Interleukin 17A Promotes Hepatocellular Carcinoma Metastasis via NF-kB Induced Matrix Metalloproteinases 2 and 9 Expression

et al. (2011) Interleukin 17A Promotes Hepatocellular Carcinoma Metastasis via NF-kB Induced Matrix Metalloproteinases 2 and 9 Expression. PLoS ONE 6(7): e21816. doi:10.1371/journal.pone.0021816 Interleukin 17A Promotes Hepatocellular Carcinoma Metastasis via NF-kB Induced Matrix Metalloproteinases 2 and 9 Expression Jian Li George Ka-Kit Lau Leilei Chen Sui-sui Dong Hui-Yao Lan Xiao-Ru Huang Yan Li John M. Luk Yun-Fei Yuan Xin-yuan Guan Terence Lee, University of Hong Kong, Hong Kong Background: IL-17A is a pro-inflammatory cytokine that plays important role in inflammatory disease pathology and tumor microenvironment. The aim of this study is to investigate the effect of IL-17A on the progression of hepatocellular carcinoma (HCC). Methodology and Principal Finding: Expression pattern of IL-17A in clinical HCC samples (n = 43) was determined by immunohistochemistry staining. Transcript levels of MMP2, MMP9 and IL-17A were measured in another 50 pairs (including tumor and related non-tumor tissues) HCC samples. Cell growth, focus formation, cell migration, invasion and western blot assays were used to characterize the functional and signaling mechanisms in IL-17A-treated HCC. Association study was used to identify clinical significance of IL-17A in HCC. Compared with paired non-tumor tissue, higher frequency of IL-17Apositive cells was detected in tumor tissues in HCCs with metastasis, and the frequency of IL-17A-positive cells was also significantly associated with poor prognosis of HCC (P = 0.01). Functional study found that IL-17A could promote HCC cell migration and invasion. Further molecular analysis also showed that IL-17A could upregulate MMP2 and MMP9 expression via NF-kB signaling activation. Conclusions: IL-17A could promote HCC metastasis by the upregulation of MMP2 and MMP9 expression via activating NF-kB signaling pathway. - Funding: This work was supported by a Hong Kong Research Grant Council Grant (HKU 7656/07M), Hong Kong RGC Collaborative Research Grants (HKU5/CRF/08 and HKU7/CRG09) and the Hundred Talents Program at Sun Yat-sen University (85000-3171311). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Hepatocellular carcinoma (HCC) is the fifth most common cancer word wide and it is also one of the poorest prognosis tumors in the world [1]. HCC often develops from chronic liver inflammation environment where plenty of leukocytes infiltrate [1,2]. Recent studies find that immune cells and their secreted cytokines can not only contribute to the elimination of cancer cells, they could also provide a proper microenvironment for tumor development as well as promote tumor progression [3,4], which is determined by the local tumor microenviroment and the function state of immune cells. For example, IFN-c producing Th1 and CD8+ cytoxtic cells are associated with good prognosis [5], while interleukin 10 (IL-10) and TGF-b producing regulatory T cells are associated with poor prognosis in HCC [6,7]. Another interesting example is the effects of macrophages on tumor development, which mainly depends on whether they secrete anti-tumor factors IL-12 and TNF-a, or pro-tumor factors IL-10, VEGF, PDGF, CXCL8, MMP-9 and TGF-b [4,8]. Interleukin 17A (IL-17A) is a pro-inflammatory cytokine secreted by helper T cells (Th17), CD8 positive T cells, neutrophils, gamma/delta T cells and NK cells [912]. IL-17A has been found to play important role in many chronic diseases such as rheumatoid arthritis [13], inflammatory bowel disease [14] and multiple sclerosis [15]. Recently IL-17A has been also frequently detected in many cancers such as ovarian cancer [16], breast cancer [17] and gastric cancer [18]. The role of IL-17A in the development and progression of cancer remains controversial. Using animal model, some studies find that IL-17A can inhibit tumor growth and metastasis through IFN-c producing NK and T cells [19,20]. While other studies show that IL-17A can promote tumor growth and metastasis through IL-6/Stat3 signaling pathway [21] or through the induction of tumor promoting microenvironment at tumor site [22]. In the present study, we found that IL-17A was frequently overexpressed in HCC with metastasis. The frequency of IL-17Apositive cells in tumor tissue was associated with HCC metastasis and prognosis. Further study found that IL-17A could increase cell motility by the upregulation of matrix metalloproteinases 2 (MMP2) and 9 (MMP9) via activating nuclear factor-kB (NF-kB) transcript factor. IL-17A-positive cells were associated with HCC metastasis The number and distribution of IL-17A-positive cells were compared by IHC staining between primary HCC specimens with and without metastasis. IL-17A-positive cells could be detected in both tumor and adjacent non-tumorous tissues (Figure 1A). In 21 HCC cases without metastasis, no significant difference (P = 0.391, paired-samples T test) was observed in the frequency of IL17A-positive cells between tumor (mean: 157698 cells, in 10 continuous fields under 4006 microscopy) and adjacent non-tumorous tissues (mean: 114622) (Figure 1B). In 22 HCC cases with metastasis, the frequency of IL-17A-positive cells was significantly higher (P = 0.001, paired-samples T test) in tumor tissue (mean: 5166182) than that in adjacent non-tumorous tissues (mean: 164631, Figure 1B). The frequency of IL-17A-positive cells in tumor tissues was significantly higher in HCC cases with metastasis than that without metastasis (P = 0.002, Independent sample T test). Interestingly, the frequency of IL-17A-positive cells in adjacent non-tumorous tissue was also higher in HCC cases with metastasis than that without metastasis (P = 0.013, Independent sample T test) (Figure 1B). IL-17A-positive cells were associated with poor prognosis of HCC To investigate the correlation of the frequency of IL-17Apositive cells in tumor tissue with clinic pathological features, the mean of IL-17A-positive cells in tumor tissue in 43 HCC cases was calculated. According to whether the frequency of IL-17A-positive cells was above the mean level (341 cells) in tumor tissue or not, HCC cases in the present study were divided into two groups: IL17A-high group (above the mean level, n = 15) and IL-17A-low group (below the mean level, n = 28). The association study showed that the frequency of IL-17A-positive cells in tumor tissue was not significantly associated with patients gender, age, HBV infection, cirrhosis, tumor size and TNM stage (Table 1). Interestingly, high frequency of IL-17A-positive cells in tumor tissue was significantly associated with patients metastasis (P = 0.002, Table 1), overall survival rate (P = 0.01, Figure 1C) and disease-free survival rate (P = 0.03, Figure 1D). Univatiate and multivariate Cox progression analysis were performed and the results showed that the frequency of I (...truncated)