Event In Hepatocarcinogenesis Research Articles

Pathology of Hepatocellular Carcinoma and Tumor-Bearing Liver Tissue in Association with hTERT Promoter Mutation.

The hTERT promoter mutation represents a common and early event in hepatocarcinogenesis, but its linkage to the morphological status of the underlying liver tissue is poorly understood. We analyzed the connection between the histopathological changes in tumor-bearing liver tissue and the occurrence of the hTERT promoter mutation in hepatocellular carcinoma (HCC), correlated with clinical data. The study cohort comprised 160 histologically confirmed HCC in patients with or without cirrhosis that were investigated for the hTERT promoter mutation. We evaluated the frequency of the hTERT promoter mutation in patients with HCC with or without cirrhosis and correlated it with potential clinical and histopathological drivers. In particular, we examined tumor-bearing noncirrhotic liver tissue regarding inflammation; the modified histological activity index (mHAI), fibrosis, and steatosis; and its correlation with the frequency of the hTERT promoter mutation in HCC. We evaluated overall survival with multivariate Cox regression. Furthermore, we compared hTERT antibody immunohistochemistry and molecular hTERT promoter mutation analysis of both HCC and background liver tissue. The hTERT promoter mutation was especially related to HCC in cirrhotic compared with noncirrhotic liver (p < 0.001) and independently of cirrhosis in patients ≥ 60 years (p = 0.005). Furthermore, the hTERT promoter mutation was associated with cirrhosis caused by alcohol toxicity and hepatitis C virus infection. In noncirrhotic liver tissue, the frequency of hTERT-promoter-mutated HCC increased with the degree of inflammation and fibrosis. Nevertheless, 25% of the hTERT-promoter-mutated HCC developed in normal liver tissue without HCC risk factors. Multivariate Cox regression analysis did not reveal an influence of the hTERT promoter mutation in HCC on overall survival at 3, 5, and 16 years. Immunohistochemical analysis with the hTERT antibodies LS-B95 and 2D8 in hTERT-promoter-mutated HCC and hTERT-wildtype HCC showed a mildly stronger immunoreaction compared with the tumor-bearing liver tissue (LS-B95: p < 0.01, 2D8: p < 0.01). Our study reveals a connection between pathological changes in tumor-bearing liver tissue and the hTERT promoter mutation in most HCC, even in noncirrhotic liver tissue. Immunohistochemical hTERT antibodies do not discriminate between hTERT-promoter-mutated and wildtype HCC.

Expression Levels of Three Key Genes CCNB1, CDC20, and CENPF in HCC Are Associated With Antitumor Immunity.

IntroductionHepatocellular carcinoma (HCC) is the most common primary liver cancer with a low 5-year survival rate. The heterogeneity of HCC makes monotherapy unlikely. The development of diagnostic programs and new treatments targeting common genetic events in the carcinogenic process are providing further insights into the management of HCC. The aim of this study was firstly to validate key genes that are involved in promoting HCC development and as biomarkers for early diagnosis and, secondly, to define their links with antitumor immunity including inhibitory checkpoints.MethodsMultiple databases including Gene Expression Omnibus (GEO), Gene Expression Profiling Interactive Analysis (GEPIA), Kaplan–Meier Plotter, UALCAN, and Oncomine were used for target gene screening and establishment of a co-expression network. Clinical data and RNAseq of 367 HCC patients were downloaded from the Cancer Genome Atlas (TCGA) database. The diagnostic and prognostic value of screened genes were tested by receiver operating characteristic (ROC) curve and correlation analysis. The links with the key genes in HCC and antitumor immunity were defined using both blood and liver tissue collected prospectively from HCC patients in our center.ResultsUpregulation of CCNB1, CDC20, and CENPF was commonly observed in HCC and are involved in the p53 signal pathway. The hepatic mRNA expression levels of these three genes were strongly associated with patients’ prognosis and expressed high value of area under the ROC curve (AUC). Further analysis revealed that these three genes were positively correlated with the gene expression levels of IFN-γ, TNF-α, and IL-17 in peripheral blood. In addition, the expression of CENPF showed positive correlation with the percentage of CD8pos T cells and negative correlation with the percentage of CD4pos T cells in the peripheral blood. In the HCC microenvironment, the transcript levels of these three genes and inhibitory checkpoint molecules including PD-1, CTLA-4, and TIM-3 were positively correlated.ConclusionThe upregulation of CCNB1, CDC20, and CENPF genes was a common event in hepatocarcinogenesis. Expression levels of CCNB1, CDC20, and CENPF showed potential for early diagnosis and prediction of prognosis in HCC patients. There is a close association between three genes and Th1/Th17 cytokines as well as the count of CD4pos and CD8pos T cells. The positive correlation between the three genes and inhibitory checkpoint genes, PD-1, CTLA-4, and TIM-3, indicates that these genes are linked with weakened antitumor immunity in HCC. Our findings may provide further insights into developing novel therapies for HCC.

