Cancer Letters

Cancer Letters

Volume 273, Issue 2, 18 January 2009, Pages 225-232
Cancer Letters

Transgenic expression of cyclooxygenase-2 in mouse intestine epithelium is insufficient to initiate tumorigenesis but promotes tumor progression

https://doi.org/10.1016/j.canlet.2008.08.012Get rights and content

Abstract

We generated mice expressing a COX-2 transgene in colon epithelium and found that they did not develop spontaneous colon tumors. But when treated with azoxymethane, a colon carcinogen, COX-2 mice had a higher tumor load compared to wild-type mice. There was no change in the number of pre-neoplastic lesions, indicating that COX-2 does not affect tumor initiation. Tumors in the COX-2 transgenic mice had higher levels of phosphorylated epidermal growth factor receptor and Akt compared to wild-type mice. Collectively, our data indicate that COX-2 promotes colon tumor progression, but not initiation, and it does so, in part, by activating EGFR and Akt signaling pathways.

Introduction

Elevated levels of COX-2 are often found in human colorectal adenomas and adenocarcinomas. COX-2 over-expression in colorectal cancer indicates a poor clinical prognosis and a generally marginal response to conventional therapy [1], [2], [3]. While not normally detected in most tissues, COX-2 is induced at sites of inflammation and neoplastic growth, and its expression levels often surpass the levels of the other COX isoform, COX-1, leading to profound increases in prostanoid secretion, particularly prostaglandin E2 (PGE2) [3]. By binding to its receptors, PGE2 promotes cell proliferation and angiogenesis, indicating that its overproduction supports tumor development [4], [5]. As a major provider of PGE2, COX-2 over-expression likely contributes to tumorigenesis. Indeed, reducing COX-2 activity with selective inhibitors, or deleting COX-2 or PGE2 receptors, attenuated colon tumor formation in experimental mouse models [5], [6], [7], [8], [9]. Collectively, these data indicate an important role for COX-2 in colorectal tumorigenesis and have stimulated interest in COX-2 as a therapeutic target. Several retrospective studies have shown that chronic administration of aspirin and other non-steroidal anti-inflammatory drugs (NSAID) confers protection from polyp formation and colorectal cancer in some populations [10], [11], [12], [13], [14]. However, NSAIDs that target both COX enzymes have side effects that limit their potential as anti-tumor agents; specific COX-2 inhibitors also appear to have limiting toxicities [15].

Given the importance of COX-2 in colorectal tumorigenesis, it is crucial to determine the contribution of COX-2 to the various stages of tumor development and to understand the signaling mechanisms that underlie its tumor promoting effects. We and others have shown in vitro that PGE2 transactivates EGFR [16], [17], [18], which is known to promote proliferation [18], [19], [20], [21], survival [22], [23], migration [17], [19], [24], and angiogenesis [25]. Collectively, these data led us to hypothesize that COX-2 might promote colorectal tumorigenesis by activating EGFR signaling. To understand how COX-2 affects colon tumor development we generated mice that expressed a human COX-2 transgene in colon epithelium. The mice did not spontaneously develop colon tumors, even when fed a high fat diet, indicating that COX-2 is not sufficient to initiate colon tumorigenesis. But when colon tumors were induced with azoxymethane (AOM), COX-2 transgenic mice developed higher tumor loads. However, there was no change in the number of pre-neoplastic aberrant crypt foci (ACF) indicating that transgenic expression of COX-2 did not affect tumor initiation. Both EGFR and Akt were excessively phosphorylated in the COX-2 transgenic mice compared to their wild-type littermates, indicating that EGFR signaling contributes to tumor formation. Collectively, these data demonstrate that in the colon, COX-2 promotes tumor progression, but not initiation, and that its activation of EGFR and Akt signaling pathways likely contributes to its tumorigenic effects.

Section snippets

Materials

AOM was purchased from Midwest Research Institute. The PGE2 assay kit was from Cayman Chemical (#514010). Antibodies to detect COX-2, EGFR, pEGFR Tyr1068, pEGFR Tyr992, Akt, pAkt Ser473, and pAkt Thr308 were purchased from Cellular Signaling (#4842, #2232, #2234, #2235, #4057, #3787, and #9266 respectively). COX-2 antibodies were also purchased from Santa Cruz Biotechnologies (sc-1475). A third custom COX-2 antibody was kindly provided by J. Maclouf, Lariboisiere Hospital, Federated Institute

Transgenic expression of COX-2 in the intestine

COX-2 is often over-expressed in both the stromal and epithelial components of human colorectal tumors and the resulting prostaglandin products promote tumorigenesis by a variety of proposed mechanisms. Its over-expression occurs early in tumorigenesis, but it is not clear if COX-2 participates in tumor initiation, tumor progression, or both. To understand the effects of COX-2 over-expression in colorectal carcinogenesis, we generated transgenic mice that over-expressed COX-2 in the colon using

Discussion

COX-2 over-expression is an early event in colorectal cancer, and several lines of evidence support a role for COX-2 in this disease. But whether COX-2 contributes to tumor initiation or progression is not yet clear. In this study, we developed an in vivo model system that allowed us to investigate the role of COX-2 in colon tumor initiation and progression and that permitted identification of key signaling pathways activated by COX-2 during these processes. We generated mice over-expressing

Conflict of interest statement

The authors report no financial or personal relationships that could have influenced this work.

Acknowledgements

We are indebted to Kate Lund and Jared Higbee for providing excellent technical assistance. This work was supported by the Huntsman Cancer Foundation, the R. Harold Burton Foundation, the National Institutes of Health Grants R01-CA95463 (to M.K.T.), and P01-CA73992 (to D.M.S.), and in part by the Cancer Center Support Grant P30 CA042014-20. M.A. Al-Salihi was supported by a Pre-doctoral Fulbright Award (2003–05). This project used azoxymethane which was purchased from the National Cancer

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    Present address: Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA.

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