Blockade of CXCR6 reduces invasive potential of gastric cancer cells through inhibition of AKT signaling

Abstract

Chemokines and their receptors have been implicated in cell migration and metastasis of multiple malignant tumors. But the function of CXCR6 signaling in gastric cancer is not comprehensively understood. In the present study, we hypothesized that CXCR6 signaling might play an essential role in the progression of gastric cancer. The expression of CXCR6 was examined by immunohistochemical assay in human gastric cancer, and lentivirus-mediated CXCR6 knockdown by shRNA (Lv-shCXCR6) was used for investigating cell migration and invasion indicated by Wound-healing and Transwell assays. Consequently, the expression level of CXCR6 was increased in gastric cancer compared with the adjacent non-tumor tissues (54.2% vs. 27.1%, P = 0.006), and was closely associated with the metastatic lymph node in gastric cancer (P = 0.021). Furthermore, blockade of the CXCR6 signaling reduced the migration and invasion of gastric cancer cells followed by decreased expression of AKT, MMP-2, and MMP-9. In conclusion, these findings demonstrate that CXCR6 may promote the development of gastric cancer cells through regulation of AKT signaling.

Introduction

Gastric cancer is the second highest cause of cancer-related deaths worldwide. Though a variety of strategies have been taken to treat gastric cancer over the decades, it is still the fourth common type of cancer in the world. ¹ Once patents are diagnosed with gastric cancer, they are already at an advanced stage of gastric cancer and receive a poor prognosis. ² To date, molecular targeted therapy has been applied to many malignant tumors. It is necessary to identify novel biomarkers for the early diagnosis and treatment of gastric cancer. ³

Chemokines and their receptors (CXCRs) have been proven to be closely involved in tumor progression and metastasis, of which CXCR6 is overexpressed in schwannomas, ⁴ nasopharyngeal carcinoma (NPC), ⁵ colorectal cancer (CRC),^6–8 and astroglial tumors. ⁹ It accelerates tumor development through stimulating tumor growth, ⁴ recruiting T cells ⁵ and promoting epithelial-mesenchymal transition (EMT), and mediates inflammation-related migration and proliferation of multiple tumor cells,^10,11 suggesting that CXCR6 acts as a novel prognostic marker predicting poor prognosis and recurrence of liver metastases in CRC.^7,8

However, few studies show that CXC ligand 16 (CXCL16) inhibits proliferation ¹² and liver metastasis via increase of tumor-infiltrating natural killer T cells ¹³ and correlates with a good prognosis in CRC. ¹⁴ In the present study, to further clarify the role and underlying molecular mechanisms of CXCR6 in gastric cancer cells, we observed the expression of CXCR6 in gastric cancer tissues and hypothesized that CXCR6 might be implicated in invasion and metastasis of gastric cancer cells, acting as a potential therapeutic target for the treatment of gastric cancer.

Materials and methods

Materials

Human gastric cancer cell line (SGC-7901) used in this study was from Institute of Biochemistry and Cell Biology (Shanghai, PR China). Lentivirus-mediated Lv-shCXCR6 was purchased from KeyGen Biotech Co., Ltd. (Nanjing, PR China). All used antibodies including AKT, MMP-2, and MMP-9 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The tissue microarray was from Outdo Biotech Co., Ltd. (Shanghai, PR China).

Drugs and reagents

Dulbecco’s Modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL (Gaithersburg, MD, USA); TRIzol Reagent and Lipofectamine 2000 were from Invitrogen (Carlsbad, CA, USA); M-MLV Reverse Transcriptase was from Promega (Madison, WI, USA); SYBR Green Master Mixture was from Takara (Otsu, Japan). ECL-PLUS/Kit was obtained from Beyotime (Hainan, PR China).

Clinical samples and data

Gastric cancer tissues and the adjacent non-tumor tissues were obtained from our hospital from March 2011 to September 2013 (Table 1). The study was approved by Medical Ethics Committee of Wenzhou Medical University and written informed consent was obtained from the patients or their parents before sample collection. Two pathologists checked and judged the cases.

Table 1.

The clinical data of patients with gastric cancer.

