Adjacent Non-tumor Liver Tissues Research Articles

Identification of a new methylation site in the Sept9 promoter region for the diagnosis of hepatocellular carcinoma

Background. Over 600,000 people die from hepatocellular carcinoma (HCC) each year worldwide. The disease is often detected at advanced stages and in many cases is not curable. Early diagnostic and monitoring of HCC recurrences remains a substantial problem in clinical oncology. That determines the need for a search for highly sensitive and specific biomarkers for the non-invasive of HCC diagnostics. The objective of the study. Identification of the hypermethylated locus in the promoter region of the septin 9 (Sept9) gene based on the annotated methylomes from the public databases. Experimental validation of methylation on a pilot panel of paired clinical samples of patients with HCC, as well as tissue samples from patients with benign liver tumors and lymphocytes from healthy donors. Materials and methods. To analyze the methyl data, samples of HCC from TCGA, hepatocellular adenoma from GEO (Gene Expression Omnibus) depository, peripheral blood cells and tissues of healthy donors from Methbank were used. Experimental validation of methylation levels of the identified site was carried out on a pilot panel of clinical samples by bisulphite pyrosequencing using PyroMark Q24.Results. Based on the analysis of methylome data, we selected cg20275528 site, which is characterized by high level of methylation in HCC tissues and minimal levels of methylation in non-tumor liver tissue, hepatocellular adenoma and peripheral blood of healthy donors. Experimental testing on a pilot panel of clinical specimens showed that the level of marker site methylation in HCC (42 % median) is significantly higher than in non-tumor liver tissues (3 % median) and benign neoplasms (1.5 % median) and exceeds the threshold value in HCC compared to paired samples of adjacent non-tumor liver tissue in 20 out of 30 studied cases (66.6 %). The general possibility for cg20275528 methylation detection in circulating DNA of plasma in HCC patients was shown.Conclusion. The obtained results indicate that the approach to the detection and experimental verification of diagnostically significant markers developed and tested in this study can be used to identify new differentially methylated sites and to establish new approaches for non-invasive HCC diagnosis.

PD1Hi CD8+ T cells correlate with exhausted signature and poor clinical outcome in hepatocellular carcinoma

BackgroundCD8+ T cells differentiate into exhausted status within tumors, including hepatocellular carcinoma (HCC), which constitutes a solid barrier to effective anti-tumor immunity. A detailed characterization of exhausted T cells and their prognostic value in HCC is lacking.MethodsWe collected fresh tumor tissues with adjacent non-tumor liver tissues and blood specimens of 56 HCC patients, as well as archived samples from two independent cohorts of HCC patients (n = 358 and n = 254), who underwent surgical resection. Flow cytometry and multiplex immunostaining were used to characterize CD8+ T cells. Patient prognosis was evaluated by Kaplan-Meier analysis and Cox regression analysis.ResultsCD8+ T cells were classified into three distinct subpopulations: PD1Hi, PD1Int and PD1−. PD1Hi CD8+ T cells were significantly enriched in tumor compared to adjacent non-tumor liver tissues. PD1Hi CD8+ T cells highly expressed exhaustion-related inhibitory receptors (TIM3, CTLA-4, etc.) and transcription factors (Eomes, BATF, etc.). In addition, PD1Hi CD8+ T cells expressed low levels of cytotoxic molecules and displayed a compromised capacity to produce pro-inflammatory cytokines while the expression of anti-inflammatory IL-10 was up-regulated following mitotic stimulation. Furthermore, PD1Hi CD8+ T cells shared features with tissue resident memory T cells and were also characterized in an aberrantly activated status with an apoptosis-prone potential. In two independent cohorts of HCC patients (n = 358 and n = 254), we demonstrated that PD1Hi or TIM3+PD1Hi CD8+ T cells were significantly correlated with poor prognosis, and the latter was positioned in close proximity to PD-L1+ tumor associated macrophages.ConclusionThe current study unveils the unique features of PD1Hi CD8+ exhausted T cells in HCC, and also suggests that exhausted T cells could act as a biomarker to select the most care-demanding patients for tailored therapies.

