Aspirin upregulates expression of urokinase type plasminogen activator receptor (uPAR) gene in human colon cancer cells through AP1

Abstract

In this study, the effects of acetylsalicylic acid (aspirin) on the expression of uPAR and the mechanism by which it regulates expression of uPAR was examined in two different colon cancer cell lines HCT116 and GEO, respectively. The study shows that under physiological concentration, aspirin upregulates steady-state level expression of uPAR mRNA as well as expression of uPAR protein. Using a transient transfection assay, a region corresponding to −1 to −398 region of uPAR promoter has been identified which shows maximum responsiveness to aspirin treatment and found that this region is sufficient for the aspirin-induced up-regulation of uPAR. A stable integration of a single copy of this region coupled to luciferase reporter gene into the HCT116 genome also behaved similarly. Using gel mobility shift assays, it is found that the distal AP1 region between −171 and −186 is responsible for the aspirin-induced up-regulation of uPAR. Mutation of this region reduced up-regulation. Supershift assays identify that the bound proteins at this region are c-Jun and Fra-1. Real-time PCR analysis showed more than 4-fold increase in the binding of c-Jun and a 1.6-fold increase in the binding of Fra-1 in this region and this up-regulation corresponds to an increased binding of acetylated histone H4 in this region. Since an increase in the expression of uPAR corresponds to an increase in the migration of the cell, a migration assay was performed and result showed a 3-fold increased migration of HCT116 cells through the vitronectin-coated layer. Thus, an AP1 mediated pathway for aspirin induced up-regulation of uPAR has been identified.