Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model

Abstract

We reported that cyclo-oxygenase (COX)-2 expression in human breast cancer stimulated cancer cell migration and invasiveness, production of vascular endothelial growth factor (VEGF)-C and lymphangiogenesis in situ, largely from endogenous PGE2-mediated stimulation of prostaglandin E (EP)1 and EP4 receptors, presenting them as candidate therapeutic targets against lymphatic metastasis. As human breast cancer xenografts in immuno-compromised mice have limitations for preclinical testing, we developed a syngeneic murine breast cancer model of spontaneous lymphatic metastasis mimicking human and applied it for mechanistic and therapeutic studies. We tested the roles of COX-2 and EP receptors in VEGF-C and -D production by a highly metastatic COX-2 expressing murine breast cancer cell line C3L5. These cells expressed all EP receptors and produced VEGF-C and -D, both inhibited with COX-2 inhibitors or EP4 (but not EP1, EP2 or EP3) antagonists. 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 the inguinal and axillary lymph nodes and the lungs. Chronic oral administration of COX-1/COX-2 inhibitor indomethacin, COX-2 inhibitor celecoxib and an EP4 antagonist ONO-AE3-208, but not an EP1 antagonist ONO-8713 at nontoxic doses markedly reduced tumor growth, lymphangiogenesis, angiogenesis, and metastasis to lymph nodes and lungs. Residual tumors in responding mice revealed reduced VEGF-C and -D proteins, AkT phosphorylation and increased apoptotic/proliferative cell ratios consistent with blockade of EP4 signaling. We suggest that EP4 antagonists deserve clinical testing for chemo-intervention of lymphatic metastasis in human breast cancer.

A practical and sensitive method of quantitating lymphangiogenesis in vivo

“…25,27 Cells were maintained in high glucose DMEM (GIBCO, Grand Island, NY, USA) supplemented with 5% fetal bovine serum, 100 U/ml penicillin G and 100 mg/ml streptomycin in a humidified incubator with 5% CO 2 at 37 1C.…”

“…Slides were rehydrated for 10 min in PBS, followed by incubation for 20 min at room temperature in blocking buffer (5% normal goat serum in PBS with 0.3% Triton X-100) to block nonspecific antibody binding. 25,29 Three hotspots per section (areas of highest vascular density in the descending order, 24 per group) were identified and examined at  400 magnification. The incidence of marker-positive vascular-like structures (irrespective of complete or incomplete lumen, depending on the plane of section) per unit area was scored after setting the threshold for background, using the Image-J software (NIH, Bethesda, MD, USA).…”

“…The incidence of marker-positive vascular-like structures (irrespective of complete or incomplete lumen, depending on the plane of section) per unit area was scored after setting the threshold for background, using the Image-J software (NIH, Bethesda, MD, USA). 25 The mean values for MVD vs LVD were computed as the staining indices for Cd31 (red) vs Lyve1 or Prox1 (far red, depicted as sky blue), or Cd31 (far red, depicted as sky blue) vs Pdpn (red). We had earlier shown that the 'hotspot' method provides results that are relatively very similar to those provided by the vascular density scores obtained by scanning the total area of the section.…”

“…These results are highly consistent with our earlier report of inhibition of tumor growth, tumor-associated lymphangiogenesis and angiogenesis and spontaneous metastasis to the lymph nodes and the lungs in this tumor model following Celecoxib therapy. 25 …”

“…To reproduce DIVLA in immunocompetent mice, we used a syngeneic murine breast cancer cell line C3L5 that produced VEGF-C and -D in a cyclo-oxygenase (COX)-2-dependent manner. 25 …”