Variables	Cases, n (%)
Patients (n)	48 (100%)
Age (years)
⩽60	16 (33.3%)
>60	32 (66.7%)
Sex
Male	34 (70.8%)
Female	14 (29.2%)
Tumor size (cm)
⩽3.5	11 (22.9%)
>3.5	37 (77.1%)
T stage
T1	10 (20.8%)
T2	24 (50.0%)
T3	14 (29.2%)
N stage
N0+N1	17 (35.4%)
N2+N3	31 (64.6%)
Metastatic lymph node
Negative	12 (25.0%)
Positive	36 (75.0%)

Immunohistochemical staining

CXCR6 antibody was used for immunohistochemical examination of protein expression in tissue microarrays. CXCR6 antibody was used at 1:200 dilutions. Endogenous peroxidase was inhibited by incubation with freshly prepared 3% hydrogen peroxide with 0.1% sodium azide. Non-specific staining was blocked with 0.5% casein and 5% normal serum. Staining was developed with diaminobenzidine substrate and sections were counterstained with hematoxylin.

Quantification of protein expression

The expression of CXCR6 was quantitatively assessed according to the total immunostaining scores calculated as the product of a proportion score and an intensity score. The proportion and intensity of the staining was judged by two observers. The proportion score reflected the fraction of positive staining cells (score 0, <5%; score 1, 5–10%; score 2, 10–50%; score 3, 50–75%; score 4, >75%), and the intensity score represented the staining intensity (score 0, no staining; score 1, weak positive; score 2, moderate positive; score 3, strong positive). CXCR6 was considered as negative expression if the score was less than 2, and positive expression if the score was more than 2.

Cell culture and infection

Gastric cancer SGC-7901 cells, placed in a humidified atmosphere containing 5% CO2 at 37°C, were cultured in DMEM medium supplemented with 10% heat-inactivated FBS, 100 U/mL of penicillin and 100 μg/mL of streptomycin. When the cells arrived at 50% confluence, they were transfected with lentivirus vector or negative control virus, and cultured at 37°C and 5% CO₂. The clone transfected with Lv-shCXCR6 was defined as Lv-shCXCR6 group, and that transfected with negative control vectors was considered as NC group.

Quantitative real-time PCR

To quantitatively detect the mRNA expression of CXCR6 in SGC-7901 cells, real-time PCR was carried out. Total RNA of each clone was extracted with TRIzol according to the manufacturer’s protocol. Reverse-transcription was performed using M-MLV and cDNA amplification was done using SYBR Green Master Mix kit. CXCR6 gene was amplified using specific oligonucleotide primer and GAPDH gene was used as an endogenous control. Data were analyzed using the 2^-∆∆CT. Three separate experiments were performed for each clone.

Western blot assay

SGC-7901 cells were harvested and extracted using lysis buffer (Tris-HCl, SDS, Mercaptoethanol, Glycerol). Cell extracts were boiled for 5 min in loading buffer and then equal amount of cell extracts were separated on 15% SDS-PAGE gels. Separated protein bands were transferred into polyvinylidene fluoride (PVDF) membranes and the membranes were blocked in 5% skim milk powder. The primary antibodies against CXCR6, AKT, MMP-2, and MMP-9 were diluted according to the instructions of antibodies and incubated overnight at 4°C. Horseradish peroxidase-linked secondary antibodies were added at a dilution ratio of 1:1000, and incubated at room temperature for 2 h. The membranes were washed with PBS for three times and the immunoreactive bands were visualized using ECL-PLUS/Kit according to the kit’s instruction. Three separate experiments were performed for each clone.

Wound-healing assay

Gastric cancer SGC-7901 cells were plated in each well of a 6-well culture plate and allowed to grow to 90% confluence. Lv-shCXCR6 was then performed to transfect into gastric cancer cells. The next day, a wound was created using a micropipette tip. The migration of cells towards the wound was monitored daily, and images were captured at time intervals of 24 h.

Transwell invasion assay

After a pretreatment with Lv-shCXCR6 for 24 h, Transwell assay was performed by using a Transwell chamber (Qiagen, German) with pore size of 8.0 μm. The Transwell chamber was coated with Matrigel. A total of 1×10⁶ cells were suspended in 200 μL serum-free medium and seeded in the upper compartment of the chamber. The lower compartment was loaded with 750 μL full culture medium containing 10% FBS. After being incubated at 37° for 12 h, the membrane was fixed with formaldehyde, and stained with hematoxylin. Then the trans-membrane cells were counted.

