The
urokinase receptor (uPAR), transcriptionally activated in several
cancers, contributes to
tumor progression by promoting cell migration and proteolysis, and repressing expression of this gene could be of therapeutic utility. Indeed, targeting regulatory
element(s) in the promoter may represent an efficient means for reducing expression because only two alleles have to be neutralized. We previously identified the -148/-124 promoter region, bound with Sp1 and Sp3, as regulatory for uPAR expression in vitro. The purpose of this study was twofold: to determine (a) the accessibility of this region in its natural
chromatin setting and (b) the efficacy of
WP631, a bisintercalator favoring GC-rich DNA sequences, in repressing endogenous uPAR expression in RKO
colon cancer cells. In these cells, DNaseI
hypersensitivity, genomic footprinting, and
chromatin immunoprecipitation experiments revealed that the -148/-124 uPAR promoter region was accessible in
chromatin and bound with Sp1, thus validating it as a therapeutic target.
WP631 treatment competed for
transcription factor binding to this regulatory region and reduced uPAR
mRNA/
protein. However, a chemically related compound (WP629), with low
DNA binding affinity, failed to diminish uPAR
protein amount. GAPDH
mRNA level was only modestly affected by
WP631, arguing against the possibility that this bisanthracycline universally represses expression of GC-rich promoter-driven genes. Further, uPAR function, as assessed by migration of cells across a
vitronectin-coated filter, was attenuated with
WP631. Thus, we have shown that the chromatinized -148/-124 regulatory region of the uPAR promoter is accessible to small molecules and that
WP631, which disrupts the interaction of
DNA binding proteins with this region, diminishes uPAR expression and function.