Cooperation between liver-specific mutations of pten and tp53 genetically induces hepatocarcinogenesis in zebrafish

BackgroundLiver cancer, mainly hepatocellular carcinoma, is one of the deadliest cancers worldwide and has a poor prognosis due to insufficient understanding of hepatocarcinogenesis. Previous studies have revealed that the mutations in PTEN and TP53 are the two most common genetic events in hepatocarcinogenesis. Here, we illustrated the crosstalk between aberrant Pten and Tp53 pathways during hepatocarcinogenesis in zebrafish.MethodsWe used the CRISPR/Cas9 system to establish several transgenic zebrafish lines with single or double tissue-specific mutations of pten and tp53 to genetically induce liver tumorigenesis. Next, the morphological and histological determination were performed to investigate the roles of Pten and Tp53 signalling pathways in hepatocarcinogenesis in zebrafish.ResultsWe demonstrated that Pten loss alone induces hepatocarcinogenesis with only low efficiency, whereas single mutation of tp53 failed to induce tumour formation in liver tissue in zebrafish. Moreover, zebrafish with double mutations of pten and tp53 exhibits a much higher tumour incidence, higher-grade histology, and a shorter survival time than single-mutant zebrafish, indicating that these two signalling pathways play important roles in dynamic biological events critical for the initiation and progression of hepatocarcinogenesis in zebrafish. Further histological and pathological analyses showed significant similarity between the tumours generated from liver tissues of zebrafish and humans. Furthermore, the treatment with MK-2206, a specific Akt inhibitor, effectively suppressed hepatocarcinogenesis in zebrafish.ConclusionOur findings will offer a preclinical animal model for genetically investigating hepatocarcinogenesis and provide a useful platform for high-throughput anticancer drug screening.

Loss of Apc Cooperates with Activated Oncogenes to Induce Liver Tumor Formation in Mice

Hepatocellular carcinoma (HCC) and hepatoblastoma are the major types of primary liver cancer in adulthood and childhood, respectively. Wnt/β-catenin signaling deregulation is one of the most frequent genetic events in hepatocarcinogenesis. APC regulator of WNT signaling pathway (APC) encodes an inhibitor of the Wnt cascade and acts as a tumor suppressor. Germline defects of the APC gene lead to familial adenomatous polyposis, and its somatic mutations occur in multiple tumor types. However, the contribution of APC in hepatocarcinogenesis remains unclear. Therefore, APC mutations and expression patterns were examined in human HCC and hepatoblastoma samples. Whether loss of Apc alone or in cooperation with other oncogenes triggers liver tumor development invivo was also investigated. sgApc alone could not drive liver tumor formation, but synergized with activated oncogenes (YapS127A, TazS89A, and c-Met) to induce hepatocarcinogenesis. Mechanistically, Apc deletion induced the activation of β-catenin and its downstream targets in mouse liver tumors. Furthermore, Ctnnb1 ablation or TCF4-mediated transcription blockade completely prevented liver tumor formation, indicating the requirement of a functional β-catenin pathway for loss of Apc-driven hepatocarcinogenesis. This study shows that a subset of HCC patients with loss-of-function APC mutations mightbenefit from therapeutic strategies targeting the Wnt/β-catenin pathway.

Pyruvate Kinase M2 Coordinates Metabolism Switch between Glycolysis and Glutaminolysis in Cancer Cells.

SummaryCancer cells alter their nutrition metabolism to cope the stressful environment. One important metabolism adjustment is that cancer cells activate glutaminolysis in response to the reduced carbon from glucose entering into the TCA cycle due to inactivation of several enzymes in glycolysis. An important question is how the cancer cells coordinate the changes of glycolysis and glutaminolysis. In this report, we demonstrate that the pyruvate kinase inactive dimer PKM2 facilitates activation of glutaminolysis. Our experiments show that growth stimulations promote PKM2 dimer. The dimer PKM2 plays a role in regulation of glutaminolysis by upregulation of mitochondrial glutaminase I (GLS-1). PKM2 dimer regulates the GLS-1 expression by controlling internal ribosome entry site (IRES)-dependent c-myc translation. Growth stimulations promote PKM2 interacting with c-myc IRES-RNA, thus facilitating c-myc IRES-dependent translation. Our study reveals an important linker that coordinates the metabolism adjustment in cancer cells.

