CCL17-CCR4 axis promotes metastasis via ERK/MMP13 pathway in bladder cancer

2.1 Cell culture and reagents

BC cell lines were purchased from the American Type Culture Collection (Manassas, VA). The cell lines, T24, 253J, were maintained in an RPMI 1640 medium, supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO). All of these cells were incubated at 37°C and 5% CO₂. U0126 (MEK inhibitor) was purchased from MCE (Shanghai, China) and used in accordance with the manufacturer’s instructions. Recombinant human CCL17 was purchased from R&D Systems (Minneapolis, MN).

2.2 Generation of gene knockdown in stable cells

pGLV-GFP-CCR4 lentiviral vector and three small interfering RNA (shRNA) plasmids targeting different regions of CCR4 mRNA were purchased from Genechem (Shanghai, China).

Lentivirus particles were transfected into the BC cells in the presence of polybrene and selected for 2 weeks using 5 μg/mL puromycin. CCR4 sequences targeted were as follows: shRNA1: 5′-TTGAAAGATACTTGGACTA-3′; shRNA2: 5′-CTTAGGGATCATGCTGTTT-3′; and shRNA3: 5′-TTTCTGTTCAGCACTTGTT-3′.

2.3 Wound-healing assay and Matrigel invasion assay

The cells were cultured in serum-free medium for 24 hours and wounded with pipette tips. The fresh medium was then replaced. After 24 hours, the wound closing procedure was observed and photographed. The invasion assays were performed as previously reported. Briefly, 200 μL serum-free medium containing 2 × 10⁵ cells was added into the upper chamber, and 600 μL medium with 10% serum and CCL17 was added into the lower chamber. The chamber coated with diluted Matrigel (BD Biosciences, Bedford, MA) was cultured in 37°C at 5% CO₂ for 24 hours and fixed with methanol. The cells that invaded to the bottom of the membrane were stained with 1% crystal violet and photographed under microscope. Three independent experiments were conducted using the same conditions.

2.4 Real-time quantitative reverse-transcription PCR

Total RNA was isolated from cell lines and tissues using Trizol (Invitrogen, Camarillo, CA) according to the manufacturers’ instructions. Complementary DNA was synthesized using a reverse-transcription kit (Invitrogen, Camarillo, CA). Quantitative polymerase chain reaction (PCR) was performed using SYBR Green PCR Master Mix (Applied Biosystems, Austin, TX).

The primers for CCR4 were as followed: forward 5′-CCCACGGATATAGCAGACACC-3′ and reverse 5′-GTGCAAGGCTTGGGGATACT-3′. Relative mRNA expression was calculated by comparative Ct method, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the control. All the experiments were carried out in triplicates.

2.5 Patients and immunohistochemical analysis

The collection of specimens from 122 patients was authorized by Shandong University Qilu Hospital. These patients were all received radical cystectomy with BC, diagnosed by imageological examinations, and pathological results, at our institution from 2009 to 2014. Patients were excluded from this study if their information missed. Patients with autoimmune disease, cancer in other systems, received neoadjuvant chemotherapy and radiotherapy were also excluded from study. The specimens were used after obtaining informed consent from the patients. The staining of these specimens was conducted according to the manufacturer’s protocol (Immunostain SP kit, DakoCytomation). The antibodies used for immunohistochemical (IHC) analysis included antibody against CCR4 (ab1669, Abcam, Cambridge, MA). For IHC staining, briefly, the paraffin-embedded sections of BC tissues were deparaffinized and then heated in a pressure pot for 3 minutes to retrieve the antigens. Then, the sections were incubated with primary antibodies overnight at 4°C. Antibody binding was detected using a peroxidase-conjugated secondary antibody at 37°C for 30 minutes. A DAB substrate kit was used to perform the chromogenic reaction. The intensity of the staining was evaluated using the following criteria: 0, negative; 1, low; 2, medium; and 3, high. The extent of staining was scored as 0, 0% stained; 1, 1% to 25% stained; 2, 26% to 50% stained; and 3, 51% to 100% stained. The final scores were calculated by multiplying the scores of the intensity with those of the extent and dividing the samples into four grades: 0, negative; 1 to 2, low staining; 3 to 4, medium staining; and 4 to 6, high staining. The following criteria were used to quantify the expression levels of CCR4 in BC tissues: high expression, the final scores were 4 to 6; low expression, the final scores were 0 to 3.

2.6 Immunofluorescence

The BC cells were seeded on coverslips and cultured until they reached 60% to 70% confluence. The cells were washed with PBS and fixed in 4% paraformaldehyde for 15 minutes at room temperature (RT). The cells were then washed three times with cold PBS before permeabilization with 0.25% Triton X-100 in PBS for 15 minutes at RT. The cells were washed three times with PBS and blocked with 1% bovine serum albumin in polybutylene succinate-co-butylene terephthalate at RT for 30 minutes and were incubated with primary antibody overnight at 4°C, as described by the manufacturer. CCR4 expression was detected by incubating the cells for 1 hour with a secondary antibody conjugated with IF488 (1:1000). The cells were counterstained with 4′, 6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO), and the images were captured using a fluorescence microscope.

