Kai Qu

Whole-exome and targeted gene sequencing of gallbladder carcinoma identifies recurrent mutations in the ErbB pathway

Individuals with gallbladder carcinoma (GBC), the most aggressive malignancy of the biliary tract, have a poor prognosis. Here we report the identification of somatic mutations for GBC in 57 tumor-normal pairs through a combination of exome sequencing and ultra-deep sequencing of cancer-related genes. The mutation pattern is defined by a dominant prevalence of C>T mutations at TCN sites. Genes with a significant frequency (false discovery rate (FDR) < 0.05) of non-silent mutations include TP53 (47.1%), KRAS (7.8%) and ERBB3 (11.8%). Moreover, ErbB signaling (including EGFR, ERBB2, ERBB3, ERBB4 and their downstream genes) is the most extensively mutated pathway, affecting 36.8% (21/57) of the GBC samples. Multivariate analyses further show that cases with ErbB pathway mutations have a worse outcome (P = 0.001). These findings provide insight into the somatic mutational landscape in GBC and highlight the key role of the ErbB signaling pathway in GBC pathogenesis.

Negative regulation of transcription factor FoxM1 by p53 enhances oxaliplatin-induced senescence in hepatocellular carcinoma.

Previous studies have demonstrated the involvement of transcriptional factor forkhead box M1 (FoxM1) in cellular senescence of hepatocellular carcinoma (HCC). In the present study, we revealed that oxaliplatin could induce senescence in HCC cells, since advanced HCC patients with lower expression of FoxM1 were more sensitive to oxaliplatin therapy. Our data indicated that due to the repression by p53, FoxM1 played a critical role in oxaliplatin-induced senescence via regulating cycle-related proteins p21, p27, cyclins B1 and D1. Furthermore, inhibition of FoxM1, combined with oxaliplatin treatment, could significantly promote the senescence of HCC cells. Taken together, our findings suggest that FoxM1 may represent a promising therapeutic target for the medication of the chemosensitivity to oxaliplatin in HCC patients.

Emodin induces human T cell apoptosis in vitro by ROS-mediated endoplasmic reticulum stress and mitochondrial dysfunction.

Aim:To elucidate the molecular mechanisms underlying the immunosuppressive effects of emodin isolated from Rheum palmatum L.Methods:Human T cells were isolated from the peripheral venous blood of 10 healthy adult donors. Cell viability was analyzed with MTT assay. AO/EB and Annexin V/PI staining and DNA damage assay were used to detect cell apoptosis. Fluorescence staining was used to detect the levels of ROS, the mitochondrial membrane potential and intracellular Ca2+. Colorimetry was used to detect the levels of MDA and total SOD and GSH/GSSG ratio. The expression and activity of caspase-3, -4, and -9 were detected with Western blotting and a fluorometric assay. Western blotting was also used to detect the expression of Bcl-2, Bax, cytochrome C, and endoplasmic reticulum (ER) markers.Results:Emodin (1, 10, and 100 μmol/L) inhibited the growth of human T cells and induced apoptosis in dose- and time dependent manners. Emodin triggered ER stress and significantly elevated intracellular free Ca2+ in human T cells. It also disrupted mitochondrial membrane potential, and increased cytosolic level of cytochrome C, and the levels of activated cleavage fragments of caspase-3, -4, and -9 in human T cells. Furthermore, emodin significantly increased the levels of ROS and MDA, inhibited both SOD level and GSH/GSSG ratio in human T cells, whereas co-incubation with the ROS scavenger N-acetylcysteine (NAC, 20 μmol/L) almost completely blocked emodin-induced ER stress and mitochondrial dysfunction in human T cells, and decreased the caspase cascade-mediated apoptosis.Conclusion:Emodin exerts immunosuppressive actions at least partly by inducing apoptosis of human T cells, which is triggered by ROS-mediated ER stress and mitochondrial dysfunction.

Pyogenic liver abscesses associated with nonmetastatic colorectal cancers: an increasing problem in Eastern Asia.