Statistical analysis

All experiments were done three times and all numerical data were presented as mean ± standard deviation. Statistical difference in each assay was determined by SPSS version 20.0, and was tested for significance using Kruskal-Wallis H test and ANOVA analysis of variance. P <0.05 was considered significant.

Results

The expression of CXCR6 in gastric cancer

The expression of CXCR6 protein was assessed by IHC staining. The positive expression of CXCR6 protein was examined in gastric cancer tissues, where different expression levels of CXCR6 were shown in Figure 1. As indicated in Table 2, the positive expression of CXCR6 was found to be in 54.2% of the gastric cancer tissues and in 27.1% of the ANCT (P = 0.006).

Figure 1.

The expression of CXCR6 in gastric cancer (×200). (a) High expression of CXCR6 in gastric cancer tissues; (b) high expression of CXCR6 in adjacent non-cancerous tissues (ANCT); (c) low expression of CXCR6 in gastric cancer tissues; (d) low expression of CXCR6 in ANCT.

Table 2.

The expression of CXCR6 in gastric cancer.

Target	Group	Total	n				Positive rate (%)	χ2	P
			–	+	++	+++
CXCR6	GC	48	22	11	9	6	54.2
	ANCT	48	35	7	4	2	27.1	7.691	0.006

ANCT: adjacent non-cancerous tissues; GC: gastric cancer.

The correlation of CXCR6 expression with clinicopathologic data of patients with gastric cancer

The association of CXCR6 expression with clinicopathologic data of patients with gastric cancer was analyzed. As shown in Table 3, high expression of CXCR6 was not found correlated with age, sex, tumor size, T stage, and N stage in patients with gastric cancer (each P >0.05). However, it interests us that increased expression of CXCR6 was related to metastatic lymph node (P = 0.021).

Table 3.

The correlation of CXCR6 expression with clinicopathologic data.

Variables	Cases (n)	CXCR6		P value
		−	+
	48	22	26
Patients (n)
Age (years)
⩽60	16	9	7
>60	32	13	19	0.311
Sex
Male	34	15	19
Female	14	7	7	0.713
Tumor size (cm)
⩽3.5	11	7	4
>3.5	37	15	22	0.182
T stage
T1	10	7	3
T2	24	9	15
T3	14	6	8	0.222
N stage
N0+N1	17	9	8
N2+N3	31	13	18	0.469
Metastatic lymph node
Negative	12	9	3
Positive	36	13	23	0.021

The effect of CXCR6 downregulation on AKT signaling

After gastric cancer cells were infected with Lv-shCXCR6 for 24 h, the effect of CXCR6 downregulation on the expression of AKT signaling was determined by real-time PCR and western blot assays, demonstrating that the expression of CXCR6 was tremendously silenced in Lv-shCXCR6 group compared with the NC group (**P <0.01, Figure 2a, b), and then that of AKT was found to be decreased in Lv-shCXCR6 group compared with the NC group (Figure 2c, d).

Figure 2.

The effect of CXCR6 silencing on AKT signaling. Real-time PCR and western blot assays were performed to detect the expression of (a, b) CXCR6 and (c, d) AKT in Lv-shCXCR6-transfected gastric cancer cells, indicating that the expression of CXCR6 and AKT was downregulated in Lv-shCXCR6 group compared with the NC group (**P <0.01).

The effect of CXCR6 downregulation on cell migration

To decide the effect of CXCR6 downregulation on the migration of gastric cancer cells, wound-healing assay was carried out indicating that the migrating capabilities of gastric cancer cells were reduced in Lv-shCXCR6 group compared to those in NC group (**P <0.01, Figure 3a, b). The protein expression of MMP-2 detected by western blotting was found to be decreased in Lv-shCXCR6 group compared with the NC group (Figure 3c).

Figure 3.

