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

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.

Figure 1

Characterization of transgenic COX-2 expression. A. COX-2 Western blots. Colon, kidney, and liver lysates or isolated colon crypt lysates were probed with a COX-2 antibody that recognizes an epitope that is identical in mouse and human COX-2 (cellular signaling #4842). Immunoblots were reprobed for actin. B. PGE₂ assay. Isolated colon crypts were reconstituted in warm PBS for 10 minutes with or without 20μM arachidonic acid. PGE₂ was assayed in the supernatants by ELISA (the assays were performed in duplicate and each point represents the average of two mice) and then normalized to total protein in the crypt lysates. The * indicates statistically significant increased PGE₂ production in COX-2 transgenic mice compared to wild type litter mates (p<0.03); ** Indicates statistically significant increased PGE₂ production in COX-2 transgenic mice compared to wild type littermates when treated with arachadonic acid (p<0.005); error bars indicate standard deviation. C. COX-2 immunohistochemistry. Paraffin-embedded mouse colons were probed with anti-COX-2 antibodies. The insets show a representative crypt.

Figure 2

Increased tumor multiplicity and size in COX-2 transgenic mice. A. Tumor count. Mice treated with AOM or saline were sacrificed 12 weeks after the last injection and tumors were counted (30). COX-2 transgenic mice treated with AOM (n=12) developed more tumors compared to wild type littermates (n=20, p<0.05). There was no difference in the saline-treated mice. B. Tumor size distribution. There was a statistically significant shift (p<0.05) towards larger diameter tumors in the AOM-treated transgenic mice compared to AOM-treated wild type littermates. *Indicates statistical significance in the >4mm category: 4 out of the 12 transgenic mice developed tumors larger than 4mm in diameter compared to none in the wild type group (p<0.03). Error bars are standard error of the mean. C. Average tumor volume per mouse was calculated. * Indicates a statistically significant increase in average tumor volume in the AOM-treated transgenic mice compared to AOM-treated wild type littermates (p<0.03). D. Total tumor load per mouse. Tumor sizes for each mouse were summed to reflect both tumor number and average tumor size. *Indicates a statistically significant higher tumor load in the AOM-treated transgenic mice compared to AOM-treated wild type littermates (p<0.03). E. Representative methylene blue-stained colons (left) and hematoxylin-stained tissue sections (right) from AOM-treated wild type and COX-2 transgenic mice.

Figure 3

Increased proliferation COX-2 transgenic mice. Tumor and surrounding normal tissue samples from age-matched, transgenic and non-transgenic mice were fixed, paraffin-embedded, and then immunostained to detect Ki67. Wild-type and transgenic sections were processed together.

Figure 4

Downstream signaling pathways in tumors from COX-2 transgenic and wild type mice. Paraffin-embedded tumors from the mid-colon of age-matched COX-2 transgenic and wild type mice were immunostained to detect EGFR, pEGFR, Akt, and pAkt. Wild type and transgenic tissue was processed together. We found similar results with antibodies specific for phospho-Tyr992-EGFR and phospho-Thr308-Akt.

Figure 5

Assessment of EGFR and Akt signaling pathways in normal appearing tissue from COX-2 transgenic and wild type mice. Paraffin-embedded tissue samples from the mid-colon of age-matched COX-2 transgenic and wild type mice were immunostained to detect EGFR, pEGFR, Akt, and pAkt. Wild type and transgenic tissue was processed together. Similar results were obtained with antibodies to detect phospho-Tyr992-EGFR and phospho-Thr308-Akt.