Proteolytic Processing of Cyclin E in Breast Cancer

描述：(改编自研究人员的摘要)Cyclin E是一个G I 细胞周期蛋白是S进入细胞周期所必需的，并在肿瘤发生中起着重要的作用。 此前，该实验室发现Cyclin E过度表达并存在于 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 病人结局不佳。细胞周期蛋白E如何转变为这些肿瘤特异性LMW形式 对于理解细胞周期中断的本质是有价值的 肿瘤表型。我们最近的研究表明，只有肿瘤细胞才有 将周期蛋白E加工成低分子形式的机械。我们已经确定了该地区 细胞周期蛋白E被蛋白水解酶切割以产生所发现的LMW亚型 在肿瘤细胞中通过突变和生化分析。我们能够 通过以下方式产生或敲除肿瘤特异性的细胞周期蛋白E的LMW模式 标志标记的细胞周期蛋白E构建物的瞬时转染 一种乳腺癌细胞系的突变。这些研究帮助确定了 细胞周期蛋白E氨基末端的假定位点，目的是产生这些 肿瘤特异性LMW亚型。这一共识序列是丝氨酸的靶标 弹性蛋白酶类的蛋白酶。我们还表明，不仅LMW形式的 Cyclin E(到目前为止已检测到的2种)具有功能，因为它们使底物磷酸化 作为组蛋白Hi和GST-Rb，但它们的活性都高于全长 最后，这些核内定位的低分子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度表达增加了 细胞进入S和G2IM期的能力是载体的2倍 转基因细胞。这些研究提出了一种假设，即有缺陷的人 肿瘤细胞进入和退出S期的部分原因是由于 细胞周期蛋白E对细胞周期的调节；细胞周期蛋白E的蛋白降解过程 结果是LMW亚型的底物选择已从 全长蛋白质。由此导致的细胞周期的放松和改变 细胞周期蛋白E的底物特异性参与肿瘤的发生过程。至 测试这一假设，我们将(A)确定所有低分子形式的细胞周期蛋白E和 在体外检测它们的生化作用，(B)确定生物学上的， 低分子形式细胞周期蛋白E在体外培养中的生化和肿瘤学特性 细胞和体内，(C)研究弹性蛋白酶在加工 Cyclin E在肿瘤细胞中以低分子形式存在，最后，(D)检查 弹性蛋白酶抑制细胞周期蛋白E在乳腺癌中的作用 细胞。这些研究旨在了解生物化学和细胞 细胞周期蛋白E的蛋白分解过程导致其 致癌潜力。对肿瘤特异性细胞周期蛋白E的新认识 放松管制可能会导致新的预后标志物的开发和 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.