Independent Prognostic Factor For Hepatocellular Carcinoma Research Articles

Prognostic significance of miR-1268a expression and its beneficial effects for post-operative adjuvant transarterial chemoembolization in hepatocellular carcinoma.

Our recent investigation has shown that the variables of microRNA-1268a may involve in hepatocellular carcinoma (HCC) tumorigenesis. Here, we attempted to identify the prognostic significance of microRNA-1268a expression in tumor tissues by a retrospective analysis in 411 patients with HCC, and analyze its effects on post-operative adjuvant transarterial chemoembolization (TACE) improving HCC prognosis. All cases received tumor resection or tumor resection plus post-operative adjuvant TACE as an initial treatment. Logistical regression analysis exhibited that microRNA-1268a expression was significantly correlated with tumor stage, tumor grade, tumor size, and microvessel density. Cox regression analysis showed that microRNA-1268a expression was an independent prognostic factor for HCC, and TACE treatment had no effects on prognosis of HCC patients with high microRNA-1268a expression. More intriguingly, TACE improved the prognosis of HCC patients with low microRNA-1268a expression. Functionally, overexpression of microRNA-1268a inhibited while its inhibitor enhanced doxorubicin-induced the death of cancer cells. These results suggest that microRNA-1268a may be an independent prognostic factor for HCC patients, and that decreasing microRNA-1268a expression may be beneficial for post-operative adjuvant TACE treatment in HCC.

Zinc finger protein ZBTB20 is an independent prognostic marker and promotes tumor growth of human hepatocellular carcinoma by repressing FoxO1.

Zinc finger and BTB domain-containing 20 (ZBTB20) is a new BTB/POZ-domain gene and a member of the POK family of transcriptional repressors. Notably, the role of ZBTB20 and its underlying mechanisms involved in hepatocarcinogenesis are poorly investigated. In this study, the expression of ZBTB20 was significantly overexpressed in hepatocellular carcinoma (HCC) tissues. The positive expression of ZBTB20 was associated with large tumor size, high Edmondson-Steiner grading and advanced tumor-node-metastasis (TNM) tumor stage. Additionally, HCC patients with positive expression of ZBTB20 had a poorer 5-year survival. Multivariate analyses revealed that ZBTB20 overexpression was an independent prognostic factor for HCC. Gain- and loss-of-function experiments demonstrated that ZBTB20 promoted HCC cell viability, proliferation, tumorigenicity, and cell cycle progression. Mechanistically, Cyclin D1 and Cyclin E were increased, while p21 and p27 were decreased by ZBTB20 in HCC cells. FoxO1 was inversely correlated with ZBTB20 protein expression in the same cohort of HCC specimens. We further revealed that FoxO1 was transcriptionally repressed by ZBTB20 in HCC. Moreover, restoration of FoxO1 expression partially abrogated ZBTB20-induced HCC cell proliferation and growth entry in vitro and in vivo. Collectively, these results indicate that ZBTB20 may serve as a prognostic marker and promotes tumor growth of HCC via transcriptionally repressing FoxO1.

Oncogenic Wnt3a expression as an estimable prognostic marker for hepatocellular carcinoma

To investigate member 3a of Wingless-type MMTV integration site family (Wnt3a) expression in cancerous and surrounding tissues and the relationship between clinicopathologic features of hepatocellular carcinoma (HCC) and Wnt3a expression. Wnt3a expression and cellular distribution and clinicopathologic characteristics in cancerous tissue and matched surrounding tissues were analyzed in 80 HCC patients from January 2006 to August 2008 by tissue microarrays and immunohistochemistry. The overall and disease-free survival rates were estimated using the Kaplan-Meier method and compared with the log-rank test. The prognostic analysis was carried out with univariate and multivariate Cox regressions models. The incidence of oncogenic Wnt3a expression in the cancerous group was up to 96.25% (77 of 80), which was significantly higher (χ(2) = 48.818, P < 0.001) than that in the surrounding group (46.25%, 37 of 80). Brown Wnt3a staining gradually increased with clinical staging that showed very strong staining in advanced HCC. The clinicopathologic features of high Wnt3a expression in HCC were related to poorly-differentiated grade (χ(2) = 20.211, P < 0.001), liver cirrhosis (χ(2) = 8.467, P < 0.004), hepatitis B virus (HBV) infection (χ(2) = 12.957, P < 0.001), higher tumor-node-metastasis stage (χ(2) = 22.960, P < 0.001), and 5-year survival rate (χ(2) = 15.469, P < 0.001). Oncogenic Wnt3a expression associated with HBV infection and cirrhotic liver might be an independent prognostic factor for HCC.