2.7 Immunoblotting

Western blot analysis was performed as previously described [1.35]. Briefly, 100 µg of protein was separated by 10% sodium dodecyl sulfate polyacrylamide-based discontinuous gel and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk for 2 hours and were incubated at 4°C overnight with primary antibodies. The primary antibodies for CCR4 were purchased from Abcam, and the antibodies against ERK (AF0155), p-ERK (AF1015), MMP3 (AF5355), and GAPDH (AF0911) were purchased from Affinity Biosciences (Affinity, Cincinnati, OH). Horseradish peroxidase–conjugated secondary antibodies were used, and the protein bands were visualized using an Odyssey scanner (LI-COR Biosciences, Lincoln, NE).

2.8 Statistical analysis

Statistical analyses were performed using the IBM SPSS Statistics Program. Each experiment was performed in triplicates, and the values are presented as the mean ±standard deviation, unless otherwise stated. The variance between the groups was statistically compared. Student t-test was used to compare the mean values. Kaplan-Meier curves were analyzed for relevant variables. The log-rank test was used to analyze the differences in survival times among the patient subgroups. The risk factors associated with the prognoses of these patients were evaluated using Cox’s proportional hazard regression model. All the probability values had a statistical power level of 90%, and a two-sided level of 5%. P < 0.05 was considered to be significant.

3.1 Correlation of CCR4 expression with clinicopathological characteristics and prognosis of bladder cancer patients

As shown in Figure 1A, the expression of CCR4 at the protein level was significantly increased in BC samples compared with paired normal tissues from seven patients. In addition, we examined the expression of the CCR4 protein by IHC staining in a retrospective cohort of 122 BC specimens from patients (Figure 1B).

To explore the clinical significance of CCR4 in BC, we analyzed the CCR4 expression pattern in 122 BC specimens by IHC. The clinicopathologic characteristics of 122 patients are shown in Table 1. In detail, higher level CCR4 expression was detected in 68 (55.7%) tumor tissues, whereas only 54 (44.3%) specimens exhibited low/absent level (Table 1). Subsequently, the correlation between CCR4 expression level and clinicopathological parameter in patients, and patients with higher expression of CCR4 (with a cutoff at median) presented advanced grade, tumor stage, and metastasis status. However, no significant difference was observed between the two groups on age, sex, tumor grade, or metastasis status. Kaplan-Meier survival analysis revealed that patients with low CCR4 expression had more favorable overall survival (P = 0.002, respectively) compared with high CCR4 expression (Figure 1C). In addition, we analyzed whether the different pathological T stages were associated with the OS (Figure 1D). No difference was observed in patients with pTis/T1 for OS (P = 0.018), and in patients with pT2-4 (P = 0.041). The results of univariate analysis and multivariate analysis by Cox proportional hazards models suggested that CCR4 expression was an independent prognostic risk factor (P = 0.003, P = 0.007, Table 2).

3.2 Expression of CCR4 in bladder cancer cell lines

To investigate whether CCR4 expression is correlated with BC development and progression, we first analyzed CCR4 expression in BC cell lines. According to the results of Western blot, real-time PCR, and immunofluorescence, CCR4 express in BC cell lines T24 and 253J (Figure 2A-C).

3.3 Induction of the migration and invasion in CCL17-treated bladder cancer cells

To elucidate the role of CCL17, which can specifically bind to CCR4, in the invasiveness and motility of BC cells, wound-healing migration and Matrigel invasion assays were performed. According to the results, treatment of T24 and 253J cells with CCL17 (100 ng/mL) for 24 hours significantly increased invaded cells compared with the control cells (Figure 3A). Besides, both 253J and T24 cells treated with CCL17 for 24 hours exhibited relative distance compared with control (Figure 3B). These results indicated that CCL17/CCR4 can enhance the motility of the T24 and 253J cells. Western blot assay results confirmed the promotion of CCR4 expression in CCL17-treated cells (Figure 3C).

3.4 CCR4 promotes cell invasion and migration in bladder cancer

To investigate the role of CCR4 in BC cells, we employed lentivirus-mediated shRNA to knock down CCR4 in T24 and 253J cells, respectively, with the treatment of CCL17 (100 ng/mL). The effect of knockdown was confirmed by Western blot analysis, and the cells infected with sh-CCR4 exhibited a lower expression (Figure 4A). Transwell assay results showed that the number of invaded cells were significantly decreased in CCR4-knockdown cells(Figure 4B) compared with cells infected with control. In addition, wound-healing assay results showed that the relative distance of wound edges of CCR4-knockdown cells was significantly wider than those of control cells (Figure 4C). To elucidate the signaling pathways involved in the cellular responses, we investigated the MAPK signaling. The results of Western blot showed that the expression of p-ERK and MMP13 was reduced in the CCR4-knockdown cells (Figure 4D).

3.5 Association between ERK1/2 signaling and MMP13 effect on the expression of CCL17-CCR4 axis induced bladder cancer cells metastasis

To further investigate the exact roles of ERK1/2 signaling in the CCL17-CCR4 axis–induced molecular events of T24 and 253J cells, ERK1/2 specific inhibitor U0126 was used. As shown in Figure 5A and 5B, migratory and invasive effects by CCL17 were enhanced by CCL17 induction. However, this effect of CCL17 was suppressed by a U0126 pretreatment, characterized by an attenuated mobility in both 253J and T24 cell lines subsequently. Besides, CCL17 could improve the expression of p-ERK and MMP13 in bladder cell lines (Figure 5C). However, ERK1/2 and MMP13 expression induced by CCL17 were then reversed by pretreating U0126 in T24 and 253J cells (Figure 5C). These results suggested that ERK1/2 signaling was involved in the migration, invasion, and MMP13 expression through the activation of the CCL17-CCR4 axis.