LncRNA SAMD12-AS1 promotes cell proliferation and inhibits apoptosis by interacting with NPM1

Chronic hepatitis B virus infection is a major risk factor for hepatocellular carcinoma. HBV infection affects lncRNA expression in infected cells, but the detailed mechanism and biological significance are not yet clear. In this study, we focused on exploring the function of the HBV-upregulated lncRNA SAMD12-AS1 in cell proliferation. We found that there is a higher level of SAMD12-AS1 expression in tumors than in adjacent nontumorous liver tissues. We showed that ectopic expression of SAMD12-AS1 promotes cell growth and blocks apoptosis, while knockdown of SAMD12-AS1 inhibits cell proliferation and enhances etoposide-induced apoptosis. Using RNA immunoprecipitation and mass spectrometry, we determined that SAMD12-AS1 interacts with NPM1 and confirmed that SAMD12-AS1(1-350) is required for the interaction with NPM1. As it is known that NPM1 interacts with the E3 ligase HDM2 and reduces HDM2-mediated p53 degradation, we examined whether SAMD12-AS1 can affect p53 stability. Overexpression of SAMD12-AS1 caused a reduction in p53 protein levels by shortening its half-life. Conversely, knockdown of SAMD12-AS1 prolonged the half-life of p53. We further demonstrated that SAMD12-AS1 increased the interaction of HDM2 and p53 and enhanced p53 ubiquitination. Our findings reveal that HBV-upregulated SAMD12-AS1 regulates cell proliferation and apoptosis via the NPM1-HDM2-p53 axis.

SOCS5 inhibition induces autophagy to impair metastasis in hepatocellular carcinoma cells via the PI3K/Akt/mTOR pathway

SOCS5 is a member of the suppressor of cytokine signaling (SOCS) protein family with important yet incompletely understood biological functions in cancer. In hepatocellular carcinoma (HCC), controversial tumor-promoting and tumor-suppressive roles of SOCS5 have been reported. Our study aims to unravel novel functions of SOCS5 in HCC, especially that affecting metastasis. We examined the expression levels of SOCS5 in HCC using publicly available datasets, and in our patient cohort, using quantitative real-time PCR, western blotting, and immunohistochemistry. The association of SOCS5 expression with clinical pathological data of HCC patients was examined and that with the mTOR pathway was predicted. We further studied the effects of SOCS5 on PI3K/Akt/mTOR activity; HCC cell autophagy, migration, and invasion; and HCC cell metastasis in vitro and in vivo. We observed that SOCS5 was significantly overexpressed in HCC tissues, compared to adjacent non-tumor liver tissues, in both the public datasets and in our patient cohort. SOCS5 overexpression was significantly and inversely correlated with HCC patient prognosis. Moreover, SOCS5 overexpression promoted HCC cell migration and invasion in vitro by inactivating PI3K/Akt/mTOR-mediated autophagy. Conversely, SOCS5 inhibition suppressed HCC cell migration and invasion in vitro by activating PI3K/Akt/mTOR-mediated autophagy. Dual inhibition of SOCS5 and mTOR further enhanced autophagy and the subsequent anti-metastatic effects on HCC cells. In vivo, stable knockdown of SOCS5 reduced HCC cell metastasis. Overall, our study revealed a novel metastasis-promoting function of SOCS5 in HCC, acting via the PI3K/Akt/mTOR-mediated autophagy pathway. Combined inhibition of SOCS5 and mTOR may be a potential therapeutic approach to inhibit HCC metastasis and prolong patient survival.

Abstract 1799: MicroRNA hsa-miR-26a expression in hepatocellular carcinoma correlates with patient survival

