Jian-Peng Cai

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.

Characterizing the Activation of the Wnt Signaling Pathway in Hilar Cholangiocarcinoma Using a Tissue Microarray Approach

Hilar cholangiocarcinoma (HCCA) is an invasive hepatic malignancy that is difficult to biopsy; therefore, novel markers of HCCA prognosis are needed. Here, the level of canonical Wnt activation in patients with HCCA, intrahepatic cholangiocarcinoma (IHCC), and congenital choledochal cysts (CCC) was compared to understand the role of Wnt signaling in HCCA. Pathology specimens from HCCA (n=129), IHCC (n=31), and CCC (n=45) patients were used to construct tissue microarrays. Wnt2, Wnt3, β-catenin, TCF4, c-Myc, and cyclin D1 were detected by immunohistochemistry. Parallel correlation analysis was used to analyze differences in protein levels between the HCCA, IHCC, and CCC groups. Univariate and multivariate analyses were used to determine independent predictors of successful resection and prognosis in the HCCA group. The protein levels of Wnt2, β-catenin, TCF4, c-Myc, and cyclin D1 were significantly higher in HCCA compared to IHHC or CCC. Wnt signaling activation (Wnt2+, Wnt3+, nuclear β-catenin+, nuclear TCF4+) was significantly greater in HCCA tissues than CCC tissues. Univariable analyses indicated that expression of cyclin D1 as well as Wnt signaling activation, and partial Wnt activation (Wnt2+ or Wnt3+ and nuclear β-catenin+ or nuclear TCF4+) predicted successful resection, but only cyclin D1 expression remained significant in multivariable analyses. Only partial Wnt activation was an independent predictor of survival time. Proteins in the canonical Wnt signaling pathway were present at higher levels in HCCA and correlated with tumor resecility and patient prognosis. These results suggest that Wnt pathway analysis may be a useful marker for clinical outcome in HCCA.

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.