Abstract 4961: Control of tumor lymphangiogenesis by epoxyeicosanoids.
Authors: Mudge Dayna K. , Serhan Karolina , Bielenberg Diane R. , Hwang Sung Hee , Hammock Bruce D. , Kieran Mark W. , Panigrahy Dipak 
Published: August 2014

Background: Epoxyeicosatrienoic acids (EETs) are lipid autacoids biosynthesized by cytochrome P450 epoxygenases and metabolized by soluble epoxide hydrolase (sEH) to less active dihydroxyeicosatrienoic acids (DHETs). EETs are autocrine and paracrine mediators of arachidonic acid-induced vasorelaxation in the cardiovascular and renal systems. Thus, inhibitors of sEH, which raise endogenous EET levels, are being considered for long-term use in hypertension, diabetes, stroke and other medical conditions. 20-hydroxeicosatetraenoic acid (20-HETE) is a small molecule biosynthesized by cytochrome P450 ω-hydroxylases. We recently demonstrated that EETs stimulate multiorgan and lymph node metastasis, a major cause of disease dissemination and death. However, the mechanism of EETs in tumor lymphangiogenesis is unknown. Thus, we hypothesize that EETs stimulate lymph node metastasis via tumor lymphangiogenesis. Methods and Results: Using two murine tumor lines transfected with VEGFC, B16F10-VEGFC melanoma and T241-VEGFC fibrosarcoma tumors, we demonstrate that EETs stimulate primary tumor growth via tumor lymphangiogenesis. Systemic administration of 14,15-EET and/or 20-HETE by osmotic minipump accelerated primary B16F10-VEGFC melanoma and T241-VEGFC fibrosarcoma tumor growth. Inhibitors of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs, elevated endogenous EET levels and also promoted tumor growth: sEH inhibitors (TUPS and AUCB) stimulated primary B16F10-VEGFC tumor growth. Conversely, systemic administration of a 20-HETE antagonist (HET0016) and/or EET antagonist (14,15-EEZE) regressed established primary B16F10-VEGFC tumors. 14,15-EET and 11,12-EET stimulated lymphatic endothelial cell proliferation, viability and migration. To determine whether tumor lymphangiogenesis contributes to the increase in B16F10-VEGFC tumor growth in 14,15-EET treated mice, we analyzed tumors for the specific lymphangiogenesis markers podoplanin and LYVE-1. Immunohistochemistry studies revealed an increase in podoplanin-positive and LYVE-1-positive vessels in B16F10-VEGFC tumors treated with 14,15-EET compared to vehicle treatment. 14,15-EET, 11,12-EET, and 20-HETE increased the production of VEGFC by B16F10-VEGFC and T241 fibrosarcoma tumor cells. Using a well-established model in which resection of a primary tumor (Lewis lung carcinoma) reproducibly stimulates the development of distant metastases 14-17 days after resection, we investigated whether EET antagonists inhibit spontaneous metastatic growth. The EET antagonist 14,15-EEZE and/or siRNA to VEGFR3 inhibited lung metastasis and the combination showed additive inhibition. Conclusion: EETs and 20-HETE stimulate tumor lymphangogenesis, offering a mechanistic rationale for using EET and 20-HETE antagonists as novel anti-cancer therapeutics to inhibit lymph node metastasis and tumor lymphangiogenesis.

Citation Format: Dayna K. Mudge, Karolina Serhan, Diane R. Bielenberg, Sung Hee Hwang, Bruce D. Hammock, Mark W. Kieran, Dipak Panigrahy. Control of tumor lymphangiogenesis by epoxyeicosanoids. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4961. doi:10.1158/1538-7445.AM2013-4961