Shun-Li Shen

A novel and accurate predictor of survival for patients with hepatocellular carcinoma after surgical resection: the neutrophil to lymphocyte ratio (NLR) combined with the aspartate aminotransferase/platelet count ratio index (APRI)

BackgroundThe occurrence and development of hepatocellular carcinoma (HCC) depends largely on such non-tumor factors as inflammatory condition, immune state, viral infection and liver fibrosis. Various inflammation-based prognostic scores have been associated with survival in patients with HCC, such as the neutrophil/lymphocyte ratio (NLR), the platelet/lymphocyte ratio (PLR) and the prognostic nutritional index (PNI). The aspartate aminotransferase/platelet count ratio index (APRI) is thought to be a biomarker of liver fibrosis and cirrhosis. This study aims to evaluate the ability of these indices to predict survival in HCC patients after curative hepatectomy, and probe the increased prognostic accuracy of APRI combined with established inflammation-based prognostic scores.MethodsData were collected retrospectively from 321 patients who underwent curative resection for HCC. Preoperative NLR, PLR, PNI, APRI and clinico-pathological variables were analyzed. Univariate and multivariate analyses were performed to identify the predictive value of the above factors for disease-free survival (DFS) and overall survival (OS).ResultsUnivariate analysis showed that NLR, PLR, PNI and APRI were significantly associated with DFS and OS in HCC patients with curative resection. Multivariate analysis showed that NLR and APRI were superior to PLR and PNI, and both were independently correlated with DFS and OS. Preoperative NLR >2 or APRI >1.68 predicted poor prognosis of patients with HCC after hepatectomy. Furthermore, the predictive range of NLR combined with APRI was more sensitive than that of either measure alone.ConclusionsPreoperative NLR and APRI are independent predictors of DFS and OS in patients with HCC after surgical resection. Higher levels of NLR or APRI predict poorer outcomes in HCC patients. Intriguingly, combining NLR and APRI increases the prognostic accuracy of testing.

Autocrine vascular endothelial growth factor signaling promotes cell proliferation and modulates sorafenib treatment efficacy in hepatocellular carcinoma.

Tumor cells express vascular endothelial growth factor (VEGF) that can activate VEGF receptors (VEGFRs) on or within tumor cells to promote growth in an angiogenesis‐independent fashion; however, this autocrine VEGF pathway has not been reported in hepatocellular carcinoma (HCC). Sorafenib, an angiogenic inhibitor, is the only drug approved for use in advanced HCC patients. Yet the treatment efficacy is diverse and the mechanism behind it remains undetermined. Our aims were to study the molecular mechanisms underlying autocrine VEGF signaling in HCC cells and evaluate the critical role of autocrine VEGF signaling on sorafenib treatment efficacy. By immunohistochemistry, we found robust nuclear and cytoplasmic staining for active, phosphorylated VEGF receptor 1 (pVEGFR1) and phosphorylated VEGF receptor 2 (pVEGFR2), and by western blotting we found that membrane VEGFR1 and VEGFR2 increased in HCC tissues. We showed that autocrine VEGF promoted phosphorylation of VEGFR1 and VEGFR2 and internalization of pVEGFR2 in HCC cells, which was both pro‐proliferative through a protein lipase C‐extracellular kinase pathway and self‐sustaining through increasing VEGF, VEGFR1, and VEGFR2 mRNA expressions. In high VEGFR1/2‐expressing HepG2 cells, sorafenib treatment inhibited cell proliferation, reduced VEGFR2 mRNA expression in vitro, and delayed xenograft tumor growth in vivo. These results were not found in low VEGFR1/2‐expressing Hep3B cells. In an advanced HCC population on sorafenib treatment for postoperative recurrence, we found that the absence of VEGFR1 or VEGFR2 expression in resected tumor tissues before sorafenib treatment was associated with poorer overall survival. Conclusion: Autocrine VEGF signaling directly promotes HCC cell proliferation and affects the sorafenib treatment outcome in vitro and in vivo, which may enable better stratification for clinical treatment decisions. (Hepatology 2014;60:1264–1277)

miR-203 Suppresses the Proliferation and Metastasis of Hepatocellular Carcinoma by Targeting Oncogene ADAM9 and Oncogenic Long Non-coding RNA HULC

MicroRNAs (miRNAs) have been integrated into tumorigenic programs by regulating genes at post-transcriptional level. Long non-coding RNAs (lncRNAs) are novel targets for miRNAs. Here, we reported that miR-203 down-regulation was closely linked to advanced clinical features and poor overall survival (OS) of patients with hepatocellular carcinoma. We also confirmed that miR-203 and oncogene ADAM9 (a disintegrin and metalloproteinase 9)/oncogenic long non-coding RNA HULC (highly up-regulated in liver cancer) were inversely expressed in hepatocellular carcinoma (HCC) tissues or cell lines. More intriguingly, up-regulation of miR-203 diminished the expression of ADAM9 and HULC in HCC cancer cells. Over-expression of miR-203 could markedly inhibit cell proliferation, invasion and induce cell apoptosis. Furthermore, we identified that miR-203 modulated ADAM9 and HULC in a novel post-transcriptional regulatory mechanism. Over-expression of HULC partly rescued the miR-203-mediated antitumor effects. These results suggested that miR-203 played tumor suppressive roles by downregulating ADAM9 and HULC and indicated its potential application in cancer treatment.

