IL‐17A promotes cell migration and invasion of glioblastoma cells via activation of PI3K/AKT signalling pathway

Abstract Glioblastomas (GBMs) are the most common of both benign and malignant primary brain tumours, in which the inflammatory and immunologic abnormalities are involved. Interleukin‐17A (IL‐17A) plays an important role in various inflammatory diseases and cancers. Several recent studies revealed that the expression of IL‐17A was overexpressed in human GBMs tissue. However, the accurate role of IL‐17A in GBMs remains unclear. In this study, we aimed to explore the effect of IL‐17A on cell migration and invasion of GBMs and the mechanism by which the effects occurred. We found that exogenous IL‐17A promoted significantly cell migration and invasion abilities in two GBMs cell lines (U87MG and U251) in a time‐dependent manner. In addition, the protein expressions of PI3K, Akt and MMP‐2/9 were increased in the GBMs cells challenged by IL‐17A. Furthermore, a tight junction protein ZO‐1 was down‐regulated but Twist and Bmi1 were up‐regulated. Treatment with a PI3K inhibitor (LY294002) significantly reduced the abilities of both migration and invasion in U87MG and U251 cells. LY294002 treatment also attenuated the IL‐17A causing increases of protein levels of PI3K, AKT, MMP‐2/9, Twist and the decreases of protein level of ZO‐1 in the U87MG and U251 cells. Taken together, we concluded that IL‐17A promotes the GBM cells migration and invasion via PI3K/AKT signalling pathway. IL‐17A and its related signalling pathways may be potential therapeutic targets for GBM.


| INTRODUCTION
Glioblastoma, known as glioblastoma multiforme (GBM), is the most common type of primary malignant tumours in adult brain 1,2 with a mean survival rate ranging from 13.2 to 14.6 months 3 and a 5-year survival rate of 9.8%. 4 Approximately 23 000 cases were newly diagnosed with GBM each year, and patients' age has been identified as the most important prognostic factor for survival in patients with GBM in the United States. 5,6 Prognosis for patients with GBM is extremely poor because of high invasiveness of GBM. Thus, to elucidate the molecular mechanisms underlying the migration and invasion of GBM will be a prerequisite for providing novel therapeutic strategies against human GBM.
A large amount of studies tried to investigate the role of immune regulatory factors in GBM. Interleukin-17A (IL-17A) is produced by the main cytokine effector of T helper (Th17) cells and has been evoked the interest since the identification of Th17 in immunology. 7,8 Th17 cells, a recently discovered IL-17A-producing T cell subtype, have been reported in several extracranial and some intracranial tumours. IL-17A has been shown to promote tumour development by several different mechanisms, including the enhancement of HIF-1α expression induced by TNF-α, the inhibited VASP expression 9 and the activation of the IL-6-STAT3 signalling pathway. 10 Recently, IL-17A is reported to promote the migration and invasion of colorectal cancer cells by the up-regulated the expression of MMP-2/9 mediated by NF-κB signalling. 11 It improves the transition from chronic pancreatitis to pancreatic cancer through the novel REG3β-JAK2-STAT3 inflammatory pathway. 12 IL-17A is frequently observed in many cancers such as ovarian cancer, 13 gastric cancer, 14 and hepatocellular carcinoma, 15 and being reported to associate with a poor clinical outcome in patients with GBM. 16 Higher level of IL-17A is found in glioma tissues as compared with trauma tissues, [17][18][19] however, the role of IL-17A in migration and invasion of GBM cells is not fully illuminated and the mechanism remains unclear.
In the present study, we found that IL-17A expression was upregulated in human GBM tissues. Exogenous IL-17A can increase cell motility of GBM cells and up-regulate MMP-2/9 expression via PI3K/ AKT signalling pathway. These data indicate that IL-17A signalling in gliomas may be the potential therapeutic target for GBM. quantified by staining score (0-12) in GBM tissues and adjacent normal tissues. The levels of IL-17A in GBM tissues are significantly higher than those in adjacent normal tissues. (C) Relative expression of IL-17A mRNA in GBM tissues and paired adjacent tissues was determined by qPCR method. The mRNA level of IL-17A in GBM tissues is significantly higher than that in adjacent normal tissues. (D) Expression levels of IL-17A protein in three adjacent tissues, three WHO I-II grade tissues and three WHO III-IV grade tissues were determined with Western Blot method. Relative expression density of IL-17A was quantified against β-actin which was used as the inner control. IL-17A expression was higher in GBM tissues than that in adjacent normal tissues. All data are shown as mean ± SD. *P < 0.05, **P < 0.

| Immunohistochemistry
Briefly, the tissue sections were consecutively deparaffinized in xylene (I, II and III) and rehydrated using a graded series of alcohol (100% alcohol, 95% alcohol, 85% alcohol and 75% alcohol). Antigen retrieval process was performed in 0.01 M sodium citrate solution (pH 6.0) in a high-pressure steam boiler for 10 minutes. Non-specific binding was blocked by incubating the sections in phosphatebuffered saline supplemented with 10% normal goat serum at room temperature for 1 hour. Immunoreactivity was evaluated separately by two experienced pathologists who were blinded to the clinicopathological data of the participants.

