Lipoxin A4 a novel therapeutic agent for the treatment of breast cancer metastases

Clinical data for solid tumours show a correlation between high-density leukocyte infiltration and poor patient outcomes. Tumour-associated neutrophils (TAN) and macrophages (TAM) can account for 50% of the total tumour mass in invasive breast carcinomas and these inflammatory cells positively influence tissue remodeling and the development of angiogenic vasculature by producing pro-angiogenic mediators and extracellular proteases. Lipoxins (LXs), trihydroxytetraene-containing eicosanoids, are endogenous “braking signals” for inflammation. LXs down regulate pro-inflammatory cytokines, inhibit matrix metalloproteinase 9 (MMP-9), vascular endothelial growth factor (VEGF), and Hypoxia Induced Factor (HIF) and may act as a selective estrogen-receptor modulator (SERM). We hypothesis that LXA4 can alter immune tolerance and augment anti-tumour immune responses in breast cancer.

Methods: The metastatic human breast adenocarcinoma cell line MDA-MB-231 and the murine 4T1 breast cancer cell line were grown in culture and tumour conditioned medium (TCM) harvested, by centrifugation at 900g, filtered (0.22µm) and stored at -20°C. Human neutrophils were analysed from healthy volunteers’ blood. Cell surface receptor expression was assessed by flow cytometry using FITC-conjugated anti-mouse IACM-1 Mab, PE–conjugated anti-human CD11b and FITC-conjugated anti-human CD62L. Tumour cell invasion was assed using CytoSelect Cell Invasion culture plates. miRNA was isolated using miRNeasy) and reverse transcribed using TaqMan. miRNA expression was normalized to the values of the internal control Sno135.

Results: LXA4 (200nm) treatment significantly reduced CD11b adhesion receptor expression on human neutrophils induced by incubation with tumour conditioned medium (985±24.88 MCF: TCM vs 806±61.23 MCF: p<0.05, ANOVA). Treatment with LXA4 also reversed L-selectin shedding from both human and murine neutrophils induced by TCM (36.21±1.86 vs 47.41±5.8 and 70.53±2.11 vs 108.59±12.06, respectively p<0.05 ANOVA). In addition, LXA4 suppressed breast cancer cell invasion (77±9 cell/field IL-1β (10ng/ml) vs 57±7 cell/field IL-1β+LXA4 (200nm) p<0.05, ANOVA), and weakened metastatic potential by modulating tumour cell ICAM-1 expression in vitro (248.06±53.0 MCF IL-1β (10ng/ml) vs 129.5±46.23 MCF IL-1β+LXA4 (10nm) p<0.05, ANOVA n=4 on MDA-MB231; 91.91+-6.5 MCF IL-1β (10ng/ml) vs 49.32±9.4 MCF IL-1β+LXA4 (200nm) p<0.01, ANOVA n=3 on 4T1). LXA4 deceased secretion of matrix metalloproteinases (5.03±0.78 IL-1β vs 3.98±0.47 (ng/ml) IL-1β+LXA4 100nm in MDA-MB231 cells and (288.7±16.9 IL-1β vs 228.7±36.48(ng/ml) IL-1β+LXA4 100nm)in 4T1 cells). Furthermore LXA4 treatment at 4 hr significantly increased human (2.5 fold change) and murine 4T1 (1.6 fold change) tumour cell expression of miRNA 146.

Discussion: Transduction of miR-146 into breast cancer cells has been shown to inhibited invasion, migration in vitro, and significantly suppressed lung metastasis in a murine model. We demonstrate for the first time that LXA4 can augment miRNA 146 expression. Taken together, these results indicate that LXA4 has the potential to reduce breast cancer metastatic spread. Combination antiangiogenic therapeutic strategies that target both tumour-related inflammation and angiogenesis may prove successful where anti VEGF strategies alone have failed. Further investigation of LXA4 as a potential anti-tumour agent is merited in an in vivo murine model of breast cancer.