Ack1 overexpression promotes metastasis and indicates poor prognosis of hepatocellular carcinoma.

Despite the substantial data supporting the oncogenic role of Ack1, the predictive value and biologic role of Ack1 in hepatocellular carcinoma (HCC) metastasis remains unknown. In this study, both correlations of Ack1 expression with prognosis of HCC, and the role of Ack1 in metastasis of HCC were investigated in vitro and in vivo. Our results showed that Ack1 was overexpressed in human HCC tissues and cell lines. High Ack1 expression was associated with HCC metastasis and determined as a significant and independent prognostic factor for HCC after liver resection. Ack1 promoted HCC invasion and metastasis in vitro and in vivo. Mechanistically, we confirmed that Ack1 enhanced invasion and metastasis of HCC via EMT by mediating AKT phosphorylation. In conclusion, our study shows Ack1 is a novel prognostic biomarker for HCC and promotes metastasis of HCC via EMT by activating AKT signaling.

Over-expression of TRIM37 promotes cell migration and metastasis in hepatocellular carcinoma by activating Wnt/β-catenin signaling

Hepatocellular carcinoma (HCC) is the most common cancer in the world especially in East Asia and Africa. Advanced stage, metastasis and frequent relapse are responsible for the poor prognosis of HCC. However, the precise mechanisms underlying HCC remained unclear. So it is urgent to identify the pathological processes and relevant molecules of HCC. TRIM37 is an E3 ligase and has been observed deregulated expression in various tumors. Recent studies of TRIM37 have implicated that TRIM37 played critical roles in cell proliferation and other processes. In the present study, we demonstrated that TRIM37 expression was notably up-regulated in HCC samples and was associated with advanced stage and tumor volume, which all indicating the poor outcomes. We also found that TRIM37 could serve as an independent prognostic factor of HCC. During the course of in vitro and in vivo work, we showed that TRIM37 promoted HCC cells migration and metastasis by inducing EMT. Furthermore, we revealed that the effect of TRIM37 mediated EMT in HCC cells was achieved by the activation of Wnt/β-catenin signaling. These finding may provide insight into the understanding of TRIM37 as a novel critical factor of HCC and a candidate target for HCC treatment.

Long non-coding RNA AOC4P suppresses hepatocellular carcinoma metastasis by enhancing vimentin degradation and inhibiting epithelial-mesenchymal transition.

Increasing evidence indicates that long non-coding RNAs (lncRNAs) regulate diverse cellular processes, including cell growth, differentiation, apoptosis, and cancer progression. However, the function of lncRNAs in the progression of hepatocellular carcinoma (HCC) remains largely unknown. We performed a comprehensive microarray analysis of lncRNA expression in human HCC samples. After validation in 108 HCC specimens, we identified a differentially expressed novel tumor suppressive lncRNA termed amine oxidase, copper containing 4, pseudogene (AOC4P). The level of AOC4P expression was significantly downregulated in 68% of HCC samples and negatively correlated with advanced clinical stage, capsule invasion and vessel invasion. Low AOC4P expression correlated with poor prognostic outcomes, serving as an independent prognostic factor for HCC. In vitro functional assays indicated that AOC4P overexpression significantly reduced cell proliferation, migration and invasion by inhibiting the epithelial-mesenchymal transition (EMT). RNA immunoprecipitation assays demonstrated that AOC4P binds to vimentin and promotes its degradation. Animal model experiments confirmed the ability of AOC4P to suppress tumor growth and metastasis. Taken together, our findings suggest that AOC4P lncRNA acts as an HCC tumor suppressor by enhancing vimentin degradation and suppressing the EMT. By clarifying the mechanisms underlying HCC progression, these findings promote the development of novel therapeutic strategies for HCC.

