Xiangsong Wu

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.

MALAT1 promotes the proliferation and metastasis of gallbladder cancer cells by activating the ERK/MAPK pathway.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non-coding RNA (lncRNA), is associated with metastasis and is an independent prognostic factor for lung cancer. Recent studies have demonstrated that MALAT1 plays an important role in other malignancies. However, little is known about the role of MALAT1 in gallbladder carcinoma (GBC), which is the most common cancer of the biliary tract and has an extremely poor prognosis. In this study, we focused on the expression, biological functions and mechanism of MALAT1 in GBC and found that MALAT1 was significantly upregulated in GBC tissues compared with corresponding non-cancerous tissues. Knockdown of MALAT1 in GBC cell lines using lentivirus-mediated RNA interference significantly inhibited the proliferation and metastasis of the GBC cells both in vitro and in vivo. Furthermore, ERK/MAPK pathway was found to be inactivated in the GBC cell lines after MALAT1 knockdown. These results indicated that MALAT1 might serve as an oncogenic lncRNA that promotes proliferation and metastasis of GBC and activates the ERK/MAPK pathway

Evaluation of two inflammation-based prognostic scores in patients with resectable gallbladder carcinoma.

BackgroundSurvival after surgery for gallbladder cancer is generally poor. A number of inflammation-based prognostic scores have been established to help predict survival after surgery for several types of cancer. The objective of this study was to analyze and compare the utility of two inflammation-based prognostic scores, the Glasgow prognostic score (GPS) and the neutrophil-to-lymphocyte ratio (NLR), for predicting survival in patients with gallbladder cancer after surgery with curative intent.MethodsWe retrospectively reviewed the medical records of 85 patients with histologically confirmed, resectable gallbladder carcinoma (GBC), who were to receive curative surgery in our department. Univariate and multivariate analyses were performed to evaluate the relationship between the variables to overall survival (OS).ResultsA significant difference was detected in OS in patients with low and high GPS and NLR scores. Univariate analyses using clinicopathological characteristics revealed that tumor differentiation; tumor invasion; lymph node metastasis; tumor, node, metastasis classification system stage; positive margin status; combined common bile duct resection; serum levels of C-reactive protein, albumin, carbohydrate antigen 19-9 (CA19-9), carcinoembryonic antigen, and CA125; white blood cell count; and GPS and NLR were all associated with OS. Among these characteristics, multivariate analysis demonstrated that a high GPS was independently associated with poorer OS, together with tumor invasion, lymph node metastasis, and positive margin status.ConclusionsGPS is superior to NLR with respect to its prognostic value for patients with GBC after surgery with curative intent. GPS is not only associated with tumor progression but is also an independent marker of poor prognosis.

SPOCK1 as a potential cancer prognostic marker promotes the proliferation and metastasis of gallbladder cancer cells by activating the PI3K/AKT pathway

BackgroundGallbladder cancer (GBC) is a leading cause of cancer-related death worldwide, and its prognosis remains poor, with 5-year survival of approximately 5%. In this study, we analyzed the involvement of a novel proteoglycan, Sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1), in the tumor progression and prognosis of human GBC.MethodsSPOCK1 expression levels were measured in fresh samples and stored specimens of GBC and adjacent nontumor tissues. The effect of SPOCK1 on cell growth, DNA replication, migration and invasion were explored by Cell Counting Kit-8, colony formation, EdU retention assay, wound healing, and transwell migration assays, flow cytometric analysis, western blotting, and in vivo tumorigenesis and metastasis in nude mice.ResultsSPOCK1 mRNA and protein levels were increased in human GBC tissues compared with those in nontumor tissues. Immunohistochemical analysis indicated that SPOCK1 levels were increased in tumors that became metastatic, compared with those that did not, which was significantly associated with histological differentiation and patients with shorter overall survival periods. Knockdown of SPOCK1 expression by lentivirus-mediated shRNA transduction resulted in significant inhibition of GBC cell growth, colony formation, DNA replication, and invasion in vitro. The knockdown cells also formed smaller xenografted tumors than control GBC cells in nude mice. Overexpression of SPOCK1 had the opposite effects. In addition, SPOCK1 promoted cancer cell migration and epithelial-mesenchymal transition by regulating the expression of relevant genes. We found that activation of the PI3K/Akt pathway was involved in the oncogenic functions of SPOCK1 in GBC.ConclusionsSPOCK1 activates PI3K/Akt signaling to block apoptosis and promote proliferation and metastasis by GBC cells in vitro and in vivo. Levels of SPOCK1 increase with the progression of human GBC. SPOCK1 acts as an oncogene and may be a prognostic factor or therapeutic target for patients with GBC.