The effect of CXCR6 silencing on cell migration. (a, b) Wound-healing assay was to assess cell migrating capabilities, demonstrating the inhibitory effect of CXCR6 silencing on migrating capabilities of gastric cancer cells (**P <0.01). (c) The protein expression of MMP-2 examined by western blotting was shown to be downregulated in Lv-shCXCR6 group compared with the NC group.

The effect of CXCR6 downregulation on cell invasion

To estimate the effect of CXCR6 downregulation on the invasive potential of gastric cancer cells, Transwell assay was implemented, revealing that the invasive potential of gastric cancer cells was lowered in Lv-shCXCR6 group compared to those in NC group (**P <0.01, Figure 4a, b). The protein expression of MMP-9 detected by western blotting was found decreased in Lv-shCXCR6 group compared with the NC group (Figure 4c).

Figure 4.

The effect of CXCR6 silencing on cell invasion. (a, b) Transwell assay was to evaluate cell invasive potential, indicating the inhibitory effect of CXCR6 silencing on invasive potential of gastric cancer cells (**P <0.01). (c) The protein expression of MMP-9 detected by western blotting was shown to be downregulated in Lv-shCXCR6 group compared with the NC group.

Discussion

CXC chemokines and their receptors such as CXCR6 have been widely involved in the pathogenesis of many tumors including human meningiomas. ¹⁵ CXCR6 overexpression modulates stem cell self-renewal of cancer cells ¹⁶ and leads to proinflammatory tumor microenvironment that promotes invasiveness and poor prognosis in hepatocellular carcinoma (HCC). ¹⁷ Increased expression of CXCR6 is related with lung metastasis and worse prognosis in Ewing sarcoma ¹⁸ and with lymph node metastasis in cervical cancer, ¹⁹ involving the pathophysiology of epithelial ovarian carcinoma and aggressive cancer behaviors such cell growth, invasion, and metastasis.^20–22 But, CXCR6 expression is reported to be not associated with lymphocyte infiltration and survival in ovarian cancer. ²³ In the present study, CXCR6 was highly expressed in gastric cancer tissues, and was closely correlated with the lymph node metastases in patients with gastric cancer, indicating that CXCR6 might promote the tumorigenesis of gastric cancer. ²⁴

Metastasis is thought to be the clear mark for many aggressive cancers. CXCR6 is correlated with aggressive cancer behavior ²⁵ and functions as a regulator in metastasis and progression of cancer. ²⁶ CXCL16/CXCR6 axis significantly results in cell proliferation, bone invasion, and metastasis and its antagonist inhibits cancer cell metastasis and disease progression. ²⁷ However, few studies show that blockade of the CXCR6 enhances the tumor metastasis in melanoma and Lewis lung carcinoma, ²⁸ and induces the cell migration in renal cancer. ²⁹ To confirm the role of CXCR6 in the invasiveness of gastric cancer, it was found that knockdown of CXCR6 suppressed the migration and invasive potential of gastric cancer cells, suggesting that CXCR6 might promote the progression of gastric cancer cells.

AKT signaling plays an important role in the formation and progression of various cancers, and is considered as a key therapeutic target for gastric cancer. ³⁰ CXCR6 contributes to invasive activities of prostate cancer cells by the AKT/mTOR signaling and blockade of the CXCR6/AKT/mTOR signaling inhibits metastasis and provide a more effective therapeutic strategy.^7,31 Moreover, targeted inhibition of Akt1 reduces the expression of MMP-2 and MMP-9, exerting inhibitory effects on gastric cancer cell proliferation and invasion. ³² CXCR6 is expressed in human prostate cancer and accelerates the invasion of prostate cancer cells through upregulation of MMP-2 and MMP-9. ³³ Interestingly, in the present study, it was shown that blockade of the CXCR6 signaling reduced the migration and invasion of gastric cancer cells followed by decreased expression of AKT, MMP-2, and MMP-9, indicating that CXCR6 might be involved in the invasion and metastasis of gastric cancer cells through regulation of AKT-mediated MMP-2/-9 expression.

In short, our findings demonstrate that increased expression of CXCR6 is correlated with tumor metastasis of patients with gastric cancer and inhibition of CXCR6 signaling may suppress the invasion of gastric cancer cells via inhibition of AKT signaling-mediated MMP-2/-9 expression.