Abstract Background: MicroRNAs (miRNAs) are small noncoding RNAs that act as important regulators of gene expression at a post-transcriptional level. Using a next-generation sequencer system, in a previous study, we have identified 49 miRNAs expressed differentially by comparing 26 pair samples of hepatocellular carcinoma (HCC) tumor tissue and nontumor liver tissue. For this validation study, we selected hsa-miR-26a, one of the candidate miRNAs. We measured hsa-miR-26a expression in the validation set HCC samples using real-time quantitative polymerase chain reaction (qPCR) and examined the correlation between miRNA expression and clinical data. Patients and Methods: Overall, 40 patients with HCC who underwent tumor resection from 1991 to 1997 at the Department of Surgery, Institute of Gastroenterology, Tokyo Women’s Medical University, Japan were enrolled. Formalin-fixed paraffin-embedded samples were prepared using the standard protocol. The paraffin blocks were cut into 10-μm sections; HCC tumor and adjacent nontumor liver tissues were collected using laser-captured microdissection, and total RNA, including miRNA fractions, were isolated. Following the synthesis of a complementary DNA (cDNA) using an miRNA-specific primer, real-time qPCR was performed using an miRNA-specific primer/probe. Results: The hsa-miR-26a expression tended to be downregulated in HCC tumor tissue compared to nontumor liver tissue (P = 0.0799). Patients in the higher hsa-miR-26a expression group showed a significantly longer overall survival time compared with those in the lower hsa-miR-26a expression group (P = 0.0409). Conclusion: Inhibition of hsa-miR-26a expression might play a role in tumorigenesis, and hsa-miR-26a expression could be a potential prognostic factor for patients with HCC. Citation Format: Go Nakajima, Kazuhiko Hayashi. MicroRNA hsa-miR-26a expression in hepatocellular carcinoma correlates with patient survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1799.

SIRT7 regulates hepatocellular carcinoma response to therapy by altering the p53-dependent cell death pathway

BackgroundOptimal therapeutic strategies for hepatocellular carcinoma (HCC) patients are still challenging due to the high recurrence rate after surgical resection and chemotherapy resistance. Growing evidence shows that genetic and epigenetic alterations are involved in HCC progression and resistance to therapy, however the molecular mechanisms underlying resistance to therapy have not been fully understood.MethodsExpression of SIRT7 in 17 paired paraffin-embedded HCC tissues and adjacent nontumoral liver tissues was examined by immunohistochemistry and Western blot. The mRNA expression of SIRT7 in 20 paired frozen HCC tissues and adjacent nontumoral liver tissues was analyzed by quantitative RT-PCR. The biologic consequences of overexpression and knockdown of SIRT7 in HCC therapy sensitivity were studied in vitro and in vivo. Interaction between SIRT7 and p53 were studied in HCC cell lines.ResultsSIRT7 expression was frequently upregulated in clinical HCC samples, and its expression was highly associated with TACE-resistance and poor survival (P = 0.008.) Depletion of SIRT7 from multiple liver cancer cell lines significantly increased doxorubicin toxicity while overexpression of SIRT7 largely abolished doxorubicin induced apoptosis. At the molecular level, we observed that SIRT7 interacts with and induces deacetylation of p53 at lysines 320 and 373. Deacetylated p53 showed significantly less affinity for the NOXA promoter and its transcription. In mouse xenografts, SIRT7 suppression increased doxorubicin induced p53 activation, inhibited tumor growth and induced apoptosis.ConclusionThe newly identified SIRT7-p53-NOXA axis partially illustrates the molecular mechanism of HCC resistance to therapy and represents a novel potential therapeutic target for HCC treatment.

Expression of ZNF207 in hepatocellular carcinoma and its significance

To investigate the expression of zinc finger protein 207 (ZNF207) in hepatocellular carcinoma (HCC) tissues, and analyze the correlation of ZNF207 with clinicopathological factors and HCC patients' survival. Methods: Real-time PCR was used to detect ZNF207 mRNA expression in 10 paired fresh HCC and adjacent non-tumor liver tissue samples. Immunohistochemical (IHC) analysis was used to detect ZNF207 protein expression in 135 cases of randomly selected paraffin-embedded HCC tissues. The correlation of ZNF207 expression with clinicopathological factors and survival of HCC was analyzed. Results: The ZNF207 mRNA expression level in HCC was significantly higher than that in the adjacent non-tumor liver tissue (P<0.01). IHC results showed that ZNF207 protein level was elevated in HCC tissues and ZNF207 expression was correlated with cirrhosis, nodule number, tumor capsule, vascular invasion, and TNM stage (P<0.05). Survival analysis showed that patients with high ZNF207 expression had poorer overall survival (OS) and disease-free survival (DFS) than those with the low ZNF207 expression (P<0.01), and ZNF207 was an independent risk factor for OS and DFS of HCC (P<0.05). Conclusion: ZNF207 expression is elevated in HCC and associated with adverse clinicopathological factors, indicating poor prognosis for HCC.

