Interleukin-8 Induces Expression of FOXC1 to Promote Transactivation of CXCR1 and CCL2 in Hepatocellular Carcinoma Cell Lines and Formation of Metastases in Mice
Interleukin-8 Induces Expression of FOXC1 to Promote Transactivation of CXCR1 and CCL2 in Hepatocellular Carcinoma Cell Lines and Formation of Metastases in Mice
复制标题
Interleukin-8 诱导 FOXC1 表达，促进肝癌细胞系中 CXCR1 和 CCL2 的反式激活以及小鼠转移瘤的形成。
DOI：
10.1053/j.gastro.2015.05.058
复制
发表时间：
2015-10-01
期刊：
影响因子：
29.4
通讯作者：
Xia, Limin
中科院分区：
文献类型：
--
作者：
Huang, Wenjie;Chen, Zhangqian;Xia, Limin
背景和目标
白介素8调节的炎症反应促进肝细胞癌的转移。转录因子叉头盒C_1(FOXC_1)通过激活上皮细胞向间充质细胞的转化促进肿瘤转移；其在肝肿瘤中的水平与患者较短的生存期有关。我们研究了FOXC1是否激活了肝癌细胞系中的炎症信号通路。
方法
我们对人肝癌细胞系Huh-7和SMMC7721以及转移细胞系MHCC97H和HCCLM3进行了研究。用IL8孵育细胞，并检测报告基因的转录；细胞也用激酶抑制剂孵育。用小干扰信使RNA下调HuH7细胞中FOXC1或IL8的表达水平；体外分析细胞的迁移和侵袭能力。为研究肿瘤转移情况，将肝癌细胞接种于BALB/C裸鼠的腹侧，4周后收集皮下肿瘤组织块种植于裸鼠肝脏，测量肿瘤的数量和大小。染色质免疫沉淀法检测转录因子基因启动子区域的结合。我们用免疫组织化学方法检测了来自中国西京医院和同济医院的两组人肝癌组织(分别为690例和312例)中FOXC1I、IL8、CXCR1和CCL2的表达水平。
结果
肝癌细胞与IL8共同孵育后，通过激活AKT的肌醇磷脂3-激酶信号和缺氧诱导因子1α，导致FOXC1Foxc1表达增加。在过度表达IL8的肝癌细胞中，与没有FOXC1基因敲除的细胞相比，下调FOXC1基因可以减少小鼠体内形成的转移瘤的数量。在带有IL8基因敲除的肝癌细胞中过表达FOXC1，与不过表达FOXC1的细胞相比，在小鼠体内形成的转移瘤数量增加。CXCR1和CCL2是FOXC1的直接转录靶点。CXCR1和CCL2的联合敲除降低了过表达FOXC1的肝癌细胞的侵袭活性和小鼠肺转移的形成，而过表达CXCR1的转基因细胞增加了FOXC1敲除后的细胞侵袭和转移能力。从高表达FOXC1的细胞生长的肝转移被肿瘤相关的巨噬细胞渗透，而CCL2基因敲除减少了肿瘤相关的巨噬细胞的渗透；耗尽小鼠的巨噬细胞显著减少了高表达FOXC1的细胞的转移生长。在人肝细胞癌组织中，FOXC1水平与IL8、CXCR1、CCL2水平及巨噬细胞对肿瘤的侵袭密切相关。在多因素分析中，FOXC1和CCL2的检测是预测肝癌术后复发和总生存期的独立预测因素。
结论
在肝癌细胞系中，IL8通过向AKT和低氧诱导因子1α传递磷脂酰肌醇3-激酶信号来激活FOXc1的表达。FOXC1的表达导致CXCR1和CCL2的反式激活，促进炎症和肝癌细胞的侵袭和转移能力。
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.