Mechanistic regulation of tumor specific forms of cyclin E in breast cancer

描述（由申请方提供）：细胞周期失调是肿瘤细胞与正常细胞的显著特征。因此，了解控制细胞周期蛋白调节的机制对于开发特异性靶向肿瘤细胞的治疗策略是不可或缺的。细胞周期蛋白E是一种介导GO释放和G1至S期转变的细胞周期蛋白，显示出与乳腺癌、卵巢癌和结直肠癌恶性肿瘤相关的独特肿瘤特异性失调。特别是，在许多类型的癌症中，全长野生型细胞周期蛋白E被加工成较低分子量的同种型。低分子量（LMW）异构体由全长细胞周期蛋白E的蛋白水解切割或选择性翻译产生。在永生化乳腺上皮细胞和乳腺癌细胞系和转基因小鼠中，LMW细胞周期蛋白E亚型诱导早期G1至S期转变、S期延长、基因组不稳定性、增加细胞增殖、转移以及改变细胞形态和发育。低分子量细胞周期蛋白E诱导的表型部分是由于增强的细胞周期蛋白依赖性激酶（Cdk）-2结合，相关的过度活跃的激酶活性，并降低对Cdk抑制剂的敏感性。因此，抑制低分子量细胞周期蛋白E活性和恢复适当的细胞周期蛋白E细胞周期调节可能会潜在地减轻多种类型癌症的恶性程度。E3泛素连接酶特异性因子Fbw 7是一种F-box蛋白，其结合并介导正常细胞中的细胞周期蛋白E降解，使Fbw 7成为抑制肿瘤细胞中LMW细胞周期蛋白E活性的候选蛋白。Fbw 7基因座的突变与一些乳腺、子宫内膜和胰腺肿瘤和细胞系相关，野生型Fbw 7表达可以恢复乳腺细胞中的细胞周期蛋白E调节。然而，Fbw 7调节肿瘤细胞中全长与LMW细胞周期蛋白E亚型的机制仍有待阐明。我们假设低分子量细胞周期蛋白E亚型改变了与细胞周期蛋白E结合蛋白（包括Fbw 7）的相互作用，并与新蛋白相互作用，从而使低分子量细胞周期蛋白E亚型致癌。我们建议确定Fbw 7的机制，全长与低分子量细胞周期蛋白E亚型的调控，包括细胞周期蛋白E的稳定性，激酶活性，细胞周期调控的影响，使用荧光化学，体外酶测定和细胞学。我们还建议进行一种新的，高通量的筛选，以确定全长和低分子量的细胞周期蛋白E相互作用，并表征这些细胞周期蛋白E复合物的细胞和生化特性。阐明调节细胞周期蛋白E稳定性和功能的信号分子或途径可以导致靶向肿瘤中异常细胞周期蛋白E表达的合理方法。

DESCRIPTION (provided by applicant): Cell cycle deregulation is a distinct characteristic of neoplastic versus normal cells. Therefore, understanding the mechanisms that govern the regulation of cell cycle proteins is integral to developing treatment strategies that specifically target tumor cells. Cyclin E, a cell cycle protein that mediates GO release and G1 to S phase transition, shows a unique tumor-specific deregulation associated with malignancy in breast, ovarian, and colorectal cancers. In particular, full length wild-type cyclin E is processed into lower molecular weight isoforms in many types of cancers. The low molecular weight (LMW) isoforms result from proteolytic cleavage or alternative translation of full length cyclin E. LMW cyclin E isoforms induce early G1 to S phase transition, prolonged S phase, genomic instability, increase cell proliferation, metastasis, and altered cell morphology and development in immortalized mammary epithelial and breast cancer cell lines and transgenic mice. LMW cyclin E-induced phenotypes are due in part to enhanced cyclin dependent kinase (Cdk)-2 binding, associated hyperactive kinase activity, and reduced sensitivity to Cdk inhibitors. Therefore, inhibiting LMW cyclin E activity and restoring proper cyclin E cell cycle regulation may potentially alleviate malignancy in multiple types of cancers. The E3 ubiquitin ligase specificity factor, Fbw7, is an F-box protein that binds to and mediates cyclin E degradation in normal cells, making Fbw7 a candidate protein to inhibit LMW cyclin E activity in tumor cells. Mutations at the Fbw7 locus have been correlated with some breast, endometrial, and pancreatic tumors and cell lines and wild type Fbw7 expression can restore cyclin E regulation in breast cells. However, the mechanism of Fbw7 regulation of full length versus LMW cyclin E isoforms in tumor cells remains to be elucidated. We hypothesize that LMW cyclin E isoforms have altered interaction with cyclin E binding proteins, including Fbw7, and interact with novel proteins, thereby rendering the LMW cyclin E isoforms oncogenic. We propose to determine the mechanism of Fbw7 regulation of full length versus LMW cyclin E isoforms including effects on cyclin E stability, kinase activity, and cell cycle regulation using immunohistochemisty, in vitro enzyme assays, and cytology. We also propose to perform a novel, high throughput screen to identify full length and LMW cyclin E interactors and characterize the cellular and biochemical properties of these cyclin E complexes. Elucidation of the signaling molecules or pathways that regulate cyclin E stability and function can lead to rational approaches to target aberrant cyclin E expression in tumors.