Zheng-Gang Ren

Activation of beta-Catenin by Hypoxia in Hepatocellular Carcinoma Contributes to Enhanced Metastatic Potential and Poor Prognosis

Purpose: Aberrant activation of β-catenin contributes to the malignant phenotype in hepatocellular carcinoma (HCC). Hypoxia is also known to promote HCC invasion and metastasis. However, the association between β-catenin and the proinvasive role of hypoxia remains unclear. We investigated the role of β-catenin in the proinvasive consequences of hypoxia in HCC. Experimental Design: We established in vitro and in vivo hypoxic models to investigate the expression of β-catenin in hypoxic HCC cells and its role in hypoxia-induced aggressiveness. The clinical significance of β-catenin and/or hypoxia-induced factor-1α (HIF-1α) was evaluated using HCC tissue microarrays. Results: Hypoxia induced β-catenin overexpression and/or intracellular accumulation in four HCC cell lines through downregulating the endogenous degradation machinery, and promoted in vitro invasion and in vivo metastasis of MHCC97 and Hep3B cells. Besides morphologic changes, hypoxic MHCC97 and Hep3B cells exhibited molecular alterations consistent with epithelial-mesenchymal transition, characterized by the loss of epithelial cell markers (E-cadherin and plakoglobin) and upregulation of mesenchymal markers (vimentin and N-cadherin), as well as the increase of matrix metalloproteinase 2. However, silencing β-catenin in these hypoxic cells reversed epithelial-mesenchymal transition and repressed metastatic potential. Positive expression of β-catenin in HCC tissue microarray was associated with the expression of HIF-1α (P = 0.034), and coexpression of β-catenin and HIF-1α in HCC was correlated with shorter overall survival and time to recurrence. Conclusion: β-Catenin in HCC is activated by hypoxia and contributes to hypoxia-induced metastatic potential. Clin Cancer Res; 16(10); 2740–50. ©2010 AACR.

Transarterial chemoembolization for hepatocellular carcinoma with portal vein tumor thrombus: a meta-analysis

BackgroundAlthough transarterial chemoembolization (TACE) has been used extensively for advanced hepatocellular carcinoma (HCC) with portal vein tumor thrombus (PVTT), no consensus has been reached and an evidence base for practice is lacking. This meta-analysis evaluated the efficacy and safety of TACE for treatment of HCC with PVTT.MethodsOvid Medline, EMBASE, Web of Knowledge, and Cochrane library databases were searched up to August 2012 for controlled trials assessing TACE in patients with PVTT. Data concerning the study design, characteristics of trials, and outcomes were extracted. Hazard ratio (HR) and 95% confidence interval (CI) were calculated using random effects models.ResultsEight controlled trials involving 1601 HCC patients were included. TACE significantly improved the 6-month (HR, 0.41; 95% CI: 0.32–0.53; z, 6.28; p = 0.000) and 1-year (HR, 0.44; 95% CI: 0.34–0.57; z, 6.22; p = 0.000) overall survival of patients with PVTT compared with conservative treatment. Subgroup analyses showed that TACE was significantly effective in HCC patients whether with main portal vein (MPV) obstruction or with segmental PVTT. Fatal complications were rare, even in patients with MPV obstruction. Temporary liver decompensation and postembolization syndrome occurred frequently. However, they could be treated successfully with conservative treatment.ConclusionsTACE, as a safe treatment, has potential for incurring a survival benefit for advanced HCC with PVTT, even with MPV obstruction. Further large randomized controlled trials may be needed to confirm this result.

Vascular endothelial cells facilitated HCC invasion and metastasis through the Akt and NF-κB pathways induced by paracrine cytokines