Hepatic CYP1A2 activity in liver tumors and the implications for preoperative volume-function analysis.

Dynamic liver function assessment by the [13C]methacetin maximal liver function capacity (LiMAx) test reflects the overall hepatic cytochrome P-450 (CYP) 1A2 activity. One proven strategy for preoperative risk assessment in liver surgery includes the combined assessment of the dynamic liver function by the LiMAx test, the volumetric analysis of the liver, and calculation of future liver remnant function. This so-called volume-function analysis assumes that the remaining CYP1A2 activity in any tumor lesion is zero. The here presented study aims to assess the remaining CYP1A2 activities in different hepatic tumor lesions and its consequences for the preoperative volume-function analysis in patients undergoing liver surgery. The CYP1A2 activity analysis of neoplastic lesions and adjacent nontumor liver tissue from resected tumor specimens revealed a significantly higher CYP1A2 activity (median, interquartile range) in nontumor tissues (35.5, 15.9-54.4 µU/mg) compared with hepatocellular adenomas (7.35, 1.2-32.5 µU/mg), hepatocellular carcinomas (0.18, 0.0-2.0 µU/mg), or colorectal liver metastasis (0.17, 0.0-2.1 µU/mg). In nontumor liver tissue, a gradual decline in CYP1A2 activity with exacerbating fibrosis was observed. The CYP1A2 activity differences were also reflected in CYP1A2 protein signals in the assessed hepatic tissues. Volume-function analysis showed a minimal deviation compared with the current standard calculation for hepatocellular carcinomas or colorectal liver metastasis (<1% difference), whereas a difference of 11.9% was observed for hepatocellular adenomas. These findings are important for a refined preoperative volume-function analysis and improved surgical risk assessment in hepatocellular adenoma cases with low LiMAx values. NEW & NOTEWORTHY The cytochrome P-450 (CYP) 1A2-dependent maximal liver function capacity test reflects the overall functional capacity of the liver. To which extent hepatocellular tumors harbor CYP1A2 activity and thus contribute to the maximal liver function capacity test outcome is unknown. We here show that hepatocellular adenomas but not hepatocellular carcinomas or colorectal liver metastasis contain significant residual CYP1A2 activity. These findings are important for an improved preoperative volume-function analysis and an accurate surgical risk assessment in hepatocellular adenoma cases.

LncRNA MIR22HG inhibits growth, migration and invasion through regulating the miR-10a-5p/NCOR2 axis in hepatocellular carcinoma cells.

Despite the rapidly identified numbers of lncRNA in humans, exploration of the molecular mechanisms of lncRNA is lagging, because the molecular mechanisms of lncRNA can be various and complex in different conditions. In this study, we found a new molecular mechanism for a versatile molecule, MIR22HG. MIR22HG is an lncRNA that contributes to the initiation and progression of many human cancers, including hepatocellular carcinoma (HCC). We report that MIR22HG was downregulated in 120 HCC samples compared with adjacent nontumor liver tissues. More interestingly, decreased expression of MIR22HG in HCC could predict poor prognosis of HCC patients. Knockdown of MIR22HG promoted the growth, migration and invasion of HCC cells. In exploring the molecular mechanism of MIR22HG, we found that MIR22HG functioned as a tumor suppressor in hepatocellular carcinomas, in part through serving as a competing endogenous RNA to modulate the miRNA‐10a‐5p level. Moreover, NCOR2 was verified to act as the downstream target gene of MIR22HG/miR‐10a‐5p. In addition, the MIR22HG/miRNA‐10a‐5p/NCOR2 axis inhibited the activation of the Wnt/β‐catenin pathway. Together, our results demonstrated that MIR22HG inhibited HCC progression in part through the miR‐10a‐5p/NCOR2 signaling axis and might act as a new prognostic biomarker for HCC patients.

