title: C-Type Lectin-Like Receptor 2 Suppresses AKT Signaling and Invasive Activities of Gastric Cancer Cells by Blocking Expression of Phosphoinositide 3-Kinase Subunits.
abstract: C-type lectin-like receptor 2 (CLEC2) is a transmembrane receptor expressed on platelets and several hematopoietic cells. CLEC2 regulates platelet aggregation and the immune response. We investigated its expression and function in normal and transformed gastric epithelial cells from human tissues.
  
  
  
  We performed tissue microarray analyses of gastric carcinoma samples collected from 96 patients who underwent surgery at Zhongshan Hospital of Fudan University in Shanghai, China and performed real-time polymerase chain reaction assays from an independent group of 60 patients; matched nontumor gastric mucosa tissues were used as the control. Full-length and mutant forms of CLEC2 were expressed in gastric cancer cell line (MGC80-3), or CLEC2 protein was knocked down using small-hairpin RNAs in gastric cancer cell lines (NCI-N87 and AGS). CLEC2 signaling was stimulated by incubation of cells with recombinant human podoplanin or an antibody agonist of CLEC2; cell migration and invasion were assessed by transwell and wound-healing assays. Immunoblot, immunofluorescence microscopy, and real-time polymerase chain reaction assays were used to measure expression of markers of the epithelial to mesenchymal transition and activation of signaling pathways. Immunoprecipitation experiments were performed with an antibody against spleen tyrosine kinase (SYK). Cells were injected into lateral tail vein of BALB/C nude mice; some mice were also given injections of the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Lung and liver tissues were collected and analyzed for metastases.
  
  
  
  Levels of CLEC2 were higher in nontumor gastric mucosa (control) than in gastric tumor samples. Levels of CLEC2 protein in gastric tumor tissues correlated with depth of tumor invasion, metastasis to lymph node, tumor TNM stage, and 5-year survival of patients. Activation of CLEC2 in gastric cancer cells reduced their invasive activities in vitro and expression of epithelial to mesenchymal transition markers; these tumor-suppressive effects of CLEC2 required SYK. CLEC2 and SYK interacted physically, and SYK maintained the stability of CLEC2 in cells. AGS cells with CLEC2 knockdown had increased levels of phosphorylated AKT and glycogen synthase kinase-3 beta, increased expression of Snail, reduced levels of E-cadherin, and formed more metastases in mice than AGS cells that expressed CLEC2; these knockdown changes were prevented by the PI3K inhibitor LY294002. Activation of CLEC2 in AGS cells reduced protein and messenger RNA levels of PI3K subunits p85 and p110; this effect was blocked by SYK inhibitor R406. Levels of CLEC2 and SYK proteins and messenger RNAs correlated in gastric tumor samples.
  
  
  
  CLEC2 suppresses metastasis of gastric cancer cells injected into mice, and prevents activation of AKT and glycogen synthase kinase-3 beta signaling, as well as invasiveness and expression of epithelial to mesenchymal transition markers in gastric cancer cell lines. CLEC2 prevents expression of PI3K subunits, in a SYK-dependent manner.
authors: Key Laboratory of Glycoconjugate Research Ministry of Public Health, School of Basic Medical Sciences, Fudan University, Shanghai, PR China; Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, PR China; Institutes of Biomedical Sciences, Fudan University, Shanghai, PR China.
authors: Lan
authors: L
authors: Wang
authors: Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai, PR China.
authors: Jie
authors: J
authors: Yin
authors: Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai, PR China.
authors: Xuefei
authors: X
authors: Wang
authors: Key Laboratory of Glycoconjugate Research Ministry of Public Health, School of Basic Medical Sciences, Fudan University, Shanghai, PR China; Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, PR China.
authors: Miaomiao
authors: M
authors: Shao
authors: Key Laboratory of Glycoconjugate Research Ministry of Public Health, School of Basic Medical Sciences, Fudan University, Shanghai, PR China; Institutes of Biomedical Sciences, Fudan University, Shanghai, PR China.
authors: Fangfang
authors: F
authors: Duan
authors: Key Laboratory of Glycoconjugate Research Ministry of Public Health, School of Basic Medical Sciences, Fudan University, Shanghai, PR China; Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, PR China.
authors: Weicheng
authors: W
authors: Wu
authors: Key Laboratory of Glycoconjugate Research Ministry of Public Health, School of Basic Medical Sciences, Fudan University, Shanghai, PR China; Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, PR China.
authors: Peike
authors: P
authors: Peng
authors: Institute of Glycobiological Engineering, Zhejiang Provincial Key Laboratory of Medical Genetics, Wenzhou Medical University, Wenzhou, Zhejiang, PR China.
authors: Jing
authors: J
authors: Jin
authors: Key Laboratory of Glycoconjugate Research Ministry of Public Health, School of Basic Medical Sciences, Fudan University, Shanghai, PR China; Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, PR China.
authors: Yue
authors: Y
authors: Tang
authors: Key Laboratory of Glycoconjugate Research Ministry of Public Health, School of Basic Medical Sciences, Fudan University, Shanghai, PR China; Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, PR China. Electronic address: yuanyuanruan@fudan.edu.cn.
authors: Yuanyuan
authors: Y
authors: Ruan
authors: Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai, PR China. Electronic address: sun.yihong@zs-hospital.sh.cn.
authors: Yihong
authors: Y
authors: Sun
authors: Key Laboratory of Glycoconjugate Research Ministry of Public Health, School of Basic Medical Sciences, Fudan University, Shanghai, PR China; Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, PR China; Institutes of Biomedical Sciences, Fudan University, Shanghai, PR China. Electronic address: jxgu@shmu.edu.cn.
authors: Jianxin
authors: J
authors: Gu
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D058543:Class Ia Phosphatidylinositol 3-Kinase / Q000235:genetics / Q000378:metabolism*; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D000071679:Glycogen Synthase Kinase 3 beta / Q000378:metabolism; D006801:Humans; D037181:Lectins, C-Type / Q000235:genetics / Q000378:metabolism*; D008113:Liver Neoplasms / Q000201:enzymology / Q000517:prevention & control / Q000556:secondary; D008175:Lung Neoplasms / Q000201:enzymology / Q000517:prevention & control / Q000556:secondary; D008297:Male; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008875:Middle Aged; D009361:Neoplasm Invasiveness; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects; D013274:Stomach Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D000072377:Syk Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; C546928:CLEC1B protein, human; D037181:Lectins, C-Type; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D058543:Class Ia Phosphatidylinositol 3-Kinase; C000606896:SYK protein, human; D000072377:Syk Kinase; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D051057:Proto-Oncogene Proteins c-akt
keywords: DNA Methylation; Inflammatory Homeostasis; Spleen Tyrosine Kinase; Tumor Suppressor