BackgroundIt is well documented that cancer cells secrete angiogenic factors to recruit and sustain tumor vascular networks. However, little is known about the effects of endothelial cells on the behavior of tumor cells. The study here was to determine the roles of endothelial cells in HCC cell growth, migration and invasion.MethodsA mixture of highly metastatic MHCC97H cells and HUVEC cells, as well as MHCC97H cells alone were subcutaneously injected into nude mice to observe the effects of HUVECs on HCC growth. The biological characteristics of MHCC97H cells respectively treated with conditioned medium (CM) derived from HUVECs and endothelial cell basal medium (EBM) in vitro, such as proliferation, migration and invasion, invasion/metastasis associated gene expression, were comparatively analyzed. Differential cytokines between CM and EBM were screened and identified using human cytokine array. Effects of the interested differential cytokine CCL2, IL-8 and CXCL16 and its related signaling pathways were further investigated in HCC cells.ResultsSubcutaneous tumorigenicity of MHCC97H cells in nude mice was promoted by HUVECs and its invasion/metastasis associated genes were significantly upregulated. The in vitro, proliferation, migration and invasion of HCC cells treated with CM were all significantly enhanced as compared to those with EBM stimulation. Simultaneously, PI3K/Akt and ERK1/2 pathway in HCC cells were activated by CM. Total of 25 differential cytokines were identified between CM and EBM such as angiopoietin-2, CCL2 (MCP-1), uPA, endostatin, CXCL16, IL-8, pentraxin 3 etc. The selected differential cytokines CCL2, IL-8 and CXCL16 all modulated the expressions of HCC invasion/metastasis genes, especially MMP2 and MMP9. In exposure to CCL2 or CXCL16 alone, upregulation in AKT phosphorylation but no change in ERK phosphorylation were found in MHCC97H cells, moreover the contents of nuclear transcription factor NF-κB were increased as compared to the control. However, no effects on the activation of Akt and ERK pathway in MHCC97H were found in exposure to IL-8.ConclusionThis study expands the contribution of endothelial cells to the progression of HCC. It unveils a new paradigm in which endothelial cells function as initiators of molecular crosstalks that enhance survival, migration and invasion of HCC cells.

Coexpression of gene Oct4 and Nanog initiates stem cell characteristics in hepatocellular carcinoma and promotes epithelial-mesenchymal transition through activation of Stat3/Snail signaling

BackgroundOct4 and Nanog are key regulatory genes that maintain the pluripotency and self-renewal properties of embryonic stem cells. We previously reported that the two stemness markers were tightly associated with cancer progression and poor outcomes of hepatocellular carcinoma. In this study, we demonstrate that coexpression of Oct4/Nanog modulates activation of signal transducer and activator of transcription 3 (Stat3), an oncogenic transcription factor that is activated in many human malignancies including hepatocellular carcinoma (HCC), as well as the expression of Snail, a key regulator implicated in epithelial-mesenchymal transition and tumor metastasis.MethodsOct4 and Nanog were ectopic expressed in MHCC97-L cell lines via lentiviral gene transfection. The stemness characteristics including self-renewal, proliferation, chemoresistance, and tumorigenicity were assessed. The effect of coexpression of Oct4 and Nanog on epithelial-mesenchymal transition change, and the underlying molecular signaling was investigated.ResultsEctopic coexpression of Oct4 and Nanog empowered MHCC97-L cells with cancer stem cell (CSC) properties, including self-renewal, extensive proliferation, drug resistance, and high tumorigenic capacity. Significantly, Oct4 and Nanog encouraged epithelial-mesenchymal transition change contributing to tumor migration, invasion/metastasis in vitro and in vivo. Following molecular mechanism investigation indicated Oct4/Nanog-regulated epithelial-mesenchymal transition change through Stat3-dependent Snail activation. Moreover, silencing Stat3 abrogates Oct4/Nanog-mediated epithelial-mesenchymal transition (EMT) change and invasion/metastasis in HCC.ConclusionsWe delineate Oct4 and Nanog initiate stem cell characteristics in hepatocellular carcinoma and promote epithelial-mesenchymal transition through activation of Stat3/Snail signaling. Our findings propose Stat3/Snail pathway as a novel therapeutic target for the treatment of progression and metastasis of HCC with CSC-like signatures and epithelial-mesenchymal transition phenotype.

Evaluation of midkine as a diagnostic serum biomarker in hepatocellular carcinoma.

