IL‐8 promotes cell migration through regulating EMT by activating the Wnt/β‐catenin pathway in ovarian cancer

Abstract Interleukin‐8 (IL‐8), as an inflammatory chemokine, has been previously shown to contribute to tumorigenesis in several malignancies including the ovarian cancer. However, little is known about how IL‐8 promotes the metastasis and invasion of ovarian cancers cells. In this study, we found that IL‐8 and its receptors CXCR1 and CXCR2 were up‐regulated in advanced ovarian serous cancer tissues. Furthermore, the level of IL‐8 and its receptors CXCR1 and CXCR2 expression were associated with ovarian cancer stage, grade and lymph node metastasis. In vitro, IL‐8 promoted ovarian cancer cell migration, initiated the epithelial‐mesenchymal transition (EMT) program and activated Wnt/β‐catenin signalling. However, when treated with Reparixin (inhibitor of both IL‐8 receptors CXCR1 and CXCR2), effect of both endogenous and exogenous IL‐8 was reversed. Together, our results indicated that IL‐8 triggered ovarian cancer cells migration partly through Wnt/β‐catenin pathway mediated EMT, and IL‐8 may be an important molecule in the invasion and metastasis of ovarian cancer.


| INTRODUC TI ON
Ovarian cancer is a disease that has the highest mortality rate among all gynaecological malignancies. It was estimated that 22 440 new cases and 14 080 deaths of ovarian cancer are expected in American in 2018, in the meanwhile a 5-year survival rate that can be as low as 30% when diagnosed after the cancer. 1 The development of ovarian cancer begins at the ovary and is more apt to through peritoneal metastasis to adjacent organs. 2 Even with the use of recommended therapies, patients diagnosed with ovarian cancer always have the high rates of relapse because the disseminating ovarian cancer cells form the secondary tumour foci in the peritoneal cavity. 3,4 Ovarian tumour microenvironment is regarded as a place rich in proinflammatory cytokines and chemokines, and this proinflammatory microenvironment is considered to be a prominent factor affecting the spread of ovarian cancer cells. 5 Physiological environment is determined by many chemical factors which direct influence tissue function and also have a great impact on diseases such as cancer. Inflammation mediators play an essential role in ovarian cancer pathogenesis, affecting ovarian cancer initiation, promotion and progression. 6 In the tumour microenvironment, related chemokines and cytokines promote the angiogenesis of tumour tissues and the phenotype transformation of cancer cells, which in turn leads to tumour invasion and metastasis. 7 In ovarian cancer, chemokines regulate the cancer cell survival, tumour angiogenesis, cancer cell proliferation and metastasis. 8 Elucidating the relationship between chemokines and ovarian cancer may reveal new target therapies. Interleukin-8 (IL-8) is an inflammatory chemokine that mediates the activity of immune cells and chemotaxis, leading to chronic inflammation. The activity of IL-8 is mainly determined by the binding of IL-8 to its receptors CXCR1 and CXCR2. 9 In ovarian cancer, increased secretion of IL-8 has been proved to promote tumour growth and upgrade metastasis. 10 However, how IL-8 enhances the metastasis and invasion of ovarian cancers cells remains unknown.
Recent studies have suggested that epithelial-mesenchymal transition (EMT) was closely related to the invasion and metastasis of ovarian cancer, 11 which may provide the molecular basis for the IL-8-triggered ovarian cancer cell migration. In the process of EMT in tumour cells, special markers of epithelial cells such as E-cadherin are down-regulated, causing a loss of the epithelial features and a acquire of some mesenchymal features. 12 When cancer cells undergoing EMT, the tumour cells locally infiltrate and distantly migrate, which ultimately leads to a decrease in patient survival rate. 13 In ovarian cancer, EMT is thought to be involved in the metastasis and be predicted of poor prognosis, the reduction in E-cadherin on the cell surface is closely correlated with poor overall survival of patients with ovarian cancer. 14 For the present study, we correlated the expression of IL-8 and its receptors (CXCR1, CXCR2) with clinical measures (staging, tumour grade and overall survival) in patients with ovarian cancer. We also found the regulatory role of IL-8-induced EMT in ovarian cell lines SKOV3 and A2780. Furthermore, our results indicated that IL-8 may induce EMT of ovarian cancer cells partly through Wnt/ β-catenin signalling pathway. Therefore, the study revealed the significant roles and the potential mechanisms of IL-8 in ovarian cancer migration and provided new therapeutic and prognostic targets.

