PROTEASE EXPRESSION IN ORAL CANCER

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Squamous cell carcinomas (SCC) of the oral cavity are often characterized by their local and regional spread thereby requiring extensive resective procedures and as a consequence costly reconstructive procedures. Studies addressing the mechanism by which SCC spreads may ultimately lead to new therapeutic strategies aimed at reducing the spread of residual disease postoperatively. We previously demonstrated that the invasive phenotype of SCC of the oral cavity required the 92 kDa type IV collagenase (MMP-9) and the urokinase-type plasminogen activator (u-PA) both of which cleave integral components of the basement membrane. The studies proposed in this renewal application will address the transcriptional requirements for the expression of both of these genes as well as determine the signaling pathways which modulate the activity and/or synthesis of transcription factors which regulate the synthesis of MMP-9 and u-PA. In Specific Aim #1, we will identify the cis- and trans-acting factors which regulate MMP-9 expression in SCC of the oral cavity placing emphasis on the role of AP-1 binding transcription factors. This will be accomplished by assaying MMP-9-secreting oral cancer cell lines using a reporter driven by 5' deleted and mutated MMP-9 promoter sequences and by identifying the transcription factor-binding regions of the promoter by DNase I footprinting. In addition, the ability of an expression construct encoding a mutated c-jun, which interferes with AP-1-dependent gene expression, to diminish MMP-9 synthesis will be determined. In Specific synthesis. This will be accomplished by assaying u-PA-producing SCC cell lines for CAT activity using a reporter construct driven by the promoter, which has been point mutated at the eAP-1 and PEA3 motifs, and by determining the ability of dominant negative expression vectors to the corresponding transcription factors to reduce u-PA secretion. The activity and/or synthesis of transcription factors which bind to AP-1 and PEA3 motifs can be regulated by multiple signaling pathways. Two of these pathways terminate in the extracellular signal-regulated kinases (ERKs) and the jun amino-terminal kinases (JNKs) and hereafter are referred to as c-raf-ERK and MEKK-JNK, respectively. The ERKs are activated by the sequential activation of c-raf, and mitogen-activated protein kinase (MEK-1) while the JNKs are stimulated by MEKK via JNNK. Since our preliminary data indicate that MMP-9 and u-PA expression in, at least, a sub-population of SCC is driven through transcription factors which bind to the AP-1 and PEA3 sites, we will, in Specific Aim # 3, determine the role of the c-raf-ERK and MEKK-JNK signaling pathways in the regulation of expression of these proteases. Towards this end, the ability of expression vectors encoding mutated molecules in these pathways as ell as a chemical inhibitor of MEK1 to downregulate MMP-9 and u-PA synthesis will be determined. In addition, the levels of these proteases will correlated with the activity of ERKs and JNKs in resected SCC. If interfering with the above-mentioned transcription factors and signaling pathways with dominant negative expression constructs or a chemical inhibitor reduces protease synthesis, the ability of these constructs/inhibitor to attenuate the in vitro invasiveness of SCC will be determined in Specific Aim #4.