Proteolytic Processing of Cyclin E in Breast Cancer

描述：（改编自研究者摘要）细胞周期蛋白E是一种G I 细胞周期蛋白是S期进入所必需的，并且在肿瘤发生中具有深远的作用。 以前，该实验室发现细胞周期蛋白E过表达，并存在于 乳腺癌细胞和肿瘤组织中的低分子量（LMW）亚型 与正常细胞和组织相比。细胞周期蛋白E的这种改变与 患者预后差。细胞周期蛋白E如何变成这些肿瘤特异性LMW形式 将有助于理解细胞周期破坏的本质， 肿瘤表型。我们最近的研究表明，只有肿瘤细胞具有 将细胞周期蛋白E加工成LMW形式。我们已经确定了 细胞周期蛋白E被蛋白水解裂解产生LMW亚型， 通过突变和生化分析在肿瘤细胞中的作用。我们能够 产生或敲除细胞周期蛋白E的肿瘤特异性LMW模式， 瞬时转染FLAG标记的细胞周期蛋白E构建体 乳腺癌细胞系中的突变。这些研究有助于确定 在细胞周期蛋白E的氨基末端的推定位点靶向产生这些 肿瘤特异性LMW亚型。该共有序列由丝氨酸靶向， 弹性蛋白酶类的蛋白酶。我们还表明，不仅是低分子量形式的 细胞周期蛋白E（迄今为止检测的2种）功能，因为它们磷酸化底物， 与Histone Hi和GST-Rb相似，但其活性均高于全长 细胞周期蛋白E。最后，这些核定位的低分子量形式的细胞周期蛋白E是 生物学功能，因为它们在正常细胞中的过表达增加了细胞的增殖。 这些细胞进入S和G2 IM期的能力是单独载体的2倍 转染细胞这些研究提出了一个假设， 肿瘤细胞进入和退出S期的部分原因是由于细胞周期的丧失。 细胞周期蛋白E对细胞周期的调节;细胞周期蛋白E的蛋白水解加工 产生LMW亚型，其底物选择已从 全长蛋白质由此导致的细胞周期失调和改变 细胞周期蛋白E的底物特异性参与肿瘤发生过程。到 为了检验这一假设，我们将（a）鉴定所有低分子量形式的细胞周期蛋白E， 在体外检查它们的生物化学作用，（B）确定生物学作用， 细胞周期蛋白E的低分子量形式的生物化学和肿瘤学特性 细胞和体内，（c）研究弹性蛋白酶在加工中的作用， 最后，（d）检查细胞周期蛋白E在肿瘤细胞中的LMW形式， 弹性蛋白酶抑制对乳腺癌细胞周期蛋白E的影响 细胞这些研究旨在了解生物化学和细胞 细胞周期蛋白E的蛋白水解过程导致其 致癌潜力这种对肿瘤特异性细胞周期蛋白E的新认识 去调控可能导致新的预后标志物的发展， 乳腺癌的治疗目标-癌症死亡的第二大原因 在这个国家的女性。

DESCRIPTION: (Adapted from the investigator's abstract) Cyclin E is a G I cyclin essential for S phase entry and has a profound role in oncogenesis. Previously this laboratory found that cyclin E is overexpressed and present in lower molecular weight (LMW) isoforms in breast cancer cells and tumor tissues compared to normal cells and tissues. Such alteration of cyclin E is linked to poor patient outcome. How cyclin E turns into these tumor specific LMW forms will be valuable in understanding the nature of the cell cycle disruption in the tumor phenotype. Our recent studies revealed that only tumor cells have the machinery to process cyclin E into its LMW forms. We have identified the region of cyclin E that is proteolytically cleaved to generate the LMW isoforms found in tumor cells through mutational and biochemical analysis. We were able to either generate or knockout the tumor specific LMW pattern of cyclin E by transient transfection of FLAG-tagged cyclin E constructs harboring specific mutations in a breast cancer cell line. These studies helped identify the putative site in the amino terminus of cyclin E targeted to generate these tumor specific LMW isoforms. This consensus sequence is targeted by a serine protease of the elastase class. We also show that not only are the LMW forms of cyclin E (2 examined so far) functional, as they phosphorylate substrates such as Histone Hi and GST-Rb, but their activities are higher than the full length cyclin E. Lastly, these nuclear localized LMW forms of cyclin E are biologically functional, as their overexpression in normal cells increases the ability of these cells to enter S and G2IM phase by 2 fold over vector alone transfected cells. These studies have raised the hypothesis that the defective entry into and exit from S phase by tumor cells is in part due to the loss of cyclin E regulation of the cell cycle; proteolytic processing of cyclin E results in LMW isoforms whose substrate selection has been modified from the full-length protein. The resulting deregulation of the cell cycle and altered substrate specificity of cyclin E contribute to the oncogenesis process. To test this hypothesis we will (a) identify all the LMW forms of cyclin E and examine their biochemical roles in vitro, (b) determine the biological, biochemical and oncological properties of the LMW forms of cyclin E in cultured cells and in vivo, (c) investigate the role of elastase in the processing of cyclin E into its LMW forms in tumor cells, and lastly, (d) examine the consequences of inhibition of elastases on cyclin E in human breast cancer cells. These studies are designed to understand the biochemical and cellular pathways through which the proteolytic processing of cyclin E leads to its oncogenic potential. This new understanding of tumor specific cyclin E deregulation may lead to development of novel prognostic markers and therapeutic targets for breast cancer-the second leading cause of cancer deaths of women in this country.