A Bisanthracycline (WP631) Represses uPAR Gene Expression and Cell Migration of RKO Colon Cancer Cells by Interfering With Transcription Factor Binding to a Chromatin-Accessible −148/−124 Promoter Region
Abstract: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.
Document Type: Research Article
Affiliations: 1: Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA 2: Department of Bioimmunotherapy, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd. Houston, TX 77030, USA
Publication date: 2005-05-01
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