18F-FDG PET CT as a prognostic factor in hepatocellular carcinoma.

To elucidate the role of 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG-PET/CT) imaging as an independent prognostic factor in hepatocellular carcinoma (HCC). A total of 104 patients with newly diagnosed HCC who underwent 18F-FDG-PET/CT imaging from 2009 to 2014 were reviewed retrospectively. The ratio of the maximal tumor standardized uptake value (SUV) to the mean mediastinum SUV (TSUVmax/MSUVmean) was evaluated as the predictive factor. A high TSUVmax/MSUVmean ratio (≥3.1) was significantly associated with tumor burden indices, including α-fetoprotein (p<0.001), amino transaminase (AST) (p=0.007), tumor size (p=0.043), Tumor, Node, and Metastasis (TNM) stage (p<0.001), and Barcelona Clinic Liver Cancer (BCLC) staging (p<0.001). The mortality rate was higher (48.1% vs. 23.1%, p<0.001) in patients with a high TSUVmax/MSUVmean ratio (≥3.1). Among the 47 patients who underwent transarterial chemoembolization (TACE), patients with a high TSUVmax/MSUVmean ratio (≥3.1) were more likely to have recurrence following TACE (18/19 vs. 18/28, p=0.016). A high TSUVmax/MSUVmean ratio on 18F-FDG-PET/CT imaging can serve as an independent prognostic factor in HCC and may predict tumor recurrence after TACE.

Prognostic significance of Cbx4 expression and its beneficial effect for transarterial chemoembolization in hepatocellular carcinoma.

Our recent investigations showed that polycomb chromobox 4 (Cbx4) promotes angiogenesis and metastasis of hepatocellular carcinoma (HCC) through its sumoylating action on hypoxia-inducible factor-1α protein. Here, we attempt to identify the prognostic significances of Cbx4 by a retrospective analyses in 727 cases of HCC patients with and without postoperative transarterial chemoembolization (TACE) or transarterial embolization (TAE). Binary logistic regression tests indicated that Cbx4 is correlated with histological grading, tumor-node-metastasis stage, microvessel density, distant metastasis and hematogenous metastasis of HCC. By univariate and multivariate analyses, we show that Cbx4 is an independent prognostic factor of HCC, and both TAE and TACE treatments have no effects on the overall survival in HCC patients with low Cbx4 expression. More intriguingly, TACE prolongs, while TAE shortens, the overall survival of HCC patients with high Cbx4 expression, indicating that Cbx4 is a good biomarker on decision-making to perform postoperative TACE in HCC patients. Moreover, Cbx4 overexpression enhances while Cbx4 silencing antagonizes doxorubicin-induced cell death of HCC cell lines. In conclusion, Cbx4 is an independent prognostic factor for HCC patients, and the patients with high Cbx4 expression should receive postoperative TACE treatment to improve their survival.

Bile duct invasion can be an independent prognostic factor in early stage hepatocellular carcinoma.

Backgrounds/AimsIn hepatocellular carcinoma (HCC), bile duct invasion occurs far more rarely than vascular invasion and is not well characterized. In addition, the pathologic finding of bile duct invasion is not considered an independent prognostic factor for HCC following surgery. In this study, we determined the characteristics of HCC with bile duct invasion, and assessed the clinical significance of bile duct invasion.MethodsWe retrospectively reviewed the medical records of 363 patients who underwent hepatic resection for HCC at Seoul National University Hospital (SNUH) from January 2009 to December 2011. Preoperative, operative, and pathological data were collected. The risk factors for recurrence and survival were analyzed. Subsequently, the patients were divided into 2 groups according to disease stage (American Joint Committee on Cancer/International Union Against Cancer 7th edition): early stage (T1 and 2) and advanced stage (T3 and 4) group; and risk factors in the sub-groups were analyzed.ResultsAmong 363 patients, 13 showed bile duct invasion on pathology. Patients with bile duct invasion had higher preoperative total bilirubin levels, greater microvascular invasion, and a higher death rate than those without bile duct invasion. In multivariate analysis, bile duct invasion was not an independent prognostic factor for survival for the entire cohort, but, was an independent prognostic factor for early stage.ConclusionsBile duct invasion accompanied microvascular invasion in most cases, and could be used as an independent prognostic factor for survival especially in early stage HCC (T1 and T2).