AIM To elaborate the clinicopathologic features of colorectal cancer-related pyogenic liver abscess (PLA). METHODS Reported cases of colorectal cancer-related PLAs were collected from the literature published up to October 2011 and evaluated for their clinicopathologic features. Data of collected cases included demographics, clinical presentation, microbial findings and treatment. Categorical variables were compared by χ² analysis and continuous variables were evaluated using Students t test. RESULTS A total 96 cases of colorectal cancer-related PLA were collected from the previous literature. Most patients (60%) were male and 40% cases occurred in the age group of 61-70 years. Apart from some special types of PLA, there were significant differences in the microbiological spectrum between Eastern Asia and non-Eastern Asian countries, which implied different risk factors and courses of the disease. Gram negative bacteria especially Klebsiella pneumoniae (K. pneumoniae) PLA was predominant in Eastern Asia (80.0%) in contrast to non-Eastern Asian countries (P < 0.01). Meanwhile, most of the Eastern Asian patients exhibited smaller size of liver abscess and atypical presentation. Sigmoid colon and rectum (72.73%) were the main sites of tumor in Eastern Asian patients, whereas tumor sites were uneven among most of the non-Easter Asian PLA patients. CONCLUSION K. pneumoniae PLA was strongly associated with colorectal cancer, especially those occurring in sigmoid colon and rectum, in elderly Eastern Asian male patients.

The Prognostic Value of Platelet Count in Patients With Hepatocellular Carcinoma: A Systematic Review and Meta-Analysis

Abstract Thrombocytopenia has been acknowledged to be a crucial risk factor for cirrhosis formation and hepatocarcinogenesis in chronic liver diseases. However, to date, the association between platelet count (PLT) and the prognosis of hepatocellular carcinoma (HCC) remains inconsistent and controversial. The aim of the present study was to determine whether PLT could be used as a useful predictor of survival in patients with HCC. We performed systematic review in online databases, including PubMed, EmBase, and Web of Science, from inception until 2014. Studies were included if a statistical relationship was investigated between PLT and survival for HCC, and hazard ratio (HR) and 95% confidence intervals (CIs) for overall survival (OS) or recurrence-free survival (RFS) were provided. The quality of each included study was assessed by Newcastle–Ottawa scale score. To synthesize these studies, a random-effects model or a fixed-effects model was applied as appropriate. Then, we calculated heterogeneity, performed sensitivity analysis, tested publication bias, and did subgrouped and meta-regression analysis. Finally, we identified 33 eligible articles (published from 1998 to 2014) involved 5545 patients by retrieval. A low level of preoperative PLT was found to be significantly associated with a poor survival of HCC. Irrespective of the therapy used, the pooled HRs for OS and RFS were 1.41 (95% CI, 1.14–1.75) and 1.44 (95% CI, 1.13–1.83), respectively. Specifically, in patients who underwent liver resection, the pooled HRs for OS and RFS were 1.67 (95% CI, 1.22–2.27) and 1.44 (95% CI, 1.04–1.99), respectively. Furthermore, patients with preoperative thrombocytopenia (PLT < 100 × 109/L) had a worse OS (HR: 1.73, 95% CI, 1.29–2.32) and RFS (HR: 1.57, 95% CI, 1.31–1.87) in comparison with patients without thrombocytopenia. All our findings showed no significant changes due to the removal of any study or the use of an opposite-effects model, and there was no significant publication bias. The limitations of this meat-analysis were nonuniform cut-off values of PLT, high between-study heterogeneities, potential confounders, and a bias of publication year. A low preoperative PLT level results in an unfavorable outcome in HCC. PLT is a simple, inexpensive, and useful predictor of survival in patients with HCC.

Down-regulation of miR-146b-5p by long noncoding RNA MALAT1 in hepatocellular carcinoma promotes cancer growth and metastasis

MicroRNAs play an important role in liver cancer genesis and progression. In this study, we identified down-regulation of miR-146b-5p associated with tumor growth, metastasis and poor survival in hepatocellular carcinoma (HCC) patients. miR-146b-5p could suppress proliferation, migration, and invasion and induced apoptosis in vitro and in vivo. Remarkably, TNF receptor associated factor 6 (TRAF6) was confirmed as a direct target of miR-146b-5p in HCC and miR-146b-5p exerted the tumor suppression roles through inhibiting the phosphorylation of Akt mediated by TRAF6. Furthermore, we identified long non-coding RNA MALAT1 as a molecular sponge of miR-146b-5p to down-regulate its expression in HCC. In general, our results indicate that miR-146b-5p inhibits tumor growth and metastasis of HCC by targeting TRAF6 mediated Akt phosphorylation.

