Xiao-Yu Yin

Mechanisms and influence of octreotide-induced regulation of somatostatin receptor 2 on hepatocellular carcinoma.

Background: Somatostatin receptors (SSTRs) belong to the family of G protein-coupled receptors. Exposure of G protein-coupled receptors to their agonists induces a rapid decrease in their initial response. The goal of this study is to investigate alteration in SSTR2 by the treatment of SSTR agonist octreotide (OCT) in hepatocellular carcinoma (HCC) and the resulting consequence. Methods: Morphology, proliferation and cell cycle of the human HCC cell line (Bel7402) were evaluated. Effect of OCT on HCC growth and development was assessed in vivo. SSTR2 expression was measured by RT-PCR and detected by immunohistochemistry. Results: Short-term OCT treatment on Bel7402 cells barely changed cell proliferation and morphology, and no apoptosis was induced. The SSTR2 protein level was markedly decreased on Bel7402 cells after exposure to OCT. However, the weight of the HCC xenograft was significantly lower in the OCT treatment group as compared with the control group. In the rat hepatocarcinogenesis model, the mortality and incidence of HCC in the OCT treatment group were remarkably less than those in the control group. Long-term OCT treatment led to increased levels of both SSTR2 mRNA and protein in hepatocytes and HCC cells. Conclusion: Short-term OCT treatment could lead to SSTR2 desensitization, resulting in a reduced inhibitory effect on HCC by OCT. However, long-term OCT treatment effectively inhibited the development and growth of HCC probably via resensitization and upregulation of SSTR2.

Monoamine oxidase A expression is suppressed in human cholangiocarcinoma via coordinated epigenetic and IL-6-driven events

The secretion of dopamine and serotonin is increased in cholangiocarcinoma, which has growth-promoting effects. Monoamine oxidase A (MAOA), the degradation enzyme of serotonin and dopamine, is suppressed in cholangiocarcinoma via an unknown mechanism. The aims of this study were to (i) correlate MAOA immunoreactivity with pathophysiological parameters of cholangiocarcinoma, (ii) determine the mechanism by which MAOA expression is suppressed and (iii) evaluate the consequences of restored MAOA expression in cholangiocarcinoma. MAOA expression was assessed in cholangiocarcinoma and nonmalignant controls. The control of MAOA expression by promoter hypermethylation was evaluated and the contribution of interleukin-6 (IL-6) signaling to the suppression of MAOA expression was determined. The effects of MAOA overexpression on cholangiocarcinoma growth and invasion were also assessed. MAOA expression is correlated with differentiation, invasion and survival in cholangiocarcinoma. The MAOA promoter was hypermethylated immediately upstream of the start codon in cholangiocarcinoma samples and cell lines but not in nonmalignant counterparts. IL-6 signaling also decreased MAOA expression via a mechanism independent of hypermethylation, involving the regulation of the balance between SP-1 transcriptional activity and its inhibitor, R1 repressor. Inhibition of both IL-6 signaling and DNA methylation restored MAOA levels to those observed in cholangiocytes. Forced MAOA overexpression inhibited cholangiocarcinoma growth and invasion. MAOA expression is suppressed by the coordinated control of promoter hypermethylation and IL-6 signaling. MAOA may be a useful prognostic marker in the management of cholangiocarcinoma, and therapies designed to increase MAOA expression might prove beneficial in the treatment of cholangiocarcinoma.

Establishment and validation of a prognostic nomogram for patients with resectable perihilar cholangiocarcinoma

As the conventional staging systems have poor prognosis prediction ability for patients with perihilar cholangiocarcinoma (pCCA), we established and validated an effective prognostic nomogram for pCCA patients based on their personal and tumor characteristics. A total of 235 patients who received curative intent resections at the Eastern Hepatobiliary Surgery Hospital from 2000 to 2009 were recruited as the primary training cohort. Age, preoperative CA19-9 levels, portal vein involvement, hepatic artery invasion, lymph node metastases, and surgical treatment outcomes (R0 or R1/2) were independent prognostic factors for pCCA patients in the primary cohort as suggested by the multivariate analyses and these were included in the established nomogram. The calibration curve showed good agreement between overall survival probability of pCCA patients for the nomogram predictions and the actual observations and the concordance index (C-index) was 0.68 (95% CI, 0.61-0.71). The C-index values and time-dependent ROC tests suggested that the nomogram is superior to the conventional staging systems including the Bismuth-Corlette, Gazzaniga, Memorial Sloan Kettering Cancer Center (MSKCC), American Joint Committee on Cancer (AJCC) TNM 7th edition, and Mayo Clinic. The nomogram also performed better than the traditional staging system in the internal cohort with 93 pCCA patients from the same institution and an external validation cohort including 84 pCCA patients from another institution in predicting the overall survival of the pCCA patients as suggested by the C-index values and the time-dependent ROC tests. In summary, the proposed nomogram has superior predictive accuracy of prognosis for resectable pCCA patients.

