It is well established that high COX-2 enzyme levels correlate with poor clinical outcome in cancer patients (26
), and inhibition of cyclooxygenase is an effective approach to reduce polyp burden in humans (33
). Moreover, genetic studies demonstrate that colorectal tumor growth and vascular density are significantly attenuated in COX-2−/−
), indicating that COX-2 plays a crucial role in tumor-associated angiogenesis (35
). Our laboratory demonstrated previously that overexpression of COX-2 in CRC cells stimulates endothelial cell migration and tube formation when endothelial cells were cocultured with CRC cells expressing COX-2 (25
). However, all of the mechanisms by which COX-2 stimulates angiogenesis are not known. In this study, we extended our previous work to assess the role of COX-2–derived PGE2
in vitro and in vivo in tumor-associated angiogenesis. We identified CXCL1 as a key downstream mediator of PGE2
in regulating tumor-associated angiogenesis. We have established a role for CXCL1 in colorectal tumor–associated angiogenesis by our results demonstrating reduced tumor growth and decreased tumor vascular density in mice treated with a neutralizing CXCL1 antibody. These findings may have clinical relevance because CXCL1 is elevated in the majority of human CRCs and correlates well with tissue PGE2
levels. Importantly, this study delineates a novel molecular mechanism by which COX-2 regulates tumorassociated angiogenesis.
The molecular events involved in progression of CRC are complex, involving dysregulation of factors that regulate cell growth and cell death, evasion of host defenses, invasion, and metastasis. Tumor growth and metastatic spread of disease are dependent on angiogenesis (37
), and substantial increases in tumor size require a dependable blood supply. For neoplasms to develop a stable blood supply, the tumor cells and/or the stromal microenvironment must secrete a variety of pro-angiogenic factors, including vascular endothelial growth factor (VEGF) and CXC chemokines that stimulate endothelial cell proliferation, migration, and tube formation (19
). Inhibition of tumor-associated angiogenesis is being pursued as a promising therapeutic strategy for treatment of patients with advanced disease. Recently, Avastin (bevacizumab), which blocks VEGF signaling, was approved for treatment of patients with advanced CRC. However, treatment with bevacizumab plus chemotherapy extends progression-free survival in patients with metastatic CRC by only 5–10 mo (45
), and this treatment is very expensive. ELISA analysis revealed that LS-174T cells do secrete VEGF at very low levels compared with the amount of CXCL1 (Fig. S1 A, available at http://www.jem.org/cgi/content/full/jem.20052124/DC1
, and ). Therefore, it is crucial to determine what other pro-angiogenic factors are involved in progression of CRC. Our in vitro and in vivo results here demonstrate that CXCL1 is an important target for PGE2
signaling in CRC cells ( and ).
Although our studies support the role of CXCL1 as an important angiogenic factor in tumor growth, other angiogenic factors are likely to be involved as well. For example, we were unable to fully inhibit basal tumor–associated angiogenesis by only targeting CXCL1 (). Moreover, the ability of CXCL1 alone to induce cell migration is less than that seen with conditioned media (), indicating that CXCL1 is not the sole factor involved in the regulation of endothelial cell migration. One plausible explanation is that human CRC cells in vivo produce other members of the CXC chemokine family, bFGF, and VEGF, which also promote neovascularization. For example, recombinant hVEGF stimulated endothelial cell migration in both Py-4-1 and BLMVECs (Fig. S1 B), although the ability of VEGF alone to induce cell migration is much less than that seen with conditioned media. However, VEGF failed to synergize with CXCL1 on stimulating endothelial cell migration in our in vitro model (Fig. S1 C). It is possible that VEGF may synergize with CXCL1 to further stimulate angiogenesis in vivo. Further work is needed to determine whether both angiogenic factors coordinate tumor-associated angiogenesis in vivo. We have performed real-time quantitative PCR assays to determine whether VEGF levels correlate with CXCL1 in human tumor samples. As shown in Fig. S1 E and , VEGF expression is elevated in 17 of 20 (85%) cancer specimens and positively correlates with CXCL1 expression (r = 0.65, P < 0.0001). In this study, we demonstrate that PGE2 accelerates tumor growth, in part, by inducing CXCL1 secretion by tumor epithelial cells. These results may point out, in part, why treating patients with only one anti-angiogenic agent may not be the most effective clinical regimen.
