Smad4 Stability by beta-TrCP1 in Pancreatic Cancer

描述（由申请方提供）：对TGF-β依赖性信号传导的敏感性丧失是胰腺肿瘤发展的重要促成因素。Smad 4是TGF-β信号传导的关键下游决定因子，其缺失或突变已在约50%的胰腺癌中被鉴定，并且Smad 4突变体的蛋白质不稳定性被认为在胰腺肿瘤发生中细胞对TGF-β的反应性丧失中起重要作用。研究Smad 4在胰腺癌中的降解机制对进一步了解胰腺癌的发病机制具有重要意义。在我们的初步研究中，我们已经确定了泛素E3连接酶SCF β-TrCP 1作为Smad 4蛋白质降解的关键决定因素。我们发现：1）在酵母和哺乳动物细胞中，E3连接酶中的F-box蛋白β-TrCP 1与Smad 4相互作用。MH 1加上Smad 4的Linker区域是介导相互作用的结构域。2)SCF beta-TrCP 1有效诱导Smad的遍在化和降解4.3）SCF beta-TrCP 1抑制TGF-β诱导的基因反式激活、细胞周期停滞和细胞凋亡。4)胰腺癌细胞系中Smad 4点突变表现出更高的Smad 4泛素化和蛋白质不稳定性介导的SCF β-TrCP 1。5)使用siRNA诱导的beta-TrCP 1基因沉默，Smad 4蛋白的表达水平升高，并且挽救了胰腺癌细胞系中TGF-β诱导的基因转录。因此，我们假设β-TrCP 1是控制肿瘤抑制因子Smad 4蛋白稳定性的关键分子，并且通过β-TrCP 1基因沉默增加Smad 4蛋白水平挽救了胰腺癌细胞中TGF-β反应的丧失。我们将追求以下具体目标：1）确定β-TrCP 1识别Smad 4并介导其降解的机制。2)确定β-TrCP 1基因沉默对胰腺癌细胞中Smad 4蛋白稳定性和TGF-β介导的细胞反应性的影响。我们相信，SCF β-TrCP 1对Smad 4降解的表征将为胰腺癌中肿瘤抑制因子Smad 4的失活机制提供重要的新见解，并能够开发胰腺癌的潜在治疗方法。

DESCRIPTION (provided by applicant): Loss of sensitivity to TGF-beta-dependent signaling is an important contributing factor in pancreatic tumor development. Deletions or mutations in Smad4, a key downstream determinant in TGF-beta signaling, have been identified in about 50% of pancreatic adenocarcinomas, and protein instability of Smad4 mutants is thought to play an important role in the loss in cellular responsiveness to TGF-beta in pancreatic tumorigenesis. Studies of the mechanism underlying the degradation of Smad4 in pancreatic carcinoma should be instructive for further understanding of the pathogenesis of this disease. In our preliminary studies, we have identified ubiquitin E3 ligase SCFbeta-TrCP1 as a critical determinant for the protein degradation of Smad4. We found that: 1) F-box protein beta-TrCP1 in this E3 ligase interacts with Smad4 in yeast and in mammalian cells. MH1 plus Linker region of Smad4 is the domain that mediates the interaction. 2) SCF beta-TrCP1 efficiently induces the ubiquitination and degradation of Smad4.3) SCFbeta-TrCP1 inhibits TGF-beta-induced gene transactivation, cell cycle arrest and apoptosis. 4) Pancreatic cancer cell lines harboring Smad4 point mutation exhibited much higher Smad4 ubiquitination and protein instability mediated by SCFbeta-TrCP1. 5) Using siRNA-induced beta-TrCP1 gene silencing, the expression level of Smad4 protein was elevated, and TGF-beta-induced gene transcription in pancreatic cancer cell lines was rescued. Therefore, we hypothesize that beta-TrCP1 is a key molecule that controls the protein stability of tumor suppressor Smad4, and increased Smad4 protein level by beta-TrCP1 gene silencing rescues the loss of TGF-beta responses in pancreatic cancer cells. We will pursue the following specific aims: 1) Determine the mechanisms by which beta-TrCP1 recognizes Smad4 and mediates its degradation. 2) Determine the effects of beta-TrCP1 gene silencing on Smad4 protein stability and TGF-beta-mediated cellular responsiveness in pancreatic cancer cells. We believe that characterization of Smad4 degradation by SCFbeta-TrCP1 would provide important new insights into the mechanism responsible for the inactivation of tumor suppressor Smad4 in pancreatic cancer, and also enable development of a potential therapeutic approach for pancreatic carcinoma.