EYA4 functions as tumor suppressor gene and prognostic marker in pancreatic ductal adenocarcinoma through β-catenin/ID2 pathway

Eye absent homolog 4 (EYA4) was initially found as key gene in controlling eye development in Drosophila. We recently found that EYA4 was an independent prognostic factor in hepatocellular carcinoma. Its biological functions in malignancies remained unknown. The present study aimed at investigating its biological functions, molecular mechanisms and prognostic values in pancreatic ductal adenocarcinoma (PDAC). Overexpression of EYA4 in PDAC cells inhibited proliferation and invasion in vitro and tumor growth in vivo. Depletion of EYA4 in PDAC cells enhanced proliferation and invasion in vitro and tumor growth in vivo. Mechanistically, armed with the serine/threonine-specific protein phosphatase activity, EYA4 dephosphorylated β-catenin at Ser675, blocked β-catenin nuclear translocation and inhibited ID2 transactivation. Consistently, EYA4 expression inversely correlated with the levels of p-Ser675-β-catenin and ID2 in tissues. EYA4 expression in PDAC tissues was significantly reduced as compared with adjacent non-tumoral tissues. EYA4 expression was an independent prognostic factor in PDAC, with a lower EYA4 level in association with shorter long-term survival and disease-free time. We showed that EYA4 functioned as tumor suppressor gene in PDAC via repressing β-catenin/ID2 activation, and was an independent prognostic factor in PDAC.

Simvastatin enhances the chemotherapeutic efficacy of S-1 against bile duct cancer: E2F-1/TS downregulation might be the mechanism.

Simvastatin has inhibitory effects on cancers. The present study aimed to investigate the interactive effects between simvastatin and S-1 against bile duct cancer and its mechanisms. The effects of simvastatin and 5-fluorouracil (5-FU) alone or in combination on the growth and apoptosis of the human cholangiocarcinoma cell line EGI-1 and the gallbladder carcinoma cell line Mz-ChA-1 cells were evaluated in vitro. Real-time PCR and western blot were used to determine E2F-1 and thymidylate synthase (TS) expressions in the treated cells. Tumoricidal efficacy of simvastatin and S-1 was further investigated in a subcutaneous bile duct cancer model in NOD/SCID mice. Simvastatin enhanced the cytotoxicity of 5-FU on bile duct cancer cells in vitro. IC50 of 5-FU alone was 4.34 &mgr;mol/l for EGI-1 and 13.9 &mgr;mol/l for MZ-ChA-1, whereas it decreased markedly to 0.90 and 2.95 &mgr;mol/l, respectively, when combined with simvastatin. The Chou and Talalay combination index of 5-FU and simvastatin was 0.41 and 0.40 at IC50 for EGI-1 and MZ-ChA-1, respectively. Simvastatin alone or plus 5-FU significantly suppressed E2F-1 and TS expressions in EGI-1 and MZ-ChA-1. Simvastatin plus 5-FU induced greater proportion of apoptotic cells on both EGI-1 and MZ-ChA-1, with an increase in cleaved caspase-3 levels, compared with simvastatin or 5-FU alone (all P<0.05). Simvastatin plus S-1 induced greater tumor inhibition than simvastatin or S-1 alone with E2F-1/TS downregulation in vivo (all P<0.05). Simvastatin and S-1 exerted synergistic effects against bile duct cancer, which might be mediated by E2F-1/TS downregulation. The combination could be a reasonable regimen in the management of bile duct cancer.

Activation of c-Jun predicts a poor response to sorafenib in hepatocellular carcinoma: Preliminary Clinical Evidence.

