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Interleukin-8 Induces Expression of FOXC1 to Promote Transactivation of CXCR1 and CCL2 in Hepatocellular Carcinoma Cell Lines and Formation of Metastases in Mice.
Gastroenterology. 2015 Oct; 149(4):1053-67.e14.G

Abstract

BACKGROUND & AIMS

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+Show Affiliations

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.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.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.Department of Gastroenterology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, People's Republic of China.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.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.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.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

26065367

Citation

Huang, Wenjie, et al. "Interleukin-8 Induces Expression of FOXC1 to Promote Transactivation of CXCR1 and CCL2 in Hepatocellular Carcinoma Cell Lines and Formation of Metastases in Mice." Gastroenterology, vol. 149, no. 4, 2015, pp. 1053-67.e14.
Huang W, Chen Z, Zhang L, et al. Interleukin-8 Induces Expression of FOXC1 to Promote Transactivation of CXCR1 and CCL2 in Hepatocellular Carcinoma Cell Lines and Formation of Metastases in Mice. Gastroenterology. 2015;149(4):1053-67.e14.
Huang, W., Chen, Z., Zhang, L., Tian, D., Wang, D., Fan, D., Wu, K., & Xia, L. (2015). Interleukin-8 Induces Expression of FOXC1 to Promote Transactivation of CXCR1 and CCL2 in Hepatocellular Carcinoma Cell Lines and Formation of Metastases in Mice. Gastroenterology, 149(4), 1053-e14. https://doi.org/10.1053/j.gastro.2015.05.058
Huang W, et al. Interleukin-8 Induces Expression of FOXC1 to Promote Transactivation of CXCR1 and CCL2 in Hepatocellular Carcinoma Cell Lines and Formation of Metastases in Mice. Gastroenterology. 2015;149(4):1053-67.e14. PubMed PMID: 26065367.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Interleukin-8 Induces Expression of FOXC1 to Promote Transactivation of CXCR1 and CCL2 in Hepatocellular Carcinoma Cell Lines and Formation of Metastases in Mice. AU - Huang,Wenjie, AU - Chen,Zhangqian, AU - Zhang,Lin, AU - Tian,Dean, AU - Wang,Daowen, AU - Fan,Daiming, AU - Wu,Kaichun, AU - Xia,Limin, Y1 - 2015/06/09/ PY - 2014/09/14/received PY - 2015/05/03/revised PY - 2015/05/30/accepted PY - 2015/6/13/entrez PY - 2015/6/13/pubmed PY - 2015/12/30/medline KW - Chemokine (C-C Motif) Ligand 2 KW - Chemokine (C-X-C Motif) Receptor 1 KW - Liver Cancer KW - Mouse Model SP - 1053 EP - 67.e14 JF - Gastroenterology JO - Gastroenterology VL - 149 IS - 4 N2 - BACKGROUND & AIMS: 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. SN - 1528-0012 UR - https://www.unboundmedicine.com/medline/citation/26065367/Interleukin_8_Induces_Expression_of_FOXC1_to_Promote_Transactivation_of_CXCR1_and_CCL2_in_Hepatocellular_Carcinoma_Cell_Lines_and_Formation_of_Metastases_in_Mice_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0016-5085(15)00787-8 DB - PRIME DP - Unbound Medicine ER -