| Reagents and antibodies
The

| Western blot
The cells were washed with ice-cold PBS and lysed in a modified inhibitor cocktail (1 tablet/10 mL; Invitrogen, Carlsbad, CA, USA).
The cell lysate was sonicated and followed by centrifugation at 12 000 g for 20 minutes at 4°C. Protein concentration was measured with BCA protein assay kit (Beyotime Biotechnology). Western blots were performed with specific antibodies to detect the corresponding proteins. After incubation at 4°C overnight, the blot was washed three times with 0.05% Tween-20 TBS (TBST), and then incubated with 1:10 000 diluted goat anti-rabbit/anti-mouse IgG conjugated with HRP for 2 hours at room temperature. After additional washing with TBST, the target proteins on the blot membrane were visualized using the ECL system. The MF-ChemiBIS 3.2 Imaging System (DNR Bio-Imaging Systems, Jerusalem, Israel) was used for image capture. To control sampling error, the same blot was also probed for β-Actin or GAPDH as an internal loading control. The integral optical density of each band was analysed using the Image-J software and the ratio of band intensities of target protein over associated control was obtained as the statistic value. Data were expressed as the mean ± SD of at least three independent experiments.

| MTT assay
U251 and U87 cells were seeded into 96-well plates (5 × 10 3 cells/ well, 60% density) and challenged with rhIL-17A at different concentrations. Then, 0.5 mg/mL MTT dye solution was added to each well and the cells were incubated at 37°C for 4 hours. Subsequently, the culture medium was discarded and 150 μL dimethyl sulphoxide was added to solubilize the precipitate. The absorbance was measured using a plate reader at 490 nm. Three dependent experiments were repeated. Data were presented as the mean ± SD.

| Colony formation assay
The cells at a density of 1 × 10 3 were seeded in 6-well culture in culture medium with 10% FBS for 1 weeks. Then, the cells were fixed with methanol for 30 minutes and stained with 1% crystal violet for 10 minutes. Colonies of more than 50 cells were counted. All experiments were performed in triplicate. Data were presented as the mean ± SD.

| Flow cytometry for the cell cycle assay
In brief, U251 and U87 cells were grown in 6-well plates were performed three times. Data were presented as the mean ± SD.

| Statistical analysis
All experiments were obtained at least in three replicates. In addition, assays producing quantitative data were run in triplicate. SPSS

| IL-17A promotes migration and invasion of GBM cells in vitro
Previous studies showed that IL-17A had an effect on cell migration and invasion in several type of cancers including lung cancer, 20 colorectal cancer, 11 gastric cancer 21 and hepatocellular carcinoma. 22 However, there is no report to show whether IL-17A could promote GBM cell migration and invasion. Therefore, the effect of IL-17A on cell motility was investigated by migration and by Matrigel invasion assays in U251 and U87 cells. The results showed that the mobility was significantly increased after IL-17A treated at different doses (5, 10 and 20 ng/mL) compared with the control group (***P < 0.001) (Figure 2A and B). Moreover, the invasion assay showed that IL-17A could also facilitate the invasion of the GBM cells (***P < 0.001) ( Figure 2C and D). The results showed that 5 ng/mL concentration of IL-17A achieved the most effective result. These data showed that IL-17A could enhance GBM cells' abilities of migration and invasion.

| IL-17A promotes activation of PI3K/AKT signalling pathway in glioma cells
MMP-2/9 plays an important role in cancer metastasis, 23 and IL-17A is widely accepted as a regulator for MMP-2 and MMP-9. 22  functioning as a double-edged sword for cancer cell growth or death.
Among these cytokines, IL-17A has been attracted much more attention. As a pro-inflammatory cytokine secreted primarily by Th17 cells, IL-17A has been found frequently to be involved in many cancer entities, such as ovarian cancer, 39 pancreatic cancer, 40 tongue squamous cell carcinoma, 41 cervical cancer 42 and hepatocellular carcinoma. 22 Malignant GBMs are primary tumours of the central nervous system characterized by diffuse infiltration into the brain, which are the one of most challenging malignancies to treat in clinic. 43 Although there are several studies carried out to investigate the potential role of IL-17A in the progression of malignant GBMs, the mechanism of IL-17A could influence glioma progression remains unclear. In GBM patients, high IL-17A infiltration is associated with poorer prognosis, suggesting it as a potential prognosis factor. 16 Previously, it has been reported by other group that IL-17A protein expression is increased in traumatic brain injury group compared with the sham-operated group in a rat model. 44 As a pro-inflammatory factor, the IL-17A In this study, we aimed to explore the effect of hIL-17A on GBM cells. However, the in vivo function of IL-17A needs to be proved and verified using animal models of GBM. In addition, it is most important to clarify and determine the potential functions of IL-17A in the tumour tissues derived from GBM patients.
Here, we demonstrated that IL-17A functioned as a pro-metastatic and pro-proliferative factor, in vitro. We revealed the molecular mechanisms that IL-17A induced GBM cell invasiveness through the PI3K/AKT mediated MMP-2/9 activation (Figure 7). In conclusion, our study provided evidence that IL-17A could promote the migration and invasion of GBM cells. Considering that tumour metastasis is often associated with poor prognosis and high mortality among GBM patients, our study is clinically relevant and IL-17A signalling could be a novel target for GBM therapy, especially GBM metastasis.
Furthermore, serum IL-17A level could be potentially a valuable marker in GBM patients who were at high risk of metastasis.