title: Prostaglandin E2 receptor EP4 as the common target on cancer cells and macrophages to abolish angiogenesis, lymphangiogenesis, metastasis, and stem-like cell functions.
abstract: 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 EP4 subtype (EP4), downstream of COX-2. Here we used the highly metastatic syngeneic murine C3L5 breast cancer model to test the role of EP4-expressing macrophages in vascular endothelial growth factor (VEGF)-C/D production, angiogenesis, and lymphangiogenesis in situ, the role of EP4 in stem-like cell (SLC) functions of tumor cells, and therapeutic effects of an EP4 antagonist RQ-15986 (EP4A). 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 EP4A. Treating murine macrophage RAW 264.7 cell line with COX-2i celecoxib and EP4A 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 (CD31/LYVE-1 and CD31/PROX1 immunostaining), and metastasis to lymph nodes and lungs. Tumors revealed high incidence of EP4-expressing, VEGF-C/D producing macrophages identified with dual immunostaining of F4/80 and EP4 or VEGF-C/D. Celecoxib or EP4A 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 EP4 in C3L5 cells or treating cells in vitro with celecoxib or EP4A 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 ALDH1A, CD44, OCT-3/4, β-catenin, and SOX-2. Thus, EP4 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.
authors: Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.
authors: Mousumi
authors: M
authors: Majumder
authors: Xiping
authors: X
authors: Xin
authors: Ling
authors: L
authors: Liu
authors: Gannareddy V
authors: GV
authors: Girish
authors: Peeyush K
authors: PK
authors: Lala
mesh_terms: D000230:Adenocarcinoma / Q000098:blood supply / Q000188:drug therapy* / Q000556:secondary; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use; D017209:Apoptosis; D001549:Benzamides / Q000494:pharmacology / Q000627:therapeutic use; D000068579:Celecoxib; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D051546:Cyclooxygenase 2 / Q000378:metabolism; D004354:Drug Screening Assays, Antitumor; D005260:Female; D008175:Lung Neoplasms / Q000098:blood supply / Q000188:drug therapy* / Q000556:secondary; D042583:Lymphangiogenesis; D008207:Lymphatic Metastasis; D008264:Macrophages / Q000378:metabolism*; D008325:Mammary Neoplasms, Experimental / Q000098:blood supply / Q000188:drug therapy* / Q000473:pathology; D051379:Mice; D008809:Mice, Inbred C3H; D058990:Molecular Targeted Therapy; D009368:Neoplasm Transplantation; D014411:Neoplastic Stem Cells; D009389:Neovascularization, Pathologic / Q000188:drug therapy*; D011720:Pyrazoles / Q000494:pharmacology / Q000627:therapeutic use; D058309:Receptors, Prostaglandin E, EP4 Subtype / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology / Q000627:therapeutic use; D047368:Tumor Burden; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: C523467:(S)-4-(1-(5-chloro-2-(4-fluorophenyoxy) benzamido)ethyl) benzoic acid; D000970:Antineoplastic Agents; D001549:Benzamides; D011720:Pyrazoles; D058309:Receptors, Prostaglandin E, EP4 Subtype; D013449:Sulfonamides; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2; D000068579:Celecoxib
keywords: Cancer stem cells; RQ-00015986; cyclooxygenase 2; macrophages; prostaglandin E2 receptor EP4 subtype