Expression of the type 3 inositol 1, 4, 5-trisphosphate receptor according to the chronic liver disease etiology in hepatocellular carcinoma.

e16604 Background: The expression of type 3 isoform of the inositol 1, 4, 5-trisphosphate receptor (ITPR-3), an intracellular calcium (Ca2+) channel reported in liver cancer cells, is important in the Ca2+ signalling. Thus, it may be involved in the many events of hepatocarcinogenesis. Here, we investigated the ITPR-3 expression in hepatocellular carcinoma (HCC) and its association with clinicopathological parameters and long-term outcomes, according to the etiology of underlying chronic liver disease (CLD). Methods: Clinical and laboratory data from patients (n = 53) who underwent orthotopic liver transplantation for HCC treatment in a Brazilian referral center were retrospectively collected. After pathological reviewing of their explanted liver samples, ITPR-3 expression in both tumor and underlying cirrhosis were assessed by immunohistochemistry, and quantified using density histograms in the ImageJ software. Event (tumor recurrence or death from any cause) occurrence and event-free survival (EFS) were analysed. Results: Hepatitis C virus (HCV) (n = 31), alcohol abuse (n = 16) and cryptogenic cirrhosis (n = 6) were the underlying CLD etiology, and the groups were, in general, well balanced regarding clinicopathological indices. Median EFS was 78.9 months (range, 63.6-94.1). The ITPR-3 expression profile was cytoplasmatic, predominantly perinuclear, and was stronger in tumor than in adjacent cirrhosis, considering all etiologies together (intensity 9.1% higher in tumors, p &lt; 0.001) However, analyzing each etiologic group, the cryptogenic was the only one in which there was no difference between tumor and underlying CLD. Comparing the ITPR-3 expression only in tumors, there was no difference regarding the etiology of CLD. The tumor ITPR-3 higher intensity was correlated with higher serum aspartate alanine-transferases (ALT) levels (p = 0.018) and lower mitotic index ( &lt; 5 per 10 high power fields) (p = 0.009). There was no association between receptor expression and event occurrence or EFS. Conclusions: The ITPR-3 was expressed in HCC, regardless of the underlying CLD etiology. Its correlation with mitotic index, a cell proliferation marker, was demonstrated, but there were no associations with clinical outcomes. Apart from cryptogenic cirrhosis, ITPR-3 expression was more intense in tumors than in underlying cirrhosis. These findings suggest that ITPR-3 could have a role in carcinogenesis. However, the prognostic and therapeutic implications need to be investigated.

Identification of genes involved in the regulation of TERT in hepatocellular carcinoma.

Telomerase reverse transcriptase (TERT) promotes immortalization by protecting telomeres in cancer cells. Mutation of the TERT promoter is one of the most common genetic alterations in hepatocellular carcinoma (HCC), indicating that TERT upregulation is a critical event in hepatocarcinogenesis. Regulators of TERT transcription are, therefore, predicted to be plausible targets for HCC treatment. We undertook a genome‐wide shRNA library screen and identified C15orf55 and C7orf43 as regulators of TERT expression in HepG2 cells. Promoter assays showed that C15orf55‐ and C7orf43‐responsive sites exist between base pairs −58 and +36 and −169 and −59 in the TERT promoter, respectively. C15orf55 upregulates TERT expression by binding to two GC motifs in the SP1 binding site of the TERT promoter. C7orf43 upregulates TERT expression through Yes‐associated protein 1. The expression levels of C15orf55 and C7orf43 also correlated with that of TERT, and were significantly increased in both HCC tissues and their adjacent non‐tumor tissues, compared to normal liver tissues from non‐HCC patients. Analysis of 377 HCC patients in The Cancer Genome Atlas dataset showed that overall survival of patients with low levels of C15orf55 and C7orf43 expression in tumor tissues was better compared with patients with high levels of C15orf55 and/or high C7orf43 expression. These results indicate that C15orf55 and C7orf43 are involved in the incidence and progression of HCC by upregulating TERT. In conclusion, we identified C15orf55 and C7orf43 as positive regulators of TERT expression in HCC tissues. These genes are promising targets for HCC treatment.

Genome sequencing analysis of liver cancer for precision medicine.

Liver cancer is the third leading cause of cancer-related death worldwide. Some thousands of liver cancer genome have been sequenced globally so far and most of driver genes/mutations with high frequency are established in liver cancer, including Wnt/β-catenin pathway, TP53/cell-cycle pathways, telomere maintenance, and chromatin regulators. HBV integration into cancer-related genes is also a driver event in hepatocarcinogenesis. These genes are affected by structural variants, copy-number alterations and virus integrations as well as point mutations. Etiological factors of liver cancer is most understood among common cancers, such as hepatitis, aflatoxin, alcohol, and metabolic diseases, and mutational signatures of liver cancer can provide evidence of the association between specific etiological factors and mutational signatures. Molecular classifications based on somatic mutations profiles, RNA expression profiles, and DNA methylation profiles are related with patient prognosis. For precision medicine, several actionable mutations with solid evidence such as targets of multi-kinase inhibitors is observed in liver cancer, but there is few molecular target therapy so far. It is possible that rare actionable mutations in liver cancer can guide other specific molecular therapy and immune therapy.