We determined the mitogen-activated protein kinase (MAPK) gene expression profile of acquired resistance in sorafenib-sensitive hepatocellular carcinoma (HCC) cells and aimed to identify c-Jun as an important molecule mediating the efficacy of sorafenib. Differences in gene expression of the MAPK signaling between untreated and sorafenib-treated HCC cell lines were investigated using real-time polymerase chain reaction array. Western blot and real-time PCR further evaluated the expression of c-Jun. Pathological specimens from 50 patients with advanced HCC were collected to measure p-c-Jun expression. Sorafenib-resistant HCC cells demonstrated greater levels of basal c-Jun mRNA and protein compared with sorafenib-sensitive HCC cells. Sorafenib activated p-c-Jun in a dose- and time-dependent manner in PLC/PRF/5 and MHCC97H cell lines. Decreased expression levels of 6 genes after sorafenib treatment suggested a robust inhibitory impact of sorafenib on MAPK signaling in HCC cells. c-Jun and p-c-Jun expression levels were inversely correlated with the efficacy of sorafenib; a high expression level of p-c-Jun was associated with resistance to sorafenib and poor overall survival in patients with clinical HCC. p-c-Jun may act as a biomarker for predicting responses of sorafenib treatment, thus advocating targeting of JNK/c-Jun signaling as an optimal therapeutic strategy in a subset of HCC.

WAVE3 Induces EMT and Promotes Migration and Invasion in Intrahepatic Cholangiocarcinoma

AbstractBackground Wiskott–Aldrich syndrome protein family verprolin-homologous protein 3 (WAVE3) plays a critical role in cancer progression and metastasis. However, the specific role of WAVE3 in intrahepatic cholangiocarcinoma (ICC) has not been studied.AimsThis study aimed to explore the role and mechanism of WAVE3 in the progression and metastasis of ICC.MethodsThe expression of WAVE3 in ICC tissues and adjacent non-cancerous tissues was detected by immunohistochemistry. Western blot analysis was utilized to detect the expression of WAVE3 in ICC cells. A transwell assay was used to assess the potential for migration and invasion. The expression of WAVE3 in CC-LP-1 cells was knocked down by small interfering RNA (siRNA) interference.ResultsThe expression of WAVE3 in ICC tissues was significantly higher than that in adjacent non-cancerous tissues. The overall survival was lower in the subgroup of ICC patients with higher WAVE3 expression compared to the subgroup with a lower level of WAVE3 expression. WAVE3 expression was an adverse prognostic factor for ICC patients. CC-LP-1 cells expressed higher levels of WAVE3 protein compared to RBE cells and human intrahepatic biliary epithelial cells, which correlated with greater migration and invasion capabilities compared with the RBE cells. After the transfection of CC-LP-1 cells with WAVE3 siRNA, the level of WAVE3 protein was significantly decreased, accompanied by a marked reduction in migration, invasion and proliferation. Moreover, after the knockdown of WAVE3 expression in CC-LP-1 cells, the protein levels of Slug and Vimentin were significantly decreased, while that of E-cadherin was significantly increased.ConclusionsWAVE3 may represent a new adverse prognostic factor for patients with ICC. This protein enhances migration and invasion capabilities in ICC, most likely through the induction of an epithelial-mesenchymal transition.

WAVE3 promotes proliferation, migration and invasion via the AKT pathway in pancreatic cancer

Alterations in Wiskott-Aldrich syndrome protein family verprolinhomologous protein 3 (WAVE3) expression play various roles in certain types of cancer. However, the roles of WAVE3 expression in pancreatic cancer remain unknown. The present retrospective study demonstrated that WAVE3 expression was higher in cancerous pancreatic tissues than in non-neoplastic tissues. Moreover, WAVE3 overexpression was related to lymphatic metastasis, a poor differentiation and high pre-operative CA19-9 levels and was an adverse prognostic factor for patients with pancreatic cancer. In vitro, the knockdown of WAVE3 inhibited the proliferative, migratory and invasive potential of pancreatic cancer cells and promoted cell apoptosis. Western blot analysis demonstrated that WAVE3 influenced the protein kinase B (PBK/AKT) pathway by suppressing the expression of pyruvate dehydrogenase kinase isoform 2 (PDK2) and then negatively inhibiting the phosphorylation of Ser473 on AKT. Furthermore, the expression of AKT pathway downstream proteins [certain epithelial-mesenchymal transition (EMT)-related proteins, p53, Bcl-2 and cyclin D1] was accordingly altered. Taken together, our findings suggest that WAVE3 influences cell proliferation, migration and invasion via the AKT pathway, and targeting WAVE3 and/or the AKT pathway may potentially serve as a treatment strategy for pancreatic cancer.