Circulating B7-H4 in serum predicts prognosis in patients with hepatocellular carcinoma.

B7-H4 is member of the B7 family that negatively regulates the immune response, which are important for fine-tuning of the tumor microenvironment. Dysregulation of B7-H4 expression has been associated with tumor progression. However, expression level of B7-H4 in hepatocellular carcinoma (HCC) tissues is still a controversial topic. In addition, whether serum B7-H4 expression of HCC patients has any clinical value is unknown. We compared serum levels of B7-H4 in patients with HCC and healthy controls by using the ELISA method. Association between serum B7-H4 expression level and clinical parameters of HCC was further investigated. Log-rank test and Kaplan-Meier method were employed to evaluate the overall survival rate of HCC patients. Univariate and multivariate analysis of prognostic factors were performed with the Cox regression model. Our results showed that HCC patients had significantly higher serum B7-H4 level as compared with healthy controls (P < 0.001). In addition, serum B7-H4 expression was correlated with HCC clinical parameters including serum AFP expression and TNM stage. HCC patients in the higher serum B7-H4 expression group had a poorer 5-year overall survival rate (P = 0.028). Moreover, serum B7-H4 expression was shown to be an independent prognostic factor for HCC (P = 0.034). The findings from this study suggest that serum B7-H4 is an independent prognostic indicator for HCC and may be a promising biomarker for early diagnosis as well as disease prognosis of HCC.

Loss of CD155 expression predicts poor prognosis in hepatocellular carcinoma

CD155 is an important ligand in triggering tumour rejection by immune cells. However, the expression of CD155 and its clinical significance in hepatocellular carcinoma (HCC) remains unknown. We examined the expression level of CD155 in 174 HCC tissue samples by immunohistochemical staining and in HCC cell lines by flow cytometry; 63.8% (111 of 174) of HCC tissue samples showed negative CD155 expression. When compared with adjacent peritumour tissues, HCC tissues exhibited a significantly lower expression of CD155 (P<0.001). Flow cytometry analysis indicated that HCC cell lines had low levels of CD155 expression. Moreover, negative CD155 expression was associated significantly with higher serum α-fetoprotein level (P=0.016) and a higher incidence of portal vein tumour thrombus (P=0.050). Importantly, patients with positive CD155 expression had better overall survival after surgery than those with negative CD155 expression (P=0.005). Furthermore, Cox regression analyses showed that CD155 expression was an independent prognostic factor for HCC (P=0.049). Our findings suggest that loss of CD155 expression may play an important role in the immune escape of HCC cells and thus CD155 may serve as a prognostic marker as well as a potential therapeutic target for HCC.

Prognostic value of RAS association domain family 1A gene methylation in patients with hepatocellular carcinoma after surgery

