C-X-C chemokine receptor 2 correlates with unfavorable prognosis and facilitates malignant cell activities via activating JAK2/STAT3 pathway in non-small cell lung cancer

Figures & data

Table 1. Baseline characteristics of NSCLC patients.

Characteristics	NSCLC patients (N = 340)
Age (years), mean±SD	61.9 ± 10.6
Gender, No. (%)
Male	273 (80.3)
Female	67 (19.7)
Smoke, No. (%)	191 (56.2)
Drink, No. (%)	132 (38.8)
Pathological differentiation, No. (%)
Well	44 (12.9)
Moderate	214 (62.9)
Poor	82 (24.2)
Tumor size (cm), mean±SD	5.3 ± 2.1
Lymph node metastasis, No. (%)	114 (33.5)
TNM stage, No. (%)
I	117 (34.4)
II	113 (33.2)
III	110 (32.4)
CEA (ng/mL), median (IQR)	7.0 (3.1–30.2)

NSCLC, non-small-cell lung cancer; SD, standard deviation; IQR, interquartile range; CEA, carcinoembryonic antigen.

Figure 1. CXCR2 expression in NSCLC tumor tissues and adjacent tissues.IHC staining examples of CXCR2 expression tumor tissues and adjacent tissues (A). IHC semi-quantitative score of CXCR2 expression in tumor tissues and adjacent tissues (B). Percentage of CXCR2 high expression and low expression in tumor tissues and adjacent tissues (C). Comparison of IHC semi-quantitative score of CXCR2 expression was done by paired-samples t-test, and comparison of CXCR2 high expression and low expression between tumor tissues and adjacent tissues was done by McNemar test. P < 0.05 was considered significant. CXCR2, C-X-C Chemokine Receptor Type 2; NSCLC, non-small cell lung cancer.

Table 2. Correlation of CXCR2 expression with tumor characteristics.

	CXCR2 expression
Items	High	Low	P value
Pathological differentiation, No. (%)			<0.001
Well	15 (34.1)	29 (65.9)
Moderate	103 (48.1)	111 (51.9)
Poor	55 (67.1)	27 (32.9)
Tumor size, No. (%)			0.001
≤5 cm	88 (43.3)	115 (56.7)
>5 cm	85 (62.0)	52 (38.0)
Lymph node metastasis, No. (%)			0.003
No	102 (45.1)	124 (54.9)
Yes	71 (62.3)	43 (37.7)
TNM stage, No. (%)			<0.001
I	42 (35.9)	75 (64.1)
II	61 (54.0)	52 (46.0)
III	70 (63.6)	40 (36.4)
CEA^a, No. (%)			0.207
Normal	64 (46.7)	73 (53.3)
Abnormal	109 (53.7)	94 (46.3)

Comparison was determined by Chi-square test or Wilcoxon rank-sum test. P value <0.05 was considered significant. CXCR2, C-X-C chemokine receptor 2; CEA, carcinoembryonic antigen. ^aAbnormal, CEA >5 ng/mL, normal, CEA ≤5 ng/mL.

Figure 2. Comparison of survival between CXCR2 high expression and low expression patients. DFS (A) and OS (B) between CXCR2 high expression and low expression patients. The Kaplan–Meier curves were plotted to show the profiles of DFS and OS between the two groups, and the log-rank test was applied to determine the difference of DFS and OS between the two groups. P < 0.05 was considered significant. CXCR2, C-X-C Chemokine Receptor Type 2; DFS, disease-free survival; OS, overall survival.

Figure 3. Comparison of survival between CXCR2 high expression and low expression patients in subgroups. DFS between CXCR2 high expression and low expression patients in patients with TNM stage I (A), II (B) and III (C). OS between CXCR2 high expression and low expression patients in patients with TNM stage I (D), II (E) and III (F). Kaplan–Meier curves were plotted to show the profiles of DFS and OS between the two groups, and the log-rank test was applied to determine the difference of DFS and OS between the two groups. P < 0.05 was considered significant. CXCR2, C-X-C Chemokine Receptor Type 2; DFS, disease-free survival; OS, overall survival.