NLK is a key regulator of proliferation and migration in gallbladder carcinoma cells

Gallbladder cancer (GBC) is one of the most lethal neoplasm and is the fifth most common malignancy of gastrointestinal tract. The prognosis of gallbladder cancer is extremely terrible partially due to metastasis. Thus, understanding the molecular pathways controlling metastasis of this lethal disease may provide new targets for targeted therapeutic approach. In this study, we investigated the function of nemo-like kinase (NLK) in GBC growth and migration. Lentivirus-mediated siRNA was employed to alleviate the expression level of NLK in GBC cell lines (GBC-SD and SGC-996). Real-time PCR and western-blot analysis demonstrated that both mRNA and protein levels of NLK in GBC-SD and SGC-996 cells were decreased after infection with NLK-siRNA-expressing lentivirus (Lv-shNLK). The proliferation and in vitro tumorigenesis (colony formation) ability as well as migration of GBC-SD and SGC-996 cells with low NLK expression decreased significantly. Our results suggested that NLK is a key regulator involved in proliferation and migration of GBC, and it could be used as a potential therapeutic target for GBC.

A role for p21-activated kinase 7 in the development of gastric cancer.

p21‐activated kinase (PAK)7 (also known as PAK5) is a member of the group B PAK family of serine/threonine protein kinases, which are effectors of the small GTPases Rac and CDC42. PAK7 can promote neurite outgrowth, induce microtubule stabilization, and activate cell survival signaling pathways. However, the role of PAK7 in cancer is still poorly understood. Here, we showed that PAK7 expression was upregulated in different gastric cancer cell lines and gastric cancer tissues, as compared with human embryonic kidney 293 cells and adjacent normal tissues, respectively. The results suggested that PAK7 expression was related to gastric cancer progression. Thus, we employed lentivirus‐mediated small interfering RNA to inhibit PAK7 expression, to investigate the role of PAK7 in human gastric carcinogenesis. RNA interference efficiently downregulated expression of PAK7 in SGC‐7901 and MGC‐803 cells at both mRNA and protein levels. Knockdown of PAK7 inhibited human gastric cancer cell proliferation by inducing cell cycle arrest in G0/G1 phase, in concordance with the downregulation of CDK2, CDC25A, and cyclin D1. Our data suggest that PAK7 is a new hallmark of gastric cancer, in which PAK7 might contribute to gain of tumor growth potential, acting by affecting the expression of cell cycle regulators. Therefore, PAK7 may be an attractive candidate as a therapeutic target in gastric cancer.

Curcumin induces apoptosis in gallbladder carcinoma cell line GBC-SD cells

BackgroundGallbladder carcinoma is a malignant tumor with a very low 5-year survival rate because of the difficulty with its early diagnosis and the very poor prognosis of the advanced cancer state. The aims of this study were to determine whether curcumin could induce the apoptosis of a gallbladder carcinoma cell line, GBC-SD, and to clarify its related mechanism.MethodsFirst, the anti-proliferative activities of curcumin-treated and untreated GBC-SD cells were determined using the MTT and colony formation assays. Then, the early apoptosis of cells was detected by the annexin V/propidium iodide double-staining assay and Hoechst 33342 staining assay. Detection of mitochondrial membrane potential was used to validate the ability of curcumin on inducing apoptosis in GBC-SD cells. Cell cycle changes were detected by flow cytometric analysis. Finally, the expressions of the apoptosis-related proteins or genes caspase-3, PARP, Bcl-2, and Bax were analyzed by western blot and quantitative real time PCR assay. Statistical analyses were performed using the Student’s t-test for comparison of the results obtained from cells with or without curcumin treatment.ResultsThe MTT assay revealed that curcumin had induced a dose- and a time-dependent decrease in cell viability. Colony counting indicated that curcumin had induced a dose-dependent decrease in the colony formation ability in GBC-SD cells. Cells treated with curcumin were arrested at the S phase, according to the flow cytometric analysis. A significant induction of both the early and late phases of apoptosis was shown by the annexin V-FITC and PI staining. Morphological changes in apoptotic cells were also found by the Hoechst 33342 staining. After treatment with curcumin fluorescence shifted from red to green as ΔΨm decreased. Furthermore, western blot and quantitative real time PCR assays demonstrated that the curcumin induced apoptosis in GBC-SD cells by regulating the ratio of Bcl-2/Bax and activating the expression of cleaved caspase-3.ConclusionsTaken together, the results indicate that curcumin may be a potential agent for the treatment of gallbladder cancer.