| Specimen and immunohistochemistry (IHC)
All ovarian cancer specimens (n = 93) were obtained from surgical patients at the department of gynaecology, West China Second University Hospital (Chengdu, China). The patient was pathologically diagnosed with serous ovarian cancer and did not receive any other treatment prior to harvesting the specimen. All patients received written informed consent, which was approved by the Ethics Committee of Sichuan University. IHC was performed to detect IL-8 and its receptors (CXCR1, CXCR2) with the using of anti-IL-8, anti-CXCR1 and anti-CXCR2 (1:100, Abcam) antibodies. Heat-mediated antigen retrieval was performed in a pH 6.0 citrate buffer in a 98°C water bath for 15 minutes. Primary antibody was incubated at 4°C overnight, and secondary antibody was incubated at room temperature for 1 hours, followed by counterstaining with haematoxylin and visualization with the DAB. Finally, the sections were mounted using neutral gum. Two investigators independently assessed and scored the immunostaining based on staining intensity. Moderate and intense staining was defined as high expression, while no and weak staining was defined as low expression.

| Bioinformatics analysis
We subjected the IL-8, CXCR1 and CXCR2 to survival analysis using the online database (Gene Expression Profiling Interactive Analysis).
P-values were calculated with log-rank (Mantel-Cox) test. Patients were classified into 'low' and 'high' expression based on auto selected best cut-off in the database.

| Cell cultures
Two human ovarian cancer cell lines (SKOV3, A2780) were purchased from Nanjing KeyGen Biotech.Inc Cells were cultured in α-MEM medium (Hyclone) supplemented with 10% fetal bovine serum (Israel) and 1% penicillin-streptomycin (Hyclone), and every 3 days, the medium was changed. All cell cultures were maintained in a humidified incubator (Heraeus) with 5% CO 2 saturation at 37°C, and the cells in their third or fourth passage were used.
To investigate whether IL-8 played a key role in facilitating cell migration, the exogenous IL-8 and the inhibitor of IL-8 receptor (Reparixin) were used. Because the CXCR1/2 binds to IL8 with high affinity and transduces the signal through a G-protein-activated second messenger system, the CXCR1/2 antagonism (Reparixin, MCE, USA) was commonly used to inhibit the IL-8 signalling pathway.

| Immunocytochemistry
The SKOV3 and A2780 cells were digested by trypsin-EDTA and seeded into 24 cell plates. Then, the cells were incubated at 37°C, washed with PBS and fixed in 4.00% paraformaldehyde for 15 minutes. After permeablization using 0.50% Triton X-100 and blocking by 5.00% normal goat serum, the cells were incubated with the using of anti-IL-8, anti-CXCR1 and anti-CXCR2 (1:100, Abcam) antibodies at 4°C overnight, and the second antibody was incubated for 1 hours at room temperature. Finally, the cells were counterstained with haematoxylin and visualized with the DAB.

| Monolayer wound healing assay
Migration ability was measured using the wound healing assay.
SKOV3 and A2780 were grown in 6-well plates. When the cells reached 80% density, a 100 μL pipette tip was used to create a horizontal line through the wells in the confluent cells, and then, the cells were incubated in fresh medium for 48 hours. Each experiment was repeated at least three times. The image was processed using ImageJ software (Rawak Software, Inc) and was calculated for the proportion of relative wound closure.

| Transwell migration assay
Cell migration assay was performed using Transwell chambers. For this assay, cells were seeded in 100 μL of FBS-free medium in the upper chamber of the well, while the lower chamber were filled with 600 μL of 20% FBS medium. Following incubation for 24 hours, the migrating cells on the bottom surface were fixed with methanol, stained with 0.1% crystal violet and imaged. The number of cells was calculated in at least five random regions of each chamber, and then, the average was calculated.

| Immunofluorescence
The SKOV3 and A2780 cells cultured on glass slides were fixed with 4% paraformaldehyde for 20 minutes and then blocked with bovine serum albumin (BSA) at room temperature for 1 hours. Next, the cells were washed three times with 0.1% Triton X-100/ PBS. Then, cells were incubated in F-actin (1:100, Solarbio, China) for 2 hour and counterstained with DAPI (1:800, Solarbio, China) for 10 minutes.
Finally, the cells were mounted on 50% glycerol/PBS and imaged using a Nikon eclipse Ti-S microscope.

| Western blot analysis
After the separation by sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE), the cellular protein was transferred to polyvinylidene fluoride (PVDF) membranes (Invitrogen Life Technologies). Followed the blocking with 5% fat-free milk, the membrane was incubated with primary antibodies against E-cadherin, vimentin, Wnt5a, p-βcatenin, β-catenin, Met, C-Jun and GAPDH (all from Cell Signaling Technology, Inc) overnight at 4°C and then incubated with a secondary antibody for 1 hours. Enhanced chemiluminescence kit (Beyotime Institute of Biotechnology) was used to visualize the blots.