CD44 standard isoform is involved in maintenance of cancer stem cells of a hepatocellular carcinoma cell line.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide. Cancer stem cells (CSCs) have attracted attention as a novel therapeutic target for cancer because they play important roles in the development and aggravation of cancer. CD44 is expressed as a standard isoform (CD44s) and several variant isoforms. CD44v is a major isoform expressed on CSCs of a variety of tumors and has been extensively studied. However, HCC tissues dominantly express CD44s, whose function in CSCs remains unclear. In the present study, we investigated the roles of CD44s in CSCs of HCC. Knock‐out of the CD44 gene in HuH7 HCC cells on which only CD44s is expressed resulted in decreased spheroid formation and increased drug sensitivity. The expression of CSC marker genes, including CD133 and EpCAM, was significantly downregulated in the spheroids of CD44‐deficient cells compared with those in the spheroids of HuH7 cells. In addition, CD44 deficiency impaired antioxidant capacity, concomitant with downregulation of glutathione peroxidase 1 (GPX1) and thioredoxin. Because GPX1 uses the reduced form of glutathione (GSH) to regenerate oxidized cellular components, GSH levels were significantly increased in the CD44‐deficient cells. We also found that NOTCH3 and its target genes were downregulated in the spheroids of CD44‐deficient cells. NOTCH3 expression in HCC tissues was significantly increased compared with that in adjacent nontumor liver tissues and was correlated with CD44 expression. These results suggest that CD44s is involved in maintenance of CSCs in a HCC cell line, possibly through the NOTCH3 signaling pathway.

RhoC/ ROCK2 promotes vasculogenic mimicry formation primarily through ERK/MMPs in hepatocellular carcinoma

Introduction: Our previous study reported that rho-associated coiled-coil containing kinases (ROCKs) were involved in vasculogenic mimicry (VM) formation in hepatocellular carcinoma (HCC). This study focused on clarifying potential mechanisms of RhoC/ROCK on VM formation in hepatocellular carcinoma (HCC). Method: In total, 80 cases of HCC tissues and adjacent non-tumorous liver tissues were used to identify VM in HCC tissues by PAS/CD34 double staining. Kaplan-Meier analysis was conducted to test the predictive relationship between RhoC/ROCK expression and patient prognosis. Furthermore, we stably overexpressed RhoC and inhibited RhoC, ROCK1 or ROCK2 expression to clarify the effect of RhoC/ROCK on VM formation in vitro and in vivo. Result: RhoC expression was up regulated in HCC tissues, especially the VM-positive (VM+) group, compared to non-cancerous tissues (P < 0.01), and patients with high expression of RhoC had shorter survival times (P < 0.001). Cell motility and VM formation significantly decreased after knockdown of RhoC, while overexpressing RhoC led to the opposite result. Compared to ROCK1 shRNA, ROCK2 shRNA could largely affects VM formation, cell motility and the key VM factors and the epithelial-mesenchymal transition (EMT) markers in vitro and in vivo. Moreover, p-ERK, p-MEK, p-FAK and p-paxillin levels clearly altered following the change of RhoC, but ROCK2 little affected expression of p-FAK. Conclusion: RhoC/ROCK2 may have a major effect on VM in HCC via ERK/MMPs signaling and might be a potential therapeutic target for the treatment of HCC.

Identification of long non-coding RNA-related and \u2013coexpressed mRNA biomarkers for hepatocellular carcinoma

BackgroundWhile changes in mRNA expression during tumorigenesis have been used widely as molecular biomarkers for the diagnosis of a number of cancers, the approach has limitations. For example, traditional methods do not consider the regulatory and positional relationship between mRNA and lncRNA. The latter has been largely shown to possess tumor suppressive or oncogenic properties. The combined analysis of mRNA and lncRNA is likely to facilitate the identification of biomarkers with higher confidence.ResultsTherefore, we have developed an lncRNA-related method to identify traditional mRNA biomarkers. First we identified mRNAs that are differentially expressed in Hepatocellular Carcinoma (HCC) by comparing cancer and matched adjacent non-tumorous liver tissues. Then, we performed mRNA-lncRNA relationship and coexpression analysis and obtained 41 lncRNA-related and -coexpressed mRNA biomarkers. Next, we performed network analysis, gene ontology analysis and pathway analysis to unravel the functional roles and molecular mechanisms of these lncRNA-related and -coexpressed mRNA biomarkers. Finally, we validated the prediction and performance of the 41 lncRNA-related and -coexpressed mRNA biomarkers using Support Vector Machine model with five-fold cross-validation in an independent HCC dataset from RNA-seq.ConclusionsOur results suggested that mRNAs expression profiles coexpressed with positionally related lncRNAs can provide important insights into early diagnosis and specific targeted gene therapy of HCC.

