Treatment with NSAIDs limits tumor growth and metastatic potential in various model systems as well as clinically in cancer patients (26
). Of the two COX isozymes, COX-2 appears to play the predominant role in tumor growth (2
), although the underlying mechanism has remained unclear. The antiapoptotic potential of PGE2
has been suggested to contribute to colorectal carcinogenesis (30
). With the use of a synthetic antagonist specific for EP1 together with mice deficient in this receptor, Watanabe et al. (17
) showed that EP1 contributes to the formation of precancerous lesions induced by the colon carcinogen azoxymethane. However, the effects of the antagonist and of receptor disruption were limited to partial inhibition, leaving open the possibility that additional COX-2–dependent mechanisms are important in carcinogenesis.
An important factor in the promotion of tumor growth is angiogenesis (31
). Substantial increases in tumor mass must be preceded by an increase in blood supply to provide the nutrients and oxygen required for tumor growth. It has been suggested that the mechanisms for promotion of angiogenesis are activated in the early stages of tumor development (33
). We have now shown with our sarcoma 180 implantation model that COX-2–selective inhibitors inhibited tumor growth and associated angiogenesis. The COX-2 blockers inhibited tumor growth by ~80%, similar to the extent of the effect of such inhibitors on the growth of COX-2–overexpressing tumor cells transplanted into nude mice (6
). These latter researchers also showed that the promotion of tube formation by human umbilical vein endothelial cells induced by cocultured Caco-2 cells that overexpress COX-2 is mediated through the production and release of proangiogenic factors by the tumor cells themselves. If such a mechanism also operates in vivo, tumor-associated angiogenesis would depend only on tumor cells. However, we have now shown that not only angiogenesis but also tumor growth were significantly reduced in EP3–/–
mice compared with those in WT mice, although the transplanted tumor cells expressed EP3 receptors. These results strongly suggested that the host stromal PGE2
-EP3 signaling was important in tumor-associated angiogenesis and tumor growth.
To mimic stromal angiogenic responses, we developed the sponge implantation model. Two advantages of this model for studies of angiogenesis are that angiogenesis can be readily quantified by measurement of the hemoglobin content of the sponge-induced granulation tissue together with histological examination, and that the effects of exogenous substances can be investigated by their direct injection into the sponge (7
). The EP3 agonist ONO-AE-248 specifically enhanced angiogenesis in this model in a dose-dependent manner, and further the angiogenic response was certainly reduced in EP3−/−
with the reduction of VEGF expression, suggesting that endogenous PGE2
facilitates angiogenesis through the EP3 signaling and the induction of VEGF in this model. Previous studies with various cell lines have indicated that PGE2
induces VEGF expression through a cAMP-dependent mechanism (34
). We previously reported that PGE2
induces VEGF through the activation of adenylate cyclase/protein kinase A signaling pathway (36
). One of the EP3 splicing variants (37
), which may couple to the elevation in intracellular cAMP levels, may enhance angiogenesis in this model.
It has been previously reported that E type PGs have a proangiogenic activity in corneal test (39
) and in the chorioallantoic membrane (CAM) technique (40
). Further, Form and Auerbach reported that PGE2
strongly induced angiogenesis on CAM of 8-d-old chicken embryos, but PGA2
, and a derivative of TXA2
did not. A recent report (41
) described that the endothelial migration was mediated by COX-2 and TXA2
, but this experiment was performed using confluent monolayer endothelial cells stimulated with PMA. The authors also reported that corneal angiogenesis was suppressed with COX-2 inhibitor and TXA2
antagonist, the former of which inhibited more strongly than the latter, suggesting the involvement of other COX-2 products than TXA2
. However, these in vivo results were obtained under bFGF-stimulated conditions. In our separate experiment, angiogenesis in the sponge implantation model under no stimulation was not reduced with either a thromboxane synthase inhibitor, OKY046 or a TP receptor antagonist, S-1452 (unpublished data). The contribution of TP receptor signaling to tumor-associated angiogenesis should be estimated carefully. In the present experiments, we tested tumor-associated angiogenesis in knockout mice of EP subtypes or IP receptors (). As shown in the present study, not only tumor growth but also tumor-associated angiogenesis are highly dependent on EP3 receptor signaling. Judging from the time course of changes in size and angiogenesis of tumors in EP3−/−
and WT mice (), the reduced tumor growth and angiogenesis in EP3−/−
mice is not a simply minor delay. However, angiogenesis and growth of polyps in mice with a mutated APC gene were recently reported to be EP2-dependent (42
). Their report described that the major elements which express COX-2 are stromal cells around the tumors or intestinal polyps. We identified that the cells which produced VEGF to facilitate angiogenesis and tumor growth were CD3 and Mac-1 double-negative fibroblasts. It is widely known that the fibroblasts exhibit heterogeneity in term of various biological factors including prostaglandin generating systems and receptor systems (44
). We have shown in the present study, not only tumor growth but also tumor-associated angiogenesis are highly EP3 dependent. They did not show the microvessel density in EP3−/−
with APC mutation in their recent report (43
), and the reduction percentage of angiogenesis in APC-mutated EP2−/−
mice was ~30% at best. As shown in A, the major EP receptor expressed in subcutaneous tissues in WT mice was EP3, which did not expressed in the intestine (43
). These suggested that tumor-associated angiogenesis may be regulated in a site-specific fashion, and may be related to the heterogeneity of the stromal fibroblasts.
