Aspirin-triggered proresolving mediators stimulate resolution in cancer

Abstract

Inflammation in the tumor microenvironment is a strong promoter of tumor growth. Substantial epidemiologic evidence suggests that aspirin, which suppresses inflammation, reduces the risk of cancer. The mechanism by which aspirin inhibits cancer has remained unclear, and toxicity has limited its clinical use. Aspirin not only blocks the biosynthesis of prostaglandins, but also stimulates the endogenous production of anti-inflammatory and proresolving mediators termed aspirin-triggered specialized proresolving mediators (AT-SPMs), such as aspirin-triggered resolvins (AT-RvDs) and lipoxins (AT-LXs). Using genetic and pharmacologic manipulation of a proresolving receptor, we demonstrate that AT-RvDs mediate the antitumor activity of aspirin. Moreover, treatment of mice with AT-RvDs (e.g., AT-RvD1 and AT-RvD3) or AT-LXA₄ inhibited primary tumor growth by enhancing macrophage phagocytosis of tumor cell debris and counter-regulating macrophage-secreted proinflammatory cytokines, including migration inhibitory factor, plasminogen activator inhibitor-1, and C-C motif chemokine ligand 2/monocyte chemoattractant protein 1. Thus, the pro-resolution activity of AT-resolvins and AT-lipoxins may explain some of aspirin's broad anticancer activity. These AT-SPMs are active at considerably lower concentrations than aspirin, and thus may provide a nontoxic approach to harnessing aspirin's anticancer activity.

Keywords: eicosanoids; inflammation; metabolomics; metastasis; resolvins.

Fig. 1.

AT-SPMs or aspirin inhibits primary tumor growth. (A) AT-RvD1, AT-RvD3, or AT-LXA₄ (0.6 μg/kg/d) with primary LLC tumor growth. Treatment was initiated on the day of tumor cell injection throughout. Values are represented as mean ± SEM; n = 5–10 mice/group. The two-tailed Student t test was used for final tumor measurements. *P < 0.05 vs. control. (B) Aspirin (30 mg/kg/d used throughout) with orthotopic LLC tumor growth. n = 4–5 mice/group. *P < 0.05 vs. control. Lungs were resected and weighed, and visible metastatic nodules were counted at 19 d after LLC injection. *P < 0.05 vs. control. (C) LC-MS/MS fragmentation spectra of AT-RvD1, AT-RvD3, and AT-LXA₄ in LLC tumor tissue from mice systemically treated with aspirin (30 mg/kg/d) for 9 d.

Fig. 2.

Antitumor activity of aspirin is resolvin-receptor dependent. WRW4 (1 mg/kg/d) and/or aspirin (30 mg/kg/d) with primary LLC (A) or spontaneous MMTV-PyMT (B) tumor growth. Values are mean ± SEM. For MMTV-PyMT mice, tumor volume represents the sum tumor volume of all visible tumors per mouse. n = 5–10 mice/group throughout. The two-tailed Student t test was used for final tumor measurements. *P < 0.05, aspirin vs. control; aspirin vs. aspirin + WRW4. (C) Aspirin (dashed lines) or control (solid lines) with LLC tumor growth in ALX/FPR2 KO mice (red lines) compared with WT mice (blue lines). Treatment was initiated once tumors reached 100–200 mm³. *P < 0.05 vs. WT control; n.s., not significant. (D) WRW4 and aspirin with primary LLC tumor resection and subsequent metastasis. Lung weights (g) and images are representative of lung metastases for each treatment group on day 14 after LLC tumor resection. *P < 0.05, aspirin vs. control; **P < 0.01, aspirin vs. aspirin + WRW4.

Fig. 3.

AT-SPMs stimulate macrophage phagocytosis of therapy-generated tumor cell debris. Shown is human monocyte-derived macrophage or RAW264.7 murine macrophage phagocytosis of carboxyfluorescein diacetate-labeled tumor cell debris following AT-SPM treatment. Phagocytosis was quantified by relative fluorescent units (RFUs) normalized to percent increase above vehicle-treated macrophages. The two-tailed Student t test was used throughout; *P < 0.05; **P < 0.01 vs. vehicle. n = 6–12/group throughout. (A) AT-RvD3 with human or murine macrophage phagocytosis of etoposide- or erlotinib-generated tumor cell debris (H460, HCC827, and LLC). (B) AT-RvD1 with murine macrophage phagocytosis of erlotinib- or etoposide-generated LLC tumor cell debris. (C) AT-LXA₄ with human macrophage phagocytosis of etoposide-generated H460 tumor cell debris.

Fig. 4.

Low-dose aspirin stimulates macrophage phagocytosis of therapy-generated tumor cell debris. Shown is human monocyte-derived macrophage or RAW264.7 murine macrophage phagocytosis of carboxyfluorescein diacetate-labeled tumor cell debris following aspirin treatment. Phagocytosis was quantified by RFUs normalized to percent increase above vehicle-treated macrophages. The two-tailed Student t test was used throughout. *P < 0.05; **P < 0.01 vs. vehicle. n = 6–12/group throughout. (A and B) Low-dose aspirin (10⁻³−10² nM) with human macrophage phagocytosis of etoposide-generated H460 tumor cell debris (A) or erlotinib-generated HCC827 tumor cell debris (B). (C) Low- or high-dose aspirin (2,000 or 5,000 nM) with murine macrophage phagocytosis of erlotinib-generated LLC tumor cell debris. (D) WRW4 (10 μM) and aspirin (1 nM) with human macrophage phagocytosis of erlotinib-generated HCC827 tumor cell debris.

Fig. 5.

AT-SPMs and low-dose aspirin suppress macrophage secretion of proinflammatory cytokines. Shown is human monocyte-derived macrophage cytokine secretion following AT-SPM or aspirin (1 nM) treatment and/or coincubation with tumor cell debris. Values are represented as mean ± SEM throughout. The two-tailed Student t test was used throughout. *P < 0.05; **P < 0.01 vs. control (A–F) or aspirin alone (B, D, and F). n.s., not significant (C). n = 3–6/group throughout. (A and B) Baseline (gray bars) or H460 tumor cell debris-stimulated (black bars) macrophage secretion of macrophage MIF following treatment with AT-LXA₄ (1 nM), AT-RvD3 (1 nM), aspirin (1 nM), or aspirin (1 nM) + WRW4 (10 μM). (C and D) Baseline (gray bars) or HCC827 tumor cell debris-stimulated (black bars) macrophage secretion of PAI-1 following treatment with AT-RvD3 (1 nM), aspirin (1 nM), or aspirin (1 nM) + WRW4 (10 μM). (E and F) Baseline (gray bars) or H460 tumor cell debris-stimulated (black bars) macrophage secretion of CCL2/MCP-1 following treatment with AT-LXA₄ (1 nM), AT-RvD3 (1 nM), aspirin (1 nM), or aspirin (1 nM) + WRW4 (10 μM).