Identification of Dermcidin as a novel binding protein of Nck1 and characterization of its role in promoting cell migration.

A distinct feature of hepatocellular carcinoma (HCC) is the tendency of tumor cells to disperse throughout the liver. Nck family adaptor proteins function to couple tyrosine phosphorylation signals to regulate actin cytoskeletal reorganization that leads to cell motility. In order to explore the role of Nck in HCC development, we performed GST pull-down assay using the SH2 domain of Nck1 as bait. The resulting precipitates were separated by 2-DE. Mass spectrometry analysis revealed a group of Nck1 SH2 domain-binding proteins that were differentially expressed in HCC. One of these proteins, dermcidin (DCD), and its interaction with Nck1, was further validated in vitro. GST pull-down assay revealed that Nck1 SH2 domain binds to the phosphotyrosine residue at position 20 (Y20) of the DCD. Pervandate treatment significantly enhanced the interaction between DCD and Nck1. Moreover, we demonstrated that forced expression of DCD could activate Rac1 and Cdc42 and promoted cell migration. Taken together, these data suggest a role of DCD in tumor metastasis.

The prognostic value of combined TGF-β1 and ELF in hepatocellular carcinoma

BackgroundTumor suppression of Transforming Growth Factor (TGF-β) signaling pathway requires an adaptor protein, Embryonic Liver Fodrin (ELF). Disruption of ELF expression resulted in miscolocalization of Smad3 and Smad4, then disruption of TGF-β signaling. However, the prognostic significance of ELF for hepatocellular carcinoma (HCC) hasn’t been clarified. This study aimed to investigate whether measuring both TGF-β1 and ELF provides a more powerful predictor for HCC prognosis than either marker alone.MethodsTGF-β1 and ELF protein were detected by immunohistochemistry. The relationship between TGF-β1/ELF expression and patients’ clinicopathologic factors was analyzed. The association between TGF-β1/ELF expression and disease-free survival and overall survival was analyzed by Kaplan-Meier curves, the log-rank test, and Multivariate Cox regression analyses.ResultsThe expression of TGF-β1 in HCC tissues was significantly higher than that in normal liver tissues. Conversely, the expression of ELF in HCC tissues declined markedly. ELF protein was correlated with HBsAg, tumor size, tumor number, TNM and recurrence. Data also indicated a significant negative correlation between ELF and TGF-β1. Patients with high TGF-β1 expression or/and low ELF expression appeared to have a poor postoperative disease-free survival and overall survival compared with those with low TGF-β1 expression or/and high ELF expression. Furthermore, the predictive range of ELF combined with TGF-β1 was more sensitive than that of either one alone.ConclusionsTGF-β1 and ELF protein are potential and reliable biomarkers for predicting prognosis in HCC patients after hepatic resection. Our current study has demonstrated that the prognostic accuracy of testing can be enhanced by their combination.

Adjuvant antiviral therapy for hepatitis B virus‐related hepatocellular carcinoma after curative treatment: A systematic review and meta‐analysis

To investigate whether adjuvant antiviral treatment could improve prognosis and entecavir is the optimal nucleoside/nucleotide analog (NA) regimen after curative therapy of hepatitis B virus (HBV)‐related hepatocellular carcinoma (HCC).

Numb Promotes Cell Proliferation and Correlates with Poor Prognosis in Hepatocellular Carcinoma

Background Numb is an evolutionary conserved protein that plays critical roles in cell fate determination, cell adhesion, cell migration and a number of signaling pathways, but evidence for a substantial involvement of Numb in HCC has remained unclear. The present study was aimed to investigate the clinical and prognostic significance of Numb and its role in hepatocellular carcinoma (HCC). Methodology The expression of Numb was detected in 107 cases of clinical paraffin-embedded hepatocellular carcinoma tissues,5 matched paris of fresh tissues and six hepatocellular cell lines by immunohistochemistry with clinicopathological analyses,RT-PCR or Western blot. Moreover, loss of function and gain of function assays were performed to evaluate the effect of Numb on cell proliferation in vitro. Conclusions We found that Numb was obviously up-regulated in HCC tissues and cell lines (p<0.05). The Numb up-regulation correlated significantly with poor prognosis, and Numb status was identified as an independent prognostic factor. Over-expression of Numb increased proliferation in SMMC-7721 and BEL-7402 cells, while knock-down of Numb showed the opposite effect. Our study indicates that Numb up-regulation significantly correlates with cell proliferation and poor prognosis in hepatocellular carcinoma patients. It may be a useful biomarker for therapeutic strategy in hepatocellular carcinoma treatment.