Chemokines and their respective receptors are classified into the CXC, CC, C, and CX3C families based on the positions of their conserved two NH2
-terminal cysteine residues. Although chemokines play a crucial role in immune and inflammatory reactions, such as allergic disorders, autoimmune diseases, and viral infections, recent studies indicate that they have an equally important role in the development of a variety of cancers, such as melanomas, ovarian, breast, lung, and prostate cancers (47
). Some of these chemokines are involved in transformation, survival, growth, metastasis, and angiogenesis. The chemokine receptors expressed on tumor cells may contribute to organ-specific metastasis. For example, CXCR4 is expressed and participates in directed migration of cancer cells to sites of metastasis in many cancers, including small cell lung cancer, pancreatic cancer, astrogliomas, myelomas, B cell lymphomas, and chronic lymphocytic leukemias (48
). Moreover, tumor cells and/or stromal cells produce inflammatory chemokines, which in turn result in the recruitment of various types of leukocytes into the tumor tissue through binding to their receptors expressed on these leukocytes. These infiltrating leukocytes in tumors promote neoplastic development. In breast cancer, it has been shown that macrophage infiltration promotes tumor invasion and metastasis (49
inhibits production of CC chemokines CCL3 (MIP-1α) and CCL4 (MIP-1β) in dendritic cells through binding to the EP2 receptor (50
) and suppresses CC chemokine CCL5 (RANTES [regulated upon activation, normal T cell expressed and secreted]) production in human macrophages through the EP4 receptor (52
). Because breast cancer cells produce CCL5, we examined whether PGE2
regulates these CC chemokines in CRC cells. Analysis of real-time quantitative PCR revealed that PGE2
up-regulated expression of CCL3, CCL4, and CCL5 in LS-174T cells (Fig. S1 D). These CC chemokines are crucial for macrophage and lymphocyte infiltration in human breast, cervix, pancreas, and gliomas cancers (53
). These results indicate that PGE2
promotes tumor growth and metastasis through, in part, by recruiting these macrophages and lymphocytes into tumor tissues. Moreover, a recent study showed that COX-2 up-regulates expression of CXC chemokines such as CXCL5 (epithelial cell–derived neutrophil activator 78) and CXCL8 (IL-8) in human non-small cell lung cancer cells (55
), indicating that PGE2
can regulate the expression of CXC chemokines in certain contexts. In this study, we present a novel mechanism by which PGE2
stimulates expression of the CXC chemokine CXCL1 in CRC cells through activation of an EGFR–MAPK cascade ( and ). Because our previous results showed that PGE2
can transactivate EGFR through activation of EP4 receptor signaling in LS-174T cells (23
), it is likely that the EP4 mediates the effects seen on the EGFR–MAPK signaling cascade.
Recent evidence demonstrates that CXCR2 is expressed in intestinal microvessels (56
) and other endothelial cells, such as lung microvascular endothelial cells, dermal microvascular endothelial cells, umbilical vein endothelial cells, and saphenous vein endothelial cells (57
). Moreover, inhibition of COX-2 by nonselective or selective nonsteroidal antiinflammatory drugs suppresses αVβ3-mediated Cdc42/Rac-depenent migration and spreading of endothelial cells (58
). Interestingly, PGE2
promotes αVβ3-Cdc42/Rac-dependent migration and spreading of endothelial cells (59
). There is strong evidence indicating that CXCL1 can promote Rac/cdc42-Pak1–dependent migration via the CXCR2 receptor (60
). Thus, it is possible that the Rac/cdc42-Pak1 cascade is required for CXCL1-induced endothelial cell migration and tube formation. Further studies are needed to address these issues more completely.
The biological significance of the PGE2-induced CXCL1 expression and secretion in CRC cells was substantiated by the results of our in vivo experiments. Administration of exogenous PGE2 promoted tumor growth and increased microvessel density. Inhibition of PGE2-induced CXCL1 signaling led to inhibition of tumor growth with decreased microvessel density (). One very interesting finding from both our in vitro and in vivo experiments is that CXCL1 expression correlates with PGE2 levels in human CRCs ().
The key finding of this study is that PGE2 stimulates tumor epithelial cells to secrete the pro-angiogenic chemokine CXCL1 via activation of the EP4-EGFR–MAPK cascade. Furthermore, we reveal the importance of PGE2–up-regulated CXCL1 in tumor-associated angiogenesis. To our knowledge, this represents the first report suggesting that the up-regulation of CXCL1 is directly involved in the pro-angiogenic effects of PGE2 in CRC.