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Liu J.,Key Laboratory of Glycoconjugate Research | Liu J.,Nantong University | Liu H.,Key Laboratory of Glycoconjugate Research | Zhang W.,Fudan University | And 6 more authors.

Elevated expression and activity of N-acetylglucosaminyltransferase V (Mgat5) in hepatocellular carcinoma (HCC) is a common early event involved in tumor invasion during hepatocarcinogenesis. A better understanding of the functional role and the molecular mechanism for Mgat5-targeted protein and downstream signaling pathway behind hepatoma invasion and metastasis is urgently needed. Here, we show that Mgat5 overexpression promoted anchorage-independent growth and inhibited anoikis in hepatoma cells. This effect was reversed by glycosyltransferase inactive mutant Mgat5 L188R transfection, α-mannosidase II inhibitor swainsonine treatment and N-acetyl glucosamine (GlcNAc) phosphotransferase (GPT) inhibitor tunicamycin administration. Mgat5 overexpression increased p21-activated kinase 1 (PAK1) expression and shRNA-mediated PAK1 knockdown and kinase inactivation with kinase dead mutant PAK1 K299R coexpression or allosteric inhibitor P21-activated kinase inhibitor III (IPA3) treatment reversed anoikis resistance in Mgat5-overexpressed hepatoma cells. Furthermore, Mgat5 overexpression upregulated β-1-6-GlcNAc branched N-glycosylation and following phosphorylation of epidermal growth factor receptor (EGFR) in hepatoma cells. EGFR tyrosine kinase inhibitors AG1478 and Iressa treatment declined anchorage-independent growth and anoikis resistance, which could be rescued by constitutive active mutant PAK1 T423E coexpression in Mgat5-overexpressed hepatoma cells. Conversely, knockdown of Mgat5 reduced EGFR/PAK1-dependent anoikis resistance, which could be reversed by PAK1 T423E. These results identified Mgat5-mediated β-1-6-GlcNAc branched N-glycosylation and following activation of EGFR as a potential novel upstream molecular event for PAK1-induced anoikis resistance in hepatoma cells, implicating that molecular targeted therapeutics against Mgat5/EGFR/PAK1 might open a new avenue for personalized medicine in advanced-stage HCC patients. © The Author 2013. Published by Oxford University Press. Source

Wu Q.,Key Laboratory of Glycoconjugate Research | Liu H.-O.,Key Laboratory of Glycoconjugate Research | Liu Y.-D.,Key Laboratory of Glycoconjugate Research | Liu W.-S.,Key Laboratory of Glycoconjugate Research | And 6 more authors.
Journal of Biological Chemistry

MicroRNA-122 (miR-122), a mammalian liver-specific miRNA, has been reported to play crucial roles in the control of diverse aspects of hepatic function and dysfunction, including viral infection and hepatocarcinogenesis. In this study, we explored the clinical significance, transcriptional regulation, and direct target of miR-122 in hepatitis B virus (HBV)-associated hepatocellular carcinoma. Reduced expression of miR-122 in patients with HBV-associated hepatocellular carcinoma was correlated with venous invasion and poor prognosis. Furthermore, UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetyl-galactosaminyltransferase-10 (GALNT10) was identified as a bona fide target of miR-122 in hepatoma cells. Ectopic expression and knockdown studies showed thatGALNT10indeed promotes proliferation and apoptosis resistance of hepatoma cells in a glycosyltransferase-dependent manner. Critically, adverse correlation between miR-122 and GALNT10, a poor prognosticator of clinical outcome, was demonstrated in hepatoma patients. Hepatocyte nuclear factor 4α (Hnf4α), a liver-enriched transcription factor that activates miR-122 gene transcription, was suppressed in HBV-infected hepatoma cells. Chromatin immunoprecipitation assay showed significantly reduced association of Hnf4α with the miR-122 promoter in HBV-infected hepatoma cells. Moreover, GALNT10 was found to intensify O-glycosylation following signal activation of the epidermal growth factor receptor. In addition, in a therapeutic perspective, we proved that GALNT10 silencing increases sensitivity to sorafenib and doxorubicin challenge. In summary, our results reveal a novel Hnf4α/miR-122/GALNT10 regulatory pathway that facilitates EGF miR-122 activation and hepatoma growth in HBV-associated hepatocarcinogenesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Source

An H.,Fudan University | Zhu Y.,Ninth Peoples Hospital | Xu L.,Fudan University | Chen L.,Fudan University | And 2 more authors.