Combined vascular resection and analysis of prognostic factors for hilar cholangiocarcinoma.

BACKGROUND Hilar cholangiocarcinoma (HCCA) is a devastating malignancy arising from the bifurcation of the hepatic duct, whether combined vascular resection benefits HCCA patients is controversial. This study was undertaken to assess the effect of combined vascular resection in HCCA patients and to analyze the prognostic factors. METHODS Clinical data of 154 HCCA patients who had been treated from January 2005 to December 2012 were retrospectively analyzed. The patients were divided into three groups based on vascular resection: those without vascular resection; those with portal vein resection alone and those with hepatic artery resection. The survival and complication rates were compared among the three groups. Multivariate analysis was made to determine prognostic factors. RESULTS No significant differences were found in survival and complication rates among the three groups (P>0.05). Multivariate analysis showed that 3 factors were related to survival: lymph node metastasis, tumor size (>2.5 cm), and positive resection margin. CONCLUSIONS Vascular resection improved the survival rate of patients with HCCA involving the hepatic artery or portal vein. Lymph node metastasis, tumor size (>2.5 cm) and positive resection margin were poor prognostic factors in patients with HCCA.

Prognostic Evaluation of Categorical Platelet-based Indices Using Clustering Methods Based on the Monte Carlo Comparison for Hepatocellular Carcinoma

OBJECTIVES To evaluate the performance of clustering methods used in the prognostic assessment of categorical clinical data for hepatocellular carcinoma (HCC) patients in China, and establish a predictable prognostic nomogram for clinical decisions. MATERIALS AND METHODS A total of 332 newly diagnosed HCC patients treated with hepatic resection during 2006-2009 were enrolled. Patients were regularly followed up at outpatient clinics. Clustering methods including the Average linkage, k-modes, fuzzy k-modes, PAM, CLARA, protocluster, and ROCK were compared by Monte Carlo simulation, and the optimal method was applied to investigate the clustering pattern of the indices including platelet count, platelet/lymphocyte ratio (PLR) and serum aspartate aminotransferase activity/platelet count ratio index (APRI). Then the clustering variable, age group, tumor size, number of tumor and vascular invasion were studied in a multivariable Cox regression model. A prognostic nomogram was constructed for clinical decisions. RESULTS The ROCK was best in both the overlapping and non-overlapping cases performed to assess the prognostic value of platelet-based indices. Patients with categorical platelet-based indices significantly split across two clusters, and those with high values, had a high risk of HCC recurrence (hazard ratio [HR] 1.42, 95% CI 1.09-1.86; p<0.01). Tumor size, number of tumor and blood vessel invasion were also associated with high risk of HCC recurrence (all p<0.01). The nomogram well predicted HCC patient survival at 3 and 5 years. CONCLUSIONS A cluster of platelet-based indices combined with other clinical covariates could be used for prognosis evaluation in HCC.

NEK2 promotes hepatocellular carcinoma migration and invasion through modulation of the epithelial-mesenchymal transition

Never in mitosis gene-A (NIMA)-related expressed kinase 2 (NEK2) has been recently reported to play a role in tumor progression, drug resistance and tumorigenesis. However, little is known about the effects of NEK2 in hepatocellular carcinoma (HCC) metastasis and the underlying mechanism. NEK2 expression levels were examined by immunochemistry, qRT-PCR and western blot analyses in HCC cell lines and HCC tissues. A Transwell assay was used to determine the migration and invasion capacity of NEK2-silenced or NEK2-overexpressing HCC cells. Cell proliferation was investigated by MTT [(3-(4,5)-dimethylthiazol(-z-y1)-3,5-di-phenytetrazolium bromide] assay. The expression levels of epithelial-mesenchymal transition (EMT) markers in NEK2-silenced or NEK2-overexpressing HCC cells were examined by western blot analyses and qRT-PCR. The correlations between NEK2 expression and clinicopathological characteristics were further analyzed. Gene microarray was further used to analyze the effect of NEK2 expression on downstream cell signals. Our study showed that NEK2 was overexpressed in human HCC (37.84%; 98/259). NEK2 overexpression was significantly associated with liver non-capsulation and predicted poor survival outcomes in HCC patients after hepatectomy. In addition, NEK2 significantly enhanced HCC cell invasive ability. Mechanistically, we found that the epithelial-mesenchymal transition (EMT) plays a pivotal role in the NEK2-mediated promotion of HCC cell invasion. Furthermore, we provided evidence that signaling through the Wnt, NF-κB, focal adhesion, VEGF, Hippo and p53 pathways may be downstream of NEK2. Our findings highlight the importance of NEK2 in HCC metastasis and suggest that NEK2 is a reliable prognostic marker for HCC patients after hepatectomy.