Vitamin D receptor gene methylation in hepatocellular carcinoma

Worldwide, hepatocellular carcinoma (HCC) is the major subtype of primary liver cancers. HCC is typically diagnosed late in its course. With respect to cancer, the genomic actions of vitamin D are mediated through binding to the Vitamin D Receptor (VDR), which allows it to modulate the expression of genes in a cell-and tissue-specific manner. Epigenetics is a rapidly evolving field of genetic study applicable to HCC. Changes in DNA methylation patterns are thought to be early events in hepatocarcinogenesis. Curcumin has great potential as an epigenetic agent. Accordingly, the current study has been designed to study the methylation status of VDR gene promoter for the first time in HCC aiming to find its clinical significance and potential screening role in chronic Liver Disease (CLD). Additionally, we aimed to investigate, the effect of Curcumin on HCC cell line, aiming to discover new therapeutic targets through epigenetics. This study was conducted on 45 formalin-fixed, paraffin-embedded liver tissue blocks including 15 HCC samples (group A), 15 CLD samples (group B) and 15 apparently normal tissue taken from around benign lesions (group C). Methylation Specific Restriction Digestion and qPCR were done on all samples after DNA extraction. The percentage of VDR gene promoter methylation was significantly higher in the HCC group compared to both CLD and control groups (p < 0.01). VDR promoter methylation by (MS-qPCR) was decreased and the relative expression of VDR by (qRT-PCR) was markedly increased in a dose-dependent fashion in cells grown in Curcumin-adequate medium. In conclusion, this study may open a new gate for the use of VDR promoter methylation as a potential biomarker in HCC.

A Gain of Function p53 Gene Mutant Promotes Growth Suppression in Human Liver Cancer Cells

Primary liver cancer is one of the most common cancer in the world with highest cancer mortality rate. The most common type of primary liver cancer is hepatocellular carcinoma (HCC). There are many risk factors for liver cancer and currently available treatments for HCC are largely inadequate. Gene mutation and dysfunction of p53 are common and is recognized as an important molecular event in hepatocarcinogenesis. Therefore, replacement of the aberrant p53 gene is an attractive approach in the treatment of HCC providing an alternative treatment for primary HCC. In this study, we assessed whether the transfection with wild-type p53 gene is able to restore the pro-apoptotic effects and evaluate the feasibility of gene therapy in fixing a faulty p53 molecule. We established a non-viral cationic lipid-based p53 gene delivery into two human HCC cell lines namely HLF and PLC/PRF/5 cells. Both cell lines have mutations in the p53 gene. We compared the results with the normal liver cell line, WRL68, that constitutively expresses the wild-type p53 gene. In this study, the introduction of wild-type p53 gene into HLF and PLC/PRF/5 cells resulted in an increased of p53 gene expression, protein expression and cells growth inhibition shown in MTS reduction cell viability assay, FITC-Annexin V and PI apoptosis assay, western blot and caspase activity assay. In summary, the study provides a promising therapeutic approach for p53 gene delivery into HCC patients. The p53 gene delivery can be instituted together with chemotherapy as a combination treatment to induce apoptosis.

Abstract 1130: Subcellular localization of the cell polarity protein Scribble defines its oncogenic activity in hepatocellular carcinoma

