ligands (IGF1/2-mAb) alone or in combination with sorafenib was tested in a xenograft model of HCC. Results Here, IGF2-overexpression occurred in 15% of HCC patients as a result of the epigenetic reactivation of IGF2-fetal promoters, mainly through loss of promoters methylation (53% of cases) and deregulation of miR-216b, miR-483-5p and miR-let7-d (35% of cases). Re-expression of IGF2 was associated with a progenitor cell-like, poorly differentiated and aggressive subtype of HCC, and poor prognosis (p&amp;lt;0.0001). In a GEMM model, IGF2-overexpression accelerated HCC progression and reduced survival (p = 0.02). Conversely, IGF2-blockage using an IGF1/2-monoclonal antibody (mAb) or specific shRNAs against IGF2 reduced viability and proliferation in vitro (p&amp;lt;0.05), and inhibited IGF-Insulin pathway activation without disturbing insulin metabolism. IGF1/2-mAb delayed tumor growth and increased survival in vivo compared to placebo or sorafenib (p&amp;lt;0.0001), through antiproliferative and antiangiogenic mechanisms. Conclusions IGF2 is the first validated epidriver in HCC and has a key role in the hepatocarcinogenic process. These results provide the rationale for testing IGF1/2-mAb in a selected subset of HCC patients. Citation Format: Iris Martinez-Quetglas, Roser Pinyol, Daniel Dauch, Sara Torrecilla, Victoria Tovar, Agrin Moeini, Clara Alsinet, Anna Portela, Augusto Villanueva, Manel Esteller, Lars Zender, Josep M Llovet. Epigenetic re-expression of fetal IGF2 as therapeutic target 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 3820.

Relation between microRNAs and Apoptosis in Hepatocellular Carcinoma.

AIM:To determine the relation between serum microRNAs and apoptotic markers as regards development of HCC to understand the underlying mechanism of HCV related hepatocarcinogenesis.PATIENTS AND METHODS:A total of 65 serum samples (25 samples from controls, 20 samples from hepatitis and 20 samples from HCC patients) were collected for miRNAs (mir 21, mir 199-a, and mir 155) detection. Human Programmed cell death protein-4 (PDCD-4) and Human Cytochrome-C (CYT-C) were determined.RESULTS:miRNAs 21 and 155 were over expressed in sera of patients with HCC compared to patients with chronic hepatitis (p < 0.0001). While serum means values of miR 199a was significantly decreased among HCC group patients when compared to patients with chronic hepatitis (p < 0.0001). The serum levels of PCDC4 and CYTC were increased in patients with HCC when compared to chronic hepatitis patients. They were also increased in patients with chronic hepatitis when compared to controls (p < 0.05, significant). There was direct correlations between apoptotic markers and oncomirs miRNAs 21 and 155 while apoptotic markers were inversely correlated with miRNA 199-a.CONCLUSION:Both microRNAs and apoptotic markers have roles in HCC pathogenesis. It seems that oncogenic microRNAs induce liver carcinogenesis in HCV patients irrespective of suppression of apoptosis.

Transcriptomic and Proteomic Investigation of HSP90A as a Potential Biomarker for HCC.

BackgroundHepatocellular carcinoma (HCC) is the third most frequent cause of cancer-related death in adults. Despite recent advances in the clinical technologies, the screening and diagnostic efficacy for HCC remains poor. Discovering novel and reliable HCC biomarkers is urgently needed.Material/MethodsWe performed a transcriptome-proteome integrated assay to track the possible HCC biomarkers from the process of HCC-derived gene expression in malignant cells to its protein product released into serum.ResultsOur screening results demonstrated that heat shock protein 90A (HSP90A), which participates in the PI3K-Akt signaling pathway and many other cancer-related pathways, warrants further investigation. The expression of HSP90A was increased in the HCC cells, serum, and tissues. Immunohistochemistry analysis on 76 clinical tissue samples also suggested the relevance between HSP90A expression and HCC metastatic behavior.ConclusionsThese findings suggest a role for HSP90A in HCC pathogenesis and the potential use of HSP90A for the screening and diagnosis of this malignancy.

Abstract IA11: Differential networks and drug networks: A computational approach to drug discovery

Abstract Genome scale expression profiles (GEPs) of tumor samples versus non-tumor sampls can be analysed via innovative computational approaches to identify altered pathways and driver genes. These results can then be used to make identify appropriate small molecule therapies. We developed and validated computational strategy, called Differential Co-expression Analysis (DiCA), to infer metabolic, signaling and regulatory pathways which are altered across two conditions (i.e. tumor vs non-tumor). DiCA is built on the hypothesis that genes in an active pathway are more likely to be regulated and hence co-expressed. On the contrary, inactive pathways will results in uncoordinated gene expression. DiCA measures how much a set of genes is co-expressed using a multidimensional information metrics. We apply DiCA to 100 Human HepatoCarcinoma samples collected within the MODHEP European Project. We demonstrate how DMI is able to correctly identify altered pathways known to be affected by HCC and to propose new hypothesis on HCC pathogenesis, which would be missed by conventional Differential Expression. We then show how GEPs from cancer samples can be used also to query a “drug network” by comparing them to transcriptional responses induced by drug treatment. The end result is a list of drugs which induce a transcriptional response similar to that of cancer samples thus hinting at which pathways are perturbed. Analogously, drugs inducing an “anti-similar” response, i.e. up regulating genes down regulated in cancer and vice-versa, could be investigated as potentially therapeutic. We tested these hypotheses on HCC samples demonstrating their potential usefulness. Citation Format: Diego di Bernardo. Differential networks and drug networks: A computational approach to drug discovery. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr IA11.