A novel high-throughput screening system identifies a small molecule repressive for matrix metalloproteinase-9 expression.
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-kappaB 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.
Center for Cell Biology and Cancer Research, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208, USA.
Rajesh R
RR
Nair
Hector
H
Avila
Xujun
X
Ma
Zhengxin
Z
Wang
Michelle
M
Lennartz
Bryant G
BG
Darnay
Douglas D
DD
Boyd
Chunhong
C
Yan
D000818:Animals; D001562:Benzimidazoles / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects; D020780:Matrix Metalloproteinase 9 / Q000235:genetics*; D061965:Matrix Metalloproteinase Inhibitors; D051379:Mice; D008957:Models, Genetic; D010957:Plasmids; D011401:Promoter Regions, Genetic; D016755:Proto-Oncogene Proteins c-jun / Q000032:analysis; D053245:RANK Ligand / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D013997:Time Factors; D018808:Transcription Factor AP-1 / Q000032:analysis / Q000037:antagonists & inhibitors; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection
C527834:5-methyl-2-(4-methylphenyl)-1H-benzimidazole; D001562:Benzimidazoles; D061965:Matrix Metalloproteinase Inhibitors; D016755:Proto-Oncogene Proteins c-jun; D053245:RANK Ligand; D011993:Recombinant Fusion Proteins; D018808:Transcription Factor AP-1; D008156:Luciferases; D020780:Matrix Metalloproteinase 9; C501830:Mmp9 protein, mouse