FoxM1 overexpression promotes epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma

AIM To investigate the expression of forkhead box protein M1 (FoxM1) in the process of epithelial mesenchymal transition in hepatocellular carcinoma (HCC) and its role in metastasis. METHODS FoxM1 and E-cadherin expression in HCC tissue microarray specimens was evaluated by immunohistochemical staining, and statistical methods were applied to analyze the correlation between FoxM1 and epithelial-mesenchymal transition (EMT). Kaplan-Meier analysis of the correlation between the FoxM1 expression level and recurrence or overall survival of HCC patients was performed. The expression of FoxM1, E-cadherin and snail homologue 1 (SNAI1) in HCC cell lines was evaluated by real-time reverse transcription-polymerase chain reaction and Western blot. Hepatocyte growth factor (HGF) was used to induce EMT and stimulate cell migration in HCC cells. The expression of FoxM1 and SNAI1 was regulated by transfection with plasmids pcDNA3.1 and siRNAs in vitro. The occurrence of EMT was evaluated by Transwell assay, morphologic analysis and detection of the expression of EMT markers (E-cadherin and vimentin). Luciferase and chromatin immunoprecipitation assays were used to evaluate whether SNAI1 is a direct transcriptional target of FoxM1. RESULTS FoxM1 expression was increased significantly in HCC compared with para-carcinoma (10.7 ± 0.9 vs 8.2 ± 0.7, P < 0.05) and normal hepatic (10.7 ± 0.9 vs 2.7 ± 0.4, P < 0.05) tissues. Overexpression of FoxM1 was correlated with HCC tumor size, tumor number, macrovascular invasion and higher TNM stage, but was negatively correlated with E-cadherin expression in microarray specimens and in cell lines. FoxM1 overexpression was correlated significantly with HCC metastasis and EMT. In vitro, we found that FoxM1 plays a key role in HGF-induced EMT, and overexpression of FoxM1 could suppress E-cadherin expression and induce EMT changes, which were associated with increased HCC cell invasiveness. Next, we confirmed that FOXM1 directly binds to and activates the SNAI1 promoter, and we identified SNAI1 as a direct transcriptional target of FOXM1. Moreover, inhibiting the expression of SNAI1 significantly inhibited FoxM1-mediated EMT. CONCLUSION FoxM1 overexpression promotes EMT and metastasis of HCC, and SNAI1 plays a critical role in FoxM1-mediated EMT.

Central obesity and nonalcoholic fatty liver disease risk after adjusting for body mass index

AIM To investigate whether central obesity is associated with nonalcoholic fatty liver disease (NAFLD) formation after adjusting for general obesity. METHODS The online databases PubMed, EMBASE, and ISI Web of Science were searched for studies estimating the influence of central obesity on NAFLD occurrence published through April 2014. Studies that did not adjust for body mass index (BMI) were excluded. In addition, the independent effect of BMI was also assessed with the included studies. The pooled effect sizes and 95% confidence intervals (CIs) were calculated using random- or fixed-effects models based on the degree of heterogeneity. Furthermore, subgroup analyses, meta-regression, sensitivity analyses, and publication bias were performed. RESULTS Twenty eligible studies were identified. The summary odds ratio (OR) values per-unit increase in waist circumference (WC) and BMI for NAFLD formation were 1.07 (95%CI: 1.03-1.10, I (2) = 73.9%, n = 11 studies) and 1.25 (95%CI: 1.13-1.38, I (2) = 88.7%, n = 11 studies), respectively. When the indices were expressed as binary variables (with the non-obesity group as reference), the pooled OR in WC, waist-to-hip ratio, and BMI were 2.34 (95%CI: 1.83-3.00, I (2) = 41.8%, n = 7 studies), 4.06 (95%CI: 1.53-10.79, I (2) = 65.7%, n = 3 studies), and 2.85 (95%CI: 1.60-5.08, I (2) = 57.8%, n = 5 studies), respectively. Using the same studies as the latter (n = 5), pooled OR in WC was 3.14 (95%CI: 2.07-4.77), which is greater than that in BMI. CONCLUSION Central obesity may pose a greater threat to national health than general obesity, although both are independently associated with increased risk of NAFLD.

