Abstract 2310: Targeting EP4 receptor on cancer cells and macrophages to abrogate stem-like functions, lymphangiogenesis and lymphatic metastasis in a breast cancer model.
Authors: Majumder Mousumi , Xin Xiping , Girish GannaReddy V. , Lala Peeyush K. 
Published: August 2014

Background: We established that high COX-2 expression resulting in elevated prostaglandin (PG) E2 level in the tumor milieu promotes breast cancer progression and metastasis via multiple events: inactivation of immune cells, stimulation of tumor cell migration/invasiveness, tumor-associated angiogenesis and lymphangiogenesis. The lymphangiogenic action of COX-2 resulted from high VEGF-C/D production by tumor cells. These events were primarily due to activation of the PGE receptor EP4 on cancer cells, making it an excellent therapeutic target. Objectives: Present study tested the additional role of tumor-infiltrating macrophages in VEGF-C/D production and lymphangiogenesis in situ, the role of EP4 in stem-like tumor cell functions, and therapeutic effects of an EP4 antagonist. Since human cancer xenografts in immuno-compromised mice have limitations for preclinical testing, we developed a COX-2 expressing syngeneic murine breast cancer model of spontaneous lymphatic metastasis mimicking human and applied it for mechanistic and therapeutic studies. Methods and results: We tested the functional roles of COX-2 and EP4 in a highly metastatic COX-2 expressing murine breast cancer cell line C3L5. These cells expressed all EP receptors, produced VEGF-C and -D and showed high tumorsphere forming ability in ultra-low attachment plates by single cells for successive generations (surrogate of stem-like cell function) in vitro. Migration, tumorsphere formation and VEGF-C/D production by these cells were inhibited with Celecoxib or NS398 (COX-2 inhibitors) and a selective EP4 antagonist RQ-15986 (RaQualia Pharma). C3H/HeJ mice, when implanted SC in both inguinal regions with C3L5 cells suspended in growth factor-reduced Matrigel, exhibited rapid tumor growth, tumor-associated angiogenesis and lymphangiogenesis (respectively measured with CD31 and LYVE-1 immunostaining), metastasis to inguinal and axillary lymph nodes and the lungs. Tumors revealed high incidence of F4/ 80 +ve macrophages which also expressed EP4 and produced VEGF-C and -D in situ as evidenced by dual immunostaining of F4/80 and EP4, VEGF-C and -D. Chronic oral administration of Celecoxib or RQ-15986 at nontoxic doses abrogated tumor growth, lymphangiogenesis, angiogenesis, and metastasis to lymph nodes and lungs. Residual tumors in treated mice revealed markedly reduced VEGF-C/D proteins and incidence of VEGF-C/D +ve macrophages in situ. Conclusions: EP4 is an excellent therapeutic target to block stem-like properties, lymphangiogenesis induced by VEGF-C/D production by cancer cells and tumor infiltrating macrophages, deserving clinical testing for chemo-intervention in human breast cancer. (MM and XX contributed equally. Supported by grants of the CBCF, Ontario Chapter and the OICR to PKL, a TBCRU fellowship to MM and a gift of RQ-15986 by RaQualia Pharma).

Citation Format: Mousumi Majumder, Xiping Xin, GannaReddy V. Girish, Peeyush K. Lala. Targeting EP4 receptor on cancer cells and macrophages to abrogate stem-like functions, lymphangiogenesis and lymphatic metastasis in a breast cancer model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2310. doi:10.1158/1538-7445.AM2013-2310