Abstract Background: Disturbance of hepatocyte polarity is frequently observed in liver cancer tissues; however, it is still controversially discussed if this phenotype represents the cause or the result of tumorigenesis. Due to their multi-functional properties, hepatocytes are characterized by distinct luminal, lateral and basal domains that are defined by specific protein complex. These cell protein complexes such as the apical Crumbs- (CRB3, Pals1, Patj), the lateral Par- (Par3, Par6, aPKC) and the basolateral Scribble-complex (Scrib, Dlg, Lgl) are essential for the formation of hepatocellular polarity. So far, it is unknown if and which protein complex constituents are involved in liver tumor development and how these factors induce possible downstream effector mechanisms. Methods: To identify complex proteins that affect hepatocellular polarity, a siRNA screen targeting all relevant polarity factors was performed in HCC cells that form bile canaliculi in vitro (HepG2). Localization of Scribble in HCC cell lines and human HCC tissues was analyzed by western immunoblotting and immunofluorescence. Expression vectors containing wildtype Scribble and an isoform lacking polarity complex binding capacity were cloned (Scrib-wt, Scrib-P305L) and stably transfected in HCC cells. The impact of these isoforms on PI3K/AKT signaling pathway activity and cell viability were analyzed. In addition, the interaction of Scrib-wt, Scrib-P305L with phosphatases regulating the PI3K/AKT pathway was tested by Co-IP analysis. Results: The siRNA screen revealed that Scribble strongly affects hepatocellular polarity. Scribble located near the membrane in polarized cells (e.g., HepG2, HuH-1) but in the cytoplasm in non-polarized cells (HLE, HLF). Scribble overexpression and cytoplasmic accumulation correlates with poor overall survival of HCC patients. In vitro, the inhibition of Scribble reduced and overexpression of cytoplasmic Scribble (Scrib-P305L) induced AKT phosphorylation. In addition, Scrib-P305L supported HCC cell viability in an AKT-dependent manner. Immunofluorescence revealed co-localization of Scribble with the AKT phosphatases PTEN and PHLPP1. Co-IP studies revealed a physical interaction between PHLPP1 and both Scribbles isoforms (wildtype and P305L mutant). Conclusion/Outlook: Our data demonstrate that the cytoplasmic accumulation of Scribble in HCC cells promotes tumor cell growth in an AKT-dependent manner probably through the interaction with AKT phosphatases. These results indicate that the disturbance of cell polarity (e.g. by Scribble overexpression) can foster oncogenic effects and therefore must be regarded as initiating event in hepatocarcinogenesis. Citation Format: Shan Wan, Anne-Sophie Meyer, Sofia Weiler, Teresa Lutz, Stephanie Roessler, Peter Schirmacher, Federico Pinna, Kai Breuhahn. Subcellular localization of the cell polarity protein Scribble defines its oncogenic activity 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 1130.

PWE-044 Inter-Tumour Heterogeneity in Hepatitis C Virus (HCV) Related Multi-Focal Hepatocellular Carcinoma (HCC)

Introduction Curative therapies improve overall survival by more than 60 months on a small proportion of patients with HCC however the majority of patients are diagnosed with non curable, mostly multi-focal HCC. There are two theories for the development of multifocal HCC. First is intrahepatic metastasis where the new cancer would inherit genetic properties from the index cancer. Second is de novo development of HCC where both cancers are expected to have genetically diverse properties. We aimed to evaluate the presence and frequency of intrahepatic metastasis versus de novo carcinogenesis in HCV related multi-focal HCC. Methods We studied DNA copy number profiles of 70 HCV-related hepatocellular cancers found in 19 surgically removed liver specimens from 18 patients. Differences in copy number patterns were objectively analysed using Pearson’s correlations between normalised, non-smoothed, non-segmented ratios (n = 27,180 per each sample). Fisher r to z to r transformation was used in calculation of the average coefficients. Results Livers containing 2 or 3 nodules (n = 10 livers) had an average cancer correlation coefficient of 0.395 and only 20% of them (n = 2/10) harboured cancers with coefficients >0.5. Livers containing 4 or more nodules (n = 9 livers) had an average cancer correlation coefficient of 0.620 and 89% of them (n = 8/9) harboured cancers with coefficients >0.5. The higher correlation coefficient may indicate that livers with a larger number of nodules are more likely to harbour genetically similar cancers then liver with a smaller number of nodules. This implies that intrahepatic metastasis starts to occur at a more advanced stage of liver disease and that de novo carcinogenesis takes place at an earlier stage. Conclusion This study confirms that both processes exist. The occurrence of de novo carcinogenesis leads to emergence of genetically diverse cancers within the same liver. Our data suggests that this occurs at an earlier stage of multifocal spread. Intrahepatic metastasis leads to occurrence of genetically similar cancers within the same liver. This possibly occurs when HCC’s are more established. More cases are needed to validate the sequence of events in hepatocarcinogenesis. This is knowledge is clinically relevant as cancers with diverse genetic profiles are practically more difficult to manage. Disclosure of Interest W. Fateen: None Declared, H. Wood: None Declared, S. Berri Employee of: Illumina, J. Wyatt: None Declared, M. El- Meteini: None Declared, C. Millson: None Declared, P. Quirke: None Declared

PARP Inhibition Suppresses Growth of EGFR-Mutant Cancers by Targeting Nuclear PKM2.

