Epoxyeicosanoids stimulate multiorgan metastasis and tumor dormancy escape in mice

Abstract

Epoxyeicosatrienoic acids (EETs) are small molecules produced by cytochrome P450 epoxygenases. They are lipid mediators that act as autocrine or paracrine factors to regulate inflammation and vascular tone. As a result, drugs that raise EET levels are in clinical trials for the treatment of hypertension and many other diseases. However, despite their pleiotropic effects on cells, little is known about the role of these epoxyeicosanoids in cancer. Here, using genetic and pharmacological manipulation of endogenous EET levels, we demonstrate that EETs are critical for primary tumor growth and metastasis in a variety of mouse models of cancer. Remarkably, we found that EETs stimulated extensive multiorgan metastasis and escape from tumor dormancy in several tumor models. This systemic metastasis was not caused by excessive primary tumor growth but depended on endothelium-derived EETs at the site of metastasis. Administration of synthetic EETs recapitulated these results, while EET antagonists suppressed tumor growth and metastasis, demonstrating in vivo that pharmacological modulation of EETs can affect cancer growth. Furthermore, inhibitors of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs, elevated endogenous EET levels and promoted primary tumor growth and metastasis. Thus, our data indicate a central role for EETs in tumorigenesis, offering a mechanistic link between lipid signaling and cancer and emphasizing the critical importance of considering possible effects of EET-modulating drugs on cancer.

Figure 1. EETs stimulate primary tumor growth and escape from tumor dormancy.

(A) Growth of B16F10 melanoma and T241 fibrosarcoma primary tumors in sEH-null, Tie2-CYP2C8-Tr, and WT mice. n = 10–14 mice/group; *P = 0.042, **P = 0.024, ^#P = 0.008, ^§P < 0.001 versus WT; unpaired Student’s t test is used throughout. (B) Primary B16F10 melanoma and T241 fibrosarcoma tumor growth is inhibited in Tie2-sEH-Tr mice on days 22–28. n = 5–6 mice/group; *P = 0.001, **P = 0.006 versus WT. (C) Systemic administration of 14,15-EET (15 μg/kg/d) via osmotic minipump stimulates primary LLC tumor growth. When 1 × 10⁴ or 1 × 10³ LLC cells were injected, 5 of 9 control mice and 9 of 9 control mice, respectively, did not exhibit visible tumor growth up to 209 days after injection. Images show representative mice on day 29 after injection with 1 × 10⁴ LLC cells. n = 6–9 mice/group; *P ≤ 0.02, **P ≤ 0.001, ^#P ≤ 0.006 versus WT. The experiment was performed 3 times with similar results. Rulers show centimeters. (D) 14,15-EET (15 μg/kg/d) stimulates tumor growth in a genetically engineered model of cancer (TRAMP). Ruler shows centimeters. At 90 days of treatment, H&E-stained section of prostate of control TRAMP mice reveals prostatic intraepithelial neoplasia (PIN) surrounded by tumor cells, while H&E-stained section of prostate of TRAMP mice treated with 14,15-EET shows glandular parenchyma surrounded by tumor cells. n = 6 mice/group; *P = 0.002 versus control. Scale bars: 100 μm. (E) 14,15-EET accelerates escape in a tumor dormancy model of human liposarcoma cells (clone 4). After treatment with 14,15-EET for 100 days, large palpable liposarcoma tumors are visible. No tumor is visible in the control mice. Ruler shows centimeters. H&E-stained section of tumors of mice treated with 14,15-EET reveal sheets of lipoblasts. n = 5–6 mice/group; *P = 0.008 versus control. Scale bar: 100 μm.

Figure 2. EETs trigger spontaneous and multiorgan metastasis.

(A) Spontaneous LLC metastasis is increased in Tie2-CYP2C8-Tr and sEH-null mice relative to WT 10 days after primary tumor removal (LLC resection). Images show representative lung metastasis in transgenic and WT mice. n = 5 mice/group; *P = 0.011, **P = 0.004, ***P ≤ 0.001 versus WT. The experiment was performed 3 times, with similar results. Scale bars: 1 cm. (B) Spontaneous LLC metastasis is decreased in Tie2-sEH-Tr relative to WT 17 days after primary tumor removal. n = 6 mice/group; *P = 0.001, **P = 0.002. (C) Systemic administration of 14,15-EET (15 μg/kg/d) via osmotic minipump increases spontaneous LLC lung metastasis and distant axillary and inguinal lymph node metastasis 12 days after LLC resection. Dashed circles indicates metastatic and normal lung; arrows show inguinal lymph node metastasis (white arrow, 14,15-EET; black arrow, control); scale bars: 1 cm. GFP-labeled LLC tumor cells (green) demonstrate bilateral axillary and inguinal lymph node metastasis in mice treated with 14,15-EET 17 days after LLC-GFP tumor resection; scale bars: 500 μm (for GFP staining). n = 10 mice/group; *P = 0.001, **P ≤ 0.001. (D) 14,15-EET (15 μg/kg/d) reduces survival and stimulates multiorgan lung, liver, and kidney metastasis of B16F10-GFP melanoma cells injected intravenously. Dashed circles indicate (top to bottom) representative lung, liver, and kidney metastasis 19 days after injection; scale bar: 1 cm. GFP-labeled melanoma tumor cells (green) confirm lung, liver, and kidney metastasis in mice treated with 14,15-EET; scale bar: 500 μm (for GFP staining). n = 7–9 mice/group; *P = 0.021. (E) 14,15-EET (15 μg/kg/d) stimulates liver metastasis in an orthotopic human prostate cancer (PC3M-LN4) model. H&E-stained section of liver metastasis of mice treated with 14,15-EET; arrows indicate nodules of tumor within hepatic parenchyma; scale bar: 100 μm. Black circles indicate representative orthotopic prostate tumors; scale bars: 1 cm. n = 5–6 mice/group.

