Abstract
Aberrant gene expression is one of the driving forces for cancer progression and is considered an ideal target for chemical intervention. Although emerging bioluminescence reporter systems allow high-throughput searches for small molecules regulatory for gene expression, frequent silencing of reporter genes by epigenetic mechanisms hinders wide application of this drug discovery strategy. Here we report a novel system that directs the integration of a promoter-reporter construct to an open chromosomal location by Flp-mediated homologous recombination, thereby overcoming reporter-gene silencing. Using this system, we have screened more than 8000 compounds in the DIVERSet chemical library for repressors of a matrix metalloproteinase-9 (MMP-9) promoter and identified 5-methyl-2-(4-methylphenyl)-1H-benzimidazole (MPBD) inhibitory for MMP-9 gene expression. Consistent with this effect, MPBD inhibits MMP-9-dependent invasion of UMSCC-1 oral cancer cells, preosteoclast migration, and receptor activator of nuclear factor-κB ligand-induced osteoclast activity over concentration ranges that repressed MMP-9 expression. Mechanistic studies indicated that MPBD antagonizes AP-1 function by inhibiting its transactivation activity. We conclude that the Flp-mediated homologous recombination system to direct reporter integration into open chromatin regions represents a novel strategy allowing for the development of high-throughput systems screening for lead compounds targeting aberrant gene expression in cancer.
Footnotes
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This work was supported in part by a Department of Defense grant PC061106 (to C.Y.) and National Institutes of Health grants R01-CA58311 and R01-DE10845 (to D.D.B.).
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ABBREVIATIONS: MMP, matrix metalloproteinase; AP-1, activator protein-1; FRT, Flp recombination target; HTS, high-throughput screening; JNK, Jun N-terminal kinase; MPBD, 5-methyl-2-(4-methylphenyl)-1H-benzimidazole; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-κB, nuclear factor κB; PBD, 2-(4-methylphenyl)-1H-benzimidazole; PMA, phorbol myristate acetate; RANKL, receptor activator of nuclear factor-κB ligand; kb, kilobase(s); DMSO, dimethyl sulfoxide; PCR, polymerase chain reaction; TRAP, telomeric repeat amplification protocol; SP600125, anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone.
- Received October 10, 2007.
- Accepted December 7, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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