Upon growth factor stimulation or in some EGFR mutant cancer cells, PKM2 translocates into the nucleus to induce glycolysis and cell growth. Here, we report that nuclear PKM2 binds directly to poly-ADP ribose, and this PAR-binding capability is critical for its nuclear localization. Accordingly, PARP inhibition prevents nuclear retention of PKM2 and therefore suppresses cell proliferation and tumor growth. In addition, we found that PAR level correlates with nuclear localization of PKM2 in EGFR mutant brain and lung cancers, suggesting that PAR-dependent nuclear localization of PKM2 likely contributes to tumor progression in EGFR mutant glioblastoma and lung cancers. In addition, some EGFR-inhibitor-resistant lung cancer cells are sensitive to PARP inhibitors. Taken together, our data indicate that suppression of PKM2 nuclear function by PARP inhibitors represents a treatment strategy for EGFR-inhibitor-resistant cancers.

Induction of autophagy promotes the growth of early preneoplastic rat liver nodules.

Although inhibition of autophagy has been implicated in the onset and progression of cancer cells, it is still unclear whether its dysregulation at early stages of tumorigenesis plays an oncogenic or a tumor suppressor role. To address this question, we employed the Resistant-Hepatocyte rat model to study the very early stages of hepatocellular carcinoma (HCC) development. We detected a different autophagy-related gene expression and changes in the ultrastructural profile comparing the most aggressive preneoplastic lesions, namely those positive for the putative progenitor cell marker cytokeratin-19 (KRT-19) with the negative ones. The ultrastructural and immunohistochemical analyses of KRT-19-positive preneoplastic hepatocytes showed the presence of autophagic vacuoles which was associated with p62, Ambra1 and Beclin1 protein accumulation suggesting that a differential modulation of autophagy occurs at early stages of the oncogenesis in KRT-19-positive vs negative lesions. We observed an overall decrease of the autophagy-related genes transcripts and a strong up-regulation of miR-224 in the KRT-19-positive nodules. Interestingly, the treatment with the autophagy inducer, Amiodarone, caused a marked increase in the proliferation of KRT-19 positive preneoplastic lesions associated with a strong increase of their size; by contrast, Chloroquine, an inhibitor of the autophagic process, led to their reduction. These results show that autophagy modulation is a very early event in hepatocarcinogenesis and is restricted to a hepatocytes subset in the most aggressive preneoplastic lesions. Our findings highlight the induction of autophagy as a permissive condition favouring cancer progression indicating in its inhibition a therapeutic goal to interfere with the development of HCC.

Abstract 2970: Whole genome sequencing analysis of multiple liver cancer nodules for determination of causal events for multi-occurrence

Abstract Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Despite the recent progress in diagnostic accuracy and therapeutic efficacy, its five-year survival rate is still low. Characteristically, HCC shows a high rate of intrahepatic metastasis (IM) or multi-centric (MC) occurrence within the liver, and discrimination of IM/MC is difficult and important for therapeutic decision. Previously we reported that there were no common somatic mutations between whole genomes of MC tumors, indicating that MC tumors independently developed in their genetic aspect (Nat Genet, 44: 760-764, 2012). Here we performed whole genome sequencing (WGS) analysis on 43 multiple liver cancer nodules derived from 20 patients, which were clinically defined as IM or MC occurrence. We first analyzed common and unique somatic mutations in each individual and divided the cases into IM or MC by using the information of common somatic mutations between the multiple nodules. Interestingly, the clinical and pathological diagnoses of MC/IM in five of 20 cases were not concordant with the judgments from WGS, indicating the usefulness of WGS for a precise IM/MC judgment. Analysis of common somatic alterations in IM tumors enabled to extract early and late event in hepatocarcinogenesis among a large number of somatic events in each cases, while MC pairs shared only a few numbers of common alterations, including false positives. Importantly, one somatic mutation in non-coding region was exceptionally detected commonly among MC tumor pairs from three cases. Sanger sequencing confirmed that this mutation was also commonly detected in 10.9% (35/321) of liver cancer samples somatically. These results indicate the significance of this function-unknown and recurrent mutation in liver cancer development. To illuminate common driver events in MC tumors, we analyzed all detectable genetic alterations, including copy number alterations and rearrangements, on multi-time point or MC tumors derived from same individuals and extracted significant genetic alterations from them, which can help the understanding of driver genetic alterations in liver cancers. Citation Format: Mayuko Furuta, Akihiro Fujimoto, Masaki Ueno, Shinya Hayami, Yoshi-iku Kawakami, Kunihito Gotoh, Tatsuhiko Tsunoda, Satoru Miyano, Hideki Ohdan, Kazuaki Chayama, Hiroki Yamaue, Hidewaki Nakagawa. Whole genome sequencing analysis of multiple liver cancer nodules for determination of causal events for multi-occurrence. [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 2970. doi:10.1158/1538-7445.AM2015-2970

Abstract 176: MicroRNA-122 is a potential regulator of DNA methylation partly by affecting the global level profile of 5hmC

