The 92 kDa type IV collagenase (MMP-9) contributes to the spread of oral cancer and understanding how its expression is regulated could ultimately yield new agents to repress its expression and diminish tumor invasiveness. Although we previously identified multiple regulatory elements (AP-1, NF-KB, PEA3, Spl) in the 670 base pair promoter, the limited number of these binding sites makes it unlikely that MMP-9 expression is solely the consequence of trans- activation through these motifs. Indeed, emerging studies indicate a role for the chromatin environment (constituted by DNA wrapped around a histone core), in the regulation of gene expression. We show herein that Mtal, which promotes histone deacetylation, represses MMP-9 expression. Further, Mtal expression is undetectable in MMP-9-producing oral cancer.
In Specific Aim # 1 using genomic footprinting and ehromatin immunoprecipitation assays (Chip) we will identify transcription factor-bound cis elements and acetylated histones localized at these sites in the 670 base pair MMP-9 promoter targeted by Mtal to achieve MMP-9 repression. Biological suppressors of MMP-9 expression may also provide a tool for identifying regulatory elements in the chromatinized promoter. We show herein that the metastasis suppressor gene KISS-1 attenuates MMP-9 transcription partly by reducing NF-KB binding to the chromatinized promoter. However, the degree to which NF-KB binding is reduced can only partly account for the diminished transcription. Therefore, in Specific Aim # 2, we will identify trans -activated cis elements and acetylated histones localized at these sites in the MMP-9 promoter that mediate KiSS-l-dependent repression of MMP-9. Similarly, the MEK1 inhibitor PD098059 represses MMP-9 expression and in Specific Aim # 3 we will determine if the transcriptional targets of this repressor are identical to, or distinct from, those of Mtal and KiSS-I. If we determine that the transcriptional targets differ, we will determine whether combining PD098059 and KISS-1 or Mtal proves superior to individual modalities in reducing MMP-9 expression and oral cancer invasiveness. Since extra-chromosomal reporters were used in our previous studies of MMP-9 transcription, regulatory elements, that depend on the chromatin environment, may have escaped detection. Thus, to identify such novel cis elements in Specific Aim # 4, we will employ DNaseI hypersensitivity, genomic footprinting and Egtll library screening to identify additional trans- activators/repressors of MMP-9 expression. Ultimately, the goal of these studies is to identify new transcriptional targets in the MMP-9 promoter that allow for therapeutic intervention to repress expression of this collagenase and oral cancer invasiveness.
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