Long non-coding RNA LINC00472 suppresses hepatocellular carcinoma cell proliferation, migration and invasion through miR-93-5p/PDCD4 pathway

Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer and the second leading cause of cancer-related deaths. In the present study, we have demonstrated that long non-coding RNA (lncRNA) LINC00472 was low expressed in human HCC tissues and cell lines compared with adjacent non-tumor liver tissues and normal liver cell lines respectively. LINC00472 was also low expressed in HCC tissues from patients with metastasis compared with tissues from patients without metastasis. Expression level of LINC00472 was positively correlated with patient overall survival (OS) rate. Forced expression of LINC00472 suppressed cell proliferation, migration, invasion and promoted cell apoptosis in HCC cells Huh-7 and SMMC-7721. MiR-93-5p was a direct target of LINC00472, and miR-93-5p directly targeted PDCD4. The miR-93-5p/PDCD4 pathway mediated the suppressing role of LINC00472 in HCC cells. Therefore, LINC00472 was an important tumor suppressor in human HCC, which could be used as a bio-marker for HCC therapy.

Unique true predicted neoantigens (TPNAs) correlates with anti-tumor immune control in HCC patients

BackgroundA novel prediction algorithm is needed for the identification of effective tumor associated mutated neoantigens. Only those with no homology to self wild type antigens are true predicted neoantigens (TPNAs) and can elicit an antitumor T cell response, not attenuated by central tolerance. To this aim, the mutational landscape was evaluated in HCV-associated hepatocellular carcinoma.MethodsLiver tumor biopsies and adjacent non-tumor liver tissues were obtained from 9 HCV-chronically infected subjects and subjected to RNA-Seq analysis. Mutant peptides were derived from single nucleotide variations and TPNAs were predicted using two prediction servers (e.g. NetTepi and NetMHCstabpan) by comparison with corresponding wild-type sequences, non-related self and pathogen-related antigens. Immunological confirmation was obtained in preclinical as well as clinical setting.ResultsThe development of such an improved algorithm resulted in a handful of TPNAs despite the large number of predicted neoantigens. Furthermore, TPNAs may share homology to pathogen’s antigens and be targeted by a pre-existing T cell immunity. Cross-reactivity between such antigens was confirmed in an experimental pre-clinical setting. Finally, TPNAs homologous to pathogen’s antigens were found in the only HCC long-term survival patient, suggesting a correlation between the pre-existing T cell immunity specific for these TPNAs and the favourable clinical outcome.ConclusionsThe new algorithm allowed the identification of the very few TPNAs in cancer cells, and those targeted by a pre-existing immunity strongly correlated with long-term survival. Only such TPNAs represent the optimal candidates for immunotherapy strategies.

Clinical significance of microRNA-21 expression in disease progression of patients with hepatocellular carcinoma.

Aberrant microRNA (miR) expression is associated with hepatocellular carcinoma (HCC). Here, we investigated the clinical significance of miR-21 and miR-31 for HCC-specific prognostic effect. HCC patients (n=93) who underwent liver biopsy or surgical resection were enrolled, and HCC tissues and paired adjacent nontumor liver tissues were collected and analyzed for miRs expression. MiR-21 expression was significantly upregulated in HCC tissues relative to nontumor tissues. Both miR-21 and miR-31 expression in HCC tissues did not predict overall survival; however, miR-21 was considered an independent predictor of disease progression together with portal vein thrombosis and higher Barcelona Clinical Liver Cancer stage. Elevated miR-21 expression might represent a biomarker for HCC prognosis.