Abstract MicroRNA-122 is a liver specific microRNA. Previously, we demonstrated that Mir122a-/- mice developed steatohepatitis, fibrosis and HCC which recapitulates the multistep hepatocarcinogenesis found in human. In addition, we detected a global change of gene expression involving upregulation of 606 genes and downregulation of 280 genes. Among the genes highly differentially expressed in Mir122a-/- livers are several classic imprinted genes H19/Igf2, Meg3, Mirg, Gdf10 and Rian. Imprinting of both H19/Igf2 and Mirg loci is controlled by CTCF, which is a direct target of miR-122. These earlier findings imply that miR-122 may have a role in epigenetic regulation. Recently, the active demethylation pathway involving 5-hydroxymethylcytosine (5hmC) and TET (tet methylcytosine dioxygenase) enzymes are found to regulate the genome epigenetically. 5hmC epigenetic mark was found to associate with human cancer development with frequent downregulation in cancers, including HCC. We detected a progressive loss of 5hmC in liver, the tumor-adjacent tissues and liver tumors of Mir122a-/- mice similar to that of human HCC. Loss of 5hmC is accompanied with downregulation of DNA dioxygenase (Tet1 and Tet3), and upregulation of DNA deaminases (Apobec1 and Apobec3) as well as DNA glycosyalse (Smug1) in Mir122a-/- tumors. A genome-wide profiling of 5hmC was performed to delineate the dynamic changes of DNA methylation. The 5hmC-containing genomic DNA fragments were collected and sequenced. Our preliminary results showed that Mir122a-/- livers appear to have significantly fewer 5hmC peaks and covered smaller portion of the genome than that of WT. Even fewer 5hmC peaks covering small portion of the genome were detected for the Mir122a-/- liver tumor samples. The number of identified 5hmC peaks follows the order of WT &amp;gt; Mir122a-/- (2-month) &amp;gt; tumor-adjacent liver tissue &amp;gt; tumor of Mir122a-/-, a trend that is in agreement with quantification of 5hmC by both IHC and DNA dot blotting. The preliminary finding that aberrant DNA demethylation was initiated in young Mir122a-/- mice suggests that active demethylation is likely an early event of hepatocarcinogenesis. Note: This abstract was not presented at the meeting. Citation Format: Ann-Ping Tsou, Kuan-Hua Wang, Hsi-Yuan Huang, Li-Jung Chen, Tze-Tze Liu. MicroRNA-122 is a potential regulator of DNA methylation partly by affecting the global level profile of 5hmC. [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 176. doi:10.1158/1538-7445.AM2015-176

Stem cell expression profile in hepatocellular carcinoma, small cell dysplasia, and cirrhosis

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with various etiologies and its incidence still continues to increase. Tumors with progenitor/stem cell (P/SC) immunophenotype are currently the main focus of interest. Human hepatic P/SCs are located in ductal plates in fetal and neonatal livers and they remain relatively constant throughout life. Enhanced self-renewal level of normal hepatic P/SCs serves as an early event in hepatocarcinogenesis. Approximately 40% of HCCs arise from P/SCs and these cells have a critical role in development and progression of HCC. Thus, CD133 expression was found to be upregulated during early liver restoration. In spite of the increasing evidence of the impact of cancer stem cells (CSCs) in hepatocarcinogenesis, the role of CSCs in the sequence of cirrhosis (Crh)-dysplastic nodules (DN)-HCC is still not clear. In our recent research, we investigated the expression rate and staining patterns of CD133 and CD90 in Crh, small cell dysplasia, and HCC. We also evaluated the relationship between the expression rates of CSC markers and the etiology of liver diseases. Among the P/SC markers, we like to attract more attention to CD133 and CD90. Both CD133 and CD90 expressions were higher in poorly differentiated HCC cases than well differentiated ones. According to the etiology, we found that the highest staining rate for CD133 in HCC cases developed in a cirrhotic background with chronic hepatitis B and D co-infection. The highest rate for CD90 was determined in HCC cases with chronic hepatitis C. Since Crh is the end-stage of chronic injury with continuing regeneration, detection of accompanying P/SC activation by CSC markers in suspicious nodules may suggest the initiation of early phases of hepatocarcinogenesis. Some other immunophenotypical features such as Glypican 3, heat shock protein-70, and glutamine synthetase stainings are also being used to diagnose precursor lesions. Facing the emerging concept of personalized treatment strategy, it is obvious that the studies in hepatocarcinogenesis related to the theory of CSCs will provide new ideas on genesis, development, and metastasis of HCC and will bring new insights for the diagnosis and treatment of these tumors.

Wnt-/-β-catenin pathway signaling in human hepatocellular carcinoma.