Objective To evaluate the prognostic significance of Notch1 activation in patients with clear-cell renal cell carcinoma (ccRCC). Methods We retrospectively enrolled 203 patients with ccRCC undergoing nephrectomy at Zhongshan Hospital of Fudan University between 2003 and 2004. Notch1 activation was assessed by immunohistochemical staining of the intracellular domain of Notch1 (ICN1) in specimens of patients. The Kaplan-Meier method, Cox regression models, and Harrell concordance index (C-index) calculation were used to evaluate the prognostic value of ICN1 expression and its association with clinicopathologic features. Results Tumor tissues from patients with advanced TNM stage and Fuhrman grade exhibited elevated ICN1 expression, which correlated positively with tumor size, Fuhrman grade, and tumor necrosis. Moreover, high ICN1 expression indicated poor overall survival and recurrence-free survival of patients with ccRCC. After backward elimination, ICN1 expression, as well as Fuhrman grade, Eastern Cooperative Oncology Group performance status, and TNM stage, was identified as an independent adverse prognostic factor for survival and recurrence. The predictive accuracy of well-established TNM, University of California Integrated Staging System, and Mayo Clinic stage, size, grade, and necrosis prognostic models was improved when ICN1 expression was added. Furthermore, a predictive nomogram was generated with identified independent prognosticators to assess patient survival at 5 years after surgery. Conclusion Notch1 activation is a potential independent adverse prognostic biomarker for recurrence and survival of patients with ccRCC after nephrectomy. © 2015 Elsevier Inc. All Rights Reserved. Source

Li X.,Fudan University | Li Z.,Fudan University | Li Z.,Key Laboratory of Molecular Medicine | Li N.,Fudan University | And 13 more authors.
International Journal of Molecular Medicine

Adaptor proteins are involved in the assembly of various intracellular complexes and the regulation of cellular functions. Membrane-associated guanylate kinase inverted 2 (MAGI2), also known as synaptic scaffolding molecule (S-SCAM), plays a critical role in signal transduction by assembling and anchoring its ligands. However, the role of MAGI2 in mediating apoptosis remains largely unknown. In the present study, BEL-7404 human hepatocellular carcinoma cells were transfected with a plasmid containing myc-MAGI2 or an empty plasmid and cell viability was then determined using the Cell Counting kit-8. Apoptosis was also detected using an Annexin V apoptosis assay. The cells were then treated with various doses of staurosporine (STS) for different periods of time. The overexpression of myc-MAGI2 was found to sensitize the BEL-7404 cells to apoptosis in response to STS in a time- and dose-dependent manner. Our results demonstrated that MAGI2 enhanced STS-induced apoptosis by increasing the protein expression of cytoplasmic phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and decreasing its protein degradation. The apoptotic sensitivity of the cells caused by the overexpression of myc-MAGI2 was reversed by the silencing of PTEN expression by PTEN siRNA, thus revealing a momentous role of PTEN in the enhancement of the sensitivity of cancer cells to STS-induced apoptosis by MAGI2. Finally, we observed that the MAGI-PTEN complex triggered by MAGI2 overexpression reduced the phosphorylation levels of AKT. These results suggest that MAGI2 overexpression enhances the sensitivity of cancer cells harboring ectopic PTEN to STS-induced apoptosis. Source

Zhang H.,Fudan University | Wang X.,Fudan University | Shen Z.,Fudan University | Xu J.,Key Laboratory of Glycoconjugate Research | And 3 more authors.
Gastric Cancer

Background: Tumor-associated macrophages (TAMs), the most predominant tumor-infiltrating immune cells, are emerging prognostic factors and therapeutic targets for personalized therapy against malignant neoplasms. We aimed to evaluate the prognostic significance of diametrically polarized TAMs in gastric cancer and generate a predictive nomogram to refine a risk stratification system. Methods: We evaluated polarized functional status of infiltrated TAMs by immunohistochemical staining of CD68, CD11c, and CD206 in 180 consecutive gastric cancer patients from Zhongshan Hospital, Shanghai, China. Prognostic values were assessed in these patients. We created a predictive nomogram by integrating polarized TAMs with the TNM staging system for overall survival of gastric cancer patients. Results: CD68+ TAMs display polarized programs comprising CD11c+ proinflammatory macrophages (M1) and CD206+ immunosuppressive macrophages (M2) that configure versatile infiltration files in gastric cancer. CD11c+ TAMs negatively correlated with lymph node metastasis (p = 0.012), whereas CD206+ TAMs correlated with the Lauren classification (p = 0.031). No prognostic difference was observed for overall survival for CD68 density (high vs low, p = 0.1031), whereas high versus low CD11c density (p < 0.0001) and low vs high CD206 density (p = 0.0105) indicate better overall survival. Multivariate Cox regression analysis identified CD11c and CD206 as independent prognostic factors (p < 0.001 and p = 0.030, respectively), which could be integrated with the TNM staging system to generate a predictive nomogram for patient outcomes. Conclusion: Infiltration of polarized TAMs, a novel identified independent prognostic factor, could be combined with the TNM stage to refine a risk stratification system and better stratify patients with different prognosis. Tipping TAMs to an antitumoral phenotype might be a promising therapeutic target for postoperative treatment. © 2014, The International Gastric Cancer Association and The Japanese Gastric Cancer Association. Source

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