title: CCR7 mediates the TNF-α-induced lymphatic metastasis of gallbladder cancer through the "ERK1/2 - AP-1" and "JNK - AP-1" pathways.
abstract: BACKGROUND
  CC-chemokine receptor 7 (CCR7), which plays an important role in cell directional movement, is highly expressed in various cancers and positively related to lymph node metastasis. The inflammatory cytokine tumour necrosis factor (TNF)-α promotes tumour progression and lymph node metastasis in gallbladder cancer (GBC). However, the expression of CCR7 in GBC is unclear, and its role in the TNF-α-induced lymphatic metastasis of GBC requires further research.
  
  
  METHODS
  The expression of CCR7 in clinical samples was detected by immunohistochemistry, and the relationship between CCR7 and clinicopathological factors or the TNF-α level of the bile was analyzed. After treatment with various concentrations of TNF-α, CCR7 expression in GBC cell lines was measured by Western blotting. The relative luciferase reporter assay, site-directed mutagenesis and chromatin immunoprecipitation were used to analyze the promoter activity and transcriptional regulation of CCR7. MAPKs inhibitors were used to explore the upstream signalling molecules of AP-1. We established a NOZ cell line stably expressing lentiviral CCR7 shRNA that effectively silenced the expression of CCR7, and to determine the role of TNF-α - CCR7 axis in the migration of GBC cells to the lymphatic system by transwell assays and animal experiments.
  
  
  RESULTS
  CCR7 was highly expressed in GBC samples. Higher expression of CCR7 was associated with American Joint Committee on Cancer (AJCC) staging and lymph node metastasis. Moreover, we found that CCR7 expression in GBC tissue was positively correlated with the levels of TNF-α in the bile, and that TNF-α enhanced the promoter activity and protein expression of CCR7 through the "ERK1/2-AP-1" and "JNK-AP-1" pathways. Finally, we revealed that TNF-α could promote GBC cell migration to lymphatic endothelial cells or lymph nodes through upregulation of CCR7 in vitro and in vivo.
  
  
  CONCLUSIONS
  Our study suggests that CCR7 is highly expressed in GBC, and mediates the TNF-α-induced lymphatic metastasis of GBC through the "TNF-α - ERK1/2 - AP-1 - CCR7" and "TNF-α - JNK - AP-1 - CCR7" pathways.
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: 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: SiYuan
authors.initials: S
authors.lastname: Zhu
authors.affiliation: Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, 1 Xueyuan Road, Minhou, Fuzhou, 350108, China.
authors.forename: YanHui
authors.initials: Y
authors.lastname: Zhang
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; D002465:Cell Movement; D005260:Female; D005706:Gallbladder Neoplasms / Q000378:metabolism* / Q000473:pathology*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008207:Lymphatic Metastasis; D020935:MAP Kinase Signaling System; D008297:Male; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009374:Neoplasms, Experimental; D011401:Promoter Regions, Genetic; D054400:Receptors, CCR7 / Q000235:genetics / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: C515370:CCR7 protein, human; D054400:Receptors, CCR7; C439524:TNF protein, human; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha
keywords: CCR7; Cancer-related inflammation; Gallbladder cancer; Lymphatic metastasis; TNF-α