PROTEASE EXPRESSION IN ORAL CANCER

口腔癌中的蛋白酶表达

• 批准号: 6350581
• 负责人: Douglas D. Boyd
• 金额: $ 19.7万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6350581
• 关键词: DNA binding protein; DNA footprinting; biological signal transduction; collagenase; enzyme activity; enzyme biosynthesis; enzyme inhibitors; gel mobility shift assay; gene expression; genetic promoter element; genetic regulation; human tissue; mitogen activated protein kinase; neoplasm /cancer genetics; neoplasm /cancer invasiveness; northern blottings; oral pharyngeal neoplasm; phenotype; site directed mutagenesis; squamous cell carcinoma; tissue /cell culture; transcription factor; transfection /expression vector; urokinase; western blottings

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.

口腔鳞状细胞癌（SCC）的特点往往是 由于它们的局部和区域扩散，因此需要广泛的切除术， 这是一个昂贵的重建程序。 研究 解决SCC传播的机制可能最终导致新的 旨在减少残留疾病传播的治疗策略 手术后。 我们以前证明，侵袭性表型 的口腔SCC需要92 kDa的IV型胶原酶（MMP-9） 和尿激酶型纤溶酶原激活物（u-PA），两者都裂解 基底膜的组成部分。 本报告中提出的研究 续期申请将解决转录的要求， 这两种基因的表达以及决定信号传导 调节转录活性和/或合成的途径 调节MMP-9和u-PA合成的因子。 具体目标 #1，我们将确定调节MMP-9的顺式和反式作用因子 在口腔SCC中的表达，强调AP-1的作用 结合转录因子。 这将通过测定 MMP-9分泌口腔癌细胞系使用5'端驱动的报告基因 缺失和突变的MMP-9启动子序列，并通过鉴定MMP-9启动子序列， 启动子的转录因子结合区通过DNA酶I 脚印 此外，表达构建体编码 一种突变的c-jun，它干扰AP-1依赖的基因表达， 减少MMP-9的合成。 具体合成。 这 将通过测定产生u-PA的SCC细胞系的CAT来完成 活性使用由启动子驱动的报告构建体，其已经被 点突变的eAP-1和PEA 3基序，并通过确定能力 显性负性表达载体对相应的转录 减少u-PA分泌的因素。 的活性和/或合成 与AP-1和PEA 3基序结合的转录因子可以被调节， 通过多种信号通路。 其中两条途径终止于 细胞外信号调节激酶（ERK）和jun氨基末端 激酶（JNK）并且在下文中称为c-raf-ERK和MEKK-JNK， 分别 ERK是通过依次激活 c-raf和丝裂原活化蛋白激酶（MEK-1），而JNK是 MEKK通过JNNK刺激。 因为我们的初步数据显示 MMP-9和u-PA在至少一个SCC亚群中的表达是受驱动的， 通过与AP-1和PEA 3位点结合的转录因子， 将在具体目标3中确定c-raf-ERK和MEKK-JNK的作用 这些蛋白酶的表达调控的信号通路。 为此，表达载体编码突变的 分子以及MEK 1的化学抑制剂， 下调MMP-9和u-PA合成。 此外该 这些蛋白酶的水平将与ERK的活性相关， 切除的SCC中的JNK。 如果干扰上述 显性负调控的转录因子和信号通路 表达构建体或化学抑制剂减少蛋白酶合成， 这些构建体/抑制剂减弱体外细胞毒性的能力 SCC的侵袭性将在具体目标#4中确定。