Yes-associated protein 1 (YAP1) promotes human gallbladder tumor growth via activation of the AXL/MAPK pathway.

The transcriptional coactivator Yes-associated protein 1 (YAP1), a key regulator of cell proliferation and organ size in vertebrates, has been implicated in various malignancies. However, little is known about the expression and biological function of YAP1 in human gallbladder cancer (GBC). In this study we examined the clinical significance and biological functions of YAP1 in GBC and found that nuclear YAP1 and its target gene AXL were overexpressed in GBC tissues. We also observed a significant correlation between high YAP1 and AXL expression levels and worse prognosis. The depletion of YAP1 using lentivirus shRNAs significantly inhibited cell proliferation by inducing cell cycle arrest in S phase in concordance with the decrease of CDK2, CDC25A, and cyclin A, and resulted in increased cell apoptosis and invasive repression in GBC cell lines in vitro. Furthermore, knockdown of YAP1 also inhibited tumor growth in vivo. Additionally, we demonstrated that the activation of the AXL/MAPK pathway was involved in the oncogenic functions of YAP1 in GBC. These results demonstrated that YAP1 is a putative oncogene and represents a prognostic marker and potentially a novel therapeutic target for GBC.

Fibronectin promotes cell proliferation and invasion through mTOR signaling pathway activation in gallbladder cancer

Fibronectin (FN), a heterodimeric glycoprotein overexpressed in several types of tumors, has been implicated in cancer progression via the activation of integrin-mediated pro-oncogenic pathways. The FN level in human bile fluid is dramatically increased in malignant biliary diseases; however, FN expression and its biological functions in gallbladder cancer (GBC) remain unknown. In this study, we found that FN was overexpressed in GBC tissues and was associated with a worse prognosis in GBC patients. In vitro experimental studies indicated that exogenous FN significantly enhanced cell proliferation, invasion and active MMP-9 secretion in human GBC cell lines (GBC-SD and NOZ). Moreover, the key kinases of the mTOR signaling pathway, including FAK, Akt, mTOR and 4E-BP1, were markedly activated in a time-dependent manner in FN-treated GBC-SD and NOZ cells. The IHC statistical analyses validated that FN expression was positively correlated with the phosphorylation levels of the 4E-BP1 protein in GBC tissues. Furthermore, rapamycin, a specific inhibitor of mTOR, almost completely blocked FN-induced phosphorylation of 4E-BP1 and also partially abrogated the stimulatory effects of FN on GBC cell proliferation and invasion. In vivo, FN treatment significantly promoted the proliferation and metastasis of GBC cells and markedly activated Akt/mTOR/4E-BP1 signaling cascade. These findings demonstrate that FN may play a critical role in the modulation of cell proliferation and invasion via mTOR signaling pathway activation during GBC progression.

Primary closure versus T-tube drainage in laparoscopic common bile duct exploration: a meta-analysis of randomized clinical trials

PurposeTo compare the safety and effectiveness of primary closure with those of T-tube drainage in laparoscopic common bile duct exploration (LCBDE) for choledocholithiasis.MethodsA comprehensive search was performed in the PubMed, EmBase, and Cochrane Library databases. Only randomized controlled trials comparing primary closure with T-tube drainage in LCBDE were considered eligible for this meta-analysis. The analyzed outcome variables included postoperative mortality, overall morbidity, biliary complication rate, biliary leak rate, reoperation, operating time, postoperative hospital stay, time to abdominal drain removal, and retained stone. All calculations and statistical tests were performed using ReviewerManager 5.1.2 software.ResultsA total of 295 patients (148 patients with primary closure and 147 patients with T-tube drainage) from three trials were identified and analyzed. No deaths occurred in any of the trials. Primary closure showed significantly better results in terms of morbidity (risk ratio (RR), 0.51; 95% confidence interval (CI), 0.30 to 0.88), biliary complication without a combination of retained stone (RR, 0.44; 95% CI, 0.20 to 0.97), reoperation (RR, 0.16; 95% CI, 0.03 to 0.87), operating time (mean difference (MD), −20.72; 95% CI, −29.59 to −11.85), postoperative hospital stay (MD, −3.24; 95% CI, −3.96 to −2.52), and time to abdominal drainage removal (MD, −0.45; 95% CI, −0.86 to −0.04). Statistically significant differences were not found between the two methods in terms of biliary leak, biliary complication, and retained stones.ConclusionThe current meta-analysis indicates that primary closure of the common bile duct is safer and more effective than T-tube drainage for LCBDE. Therefore, we do not recommend routine performance of T-tube drainage in LCBDE.