Table 3. Analysis of factors affecting DFS by univariate and multivariate Cox’s proportional hazards regression model.

	Univariate Cox’s regression				Multivariate Cox’s regression
			95% CI				95% CI
Items	P value	HR	Lower	Higher	P value	HR	Lower	Higher
CXCR2 expression (high)	<0.001	1.814	1.421	2.317	<0.001	1.678	1.296	2.174
Age (>60 years)	0.184	1.179	0.925	1.502	0.744	1.045	0.803	1.361
Gender (male)	0.599	0.921	0.677	1.253	0.115	0.767	0.551	1.067
Smoke	0.301	1.137	0.891	1.451	0.275	1.152	0.893	1.485
Drink	0.835	0.974	0.76	1.247	0.394	1.117	0.867	1.439
Pathological differentiation (poor)	0.011	1.424	1.085	1.868	0.126	1.262	0.937	1.699
Tumor size (>5 cm)	<0.001	1.665	1.306	2.124	0.231	0.812	0.578	1.141
Lymph node metastasis	<0.001	2.965	2.301	3.82	<0.001	2.834	2.049	3.92
TNM stage (III)	<0.001	2.118	1.652	2.716	0.132	1.315	0.921	1.878
CEA^a (abnormal)	0.159	1.192	0.933	1.523	0.154	1.201	0.934	1.546

DFS, disease-free survival; HR, hazard ratio; CI, confidence interval; CXCR2, C-X-C chemokine receptor 2; CEA, carcinoembryonic antigen; *Abnormal, CEA >5 ng/mL, normal, CEA ≤5 ng/mL.

Table 4. Analysis of factors affecting OS by univariate and multivariate Cox’s proportional hazards regression model.

	Univariate Cox’s regression				Multivariate Cox’s regression
			95% CI				95% CI
Items	P value	HR	Lower	Higher	P value	HR	Lower	Higher
CXCR2 expression (high)	<0.001	1.920	1.471	2.507	<0.001	1.887	1.422	2.503
Age (>60 years)	0.726	1.048	0.807	1.361	0.216	0.835	0.627	1.111
Gender (male)	0.259	0.830	0.600	1.147	0.051	0.706	0.498	1.001
Smoke	0.727	1.048	0.806	1.362	0.641	1.068	0.811	1.406
Drink	0.824	0.970	0.742	1.268	0.330	1.146	0.871	1.509
Pathological differentiation (poor)	0.081	1.301	0.968	1.748	0.490	1.121	0.810	1.550
Tumor size (>5 cm)	<0.001	1.872	1.442	2.430	0.763	1.054	0.750	1.482
Lymph node metastasis	<0.001	3.518	2.688	4.605	<0.001	3.602	2.561	5.065
TNM stage (III)	<0.001	2.062	1.577	2.696	0.931	0.984	0.680	1.424
CEA^a (abnormal)	0.024	1.362	1.041	1.781	0.018	1.399	1.061	1.846

OS, overall survival; HR, hazard ratio; CI, confidence interval; CXCR2, C-X-C chemokine receptor 2; CEA, carcinoembryonic antigen; ^aAbnormal, CEA >5 ng/mL, normal, CEA ≤5 ng/mL.

Figure 4. CXCR2 mRNA and protein expression in different cell lines. The mRNA expression (A) and protein relative expression (B, C) of CXCR2 in NCI-H650, NCI-H1299, NCI-H1437, A549 cells compared with BEAS-2B cells. Comparison of CXCR2 mRNA and protein relative expression between cell lines was conducted by Dunnett’s t-test. P < 0.05 was considered significant. CXCR2, C-X-C Chemokine Receptor Type 2; mRNA, messenger RNA.