Objective To evaluate the prognostic value of RAS association domain family 1A gene (RASSF1A) methylation in patients after hepatocellular carcinoma (HCC) hepatectomy.Methods A total of 260 patients with HCC who underwent hepatectomy were enrolled.HCC tissues and tumor adjacent tissues which were 2 cm away from the tumor edge of the patients were obtained.The clinicopathological data of patients were collected.The methylation of RASSF1A in HCC tissues and corresponding tumor adjacent tissues was determined by methylation specific polymerase chain reaction (PCR).The correlation between the expression rate of RASSF1A methylation and clinicopathological characteristics was analyzed by chi-square test.Log-rank test was performed to analyze the relation between RASSF1A methylation and overall survival rate.Univariate and multivariate Cox statistical techniques were used to identify the influence factors in the prognosis of HCC.Results Among 260 HCC tissues and corresponding tumor adjacent tissues,RASSF1A promoter hypermethylation was detected in 214 HCC tissues (82.3 ％) and 101 corresponding tumor adjacent tissues (38.8％),and the difference was statistically significant (x2 =102.824,P ＜ 0.01).There was no correlation between RASSF1A methylation and age,gender,liver cirrhosis,α-fetoprotein level,maximum diameter of tumor,Barcelona Clinic Liver Cancer (BCLC) stage,hepatitis B virus (HBV) infection,smoking and alcohol drinking (all P＞0.05).The 5-year overall survival rate of patients with negative RASSF1A methylation was 93％,while that of patients with positive RASSF1A methylation was 51 ％,and the difference in overall survival rate between the two groups was statistically significant (x2 =26.556,P ＜ 0.01).Univariate and multivariate Cox regression analysis indicated that liver cirrhosis,BCLC stage and RASSF1A methylation were the main influence factors in the death of patients with HCC after surgery (Wald=16.767,8.791,16.286; all P＜0.01).Conclusion RASSF1A methylation is not only one of the predictive factors of survival rate in patients with HCC after hepatectomy,but also an independent prognostic factor of HCC. Key words: Carcinoma hepatocellular; RASSF1A gene; Methylation; Prognosis

Up-regulation of miR-9 expression predicate advanced clinicopathological features and poor prognosis in patients with hepatocellular carcinoma.

BackgroundMicroRNAs (miRNAs) are endogenous small (19–24 nt long) noncoding RNAs that regulate gene expression in a sequence specific manner. An increasing association between miRNA and cancer has been recently reported. Hepatocellular carcinoma (HCC), as the fifth most common cancer and the most common cause of death in men, has become the third leading cause of cancer-related deaths globally. In this study, we investigated the miR-9 expression in HCC to evaluate their value in prognosis of this tumor.MethodsThe expression of miR-9 in matched normal and tumor tissues of HCC was evaluated using a quantitative real-time RT-PCR. A Kaplan–Meier survival curve was generated following a log-rank test.ResultsIt was observed that miR-9 expression was upregulated in HCC tissues compared with noncancerous liver tissues (7.26 ± 1.30 vs. 3.14 ± 1.08, P < 0.001). The up-regulation of miR-9 in HCC cancer tissues was also significantly correlated with aggressive clinicopathological features. We found that the patients with high miR-9 expression have a higher tumor staging (P = 0.0389) and are in higher risk of venous infiltration (P < 0.0001). Moreover, the results of Kaplan–Meier analyses showed that HCC patients with the high miR-9 expression tend to have shorter overall survival (P < 0.0001). The multivariate analysis clearly indicated that the high miR-9 expression in biopsy samples may be considered as an independent prognostic factor in HCC for decreased survival (4.28; 95%CI, 2.77-7.23, P < 0.001).ConclusionOur data indicate the potential of miR-9 as a novel prognostic biomarker for HCC. Large well-designed studies with diverse populations and functional evaluations are warranted to confirm and extend our findings.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_228

B‑cell translocation gene 1 serves as a novel prognostic indicator of hepatocellular carcinoma.

