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

Abstract

This study aimed to investigate the correlation of C-X-C chemokine receptor 2 (CXCR2) with clinicopathological characteristics and survival in non-small cell lung cancer (NSCLC) patients and further explore its effect on proliferation, apoptosis, invasion, stemness, chemosensitivity as well as JAK2/STAT3 pathway in NSCLC cells. The expression of CXCR2 in tumor tissues and adjacent tissues from 340 NSCLC patients received surgery was detected by immunohistochemistry. CXCR2 overexpression and knockdown were constructed through plasmid transfection and the effect of CXCR2 dysregulation on cell proliferation, apoptosis, invasion, stemness, chemosensitivity as well as its regulatory effect on JAK2/STAT signaling pathway was assessed in NCI-H1437 cells and NCI-H1299 cells. CXCR2 expression was higher in tumor tissues than that in paired adjacent tissues, and it was correlated with poor pathological differentiation, greater tumor size, lymph node metastasis, higher TNM stage and poor survival in NSCLC patients. In vitro, CXCR2 expression was increased in human NSCLC cell lines compared with human normal lung bronchus epithelial cells. CXCR2 promoted cell proliferation and invasion, while suppressed cell apoptosis in NCI-H1437/NCI-H1299 cells. Additionally, CXCR2 increased CD133⁺ cell rate and cell sphere-forming ability, while reduced chemosensitivity to cisplatin and gemcitabine in NCI-H1437/NCI-H1299 cells. Besides, CXCR2 activated the JAK2/STAT3 signaling pathway in NCI-H1437/NCI-H1299 cells. In conclusion, the clinical implication and the molecular function of CXCR2 discovered in our study reveal the potential of CXCR2 as a future target for disease monitoring and treatment of NSCLC.

Keywords: C-X-C chemokine receptor 2; JAK2/STAT3 pathway; non-small cell lung cancer.

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.

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.

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.

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.