Supplementary Material for: Inhibition of IL-8/CXCR2 signaling axis prevents tumor growth and metastasis in triple negative breast cancer cells

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, CXCR1 and CXCR2, which are activated by IL-8. Methods: The survival rates of 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 FACS. Results: Aberrant IL-8 expression is involved with the prognosis of triple-negative breast cancer (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.