Although the B‑cell translocation gene 1 (BTG1) plays an important role in apoptosis and negatively regulates cell proliferation, BTG1 expression in hepatocellular carcinoma (HCC) has not been evaluated. In this study expression analysis of BTG1 was conducted to clarify the role of BTG1 in the initiation of HCC carcinogenesis and progression. BTG1 mRNA expression levels were determined for HCC cell lines and 151 surgical specimen pairs using quantitative real‑time reverse transcription polymerase chain reaction (RT‑qPCR) assay. The mutational and methylation status of HCC cell lines were analyzed via high resolution melting (HRM) analysis and direct sequencing analysis to elucidate the regulatory mechanisms of BTG1 expression. The expression and distribution of the BTG1 protein in liver tissues were evaluated using immunohistochemistry (IHC). Decreased expression of BTG1 mRNA was confirmed in the majority of HCC cell lines (89%) and clinical HCC tissues (85%) compared with non‑cancerous liver tissues. Mutations or promoter hypermethylation were not identified in HCC cell lines. BTG1 mRNA expression levels were not influenced by background liver status. The pattern of BTG1 protein expression was consistent with that of BTG1 mRNA. Downregulation of BTG1 mRNA in HCC was significantly associated with shorter disease‑specific and recurrence‑free survival rates. Multivariate analysis of disease‑specific survival rates identified BTG1 mRNA downregulation as an independent prognostic factor for HCC (hazard ratio 2.12, 95% confidence interval 1.12‑4.04, P=0.022). Our results indicate that altered BTG1 expression might affect hepatocarcinogenesis and may represent a novel biomarker for HCC carcinogenesis and progression.

Abstract 3882: Downregulation of PRRX1 confers cancer stem cell-like properties and poor prognosis in hepatocellular carcinoma

Abstract Background: Recent studies have been focused on the relationship between cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT). Unlike classical EMT inducers, PRRX1 is involved in the acquisition of CSC-like properties when its down-regulation induces mesenchymal-epithelial transition in breast cancer. Meanwhile, hepatocellular carcinoma (HCC) is one of the most intractable malignancies, and it is required to identify a bona-fide molecular target to regulate tumor progression and the treatment resistance. The purpose of this study is to investigate the role of PRRX1 expression on clinical significance and CSC-like properties in HCC. Methods: (1) PRRX1 expression was analyzed in 62 resected primary HCC cases by quantitative RT-PCR (qRT-PCR). Clinicopathological factors and overall survival (OS) according to PRRX1 expression were analyzed. (2) The gene set enrichment analysis (GSEA) on the published clinical data set of 242 HCC samples (GSE14520) was applied to investigate the association of PRRX1 expression levels and the stem cell-like properties. (3) PRRX1 stably expressing HCC cell lines (HuH7) were established using a lentiviral vector. The expression levels of the hepatic CSC surface markers (CD13, CD133 and EpCAM) were analyzed by flowcytometry and qRT-PCR. Furthermore, the ability of sphere formation and in vitro chemosensitivity to 5-FU were evaluated to assess the CSC-like properties. Results: (1) Univariate analysis showed that 5-year OS of the low PRRX1 expression group was significantly shorter than that of the high PRRX1 expression group (49.8% vs. 80.3%, P=0.014). On multivariate analysis, low PRRX1 expression was an independent prognostic factor for HCC (P=0.026). (2) GSEA analysis on the 247 HCC data set indicated that the down-regulation of PRRX1 was significantly correlated with the stem cell signature (P=0.039). Furthermore, using gene sets of polycomb repressive complex 2 (PCR2) targets and H3 lysine-27 trimethylation mediated by PCR2, HCC with low PRRX1 expression showed parallel enrichment patterns with human embryonic stem cells (P=0.002 and P&amp;lt;0.001, respectively). These results suggested that down-regulation of PRRX1 in HCC induces the acquisition of CSC-like properties through modification of chromatin structure. (3) The level of CSC markers (CD13, CD133 and EpCAM) and the number of spheres were significantly decreased in PRRX1-expressing HuH7 cells compared with the controls. Moreover, PRRX1 impaired resistance to 5-FU in HCC cells. Conclusions: The down-regulation of PRRX1 expression contributes to the poor prognosis through the acquisition of CSC-like properties in HCC patients. We might apply the aberrant expression of PRRX1 as a novel clinical biomarker for prediction of the malignant potential in HCC cases. Citation Format: Hidenari Hirata, Keishi Sugimachi, Ryutaro Uchi, Junji Kurashige, Tae Matsumura, Yuki Takano, Hiroki Ueo, Masami Ueda, Shotaro Sakimura, Yoshiaki Shinden, Hidetoshi Eguchi, Tomoya Sudo, Masakazu Hirakawa, Hiroshi Honda, Koshi Mimori. Downregulation of PRRX1 confers cancer stem cell-like properties and poor prognosis in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3882. doi:10.1158/1538-7445.AM2014-3882

