Inhibition of Interleukin-8/C-X-C Chemokine Receptor 2 Signaling Axis Prevents Tumor Growth and Metastasis in Triple-Negative Breast Cancer Cells

Abstract

Introduction: Previously, we reported that interleukin-8 (IL-8) was associated with poor prognosis of basal-like breast cancer patients and has been identified as a pro-tumorigenic factor, facilitating cell invasion and migration. Here, we investigated the pharmacological impact of inhibitors targeting the chemokine receptors, C-X-C chemokine receptor 1 (CXCR1) and C-X-C chemokine receptor 2 (CXCR2), which are activated by IL-8.

Methods: The survival rates of triple-negative breast cancer (TNBC) patients by IL-8 were analyzed by the Kaplan-Meier plotter. The levels of mRNA and protein expression were analyzed by real-time PCR and Western blotting. The alteration of apoptotic cell death-related proteins by SB225002 was analyzed by the Proteome Profiler Human Apoptosis Array. Cell growth was analyzed by MTT and colony-forming assay. Apoptosis and cell cycle were analyzed by fluorescence-activated cell sorting.

Results: Aberrant IL-8 expression is involved with the prognosis of TNBC patients. Basal IL-8 levels are markedly elevated in TNBC cells compared to those in HER2+ and/or ER+ breast cancer cells. Furthermore, recombinant human IL-8 treatment enhanced cell invasiveness in TNBC cells. To counteract the tumor-promoting effects of IL-8, we assessed the therapeutic potential of CXCR1 and CXCR2 inhibitors. Notably, while reparixin, a CXCR1-specific inhibitor, exhibited no impact on cell viability, SB225002, a CXCR2-specific inhibitor, significantly reduced cell viability in a dose-dependent manner. There was a noticeable reduction in the levels of anti-apoptotic biomarkers, including Bcl-2, cIAP-1, cIAP-2, Survivin, XIAP, HIF-1α, and HO-1, following SB225002 treatment. Our findings indicate an increase in the apoptotic cell population with SB225002 treatment in TNBC cells. In xenograft models, SB225002 effectively diminished the metastatic potential of 4T1 cells, which are known to metastasize to the lung and liver.

Conclusion: Our results underscore the significant role of the IL-8/CXCR2 signaling axis in the metastasis of TNBC and suggest that CXCR2 inhibitors such as SB225002 may be promising therapeutic agents for TNBC patients.

Keywords: Apoptosis; C-X-C chemokine receptor 1; C-X-C chemokine receptor 2; Interleukin-8; Triple-negative breast cancer.

Fig. 1.

Aberrant IL-8 expression is associated with poor prognosis in TNBC patients. Analysis of recurrence-free survival (RFS, low, n = 283; high, n = 109) (a), overall survival (OS, low, n = 93; high, n = 60) (b), and distant metastasis-free survival (DMFS, low, n = 160; high, n = 146) (c) in relation to IL-8 expression levels, evaluated through the Kaplan-Meier plotter database for TNBC patients. Statistical significance determined via the log-rank test.

Fig. 2.

The levels of IL-8 expression in breast cancer cells and the effect of IL-8 on cell invasiveness in TNBC cells. a Determination of IL-8 transcript levels across ER-α+ (ER+/PR+/HER2−: MCF7, T47D or ER+/PR+/HER2+: BT474), HER2+ (ER−/PR−/HER2+: HCC1419, SKBR3, MDA453), and TNBC (ER−/PR−/HER2−: HCC1143, Hs578T, MDA231, MDA468) cells through quantitative reverse transcription PCR. b Quantification of secreted IL-8 proteins in conditioned media from breast cancer cells using ELISA. c Assessment of cell invasion in TNBC cells with or without treatment of 20 ng/mL IL-8. Results presented are from three separate experiments.

Fig. 3.

A specific CXCR2 inhibitor, SB225002, dose-dependently decreases the cell viabilities in TNBC cells. a Depiction of chemical structures for reparixin and SB225002. b Analysis of cell viabilities post reparixin treatment via MTT assay. c Evaluation of cell viabilities following SB225002 treatment through MTT assay. d Investigation of cell proliferation following treatment with reparixin or SB225002 through colony forming assay. Data represent three independent trials. Values are expressed as mean ± SEM. Significance indicated by *p < 0.05, **p < 0.01 compared to the untreated group.

Fig. 4.

SB225002 induces the apoptotic cell death in Hs578T TNBC cells. a Proteomic array analysis of apoptosis-related proteins using Proteome Profiler Human Apoptosis Array Kit. We treated with 10 µm SB225002 for 24 h in Hs578T cells. After 24 h, whole cell lysates were harvested using lysis buffer from the kit. Dot blotting was performed as described in the methods. b FACS-based quantification of apoptotic cell death via Annexin V-PE and PE-Cy7 staining, as outlined in methods. c Graphical representation of data from b. d, e We treated with 10 µm SB225002 or reparixin for 24 h in Hs578T cells. After 24 h, whole cell lysates were harvested. Levels of pro-, cleaved-PARP-1, caspase3, total (t)-, phospho (p)-STAT-3, AKT, and ERK proteins were analyzed by Western blotting. β-actin was used as a loading control. Represented results are from three independent experiments. Values are mean ± SEM, with **p < 0.01 against control (Con).

Fig. 5.

SB225002 suppresses the metastatic potential of TNBC. a FACS-based analysis of apoptotic cell death in 4T1 cells employing Annexin V-PE and PE-Cy7 staining, following the described methods. b Quantification and graphical representation of findings from a. Data stem from three separate studies. Values presented as mean ± SEM, **p < 0.01 versus control (Con). c Examination of cell proliferation via colony forming assay post-treatment with reparixin or SB225002. d We treated with 10 µm SB225002 or reparixin for 24 h in 4T1 cells. After 24 h, whole cell lysates were harvested. Levels of pro-, and cleaved-PARP-1, caspase3 proteins were analyzed by Western blotting. β-actin was used as a loading control. e Illustrative scheme of the experimental approach. f Evaluation of primary tumor size in the 2nd fat pad following vehicle or SB225002 treatment, expressed as mean ± SEM, p = 0.0046 (vehicle vs. SB225002). g Analysis of lung metastasis of 4T1 cells post vehicle or SB225002 treatment. Control identified as Con.