Prostaglandin E2 receptor EP 4 as the common target on cancer cells and macrophages to abolish angiogenesis, lymphangiogenesis, metastasis, and stem‐like cell functions

We previously established that COX ‐2 overexpression promotes breast cancer progression and metastasis. As long‐term use of COX ‐2 inhibitors ( COX ‐2i) can promote thrombo‐embolic events, we tested an alternative target, prostaglandin E2 receptor EP 4 subtype ( EP 4), downstream of COX ‐2. Here we used the highly metastatic syngeneic murine C3L5 breast cancer model to test the role of EP 4‐expressing macrophages in vascular endothelial growth factor ( VEGF )‐C/D production, angiogenesis, and lymphangiogenesis in situ , the role of EP 4 in stem‐like cell ( SLC ) functions of tumor cells, and therapeutic effects of an EP 4 antagonist RQ ‐15986 ( EP 4A). C3L5 cells expressed all EP receptors, produced VEGF ‐C/D, and showed high clonogenic tumorsphere forming ability in vitro , functions inhibited with COX ‐2i or EP 4A. Treating murine macrophage RAW 264.7 cell line with COX ‐2i celecoxib and EP 4A significantly reduced VEGF ‐A/C/D production in vitro , measured with quantitative PCR and Western blots. Orthotopic implants of C3L5 cells in C3H/HeJ mice showed rapid tumor growth, angiogenesis, lymphangiogenesis ( CD 31/ LYVE ‐1 and CD 31/ PROX 1 immunostaining), and metastasis to lymph nodes and lungs. Tumors revealed high incidence of EP 4‐expressing, VEGF ‐C/D producing macrophages identified with dual immunostaining of F4/80 and EP 4 or VEGF ‐C/D. Celecoxib or EP 4A therapy at non‐toxic doses abrogated tumor growth, lymphangiogenesis, and metastasis to lymph nodes and lungs. Residual tumors in treated mice revealed markedly reduced VEGF ‐A/C/D and phosphorylated Akt/ ERK proteins, VEGF ‐C/D positive macrophage infiltration, and proliferative/apoptotic cell ratios. Knocking down COX ‐2 or EP 4 in C3L5 cells or treating cells in vitro with celecoxib or EP 4A and treating tumor‐bearing mice in vivo with the same drug reduced SLC properties of tumor cells including preferential co‐expression of COX ‐2 and SLC markers ALDH 1A, CD 44, OCT ‐3/4, β‐catenin, and SOX ‐2. Thus, EP 4 is an excellent therapeutic target to block stem‐like properties, angiogenesis, and lymphangiogenesis induced by VEGF ‐A/C/D secreted by cancer cells and tumor infiltrating macrophages.