A Novel High-Throughput Screening System Identifies a Small Molecule Repressive for Matrix Metalloproteinase-9 Expression

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-B 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.

The Activating Transcription Factor 3 Protein Suppresses the Oncogenic Function of Mutant p53 Proteins

“…Cells (2-5 ϫ 10 5 ) suspended in 0.1 ml of medium (containing 0.1% BSA) were then dispensed into the upper chambers of Transwells and incubated for 5 h. Cells migrated to the lower membrane surface were fixed, stained with hematoxylin/eosin (Fisher Scientific), and counted under a microscope. For invasion assays, 10 6 cells were plated in Transwells coated with Matrigel and cultured for 24 h. Invaded cells were stained and counted as described previously (29).…”

Matrix metalloproteinases in head and neck cancer: current perspectives

“…In consequence, patients carrying the CC genotype had almost twice the risk compared with the CT or TT genotype. 69 …”

“…50 AP1 has been reported to increase the transcription of MMP9, 69,70 while EGFR and integrins enhance MMP9 activity. [71][72][73][74][75] Furthermore, MMP9 degrades type IV collagen and promotes HNSCC invasion.…”

Meganuclease-Driven Targeted Integration in CHO-K1 Cells for the Fast Generation of HTS-Compatible Cell-Based Assays

“…These results suggest that the cGPS system could address another important issue linked with random integration techniques, for unbalanced expression of 2 monomers expressed from 2 different insertion sites can have far-reaching consequences. Altogether, these results, which show that cGPS can easily match other systems based on integrases, [9][10][11][12] demonstrate that the landing pad we selected is appropriate for expression of GOIs in cellbased assays.…”

“…The recombinase-based Flp-In system commercialized by Life Technologies (Invitrogen, Carlsbad, CA) allows for the integration into a landing pad inserted into the target cell. 9,10 Recently, a dual system based on the PhiC31 and R4 integrases was used to integrate GOIs into pseudo att target sites into human cell lines, CHOS Chinese hamster ovary cells, strain S, and human embryonic stem cells. 11,12 The other option is to target foreign DNA integration via homologous gene targeting (HGT).…”