EYA4 inhibits hepatocellular carcinoma growth and invasion by suppressing NF-κB-dependent RAP1 transactivation

BackgroundOur previous studies demonstrated that eyes absent homolog 4 (EYA4), a member of the eye development-related EYA family in Drosophila, is frequently methylated and silenced in hepatocellular carcinoma (HCC) specimens and associated with shorter survival. The current work aimed to explore the mechanisms through which EYA4 functions as a tumor suppressor in HCC.MethodsStable EYA4-expressing plasmid (pEYA4) transfectants of the human HCC cell lines Huh-7 and PLC/PRF/5 (PLC) were established. Xenografts tumors were established via subcutaneous injection of the stable transfectants into BALB/c nude mice. Tissue samples were obtained from 75 pathologically diagnosed HCC patients. Quantitative real-time polymerase chain reaction, Western blotting and immunohistochemistry were performed to determine the expression of EYA4 in cell lines, xenografts and clinical specimens. The cell proliferation, colony formation, invasiveness and tumor formation of stable transfectants were studied. A gene expression microarray was utilized to screen genes regulated by EYA4 expression. The effect of EYA4 on nuclear factor-κB (NF-κB)/RAS-related protein 1 (RAP1) signaling was demonstrated through the co-transfection of pEYA4 and Flag-tagged RAS-related protein 1A gene-expressing plasmid (Flag-RAP1A), functional studies, chromatin immunoprecipitation, immunofluorescence staining and cellular ubiquitination assay.ResultsThe restoration of EYA4 expression in HCC cell lines suppressed cell proliferation, inhibited clonogenic outgrowth, reduced cell invasion and restrained xenograft tumor growth, and Flag-RAP1A reversed the suppressive effects of pEYA4 in vitro. Activation of NF-κB with tumor necrosis factor-α (TNF-α) increased the binding of p65 to the RAP1A gene promoter and up-regulated RAP1 protein expression. The inhibition of NF-κB with BAY 11-7085 and p65 siRNA successfully blocked TNF-α-induced RAP1 up-regulation. EYA4 antagonized the TNF-α-induced phosphorylation and ubiquitination of inhibitor of NF-κBα (IκBα) as well as the nuclear translocation and transactivation of p65, resulting in repressed NF-κB activity and RAP1 expression. Blocking the serine/threonine phosphatase activity of EYA4 with calyculin A notably abrogated its suppressive effect on NF-κB activity. In addition, EYA4 expression was inversely correlated with IκBα/RAP1 activity in clinical HCC specimens.ConclusionOur findings provide a functional and mechanistic basis for identifying EYA4 as a bona fide tumor suppressor that disrupts aberrant activation of the NF-κB/RAP1 signaling pathway and thus orchestrates a physiological impediment to HCC growth and invasion.

BTG2 Is Down-Regulated and Inhibits Cancer Stem Cell-Like Features of Side Population Cells in Hepatocellular Carcinoma.

BackgroundOur previous study found that B cell translocation gene 2 (BTG2) was hyper-methylated and down-regulated in side population (SP) cells of hepatocellular carcinoma (HCC) cell line. However, its clinical significances and biological impacts on HCC SP cells remained unclear.AimsTo investigate the prognostic value of BTG2 gene in HCC and its influences on cancer stem cells (CSCs)-like traits of HCC cell line SP cells.MethodsBTG2 expression in human HCC and adjacent non-cancerous tissues was detected by immunohistochemical staining and quantitative real-time PCR, and also obtained from GEO and TCGA data. Its prognostic values were assessed. Its biological influences on HCC cell line SP cells were evaluated using cell viability, cell cycle, plate clone-forming assay, and chemoresistance in vitro and tumorigenicity in vivo.ResultsBTG2 expression was significantly suppressed in human HCC compared to adjacent non-cancerous tissues. BTG2 expression was correlated with TNM stage, tumor size and vascular invasion. Lower expression of BTG2 was associated with poorer overall survival and disease-free survival. In vitro, overexpression of BTG2 substantially suppressed cell proliferation and accumulation of HCC cell line SP cells in G0/G1 phase. Colony formation ability was markedly suppressed by BTG2 overexpression. Moreover, sensitivity of HCC cell line SP cells to 5-fluorouracil was substantially increased by overexpression of BTG2. Furthermore, tumorigenicity of HCC cell line SP cells transfected with BTG2 plasmids was significantly reduced in vivo.ConclusionsBTG2 gene could regulate the CSC-like traits of HCC cell line SP cells, and it represented as a molecular prognostic marker for HCC.