PROTEASE EXPRESSION IN ORAL CANCER

口腔鳞状细胞癌(SCC)通常是以 由其局部和区域扩散，因此需要广泛切除 程序和结果是昂贵的重建程序。研究 解决鳞状细胞癌传播的机制最终可能会导致新的 旨在减少残留病传播的治疗战略 手术后。我们之前证明了侵袭性表型 口腔鳞状细胞癌需要92 kDa的IV型胶原酶(MMP9) 和尿激酶型纤溶酶原激活物(u-PA)，两者都能裂解 基底膜的组成部分。在这篇文章中提出的研究 续签申请将满足以下内容的转录要求 这两个基因的表达以及决定信号转导 调控转录活性和/或合成的途径 调节基质金属蛋白酶-9和u-PA合成的因素。以特定的目标 #1，我们将确定调节基质金属蛋白酶-9的顺式和反式作用因子 AP-1在口腔鳞癌中的表达及其意义 结合转录因子。这将通过分析来完成 利用5‘端驱动的报告基因表达基质金属蛋白酶-9的口腔癌细胞株 缺失和突变的基质金属蛋白酶-9启动子序列 DNA酶I对启动子转录因子结合区的影响 脚印。此外，表达式构造编码的能力 干扰AP-1依赖基因表达的c-jun突变 减少基质金属蛋白酶-9的合成将被测定。在特定的合成中。这 将通过检测产生u-PA的SCC细胞系来实现CAT 使用由启动子驱动的报告构建的活动，这已经被 点突变在EAP-1和PEA3基序，并通过确定能力 显性负向表达载体到相应的转录 减少u-PA分泌的因素。活性和/或合成 可调节与AP-1和PEA3基序结合的转录因子 通过多条信号通路。其中两条路径终止于 细胞外信号调节蛋白(ERKs)与Jun氨基末端 激酶(JNK)及以下称为c-raf-ERK和Mekk-JNK， 分别进行了分析。ERK通过以下顺序激活 C-RAF和丝裂原活化蛋白激酶(MEK-1)，而JNK是 由Mekk通过JNNK刺激。因为我们的初步数据显示 基质金属蛋白酶-9和u-PA在至少一个鳞癌亚群中的表达被驱动 通过与AP-1和PEA3位点结合的转录因子，我们 将在具体目标3中确定c-raf-erk和mekk-jnk的作用 调控这些蛋白水解酶表达的信号通路。 为此，表达载体编码的能力发生了突变 这些途径中的分子就像MEK1的化学抑制剂一样 下调基质金属蛋白酶-9和u-PA的合成将被确定。此外， 这些蛋白水解酶的水平将与ERKs的活性和 切除的鳞状细胞癌中的JNK。如果干扰上述行为， 显性负值的转录因子和信号通路 表达构建体或化学抑制物减少了蛋白酶的合成， 这些构建物/抑制物在体外减弱血管内皮细胞生长的能力 鳞状细胞癌的侵袭性将在具体目标4中确定。

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.