| Statistical analysis
All of the statistical analyses were performed with SPSS 18.0 software (SPSS Inc). Results were expressed as mean ± SD of at least three independent experiments performed. Treatment groups were compared using one-way analysis of variance (ANOVA), and Student's t test. P-values < .05 were considered to indicate a statistically significant difference.

| IL-8 and its receptors expression in ovarian cancer
To identify the expression of IL-8, CXCR1 and CXCR2 in serous ovarian cancer, we collected patients with ovarian cancer from West China Second University Hospital. The clinical-pathological characteristics of the case (age, tumour grade, distant metastasis and stage) and their relationship with the expression of IL-8, CXCR1 and CXCR2 were reported in Table 1, and the high expression of IL-8 and its receptors in high-stage serous ovarian cancer were shown in Figure 1A. We found that the high expression of IL-8 and CXCR2 were closely correlated with high tumour grade, tumour distant metastasis and high tumour stage, and the high expression of CXCR1 was closely related to high tumour grade and tumour distant metastasis. To further determine whether the IL-8 and its receptors were associated with prognosis of patients with ovarian cancer, we subjected the IL-8, CXCR1 and CXCR2 to survival analysis using the online database (Gene Expression Profiling Interactive Analysis).
Data showed that CXCR2 was significantly associated with poor overall survival (OS) ( Figure 1B). Thereby, the IL-8 and its receptors, especially CXCR2, may be important mediators of ovarian cancer metastasis.

| Expression of IL-8 and its receptors in ovarian cancer cell lines
To investigate the in vitro expression and sub-cellular localization of IL-8 and its receptors, the ovarian cancer cells were studied by immunocytochemistry. By studying the expression levels of IL-8,

| Inhibition of IL-8 receptors attenuated migration of ovarian cancer cells
To investigate whether IL-8 played a key role in facilitating cell migration, wound healing and Transwell assays were performed. As was shown in Figures 2 and 3, the wound healing percentage and the mi-

| Influence of IL-8 on the cytoskeleton of ovarian cancer cells
To investigate the effect of IL-8 on the cytoskeleton of ovarian cancer cells, F-actin was detected by immunofluorescence assay. The phalloidin staining showed more protrusions formed when stimulated by the IL-8, while the inhibitor of IL-8 could significantly block the effect of endogenous and exogenous IL-8 ( Figure 4). This result suggested that the IL-8 possibly rearranged cytoskeletal F-actin assembly and promoted cellular filopodia, and ultimately retarded cellular motility.

| IL-8 induced EMT activation in ovarian cancer cells
To obtain further insights into the EMT-promoting capability of IL-8 in ovarian cancer, E-cadherin and vimentin were detected using Western blotting. As shown in Figure 5, compared with the control, E-cadherin level was markedly decreased and the vimentin level was markedly

| IL-8 promoted EMT by up-regulating the Wnt/β-catenin pathway
To clearly understand the mechanisms of IL-8 in ovarian cancer, we tested whether suppressing IL-8 affected Wnt signalling pathway whose aberration played a crucial role in ovarian cancer progression.
As shown in Figure 6, with the addition of IL-8, Wnt5a, β-catenin

| CONCLUSION
Collectively, this study demonstrated that IL-8 and IL-8 receptors CXCR1 and CXCR2 were up-regulated in advanced ovarian serous cancer tissues. Furthermore, we revealed that IL-8 enhanced ovarian cancer by initiating an EMT program to promote cancer cell migration, and we also found that the Wnt/β-catenin pathway may be F I G U R E 6 Expression of key proteins in Wnt signalling pathway in ovarian cancer cells. A, The expression levels of Wnt5a, p-β-catenin, β-catenin, Met and C-Jun in SKOV3 cells were quantified by image analysis of the Western blot bands. The expression of GAPDH in each group was taken as intrinsic controls, and relative expressions of each protein were calculated. B, The expression levels of Wnt5a, p-βcatenin, β-catenin, Met and C-Jun in A2780 cells were quantified by image analysis of the Western blot bands. Values represented the Mean ± SD from three independent experiments. *P < .05, **P < .01 vs Control. C, Diagram showed the effect of IL-8 on the migration ability of ovarian cancer cells. IL-8 up-regulated the Wnt signalling ligand Wnt5a expression and subsequently activated a β-catenin-dependent activation of Wnt pathway, which increased the expression of Wnt pathway cascade protein Met and c-Jun, to finally induce a EMT process to promote the migration of ovarian cancer cells involved in mediating the EMT process. These results suggested that IL-8 and IL-8 receptors CXCR1 and CXCR2 were potential biomarkers and therapeutic targets for ovarian cancer.

ACK N OWLED G EM ENTS
The

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data are available from the corresponding author on reasonable request.