MicroRNA‐766 promotes cancer progression by targeting NR3C2 in hepatocellular carcinoma

MicroRNAs (miRNAs) have been reported to play important roles in tumor progression of various cancers. However, the clinical significance and biologic function of miR-766 in hepatocellular carcinoma (HCC) remain unknown. In this study, we investigated the roles of miR-766 in HCC progression using HCC cell lines and a xenograft mouse model. miR-766 expression in tumor tissues and adjacent nontumorous liver tissues of patients with HCC was evaluated by quantitative RT-PCR. Our results showed that miR-766 promoted proliferation and metastasis of HCC cells in vitro and in vivo and that NR3C2 was a direct target of miR-766 and involved in miR-766-mediated proliferation and metastasis of HCC cells. We also found that miR-766 affected the β-catenin signaling pathway by targeting NR3C2. Furthermore, miR-766 was significantly up-regulated in HCC tissues and was correlated with the prognosis of patients with liver cancer. Taken together, our results show that miR-766 affects HCC progression by modulating NR3C2 expression and is a possible new therapeutic target for patients with HCC.-Yang, C., Ma, X., Guan, G., Liu, H., Yang, Y., Niu, Q., Wu, Z., Jiang, Y., Bian, C., Zang, Y., Zhuang, L. MicroRNA-766 promotes cancer progression by targeting NR3C2 in hepatocellular carcinoma.

Lower expression of tumor microRNA-26a is associated with higher recurrence in patients with hepatocellular carcinoma undergoing surgical treatment.

Hepatocellular carcinoma (HCC) in patients with hepatitis B virus (HBV) exhibit lower tumor microRNA-26a (miR-26a) expression which is associated with worse outcomes. It is unknown if similar miR-26a loss occurs inHCC developed in other liver diseases. We examined tumor miR-26a expression and its impact on recurrence and mortality in a North American HCC cohort. MiR-26a levels from tumor and surrounding nontumor liver tissue in 186 subjects were collected. We defined lower tumor expression of miR-26a as <1-fold that of the adjacent nontumor liver tissue. Viral hepatitis (42%; 40% hepatitis C and 2% HBV), alcohol (19%), and nonalcoholic fatty liver disease (NAFLD) (18%) were the most common causes of liver disease. The prevalence of lower tumor miR-26a expression was 68%, and it was evident in HCCs arising in all etiologies (viral hepatitis 60%, alcohol 61%, and NAFLD 76%). Subjects with lower tumor miR-26a expression had significantly higher tumor recurrence (hazard ratio [HR], 2.45; 95% confidence interval[CI], 1.18 to 5.1; P = 0.016) and higher mortality of borderline significance (HR, 1.51; 95% CI, 0.94 to 2.41; P = 0.086). Reduced miR-26a expression is a common phenomenon in HCC arising in North American patients with different underlying liver diseases and may increase recurrence and mortality after surgery.

Abstract 5414: The correlation between the expression of microRNA hsa-miR-101 and the prognosis of the patient with hepatocellular carcinoma

Abstract Aim: MicroRNAs (miRNA) are small RNAs that regulate gene expression at the post-transcriptional level, and they play a various role in cancer cell such as proliferation, migration and metastasis. Our previous study identified forty-nine miRNAs that are differentially expressed by comparing 26 pair samples of hepatocellular carcinoma (HCC) tumor and adjacent liver tissue using next-generation sequencer system. One of them, hsa-miR-101 is chosen in this validation study. The hsa-miR-101 expression in HCC tumor samples and its adjacent liver tissues were measured by real-time PCR, and were examined the correlation between the miRNA expression and clinical data. Patients and Methods: Forty-three patients diagnosed as HCC had undergone surgical removal from 1991 to 1997 at the Department of Surgery, Institute of Gastroenterology, Tokyo Women's Medical University, Japan. The formalin-fixed paraffin embedded samples were prepared using the standard protocol. The paraffin blocks were cut into 10 micrometer sections, and the tumor and its adjacent non-tumor liver tissue were collected using laser-captured microdissection and total RNA including miRNA fraction were isolated. After synthesized cDNA using miRNA specific primer, real-time PCR was performed using miRNA specific primer/probe. Results: The expression of hsa-miR-101 in tumor tissue was downregulated compared to adjacent liver tissue (P=0.003). No significant differences were obtained in survival time between higher or lower intratumoral expression level (P=0.8039). The patients were divided into two groups, higher/lower hsa-miR-101 expression in the tumor or in the adjacent liver tissue, the group of the higher expression in adjacent liver tissue and the lower in tumor was longer survival time compared to the other groups (P=0.0184). Conclusion: The hsa-miR-101 expression may be related to tumorigenesis, and both the expression in the tumor and in the adjacent liver tissue may be potential prognostic factor for HCC patients. Citation Format: Go Nakajima, Kazuhiko Hayashi. The correlation between the expression of microRNA hsa-miR-101 and the prognosis of the patient with hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5414.

