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.

Aspirin blocks growth of breast tumor cells and tumor-initiating cells and induces reprogramming factors of mesenchymal to epithelial transition

“…However, these studies also showed that prostaglandin E receptor, which is a downstream regulator of COX-2 enzyme, plays critical role in regulation of stemness of BC cells. 60,61 We found that ASA was unable to regulate COX-2 expression at the protein levels in Aspirin prevents breast cancer cell growth G Maity et al Figure S3). Therefore, from these collective studies, we neither strongly support the involvement of COX-2 signaling nor are able to exclude that the COX-2 protein might not play any role in the growth of BC cells or breast tumor-initiating cells or both.…”

COX-2 Elevates Oncogenic miR-526b in Breast Cancer by EP4 Activation

“…The role of EP4 in tumor progression has also been reported in colonic tumors (21). Although EP4 activity on breast cancer cells promoted SLC phenotype (19), VEGF-C and -D production by cancer cells as well as tumor-infiltrating macrophages in situ was also attributed to EP4, explaining strong antitumor, antimetastatic, and SLC-reductive effects of EP4 antagonists in a murine breast cancer model (19). Thus, contribution of EP4 in both tumor and host cell-mediated events make it a promising therapeutic target in breast cancer.…”

“…For testing the ability of single cells to form spheroids or tumorspheres (in vitro surrogate of SLC), cells (1 Â 10 4 cells/mL) were passed through 8-mm filter (Falcon; BD Biosciences) and a syringe fitted with a 27-gauge needle, and plated in 6-well ultralow attachment plates (Corning), as previously described (19,39). All tumorspheres were grown in HuMEC (GIBCO) media supplemented with EGF (20 ng/mL; Invitrogen), basic fibroblast growth factor (FGF; 20 ng/mL; Invitrogen), and B27 (1:50 dilution; Life Technologies), and allowed to grow for 7 to 10 days, or until the majority of spheroids reached a diameter of 60 mm (19,39).…”

“…All tumorspheres were grown in HuMEC (GIBCO) media supplemented with EGF (20 ng/mL; Invitrogen), basic fibroblast growth factor (FGF; 20 ng/mL; Invitrogen), and B27 (1:50 dilution; Life Technologies), and allowed to grow for 7 to 10 days, or until the majority of spheroids reached a diameter of 60 mm (19,39). Tumorspheres were harvested followed by miRNA extraction; quantification and real-time qPCR to assess miR-526b expression for treated cell lines.…”

“…Indeed thrombo-embolic side effects of COX-2 inhibitors (11,12), resulting from inhibition of cardioprotective prostanoids such as PGI2 (13) may possibly be avoided by targeting EP4. We and others have shown that EP4 activity contributes to multiple mechanisms in breast cancer progression, including: inactivation of host antitumor immune cells (14,15); stimulation of tumor cell migration (16); invasiveness (16); angiogenesis (16,17); and lymphangiogenesis due to EP4-mediated upregulation of lymphangiogenic factors VEGF-C (17,18) or VEGF-D (19); induction of stem-like cell (SLC) phenotype in vitro (19,20) and in vivo (19). The role of EP4 in tumor progression has also been reported in colonic tumors (21).…”