Abstract 1601: COX-2 and EP4 receptor as targets to ameliorate tumor growth, lymphangiogenesis, and metastasis in a murine breast cancer model

Abstract

We had earlier shown that elevated cyclo-oxygenase (COX)-2 expression by breast cancer cells promotes tumor progression and metastasis by multiple mechanisms: inactivation of host immune cells, stimulation of tumor cell migration and tumor-associated angiogenesis. Furthermore, COX-2 was causally associated with increased VEGF-C expression/secretion in human breast cancer cell lines, corroborated with in situ studies on breast cancer tissue specimens, showing a strong correlation of COX-2 with VEGF-C and lymphangiogenic markers or lymphovascular density, implicated in lymphatic metastasis. VEGF-C upregulation resulted from PGE₂-mediated stimulation of EP4, and to a smaller extent, EP1 receptors on breast cancer cells. Since human tumor xeno-transplants in immno-compromised hosts are less than ideal for a therapeutic testing of these findings, in the present study, we developed a syngeneic murine breast cancer model exhibiting lymphangiogenesis and lymphatic metastasis in C3H/HeJ mice, using a highly metastatic, COX-2 expressing C3L5 cell line. This cell line was found to express VEGF-C and VEGF-D mRNA and proteins, and all four EP receptors. Expression of VEGF-C and VEGF-D were suppressed by treatment of cells with COX-2 inhibitors or EP4 antagonist in vitro. C3L5 cells suspended in growth factor-reduced Matrigel were implanted in both inguinal regions in mice that received oral treatments with COX-1/2 inhibitor indomethacin, COX-2 inhibitor celecoxib, EP1 antagonist ONO-8713 or EP4 antagonist ONO-AE3-208, to evaluate tumor growth, metastasis to lymph nodes and the lungs and tumor-associated angiogenesis (CD31 immuno-staining) and lymphangiogenesis (LYVE1 immunostaining). Mice were killed on days 8, 12 and 16. Tumor growth in control vehicle-treated mice was associated with high levels of angiogenesis and lymphangiogenesis at all intervals. VEGF-C protein expression was noted in 20-30% of tumor cells as well as macrophages in situ. Micro-metastasis to the lungs and local lymph nodes was noted as early as day 8 and increased thereafter. Metastasis to distant axillary nodes was seen at day16. Significant reduction in tumor growth, tumor-associated angiogenesis and lymphangiogenesis, and metastasis to the lungs and lymph nodes was achieved by therapies with indomethacin, celecoxib and ONO-AE3-208 but not ONO-8713 as compared to respective vehicle treatments. In conclusion, we have devised a syngeneic mouse breast cancer model mimicking human, showing successful chemo-intervention of tumor growth, tumor-associated angiogenesis/lymphangiogenesis and metastasis to the lungs and lymph nodes with COX inhibitors and EP 4 antagonist. (Supported by grants from the Canadian Breast Cancer Foundation, Ontario chapter and the Ontario Institute of Cancer Research to PKL. EP antagonists were kindly provided by ONO pharmaceuticals, Japan and Celecoxib by Pfizer.)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1601.