735P - B-Cell Translocation Gene 1 Serves As a Novel Prognostic Indicator of Hepatocellular Carcinoma

ABSTRACT Aim: Although B-cell translocation gene 1 (BTG1) plays an important role in apoptosis and negatively regulates cell proliferation, BTG1 expression in hepatocellular carcinoma (HCC) has not been evaluated. The aim of this study was to clarify the role of BTG1 in the initiation of HCC carcinogenesis and progression. Methods: BTG1 mRNA expression levels were determined for HCC cell lines and 151 surgical specimen pairs using a quantitative real-time reverse transcription polymerase chain reaction assay. The mutational and methylation statuses of HCC cell lines were analyzed via high-resolution melting analysis and direct sequencing analysis to explore the regulatory mechanisms of BTG1 expression. The expression and distribution of the BTG1 protein in liver tissues were evaluated using immunohistochemistry. Results: Decreased expression of BTG1 mRNA was confirmed in the majority of HCC cell lines (89%) and clinical HCC tissues (85%) compared with non-cancerous liver tissues. Mutations or promoter hypermethylation of BTG1 were not identified in HCC cell lines. BTG1 mRNA expression levels were not influenced by background liver status. The pattern of BTG1 protein expression was consistent with that of BTG1 mRNA. Downregulation of BTG1 mRNA in HCC was significantly associated with shorter disease-specific and recurrence-free survival rates. Multivariate analysis of disease-specific survival rates identified BTG1 mRNA downregulation as an independent prognostic factor for HCC (hazard ratio 2.12, 95% confidence interval 1.12 – 4.04, P = 0.022). Conclusions: Our results indicate that altered BTG1 expression might affect hepatocarcinogenesis and may represent a novel biomarker for HCC carcinogenesis and progression. Disclosure: All authors have declared no conflicts of interest.

Association between ephrin-A1 mRNA expression and poor prognosis after hepatectomy to treat hepatocellular carcinoma.

Hypoxia regulates the expression of genes that promote tumor growth, angiogenesis and invasion. We previously studied hypoxic tumor cells in vitro and from hepatic metastases of colorectal cancer and determined several potential prognostic factors for hepatocellular carcinoma (HCC). In this study, we evaluated the prognostic impact of the expression of ephrin-A1 (EFNA1) and its receptor, EPHA2, in patients with HCC after curative resection. Samples from a total of 139 HCC patients were analyzed by either microarray alone (n=86) or by microarray and quantitative PCR (n=53). There was no correlation between EFNA1 expression and clinicopathological factors. EPHA2 expression was not significantly correlated with any clinicopathological factors, except for microscopic portal invasion. EFNA1 was an independent prognostic factor for HCC (p=0.0277). These findings suggest that EFNA1 expression may be a useful marker for predicting high risk of recurrence in patients who have undergone curative resection for HCC.

MiR-372 down-regulates the oncogene ATAD2 to influence hepatocellular carcinoma proliferation and metastasis

BackgroundATAD2 is associated with many cellular processes, such as cell growth, migration and invasion. However, no studies have been conducted on the molecular biological function of the ATAD2 gene in hepatocellular carcinoma (HCC).MethodsThe protein and mRNA level expression of ATAD2 was examined in tissues and cell lines. Prognostic significance was analyzed by the Kaplan-Meier survival method and Cox regression. ATAD2 knockdown was used to analyze cell proliferation and invasion. The upstream and downstream of ATAD2 was analyzed by RT2 Profiler™ PCR array and luciferasex fluorescence system.ResultsATAD2 was highly expressed in liver cancer samples and correlated with poor survival. High ATAD2 expression was positively correlated with metastasis (P = 0.005) and was an independent prognostic factor in HCC (P = 0.001). ATAD2 depletion by RNA interference reduced their capacity for invasion and proliferation and led to a G1 phase arrest in vitro. Further study revealed that miR-372 was an upstream target of ATAD2 as miR-372 was bound directly to its 3′ untranslated region (3′ UTR). In addition, ATAD2 knockdown was found to extremely up-regulate APC expression and down-regulate CTNNA1 at the mRNA level.ConclusionsThe findings demonstrated that miR-372 suppressed the expression of ATAD2, which was highly expressed in HCC and exerted a proto-oncogene effect in hepatic carcinogenesis. In conclusion, ATAD2 may promote HCC progression.

