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.
Nair
Rajesh R
RR
Center for Cell Biology and Cancer Research, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208, USA.
Avila
Hector
H
Ma
Xujun
X
Wang
Zhengxin
Z
Lennartz
Michelle
M
Darnay
Bryant G
BG
Boyd
Douglas D
DD
Yan
Chunhong
C
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