Purpose: To evaluate the value of serum midkine (MDK) as a diagnostic biomarker in hepatocellular carcinoma, particularly for those with negative alpha-fetoprotein (AFP) and at an early stage. Experimental Design: MDK expression in tumors was assessed by immunohistochemistry from 105 patients with hepatocellular carcinomas or liver cirrhosis. Serum MDK levels were detected by ELISA in 933 participants including hepatocellular carcinomas and hospital controls from different medical centers. Sensitivities and specificities of serum MDK in diagnosing hepatocellular carcinoma according to AFP level and Barcelona Clinic Liver Cancer (BCLC) stage were analyzed. Results: MDK levels were significantly elevated in hepatocellular carcinoma tissues as well as serum samples. The sensitivity of serum MDK for hepatocellular carcinoma diagnosis was much higher than that of AFP (86.9% vs. 51.9%) with similar specificities (83.9% vs. 86.3%). Notably, serum MDK had an outstanding performance in distinguishing AFP-negative hepatocellular carcinomas from different controls: In those AFP-negative hepatocellular carcinomas, the sensitivity could reach as high as 89.2%. Moreover, receiver operating characteristic (ROC) curve analysis also showed that serum MDK had a better performance compared with AFP in distinguishing early-stage hepatocellular carcinomas as well as small hepatocellular carcinomas. Even in very early-stage hepatocellular carcinomas, MDK showed an obviously higher sensitivity compared with AFP (80% vs. 40%). Furthermore, serum MDK level was significantly decreased in patients with hepatocellular carcinomas after curative resection and re-elevated when tumor relapse occurred. Conclusions: Serum MDK is significantly elevated in most hepatocellular carcinomas, including those with negative AFP and at an early stage, which may serve as a novel diagnostic marker in early diagnosis and postoperative monitoring of hepatocellular carcinomas. Clin Cancer Res; 19(14); 3944–54. ©2013 AACR.

Prognostic significance of the neutrophil-to-lymphocyte ratio in primary liver cancer: a meta-analysis.

The neutrophil-to-lymphocyte ratio (NLR) is a useful biomarker that reflects systemic inflammation responses. However, the prognostic value of the NLR in patients with primary liver cancer (PLC) remains controversial. We performed a meta-analysis of 26 studies (comprising 4,461 patients) to evaluate the association between the pre-treatment NLR and clinical outcomes of overall survival (OS) and disease-free survival (DFS) in patients with PLC. The correlation between NLR and tumor characteristics or other inflammation-related parameters was also assessed. Data were synthesized using the random-effects model of DerSimonian and Laird, and the hazard ratio (HR) or odds ratio (OR) with 95% confidence interval (CI) was used to estimate effect size. Our analysis indicated that a high NLR predicted poor OS (HR, 2.102; 95% CI: 1.741–2.538) and DFS (HR, 2.474; 95% CI: 1.855–3.300) for PLC. A high NLR was associated with the presence of tumor vascular invasion (OR: 1.889, 95% CI: 1.487–2.400; p<0.001) and an elevated alpha-fetoprotein level (OR: 1.536; 95% CI: 1.152–2.048; p = 0.003). Thus, we conclude that a high NLR indicates a poor prognosis for patients with PLC and may also be predictive for PLC invasion and metastasis. Subgroup analysis suggested that the predictive role of NLR in cholangiocarcinoma is limited, and a further large study to confirm these findings is warranted.

PROX1 promotes hepatocellular carcinoma metastasis by way of up-regulating hypoxia-inducible factor 1α expression and protein stability.

Hepatocellular carcinoma (HCC) is one of the most common cancers and the third leading cause of death from cancer worldwide. HCC has a very poor prognosis because of tumor invasiveness, frequent intrahepatic spread, and extrahepatic metastasis. The molecular mechanism of HCC invasiveness and metastasis is poorly understood. The homeobox protein PROX1 is required for hepatocyte migration during mouse embryonic liver development. In this study, we show that high PROX1 protein expression in primary HCC tissues is associated with significantly worse survival and early tumor recurrence in postoperative HCC patients. Knockdown of PROX1 expression in HCC cells inhibited cell migration and invasiveness in vitro and HCC metastasis in nude mice while overexpression of PROX1 in HCC cells promoted these processes. PROX1s pro‐metastasis activity is most likely attributed to its up‐regulation of hypoxia‐inducible factor 1α (HIF‐1α) transcription and stabilization of HIF‐1α protein by recruiting histone deacetylase 1 (HDAC1) to prevent the acetylation of HIF‐1α, which subsequently induces an epithelial‐mesenchymal transition response in HCC cells. We further demonstrated the prognostic value of using the combination of PROX1 and HDAC1 levels to predict postoperative survival and early recurrence of HCC. Conclusion: PROX1 is a critical factor that promotes HCC metastasis. (Hepatology 2013;58:692‐705)

Prognostic significance of serum gamma-glutamyl transferase in patients with intermediate hepatocellular carcinoma treated with transcatheter arterial chemoembolization.

