PGE₂ induces cyclin D1 in colon cancer via the Wnt pathway Free

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Inhibition of cyclooxygenase-2 (COX-2), a key enzyme for conversion of arachidonic acid to prostaglandins including PGE₂, is often associated with reduced proliferation of cancer cells; the underlying mechanism is not completely understood. A large body of studies demonstrates a strong link between COX-2/PGE₂ signaling and the APC/β-catenin/TCF pathway in intestinal neoplasia. The most convincing evidence was provided by clinical trials conducted in Familiar Adenomatous Polyposis (FAP) patients that result from germline mutations of the APC gene. Administration of sulindac or celecoxib, which inhibit COX enzyme activity, significantly reduces size and number of polyps in these patients. In the present study, we show that PGE₂ activated the Wnt signaling pathway and induced the expression of a Wnt target gene cyclin D1 in vitro and in vivo. Exposure to PGE₂ rapidly increased levels of cyclin D1 protein in colon cancer LS-174 cells. PGE₂ increased the activity of cyclin D1 promoter driving reporter vector 4.5-fold. Furthermore, expression of stabilized β-catenin (Δ-89) increased the activity of the cyclin D1 promoter; however, in combination, PGE₂ and mutated β-catenin (Δ-89) induced the activity of cyclin D1 promoter in a synergistic manner. Cyclin D1 promoter contains both TCF and CRE binding sites; mutation of the CRE did not attenuate PGE₂ induced cyclin D1 transcription. In contrast, TCF elements were critical for PGE₂-induced cyclin D1 promoter activity. In animal experiments, administration of PGE₂ significantly increased the levels of cyclin D1 protein in APC^min/+ mouse polyps. To confirm that PGE₂ was able to activate the Wnt pathway directly, LS-174 cells were transfected with a TCF reporter vector, TOPflash, which contains a combination of TCF binding sites. Exposure to PGE₂ increased in the transcriptional activity of TCF ∼10-fold. PGE₂ induced TCF activity in a concentration-dependent fashion; as low as 1 nM PGE₂ clearly activated TCF elements. To investigate the effect of PGE₂ on endogenous activity of the Wnt pathway, HCA-7 colon cancer cells were treated with low concentration celecoxib to block the generation of endogenous PGE₂. Celecoxib at 0.1 μM reduced TCF activity by ∼ 50%, suggesting that HCA-7 generated PGE₂ was partially responsible for the endogenous activity of the Wnt pathway. Activation of the cAMP/PKA pathway was required for PGE₂ stimulation of the Wnt pathway, as a specific PKA inhibitor, H-89, completely attenuated the PGE₂ induced Wnt activity. In summary, COX-2 generated PGE₂ may stimulate the Wnt pathway directly and activate Wnt target genes. Given the critical roles of Wnt target genes in regulation of cell proliferation, migration, transformation, and neo-angiogenesis, our results provide a novel mechanism by which COX-2/PGE₂ signaling exerts pro-oncogenic effects in colorectal carcinogenesis.