title: TNF-alpha promotes lymphangiogenesis and lymphatic metastasis of gallbladder cancer through the ERK1/2/AP-1/VEGF-D pathway.
abstract: BACKGROUND
  Tumor necrosis factor-alpha (TNF-α), a key player in cancer-related inflammation, was recently demonstrated to be involved in the lymphatic metastasis of gallbladder cancer (GBC). Vascular endothelial growth factor D (VEGF-D) is a key lymphangiogenic factor that is associated with lymphangiogenesis and lymph node metastasis in GBC. However, whether VEGF-D is involved in TNF-α-induced lymphatic metastasis of GBC remains undetermined.
  
  
  METHODS
  The expression of VEGF-D in patient specimens was detected by immunohistochemistry and the relationship between VEGF-D in the tissue and TNF-α in the bile of the matching patients was analyzed. The VEGF-D mRNA and protein levels after treatment with exogenous TNF-α in NOZ, GBC-SD and SGC-996 cell lines were measured by real-time PCR and ELISA. The promoter activity and transcriptional regulation of VEGF-D were analyzed with the relative luciferase reporter assay, mutant constructs, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assay, RNA interference and Western blotting. Inhibitors of JNK, p38 MAPK and ERK1/2 were used to explore the upstream signaling effector of AP-1. We used lentiviral vector expressing a VEGF-D shRNA construct to knockdown VEGF-D gene in NOZ and GBC-SD cells. The role of the TNF-α-VEGF-D axis in the tube formation of human dermal lymphatic endothelial cells (HDLECs) was determined using a three-dimensional coculture system. The role of the TNF-α - VEGF-D axis in lymphangiogenesis and lymph node metastasis was studied via animal experiment.
  
  
  RESULTS
  TNF-α levels in the bile of GBC patients were positively correlated with VEGF-D expression in the clinical specimens. TNF-α can upregulate the protein expression and promoter activity of VEGF-D through the ERK1/2 - AP-1 pathway. Moreover, TNF-α can promote tube formation of HDLECs, lymphangiogenesis and lymph node metastasis of GBC by upregulation of VEGF-D in vitro and in vivo.
  
  
  CONCLUSIONS
  Taken together, our data suggest that TNF-α can promote lymphangiogenesis and lymphatic metastasis of GBC through the ERK1/2/AP-1/VEGF-D pathway.
authors.affiliation: Department of Hepatobiliary Surgery and Fujian Institute of Hepatobiliary Surgery, Fujian Medical University Union Hospital, 29 Xinquan Road, Fuzhou, 350001, China.|Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, 1 Xueyuan Road, Minhou, Fuzhou, 350108, China.
authors.forename: HaiJie
authors.initials: H
authors.lastname: Hong
authors.affiliation: Department of Hepatobiliary Surgery and Fujian Institute of Hepatobiliary Surgery, Fujian Medical University Union Hospital, 29 Xinquan Road, Fuzhou, 350001, China.|Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, 1 Xueyuan Road, Minhou, Fuzhou, 350108, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Jiang
authors.affiliation: Department of Hepatobiliary Surgery and Fujian Institute of Hepatobiliary Surgery, Fujian Medical University Union Hospital, 29 Xinquan Road, Fuzhou, 350001, China.|Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, 1 Xueyuan Road, Minhou, Fuzhou, 350108, China.
authors.forename: YanFei
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Department of Hepatobiliary Surgery and Fujian Institute of Hepatobiliary Surgery, Fujian Medical University Union Hospital, 29 Xinquan Road, Fuzhou, 350001, China.|Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, 1 Xueyuan Road, Minhou, Fuzhou, 350108, China.
authors.forename: CaiLong
authors.initials: C
authors.lastname: He
authors.affiliation: Department of Hepatobiliary Surgery and Fujian Institute of Hepatobiliary Surgery, Fujian Medical University Union Hospital, 29 Xinquan Road, Fuzhou, 350001, China.|Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, 1 Xueyuan Road, Minhou, Fuzhou, 350108, China.
authors.forename: GuangWei
authors.initials: G
authors.lastname: Zhu
authors.affiliation: Department of Hepatobiliary Surgery and Fujian Institute of Hepatobiliary Surgery, Fujian Medical University Union Hospital, 29 Xinquan Road, Fuzhou, 350001, China.|Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, 1 Xueyuan Road, Minhou, Fuzhou, 350108, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Du
authors.affiliation: Department of Hepatobiliary Surgery and Fujian Institute of Hepatobiliary Surgery, Fujian Medical University Union Hospital, 29 Xinquan Road, Fuzhou, 350001, China.
authors.forename: XiaoQian
authors.initials: X
authors.lastname: Wang
authors.affiliation: Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, 1 Xueyuan Road, Minhou, Fuzhou, 350108, China. shefeifei@yeah.net.|Fujian Key Laboratory of Tumor Microbiology, Fujian Medical University, 1 Xueyuan Road, Minhou, Fuzhou, 350108, China. shefeifei@yeah.net.
authors.forename: FeiFei
authors.initials: F
authors.lastname: She
authors.affiliation: Department of Hepatobiliary Surgery and Fujian Institute of Hepatobiliary Surgery, Fujian Medical University Union Hospital, 29 Xinquan Road, Fuzhou, 350001, China. drchenyl@126.com.|Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, 1 Xueyuan Road, Minhou, Fuzhou, 350108, China. drchenyl@126.com.
authors.forename: YanLing
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D005260:Female; D005706:Gallbladder Neoplasms / Q000235:genetics* / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D042583:Lymphangiogenesis / Q000235:genetics*; D008207:Lymphatic Metastasis; D020935:MAP Kinase Signaling System / Q000235:genetics; D008297:Male; D051379:Mice; D034622:RNA Interference; D018808:Transcription Factor AP-1 / Q000096:biosynthesis* / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism; D042643:Vascular Endothelial Growth Factor D / Q000096:biosynthesis* / Q000235:genetics; D023041:Xenograft Model Antitumor Assays
chemical_list: D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; C579459:VEGFD protein, human; D042643:Vascular Endothelial Growth Factor D
keywords: Gallbladder cancer; Lymphatic metastasis; TNF-α; VEGF-D