Objectives Not every unresectable hepatocellular carcinoma (HCC) could receive survival benefits from transcatheter arterial chemoembolization (TACE), even for intermediate HCC (Barcelona Clinic Liver Cancer stage B). The aim of this study was to investigate prognostic significance of serum &ggr;-glutamyl transferase (GGT) in patients with intermediate HCC treated with TACE. Methods A total of 277 patients with intermediate HCC were consecutively treated with TACE and overall survival (OS) was evaluated with the Kaplan–Meier method. Significant difference was estimated with the Log rank method according to GGT value before treatment. Univariate and multivariate analyses were used for the study of significance of prognostic factor. Results The median follow-up period was 18.7 months. The 1-year and 3-year OS rates were 71.6 and 38.5% in patients with normal GGT and 48.8 and 16.9% in patients with high GGT (P=0.002). High GGT, correlating with higher tumor size, &agr;-fetoprotein (AFP), and alanine aminotrasferase, was an independent prognostic factor for OS (P=0.009). Others included tumor size and ascites. Furthermore, in small HCC and normal AFP subgroup, serum GGT was also correlated with OS (P=0.013 and 0.041, respectively). The combination of GGT and AFP had a better power to predict the TACE effects. Conclusion GGT level was an important prognostic factor to predict prognosis of patients with intermediate HCC treated with TACE.

Residual hepatocellular carcinoma after oxaliplatin treatment has increased metastatic potential in a nude mouse model and is attenuated by Songyou Yin

BackgroundThe opposite effects of chemotherapy, which enhance the malignancy of treated cancers such as hepatocellular carcinoma (HCC), are not well understood. We investigated this phenomenon and corresponding mechanisms to develop a novel approach for improving chemotherapy efficacy in HCC.MethodsHuman hepatocellular carcinoma cell lines HepG2 (with low metastatic potential) and MHCC97L (with moderate metastatic potential) were used for the in vitro study. An orthotopic nude mouse model of human HCC was developed using MHCC97L cells. We then assessed the metastatic potential of surviving tumor cells after in vitro and in vivo oxaliplatin treatment. The molecular changes in surviving tumor cells were evaluated by western blot, immunofluorescence, and immunohistochemistry. The Chinese herbal extract Songyou Yin (composed of five herbs) was investigated in vivo to explore its effect on the metastatic potential of oxaliplatin-treated cancer cells.ResultsMHCC97L and HepG2 cells surviving oxaliplatin treatment showed enhanced migration and invasion in vitro. Residual HCC after in vivo oxaliplatin treatment demonstrated significantly increased metastasis to the lung (10/12 vs. 3/12) when re-inoculated into the livers of new recipient nude mice. Molecular changes consistent with epithelial-mesenchymal transition (EMT) were observed in oxaliplatin-treated tumor tissues and verified by in vitro experiments. The Chinese herbal extract Songyou Yin (4.2 and 8.4 g/kg) attenuated EMT and inhibited the enhanced metastatic potential of residual HCC in nude mice (6/15 vs. 13/15 and 3/15 vs. 13/15, respectively).ConclusionsThe surviving HCC after oxaliplatin treatment underwent EMT and demonstrated increased metastatic potential. Attenuation of EMT by Songyou Yin may improve the efficacy of chemotherapy in HCC.

Risk factors for recurrence of small hepatocellular carcinoma after long-term follow-up of percutaneous radiofrequency ablation

Radiofrequency ablation (RFA) as a local therapy for liver cancer is widely used. The study is to evaluate the therapeutic efficacy of RFA on hepatocellular carcinoma (HCC) and identify the risk factors for recurrence. Clinical records of 124 patients with 135 small HCC with percutaneous RFA as a first-line treatment modality were evaluated in Liver Cancer Institute, Zhongshan Hospital from October 2001 to December 2006. With a median follow-up period of 46 months after RFA therapy, the 1-, 2-, 3-, 4-, and 5-year cumulative survival rates and disease-free survival rates were 91, 70, 61, 48 and 40% and 64, 44, 31, 24 and 24%, respectively. The total recurrence and metastasis rates were 50 and 6.5%, respectively. Independent risk factors for recurrence after RFA included tumor with diameter more than 3 cm, located near the intrahepatic blood vessels, subcapsular locations and PT prolonged more than 3s. Severe complications occurred in 2 cases (1.6%), including biliary tract hemorrhage and subphrenic effusion. RFA appears to be a safe and effective treatment for HCC. It will benefit the efficacy of RFA therapy if those risk factors are considered during the clinical practice.