Regulation of COX-2 protein expression by Akt in endometrial cancer cells is mediated through NF-kappaB/IkappaB pathway.

BACKGROUND: Cyclooxygenase-2 (COX-2) has been shown to be highly expressed in a broad series of primary endometrial tumors and its expression may be closely associated with parameters of tumor aggressiveness. In human endometrial cancer, tumor suppressor phosphatase tensin homologue (PTEN) is frequently mutated. In the presence of a mutated PTEN protein, Akt phosphorylation levels increase leading to the activation of this survival pathway. The nuclear transcription factor kappaB (NF-kappaB) is a well establish regulator of genes encoding cytokines, cytokine receptors, and cell adhesion molecules that drive immune and inflammatory responses. More recently, NF-kappaB activation has been connected with multiple aspects of oncogenesis, including the control of apoptosis, cell cycle, differentiation, and cell migration. It is known that Akt may act through NF-kappaB pathway and that COX-2 gene has been shown to be regulated at the promoter level by NF-kappaB. Recently, we showed that Akt regulates COX-2 gene and protein expressions in phospho-Akt expressing endometrial cancer cells. The present study was undertaken to determine the involvement of NF-kappaB pathway and IkappaB (an inhibitor of NF-kappaB) in the regulation of COX-2 expression and to determine more precisely the downstream targets of Akt involved in this process. RESULTS: Three different human endometrial cancer cell lines known to have wild type PTEN (HEC 1-A) or a mutated inactive PTEN protein (RL 95-2 and Ishikawa) were used for these studies. Expression IkappaB and Phospho-IkappaB were evaluated by Western analysis. The presence of IkappaB phosphorylation was found in all cell lines studied. There was no difference between cell lines in term of NF-kappaB abundance. Inhibition of PI 3-K with Wortmannin and LY294002 blocked IkappaB phosphorylation, reduced NF-kappaB nuclear activity, reduced COX-2 expression and induced apoptosis. Transfection studies with a dominant negative Akt vector blocked IkappaB phosphorylation and reduced COX-2 expression. On the opposite, constitutively active Akt transfections resulted in the induction of IkappaB phosphorylation and up-regulation of COX-2. CONCLUSION: These results demonstrate that Akt signals through NF-kappaB/IkappaB pathway to induce COX-2 expression in mutated PTEN endometrial cancer cells.