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.

引言：既往本团队报道，白细胞介素8（interleukin-8, IL-8）与基底样乳腺癌患者的不良预后相关，且已被证实为促肿瘤发生因子，可促进细胞侵袭与迁移。本研究旨在探究靶向IL-8激活的趋化因子受体CXCR1与CXCR2的抑制剂的药理学效应。
方法：采用凯普兰-迈耶绘图法（Kaplan-Meier plotter）分析IL-8表达与三阴性乳腺癌（triple-negative breast cancer, TNBC）患者生存率的相关性。通过实时荧光定量PCR（real-time PCR）与蛋白质免疫印迹（western blotting）检测mRNA与蛋白表达水平。采用人细胞凋亡蛋白质组检测芯片（Proteome Profiler Human Apoptosis Array）分析SB225002对细胞凋亡相关蛋白的调控作用。采用MTT比色法与细胞集落形成实验检测细胞增殖活性。采用荧光激活细胞分选术（Fluorescence Activated Cell Sorting, FACS）分析细胞凋亡与细胞周期情况。
结果：异常IL-8表达与三阴性乳腺癌患者的预后密切相关。与HER2阳性及/或雌激素受体阳性乳腺癌细胞相比，三阴性乳腺癌细胞中的基础IL-8水平显著升高。此外，重组人IL-8处理可增强三阴性乳腺癌细胞的侵袭能力。为抵消IL-8的促肿瘤效应，本研究评估了CXCR1与CXCR2抑制剂的治疗潜力。值得注意的是，CXCR1特异性抑制剂reparixin对细胞活力无明显影响，而CXCR2特异性抑制剂SB225002可呈剂量依赖性显著降低细胞活力。经SB225002处理后，包括Bcl-2、cIAP-1、cIAP-2、Survivin、XIAP、HIF-1α及HO-1在内的抗凋亡生物标志物水平均显著下调。本研究发现，SB225002处理可增加三阴性乳腺癌细胞的凋亡细胞比例。在异种移植模型中，SB225002可有效降低已知可向肺与肝脏转移的4T1细胞的转移潜能。
结论：本研究结果证实IL-8/CXCR2信号轴在三阴性乳腺癌转移中发挥关键作用，且提示SB225002等CXCR2抑制剂有望成为三阴性乳腺癌患者的潜在治疗药物。