The molecular basis of the carcinogenesis of hepatocellular carcinoma (HCC) has not been adequately clarified, which negatively impacts the development of targeted therapy protocols for this overwhelming neoplasia. The aberrant activation of signaling in the HCC is primarily due to the deregulated expression of the components of the Wnt-/-β-catenin. This leads to the activation of β-catenin/T-cell factor-dependent target genes that control cell proliferation, cell cycle, apoptosis, and cell motility. The deregulation of the Wnt pathway is an early event in hepatocarcinogenesis. An aggressive phenotype was associated with HCC, since this pathway is implicated in the proliferation, migration, and invasiveness of cancer cells, regarding the cell's own survival. The disruption of the signaling cascade Wnt-/-β-catenin has shown anticancer properties in HCC's clinical evaluations of therapeutic molecules targeted for blocking the Wnt signaling pathway for the treatment of HCC, and it represents a promising perspective. The key to bringing this strategy in to clinical practice is to identify new molecules that would be effective only in tumor cells with aberrant signaling β-catenin.

Investigation of diagnostic utility and expression profiles of stem cell markers (CD133 and CD90) in hepatocellular carcinoma, small cell dysplasia, and cirrhosis

The aim of this study was to investigate the expression rates of CD133 and CD90 in cirrhosis–dysplastic nodule–HCC (Crh–DN–HCC) sequence related to the etiologic background.Thirty-five HCC, 8 small cell dysplasia (SCD), and 63 cases of cirrhosis having different etiologies were collected. Immunohistochemical expressions of CD133 and CD90 were evaluated.CD133 positivity was higher in HCC cases with chronic hepatitis B and CD90 with chronic hepatitis C. The highest staining rate was seen in poorly differentiated HCC cases. Only one SCD case and almost half of the cirrhotic cases which were stained for CD133 were associated with hepatitis B; none was stained for CD90. Increased CD133 expression indicated a significantly shorter overall survival rate. No significant relationship was detected between the expression rates of CD133 or CD90 and other parameters.In this study, which investigates the immunohistochemical expression profiles of CD133 and CD90 through Crh–DN–HCC sequence, the highest staining rate was detected in HCC. It is rational to try to elucidate the earliest events in hepatocarcinogenesis by studying SCD. It is important to be aware of this issue in daily practice, which will provide a deeper insight into the understanding of the effects of CSCs in the progression and management of HCC.

Disease Progression from Chronic Hepatitis C to Cirrhosis and Hepatocellular Carcinoma is Associated with Increasing DNA Promoter Methylation

Changes in DNA methylation patterns are believed to be early events in hepatocarcinogenesis. A better understanding of methylation states and how they correlate with disease progression will aid in finding potential strategies for early detection of HCC. The aim of our study was to analyze the methylation frequency of tumor suppressor genes, P14, P15, and P73, and a mismatch repair gene (O6MGMT) in HCV related chronic liver disease and HCC to identify candidate epigenetic biomarkers for HCC prediction. 516 Egyptian patients with HCV-related liver disease were recruited from Kasr Alaini multidisciplinary HCC clinic from April 2010 to January 2012. Subjects were divided into 4 different clinically defined groups - HCC group (n=208), liver cirrhosis group (n=108), chronic hepatitis C group (n=100), and control group (n=100) - to analyze the methylation status of the target genes in patient plasma using EpiTect Methyl qPCR Array technology. Methylation was considered to be hypermethylated if >10% and/or intermediately methylated if >60%. In our series, a significant difference in the hypermethylation status of all studied genes was noted within the different stages of chronic liver disease and ultimately HCC. Hypermethylation of the P14 gene was detected in 100/208 (48.1%), 52/108 (48.1%), 16/100 (16%) and 8/100 (8%) among HCC, liver cirrhosis, chronic hepatitis and control groups, respectively, with a statistically significant difference between the studied groups (p-value 0.008). We also detected P15 hypermethylation in 92/208 (44.2%), 36/108 (33.3%), 20/100 (20%) and 4/100 (4%) , respectively (p-value 0.006). In addition, hypermethylation of P73 was detected in 136/208 (65.4%), 72/108 (66.7%), 32/100 (32%) and 4/100 (4%) (p-value <0.001). Also, we detected O6MGMT hypermethylation in 84/208 (40.4%), 60/108 (55.3%), 20/100 (20%) and 4/100 (4%), respectively (p value <0.001. The epigenetic changes observed in this study indicate that HCC tumors exhibit specific DNA methylation signatures with potential clinical applications in diagnosis and prognosis. In addition, methylation frequency could be used to monitor whether a patient with chronic hepatitis C is likely to progress to liver cirrhosis or even HCC. We can conclude that methylation processes are not just early events in hepatocarcinogenesis but accumulate with progression to cancer.