Decreased expression of PTH1R is a poor prognosis in hepatocellular carcinoma.

Hypercalcemia is a potentially fatal and not rare complication of hepatocellular carcinoma (HCC), and its underlying mechanism remains unclear. Parathyroid hormone (PTH) is the most important regulator of the concentrations of calcium and phosphate in blood; parathyroid hormone-related protein (PTHrP) was the most frequent cause of humoral hypercalcemia of malignancy; parathyroid hormone 1 receptor (PTH1R) is the common receptor for PTH and PTHrP. The aim of this study is to investigate the expression of PTH, PTHrP, and PTH1R in HCC tissues, and their relationship with clinical pathological characters in HCC. First, a meta-analysis based on online Oncomine Expression Array database was conducted to compare the different mRNA expression of PTH1R, PTH and PTHrP between hepatocellular carcinoma and normal tissues. Then, the protein expression level of differentially expressed gene was examined by immunohistochemistry staining in 223 HCC tissues and 102 non-tumorous liver tissues controls. The relationship between the protein expression and clinicopathological parameters was analyzed by χ2 test, and overall survival analysis was performed using Kaplan-Meier survival analysis. PTH1R mRNA expression was significantly lower in HCC tissues compared with normal tissues, while the expression of PTH and PTHrP showed no significant difference between HCC tissues and normal tissues. High PTH1R protein expression was found in 90/102 cases of adjacent non-tumorous liver tissues, and in 91 of 223 cases of HCC tissues. PTH1R expression was significantly related to tumor size, Edmondson Grade, AFP, and overall survival. PTH1R may be the major cause of hypercalcemia in HCC, and the decreased PTH1R expression was a poor prognosis in HCC.

Whole-exome mutational and transcriptional landscapes of combined hepatocellular cholangiocarcinoma and intrahepatic cholangiocarcinoma reveal molecular diversity

BackgroundPrimary liver cancer (PLC) is the third largest contributor to cancer mortality in the world. PLC is a heterogeneous disease that encompasses several biologically distinct subtypes including hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular-cholangiocarcinoma (CHC). CHC is a distinct, albeit rare, subtype of PLC and is comprised of cells with histopathological features of both HCC and ICC. Several studies have focused on the mutation and expression landscapes of HCC and ICC. However, studies of CHC were rare. ObjectiveThe aim of the current study was to identify genetic and gene expression alterations in the carcinogenesis and development of CHC and ICC in the Chinese population. Unraveling both similar and differing patterns among these subtypes may help to identify personalized medicine approaches that could improve patient survival. MethodsWhole genome sequencing (WGS), whole exome sequencing (WES) and RNA-seq were performed on 10 ICC and 10 CHC samples, matched with adjacent non-tumor liver tissue specimens. Comparative analysis was performed using HCC datasets from The Cancer Genome Atlas (TCGA). ResultsMutational and transcriptional landscapes of CHC and ICC were clearly delineated. TP53 and CTNNB1 were identified as exhibiting mutations in CHC. ARID1A, PBRM1, and IDH1 were frequently mutated in ICC. RYR3, FBN2, and KCNN3 are associated with cell migration and metastasis and might be driver genes in CHC. KCNN3 was identified as also exhibiting mutations in ICC. The ECM-receptor interaction pathway associated fibrogenic hepatic progenitor cell differentiation and liver fibrosis may play an important role in carcinogenesis of PLC. Chromatin remodeling and chromosome organization are key processes in carcinogenesis and development in PLC. P53 related pathways showed alterations in CHC and HCC. Inflammation may be a key factor involved in ICC carcinogenesis. ConclusionCHC and ICC are different subtypes of PLC. This study discusses predominantly the molecular genetic details of PLC subtypes and highlights the need for an accurate diagnosis and treatment of specific PLC subtypes to optimize patient management.