Abstract LB-67: Breast cancer-associated lymphangiogenesis: roles of PGE2 and EP4 receptor on lymphatic endothelial cells

Abstract

Abstract Background: Lymphatic metastasis is a common event in breast cancer, facilitated by tumor-associated lymphangiogenesis, molecular mechanisms of which remain poorly defined. We had earlier shown that endogenous PGE2 resulting from elevated COX-2 expression by breast cancer cells promotes tumor progression and metastasis by multiple mechanisms: inactivation of host immune cells, stimulation of tumor cell migration, tumor-associated angiogenesis and lymphangiogenesis. The latter was due to upregulation of VEGF-C or -D secretion due to activation of EP4 receptors on breast cancer cells and cancer-infiltrating macrophages. Objectives: To examine whether PGE2 in the tumor micro-environment directly promotes lymphangiogenesis by stimulating EP4 receptors on lymphatic endothelial cells (LEC).Approaches: In vitro studies utilized a rat mesenteric lymphatic endothelial cell line (RMLEC) and a COX-2 expressing, VEGF-C/-D producing murine breast cancer cell line C3L5; in vivo studies measured lymphangiogenesis in nude mice. Results RMLEC expressed all EP receptors and responded to PGE2, an EP4 agonist (EP4a) PGE1-OH, or C3L5 cell conditioned media (C3L5-CM) by increased proliferation, migration and accelerated tube formation on growth factor-reduced matrigel. Tube formation on matrigel alone was completely abrogated in the presence of COX 1/2 inhibitor indomethacin (10uM), COX-2 inhibitor (COX-2i) NS-398 (15uM), and a selective EP4 antagonist (EP4A) RQ15986 (2.5 nM), indicating the roles of COX-2 and EP4. Addition of PGE2, EP4a, or C3L5-CM individually in presence of the COX-2i, or EP4A, partially restored the tube formation, reinforcing the role of EP4 on RMLEC. RMLEC grown in the presence of PGE2 showed upregulation of COX2, EP4, VEGF-C and VEGF-D mRNAs. Knocking down EP4 with shRNA in RMLEC abrogated tube formation on matrigel in the absence or presence of PGE-2, EP4a, or C3L5-CM. EP4 silenced RMLEC also became unresponsive to these agents in stimulating PGE2, VEGF-C, and -D production. Finally in a directed in vivo lymphangiogenesis assay (DIVLA) using implants of angioreactors including PGE-2, EP4a or VEGF-C in the dorsal flanks of nude mice, we simultaneously measured lymphangiogenesis and angiogenesis using dual immunostaining of the tissue contents for Lyve-1 and cd31, or prox-1 and cd31. Results demonstrated the lymphangiogenic as well as angiogenic capacity of PGE2 and EP4a in vivo by recruitment of new vessels. Conclusions: Tumor or host-derived PGE2 stimulates lymphangiogenesis, at least in part, by activating EP4 on the LEC. Combined with earlier studies we show that EP4 is a common therapeutic target on tumor and host cells (macrophages and LEC) in abrogating multiple events in breast cancer progression. This may spare cardio-protective prostanoids like PGI2, inhibited by COX-2 inhibitors Citation Format: Peeyush K. Lala, Gannareddy V. Girish, Xiping Xin, Mousumi Majumder, Elena Tutunea-Fatan, Pinki Nandi. Breast cancer-associated lymphangiogenesis: roles of PGE2 and EP4 receptor on lymphatic endothelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-67. doi:10.1158/1538-7445.AM2014-LB-67