title: Interleukin-8 Induces Expression of FOXC1 to Promote Transactivation of CXCR1 and CCL2 in Hepatocellular Carcinoma Cell Lines and Formation of Metastases in Mice.
abstract: OBJECTIVE
  Inflammation regulated by interleukin (IL) 8 promotes metastasis of hepatocellular carcinoma (HCC). The transcription factor forkhead box C1 (FOXC1) promotes metastasis by activating the epithelial to mesenchymal transition; its levels in liver tumors have been associated with shorter survival times of patients. We investigated whether FOXC1 activates inflammation signaling pathways in HCC cell lines.
  
  
  METHODS
  We performed studies in the human HCC cell lines Huh-7 and SMMC7721, as well as the metastatic cell lines MHCC97H and HCCLM3. Cell lines were incubated with IL8 and transcription of reporter genes was measured; cells were also incubated with kinase inhibitors. Levels of FOXC1 or IL8 were knocked down with small interfering messenger RNAs in Huh7 cells; cells were analyzed in vitro in migration and invasion assays. To study metastasis, HCC cells were injected into flanks of BALB/C nude mice; 4 weeks later, the subcutaneous tumor fragments were collected and implanted into livers of the nude mice, and number and size tumors formed were measured. Chromatin immunoprecipitation assays were used to measure binding of transcription factors promoter regions of genes. We measured levels of FOXC1, IL8, CXCR1, and CCL2 in 2 groups of human HCC tissues collected from the Xijing or Tongji Hospitals in China (n = 690 and n = 312 samples, respectively) using immunohistochemistry.
  
  
  RESULTS
  Incubation of HCC cells with IL8 led to increased expression of FOXC1, via activation of phosphoinositide 3-kinase signaling to AKT and hypoxia-inducible factor 1α. Knockdown of FOXC1 in HCC cells that overexpressed IL8 reduced the numbers of metastases formed in mice, compared with cells without FOXC1 knockdown. Transgenic overexpression of FOXC1 in HCC cells with IL8 knockdown increased the numbers of metastases formed in mice compared with cells without FOXC1 overexpression. CXCR1 and CCL2 were direct transcriptional targets of FOXC1. Knockdown of the combination of CXCR1 and CCL2 reduced the invasive activities of HCC cells that overexpress FOXC1 and formation of lung metastases in mice, and transgenic overexpression of CXCR1 increased cell's invasive and metastatic abilities after knockdown of FOXC1. Liver metastases grown from cells that overexpressed FOXC1 were infiltrated by tumor-associated macrophages, and CCL2 knockdown decreased tumor-associated macrophage infiltration; depletion of macrophages from mice significantly reduced growth of metastases by cells that overexpressed FOXC1. In human HCC tissues, level of FOXC1 correlated with levels of IL8 and CXCR1 and CCL2 and infiltration of tumors by macrophage. In multivariate analysis, detection of FOXC1 and CCL2 were independent predictors for postoperative recurrence of HCC and overall survival.
  
  
  CONCLUSIONS
  In HCC cell lines, IL8 activates expression of FOXC1 via the phosphoinositide 3-kinase signaling to AKT and hypoxia-inducible factor 1α. FOXC1 expression leads to transactivation of CXCR1 and CCL2, promoting inflammation and the invasive and metastatic abilities of HCC cells.
authors.affiliation: State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, Shaanxi Province, People's Republic of China; Department of Gastroenterology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, People's Republic of China.
authors.forename: Wenjie
authors.initials: W
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, Shaanxi Province, People's Republic of China.
authors.forename: Zhangqian
authors.initials: Z
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, Shaanxi Province, People's Republic of China.
authors.forename: Lin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Gastroenterology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, People's Republic of China.
authors.forename: Dean
authors.initials: D
authors.lastname: Tian
authors.affiliation: Department of Gastroenterology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, People's Republic of China.
authors.forename: Daowen
authors.initials: D
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, Shaanxi Province, People's Republic of China.
authors.forename: Daiming
authors.initials: D
authors.lastname: Fan
authors.affiliation: State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, Shaanxi Province, People's Republic of China.
authors.forename: Kaichun
authors.initials: K
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, Shaanxi Province, People's Republic of China; Department of Gastroenterology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, People's Republic of China. Electronic address: xialimin@fmmu.edu.cn.
authors.forename: Limin
authors.initials: L
authors.lastname: Xia
mesh_terms: D000818:Animals; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000556:secondary; D002465:Cell Movement; D018932:Chemokine CCL2 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018920:Coculture Techniques; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D056945:Hep G2 Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D018836:Inflammation Mediators / Q000276:immunology / Q000378:metabolism*; D016209:Interleukin-8 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008113:Liver Neoplasms / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D008264:Macrophages / Q000276:immunology / Q000378:metabolism; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009361:Neoplasm Invasiveness; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D023062:Receptors, Interleukin-8A / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: C111835:CCL2 protein, human; C508606:CXCL8 protein, human; D018932:Chemokine CCL2; C112743:FOXC1 protein, human; D051858:Forkhead Transcription Factors; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D018836:Inflammation Mediators; D016209:Interleukin-8; D023062:Receptors, Interleukin-8A; D058539:Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt
keywords: Chemokine (C-C Motif) Ligand 2; Chemokine (C-X-C Motif) Receptor 1; Liver Cancer; Mouse Model