Prostaglandin E2 receptor EP4 as the common target on cancer cells and macrophages to abolish angiogenesis, lymphangiogenesis, metastasis, and stem-like cell functions

Cancer Sci. 2014 Sep;105(9):1142-51. doi: 10.1111/cas.12475. Epub 2014 Sep 11.


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.

Keywords: Cancer stem cells; RQ-00015986; cyclooxygenase 2; macrophages; prostaglandin E2 receptor EP4 subtype.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenocarcinoma / blood supply
  • Adenocarcinoma / drug therapy*
  • Adenocarcinoma / secondary
  • Animals
  • Antineoplastic Agents / pharmacology
  • Antineoplastic Agents / therapeutic use
  • Apoptosis
  • Benzamides / pharmacology
  • Benzamides / therapeutic use
  • Celecoxib
  • Cell Line, Tumor
  • Cell Proliferation
  • Cyclooxygenase 2 / metabolism
  • Drug Screening Assays, Antitumor
  • Female
  • Lung Neoplasms / blood supply
  • Lung Neoplasms / drug therapy*
  • Lung Neoplasms / secondary
  • Lymphangiogenesis
  • Lymphatic Metastasis
  • Macrophages / metabolism*
  • Mammary Neoplasms, Experimental / blood supply
  • Mammary Neoplasms, Experimental / drug therapy*
  • Mammary Neoplasms, Experimental / pathology
  • Mice
  • Mice, Inbred C3H
  • Molecular Targeted Therapy
  • Neoplasm Transplantation
  • Neoplastic Stem Cells
  • Neovascularization, Pathologic / drug therapy*
  • Pyrazoles / pharmacology
  • Pyrazoles / therapeutic use
  • Receptors, Prostaglandin E, EP4 Subtype / antagonists & inhibitors*
  • Receptors, Prostaglandin E, EP4 Subtype / metabolism
  • Sulfonamides / pharmacology
  • Sulfonamides / therapeutic use
  • Tumor Burden
  • Vascular Endothelial Growth Factor A / metabolism


  • (S)-4-(1-(5-chloro-2-(4-fluorophenyoxy) benzamido)ethyl) benzoic acid
  • Antineoplastic Agents
  • Benzamides
  • Pyrazoles
  • Receptors, Prostaglandin E, EP4 Subtype
  • Sulfonamides
  • Vascular Endothelial Growth Factor A
  • vascular endothelial growth factor A, mouse
  • Ptgs2 protein, mouse
  • Cyclooxygenase 2
  • Celecoxib