Sorafenib inhibits proliferation and invasion of human hepatocellular carcinoma cells via up-regulation of p53 and suppressing FoxM1.

Aim:Forkhead box M1 (FoxM1) is a transcription factor that plays important roles in the pathogenesis and progression of human cancers, including hepatocellular carcinoma (HCC). The aim of this study was to examine the involvement of FoxM1 in the anti-cancer action of sorafenib, a multikinase inhibitor, in human HCC cells.Methods:HCC cell lines HepG2 and HuH-7 were tested. Cell viability was examined using MTT assay and cell invasion was determined with Transwell migration assay. The relevant mRNA expression was determined with RT-PCR, and the proteins were detected using Western blotting and immunofluorescence assays. RNA interference was used to modify the expression of p53 and FoxM1. HuH-7 cell line xenograft mice were used for in vivo study, which were treated with sorafenib (40 mg/kg, po) daily for 3 weeks.Results:Sorafenib (2–20 μmol/L) inhibited the proliferation of the cells in dose- and time-dependent manners with an IC50 value of nearly 6 μmol/L at 48 h. Sorafenib (6 μmol/L) markedly suppressed the cell invasion. Furthermore, sorafenib (2−6 μmol/L) dose-dependently decreased the expression of FoxM1, MMP-2, and Ki-67, and up-regulated that of p53 in the cells. Silencing p53 abolished the decrease of FoxM1 and increase of p53 in sorafenib-treated cells. Silencing FoxM1 significantly reduced the expression of MMP-2 and Ki-67, and enhanced the anti-proliferation action of sorafenib in the cells, whereas overexpression of FoxM1 increased the expression of MMP-2 and Ki-67, and abrogated the anti-proliferation action of sorafenib. In the xenograft mice, sorafenib administration decreased the tumor growth by 40%, and markedly increased the expression of p53, and decreased the expression of FoxM1, MMP-2, and Ki-67 in tumor tissues.Conclusion:Sorafenib inhibits HCC proliferation and invasion by inhibiting MMP-2 and Ki-67 expression due to up-regulation of P53 and suppressing FoxM1.

Reactive oxygen species generation is essential for cisplatininduced accelerated senescence in hepatocellular carcinoma

Accelerated senescence is important because this process is involved in tumor suppression and has been induced by many chemotherapeutic agents. The platinum-based chemotherapeutic agent cisplatin displays a wide range of antitumor activities. However, the molecular mechanism of cisplatin-induced accelerated senescence in hepatocellular carcinoma (HCC) remains unclear. In the present study, the growth inhibitory effect of cisplatin on HepG2 and SMMC-7721 cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cellular senescence was then assessed by β-galactosidase assay. Senescence-related factors, including p53, p21, and p16, were evaluated by quantitative reverse transcription-polymerase chain reaction. Reactive oxygen species (ROS) was analyzed by flow cytometry. Our results revealed that cisplatin reduced the proliferation of HepG2 and SMMC-7721 cells in a dose- and time-dependent manner. Senescent phenotype observed in cisplatintreated hepatoma cells was dependent on p53 and p21 activation but not on p16 activation. Furthermore, cisplatininduced accelerated senescence depended on intracellular ROS generation. The ROS scavenger N-acetyl-L-cysteine also significantly suppressed the cisplatin-induced senescence of HepG2 and SMMC-7721 cells. In conclusion, our results revealed a functional link between intracellular ROS generation and cisplatin-induced accelerated senescence, and this link may be used as a potential target of HCC.