MiR-141 Suppresses the Migration and Invasion of HCC Cells by Targeting Tiam1

BackgroundWe have demonstrated that T lymphoma invasion and metastasis 1 (Tiam1) gene is associated with the poor prognosis of patients with hepatocellular carcinoma (HCC), and we used a computational approach to identify miR-141 as a Tiam1-targeting microRNA (miRNA). Here, we explored the function of miR-141 and the relationship between miR-141 and Tiam1 gene in HCC.MethodsThe miR-141 expression in HCC tissues and cell lines was detected and its roles in regulation of HCC cell proliferation, migration and invasion and target gene expression was investigated. Tiam1 was identified as a novel target of miR-141. Ethics statement: our study was approved by the Nanfang Hospital Medical Ethics Committee Ethics statement. Written informed consent was obtained before collection.ResultsBased on in situ hybridization (ISH) analysis, miR-141 was down-regulated in the same HCC samples. Kaplan-Meier analysis demonstrated that patients with low miR-141 expression had poorer overall survival rate than that of the patients with high miR-141 expression. Furthermore, multivariate Cox regression analysis indicated that miR-141 could serve as an independent prognostic factor in HCC. MiR-141 significantly inhibited in vitro cell proliferation, migration and invasion as proved by gain- and loss- of function studies, while the mRNA and protein levels of Tiam1 were reduced in cells over-expressing miR-141. Moreover, Tiam1 treatment antagonized this effect, while knockdown of Tiam1 by Tiam1 short hairpin RNA (shTiam1) induced inhibitory effects.ConclusionsThese findings indicated that miR-141 functions as a tumor suppressor and inhibits the migration and invasion of HCC cells by targeting Tiam1, which may provide novel prognostic and treatment strategies for HCC patients.

Aberrant expression of monocarboxylate transporter 4 in tumour cells predicts an unfavourable outcome in patients with hepatocellular carcinoma

The tumour cell microenvironment, which includes local oxygen saturation, pericellular pH and stromal cells, can modulate tumour progression. This study determined the prognostic impact of infiltrating tumour-associated macrophages and the expression of monocarboxylate transporter 4 (MCT4) and glypican 3 (GPC3) in hepatocellular carcinoma (HCC) clinical specimens. A total of 225 cases of resected HCC were subjected to immunohistochemical analyses of CD68, CD204, MCT4 and GPC3. Immunoreactivities and other common clinicopathological parameters were subjected to univariate prognostic analyses for overall survival (OS, n=225) and disease-free survival (DFS, n=222). All variables with prognostic impact were further analysed in multivariate analysis. Increased intratumoural infiltration of CD204-positive or MCT4-positive macrophages suggested shorter OS (P=0.015 or P=0.001 respectively), but DFS was not altered. The GPC3 score (with an emphasis on circumferential immunoreactivity) was correlated with shorter OS and DFS. Aberrant expression of MCT4 in HCC cells was observed in a subset of HCC cases (21%, 47/225). In those cases, significantly poorer OS (P<0.0001) and DFS (P=0.0003) were observed, and there was a positive correlation with the intratumoural infiltration of CD204- or MCT4-positive macrophages and the GPC3 score. Multivariate analysis showed that aberrant MCT4 expression in HCC cells was an independent prognostic factor for shorter OS (P=0.018) and DFS (P=0.006) after resection of HCC. Aberrant expression of MCT4 in carcinoma cells serves as a novel, independent prognostic factor for HCC, indicating a poorer patient outcome.