Figure 3. Pharmacological modulation of EET levels controls primary tumor growth and metastasis.

(A) Systemic administration of an sEH inhibitor (tAUCB) stimulates primary LLC-GFP tumor growth. Images show representative tumors after 13 days of treatment. n = 6 mice/group; *P = 0.007. Scale bar: 1 cm. (B) Immunofluorescence double staining for VEGF and GFP (tumor cells) shows increased tumor cell expression of VEGF in LLC-GFP tumor cells from tAUCB- versus vehicle-treated mice. Green, GFP-stained tumor cells; red, VEGF-containing cells. Colocalization of red and green fluorescence (yellow) indicates tumor cells expressing VEGF (arrows). tAUCB-treated tumors have an increase in MECA-32–positive ECs (green). Scale bars: 20 μm. (C) Systemic administration of tAUCB and TUPS (10 mg/kg/d each) increases spontaneous B16F10 axillary lymph node metastasis 21 days after B16F10 resection. Representative axillary lymph nodes after 21 days of treatment are shown. Scale bars: 1 cm. n = 6 mice/group; *P = 0.029 versus control. (D) The EET antagonist 14,15-EEZE (0.21 mg/mouse) inhibits primary LLC growth (left panel), prolongs survival (middle panel), and reduces plasma VEGF levels (right panel) in a spontaneous LLC lung metastasis model. n = 5 mice/group; *P = 0.017, **P = 0.035, ***P = 0.044. (E) The EET antagonist 14,15-EEZE-mSI (0.21 mg/mouse) inhibits 14,15-EET–induced (15 μg/kg/d) spontaneous LLC metastasis. The stable EET metabolite 14,15-DHET (15 μg/kg/d) does not stimulate metastasis. Representative photographs on day 12 after LLC resection are shown. Scale bars: 1 cm. n = 5 mice/group; *P < 0.001, **P = 0.003 versus 14,15-EET.

Figure 4. Pro-tumorigenic activity of endothelium-derived EETs is mediated by VEGF induction and loss of TSP1.

(A) EC migration is decreased in Tie2-sEH-Tr mice (left panel) but increased in Tie2-CYP2J2-Tr and Tie2-CYP2C8-Tr relative to WT mice (middle). tAUCB and TUPS stimulate VEGF-mediated endothelial migration (right). n = 3–4/group; *P = 0.0139, **P = 0.029, and ***P < 0.001 versus WT; ^†P < 0.05 versus VEGF alone. (B) 14,15-EEZE inhibits VEGF-induced EC (left panel) but not tumor cell (LLC) migration (right). n = 3–4/group; *P = 0.038, **P = 0.012, ***P < 0.001 versus control. (C) VEGF ELISA of plasma of Tie2-CYP2C8-Tr and sEH-null mice 17 days after B16F10 resection shows an increase in VEGF levels. n = 5/group (left panel). Systemic administration of 14,15-EET regulates Vegfr2 mRNA levels in primary LLC tumors in mice compared with size matched control tumors, but has no effect on Vegfr1 mRNA levels (right). n = 5/group. *P < 0.05 versus WT; **P = 0.0095 versus control. (D) VEGF depletion with Ad-sFlt suppresses B16F10 tumor growth in Tie2-CYP2J2-Tr and sEH-null but not in WT mice. Plasma levels of sFlt1 on day 10 were 35,999 ± 10,225 pg/ml (Tie2-CYP2J2-Tr) and 36,680 ± 1,308 pg/ml (sEH-null). n = 5 mice/group; *P = 0.016, **P = 0.023. (E) tAUCB does not promote spontaneous LLC metastasis in mice depleted of VEGF with Ad-sFlt (tAUCB + Ad-sFlt). n = 5 mice/group; *P = 0.004, **P = 0.002 versus tAUCB + Ad-null. (F) The angiogenesis inhibitor TSP1 is downregulated in plasma of Tie2-CYP2C8-Tr, sEH-null, and Tie2-CYP2J2-Tr relative to WT mice on day 13 after LLC injection. rTSP1, recombinant TSP1. (G) 14,15-EEZE (0.21 mg/mouse) does not significantly inhibit primary LLC tumor growth in TSP1 null mice. n = 5 mice/group.

Figure 5. Pro-tumorigenic activity of EETs is mediated by VEGF production in the tumor stroma and loss of sEH.

(A) LLC tumors in VEGF-LacZ-Tr mice treated with 14,15-EET (15 μg/kg/d) show β-galactosidase staining (marker of VEGF production) in tumor endothelium and stromal fibroblasts (arrows). Scale bars: 20 μm. (B) Expression of sEH, but not CYP2J and CYP2C, is downregulated in tumor (TEC) versus normal (NEC) ECs (two left panels). Control tissue: mouse liver. Expression of sEH, but not CYP2J and CYP2C, is downregulated in tumor lysates from larger LLC tumors (>5 cm³) versus smaller LLC tumors (<1 cm³) (third panel). sEH expression (brown staining) is also downregulated in B16F10 melanoma liver metastasis compared with normal adjacent liver (far right panel). Scale bar: 100 μm.