The mouse strains, ddy mouse and C57BL/6 mouse are allogeneic for S-180 sarcoma cell line used in the present study. However, we tested another cell line, Lewis lung carcinoma, for which C57BL/6 mouse is syngeneic ( F). The difference of tumor growth in EP3−/− mice and WT counterparts was observable in both tumor cell lines. These suggested that EP3 receptor signaling to facilitate the tumor growth was important not only in allogeneic tumors but also in syngeneic tumors.
The host microenvironment is thought to influence tumor progression (47
). Examination of human colorectal cancer tissue has revealed marked COX-2 expression not only in cancer cells but also in inflammatory cells and fibroblasts that surround the cancer cells (34
). Ohshima et al. (2
) also showed that COX-2 is abundant in the stromal cells that surround intestinal polyps in mice with a mutated APC gene. Recent results using COX-2 knockout mice also supported the significance of stromal COX-2 in tumor-induced angiogenesis and tumor growth (49
). In the present study, we have focused on the down-stream signaling pathway after COX-2 induction. We detected marked VEGF immunoreactivity in host stromal cells, including fibroblast-like cells, that surrounded the implanted sarcoma cells in EP3+/+
mice but not in the corresponding cells of EP3−/−
mice, suggesting that these stromal cells express VEGF in response to activation of EP3 receptors by endogenous PGE2
. We therefore propose that COX-2 expressing stromal cells around the tumors and/or tumor cells themselves synthesize and release PGE2
into the tumor microenvironment, and that PGE2
then acts on the stromal cells expressing EP3 receptors to induce the production of proangiogenic factors and consequent angiogenesis.
It is interesting to see whether or not EP3 receptor signaling enhances the stability of the newly formed vessels by modulating periendothelial cells which invest the vessels. Desmin was reported to be expressed in the pericytes on the tumor blood vessels, where the loose association of pericytes with endothelium (50
). We performed real time PCR to determine a ratio of the expression of desmin mRNA to that of CD31 mRNA ( B). As the expression of CD31 is dependent on the proliferation of the endothelial cells on newly formed vessels, it is plausible that this ratio neatly correlates with the development of investment of pericytes on the blood vessels. The ratio determined in the samples isolated from the normal subcutaneous tissue (H) was not different between EP3−/−
mice and WT mice. But, in the samples of S-180 tumors including surrounding stroma (T+ST), the ratio was reduced in both EP3−/−
mice and WT mice, compared with that in the normal subcutaneous tissues isolated from each mouse. Further, the reduction was more significant in EP3−/−
mice in comparison with that in WT mice. These results taken together suggest that the development of pericytes on the newly formed vessels in association to tumors is EP3 receptor signaling-dependent, and that the equipment of the pericytes in the vessels (arterioles and venules) in normal tissues is not.
Among the various factors, VEGF may be a candidate to enhance angiogenesis in sponge model judging from the results using VEGF neutralizing antibody (). Further, daily topical injections of VEGF antibody significantly reduced tumor growth and angiogenesis in WT mice, but not in EP3−/− mice ( D), suggesting that VEGF is a predominant factor to induce tumor growth and angiogenesis in host microenviroment. The results from VEGF receptor tyrosine kinase inhibitor concrete this finding ( E).
Gel shift assays of several transcriptional factors in stromal fibroblasts, revealed that AP-1 activation was significantly reduced in EP3−/−
mice, compared with EP3+/+
mice with an EP3 agonist ( I), suggesting that AP-1–dependent up-regulation of VEGF may be important in tumor-associated angiogenesis mediated by EP3 receptors. AP-1–dependent VEGF expression was also reported in several kinds of cells (51
To concrete the results so far obtained with EP3−/− mice, we topically injected an EP3 receptor antagonist, which we recently developed. Daily topical injections of an EP3 antagonist to the subcutaneous tissues around the tumor, where functionally active EP3 receptors are localized, significantly suppressed tumor-associated angiogenesis and tumor growth in WT mice, whereas those of an EP1 antagonist and an EP4 antagonist did not ( A). This chemopreventive effect of an EP3 antagonist was not seen in EP3−/− mice ( B). These results certainly confirm the results from EP3−/− mice.
In conclusion, as shown in C, host stromal PGE2-EP3 signaling appears critical for tumor-associated angiogenesis and tumor growth. EP3 signaling on the stromal cells was relevant to the induction of a potent proangiogenic growth factor, VEGF in stromal cells. Up-regulated VEGF certainly has a proangiogenic action, and facilitates tumor growth. A highly selective EP3 antagonist therefore exhibits chemoprevetive action on the stromal cells, and will become a novel therapeutic tool for cancer. The results presented here neatly provide an answer to the question how NSAIDs prevent tumor development.