Figure 5. Effect of CXCR2 on cell proliferation and apoptosis. The effect of CXCR2 overexpression and knockdown on CXCR2 mRNA expression (A) and protein relative expression (B,C) in NCI-H1437 cells. The effect of CXCR2 overexpression and knockdown on cell proliferation (D) and apoptosis (E,F,G) in NCI-H1437 cells. The effect of CXCR2 overexpression and knockdown on CXCR2 mRNA expression (H) and protein relative expression (I,J) in NCI-H1299 cells. The effect of CXCR2 overexpression and knockdown on cell proliferation (K) and apoptosis (L,M,N) in NCI-H1299 cells. Comparison of cell proliferation and apoptosis between the two groups was conducted by independent sample’s t-test. P < 0.05 was considered significant. CXCR2, C-X-C Chemokine Receptor Type 2; mRNA, messenger RNA.

Figure 6. Effect of CXCR2 on cell invasion. The effect of CXCR2 overexpression and knockdown on cell invasion in NCI-H1437 cells (A,B). The effect of CXCR2 overexpression and knockdown on cell invasion in NCI-H1299 cells (C,D). Comparison of invasive cell count between the two groups was conducted by independent sample’s t-test. P < 0.05 was considered significant. CXCR2, C-X-C Chemokine Receptor Type 2.

Table 5. Limiting dilution data.

Groups	Limiting dilution (cells per well)			Frequency (1 in cells)			P value
	1000	100	10	Lower	Estimate	Upper
NCI-H1437 cell
OE-NC	23/24	16/24	8/24	183.8	112.1	68.4	0.001
OE-CXCR2	24/24	19/24	12/24	65.2	41.7	26.7
KD-NC	23/24	17/24	9/24	163	101	62.2	0.001
KD-CXCR2	20/24	15/24	6/24	419	257	158.2
NCI-H1299 cell
OE-NC	20/24	11/24	9/24	491	308	193.1	0.001
OE-CXCR2	23/24	14/24	10/24	192	117	70.9
KD-NC	21/24	11/24	8/24	402	245	150	0.006
KD-CXCR2	18/24	7/24	2/24	843	543	350

Figure 7. Effect of CXCR2 on CD133⁺ cell rate and sphere formation efficiency. The effect of CXCR2 overexpression and knockdown on CD133+ cell rate (A,B) and sphere formation efficiency (C,D) in NCI-H1437 cells. The effect of CXCR2 overexpression and knockdown on CD133+ cell rate (E,F) and sphere formation efficiency (G,H) in NCI-H1299 cells. Comparison of CD133+ cell rate and sphere number/1000 cells between the two groups was conducted by independent sample’s t-test. P < 0.05 was considered significant. CXCR2, C-X-C Chemokine Receptor Type 2.

Figure 8. Effect of CXCR2 on chemosensitivity. The effect of CXCR2 overexpression and knockdown on NCI-H1437 cells treated with cisplatin of different concentrations (A) and NCI-H1437 cells treated with gemcitabine of different concentration (B). The effect of CXCR2 overexpression and knockdown on IC₅₀ value of cisplatin or gemcitabine in NCI-H1437 cells (C). The effect of CXCR2 overexpression and knockdown on NCI-H1299 cells treated with cisplatin of different concentrations (D) and NCI-H1299 cells treated with gemcitabine of different concentration (E). The effect of CXCR2 overexpression and knockout on IC₅₀ value of cisplatin or gemcitabine in NCI-H1299 cells (F) Comparison of relative cell viability and IC₅₀ value between the two groups was conducted by independent sample’s t-test. P < 0.05 was considered significant. CXCR2, C-X-C Chemokine Receptor Type 2.

Figure 9. Effect of CXCR2 on JAK2/STAT3 pathway. The effect of CXCR2 overexpression and knockdown on JAK2 mRNA (A) as well as JAK2 and P-STAT3 protein expression (B,C) in NCI-H1437 cells. The effect of CXCR2 overexpression and knockdown on JAK2 mRNA (D) as well as JAK2 and P-STAT3 protein expression (E,F) in NCI-H1299 cells. Comparison of mRNA expression and protein relative expression between groups was conducted by independent sample’s t-test. P < 0.05 was considered significant. CXCR2, C-X-C Chemokine Receptor Type 2; JAK2, janus kinase 2; STAT3, signal transductor and activator of transcription 3.

Data availability statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.