Prostacyclin-mediated activation of peroxisome proliferator-activated receptor delta in colorectal cancer

Abstract

There is evidence from both genetic and pharmacologic studies to suggest that the cyclooxygenase-2 (COX-2) enzyme plays a causal role in the development of colorectal cancer. However, little is known about the identity or role of the eicosanoid receptor pathways activated by COX-derived prostaglandins (PG). We previously have reported that COX-2-derived prostacyclin promotes embryo implantation in the mouse uterus via activation of the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) delta. In light of the recent finding that PPARdelta is a target of beta-catenin transactivation, it is important to determine whether this signaling pathway is operative during the development of colorectal cancer. Analysis of PPARdelta mRNA in matched normal and tumor samples revealed that expression of PPARdelta, similar to COX-2, is up-regulated in colorectal carcinomas. In situ hybridization studies demonstrate that PPARdelta is expressed in normal colon and localized to the epithelial cells at the very tips of the mucosal glands. In contrast, expression of PPARdelta mRNA in colorectal tumors was more widespread with increased levels in transformed epithelial cells. Analysis of PPARdelta and COX-2 mRNA in serial sections suggested they were colocalized to the same region within a tumor. Finally, transient transfection assays established that endogenously synthesized prostacyclin (PGI(2)) could serve as a ligand for PPARdelta. In addition, the stable PGI(2) analog, carbaprostacyclin, and a synthetic PPARdelta agonist induced transactivation of endogenous PPARdelta in human colon carcinoma cells. We conclude from these observations that PPARdelta, similar to COX-2, is aberrantly expressed in colorectal tumors and that endogenous PPARdelta is transcriptionally responsive to PGI(2). However, the functional consequence of PPARdelta activation in colon carcinogenesis still needs to be determined.

Figure 1

Expression of PPARδ mRNA in matched normal and tumor colon tissue from (A) rats treated with the carcinogen AOM and (B) human surgical specimens. In each case, total RNA (20 μg) was isolated from six paired samples and analyzed for relative levels of PPARδ mRNA levels by Northern blot hybridization. Each blot was subsequently probed for 1B15 (A) or 18S (B) to evaluate RNA loading.

Figure 2

Localization of PPARδ and COX-2 mRNA in normal human colon and colorectal carcinoma. In situ hybridization analysis of PPARδ and COX-2 expression in two different matched pairs of normal (N) and cancer tissue (T).

Figure 3

PPARδ (A) mRNA and (B) protein expression in a panel of colon cancer cell lines. (A) Total RNA (20 μg) was isolated from eight different indicated colon carcinoma cell lines and analyzed for PPARδ mRNA expression by Northern blot hybridization. (B) Whole-cell protein lysates from the indicated cell lines (50 μg) were analyzed for PPARδ protein expression by using immunoblot analysis.

Figure 4

Endogenous production of PGI₂ correlates with PPARδ transactivation. U2OS cells were transiently transfected with UAS-tk-luciferase, pRL-TK, PPARδ-GAL4, and combinations of expression vectors for COX-2 and PGI synthase (PGIS). Cells were then treated with vehicle (0.1% ethanol), AA (40 μM), or AA + the selective COX-2 inhibitor celecoxib (2 μM) for 24–36 h. (A) Media were harvested and used to measure 6-keto PGF_1α levels (represented as the mean from two independent transfections ± SEM). (B) Cells were harvested and the dual luciferase assay was performed as described in Materials and Methods. Data are presented as -fold activation and represent the mean from three independent transfections. Error bars equal SEM. No significant activation was seen in cells transfected with pGAL4, PPARα-GAL4, or PPARγ-GAL4.

Figure 5

Transactivation of endogenous PPARδ in human colon cancer cells. (A) HCA-7 cells were transiently transfected with PPRE3-tk-luciferase and pRL-TK plasmids followed by treatment with increasing doses of the following ligands (published PPAR isoform selectivity in parenthesis): cPGI (PPAR α/δ), GW 2433 (PPAR α/δ), or GW 7647 (PPAR α) for 24 h. Cells were harvested and the dual luciferase assay was performed as described in Materials and Methods. Data are presented as -fold activation relative to vehicle treated (0.1% DMSO) and represent the mean from three independent transfections. Error bars equal SEM.