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

描述（由申请人提供）：细胞周期失调是肿瘤细胞相对于正常细胞的一个明显特征。因此，了解细胞周期蛋白调控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。Cyclin E是一种介导氧化石墨烯释放和G1期到S期转变的细胞周期蛋白，在乳腺癌、卵巢癌和结直肠癌的恶性肿瘤中显示出独特的肿瘤特异性失调。特别是，在许多类型的癌症中，全长野生型细胞周期蛋白E被加工成低分子量的同种异构体。低分子量（LMW）亚型是由全长周期蛋白E的蛋白水解裂解或替代翻译产生的。LMW周期蛋白E亚型在永生化的乳腺上皮细胞和乳腺癌细胞系以及转基因小鼠中诱导早期G1期到S期转变、延长S期、基因组不稳定、增加细胞增殖、转移、改变细胞形态和发育。LMW细胞周期蛋白e诱导的表型部分是由于细胞周期蛋白依赖性激酶(Cdk)-2结合增强，相关的激酶活性过度活跃，以及对Cdk抑制剂的敏感性降低。因此，抑制LMW细胞周期蛋白E活性和恢复适当的周期蛋白E细胞周期调节可能潜在地减轻多种类型癌症的恶性肿瘤。E3泛素连接酶特异性因子Fbw7是一种F-box蛋白，在正常细胞中结合并介导细胞周期蛋白E的降解，使Fbw7成为抑制肿瘤细胞中LMW细胞周期蛋白E活性的候选蛋白。Fbw7位点的突变与一些乳腺、子宫内膜和胰腺肿瘤和细胞系相关，野生型Fbw7的表达可以恢复乳腺细胞中周期蛋白E的调节。然而，Fbw7调控肿瘤细胞周期蛋白E全长亚型和LMW亚型的机制仍有待阐明。我们假设LMW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Fbw7）的相互作用，并与新的蛋白相互作用，从而使LMW细胞周期蛋白E亚型致癌。我们建议利用免疫组织化学、体外酶分析和细胞学来确定Fbw7对全长和LMW周期蛋白E亚型的调控机制，包括对周期蛋白E稳定性、激酶活性和细胞周期调控的影响。我们还建议采用一种新颖的高通量筛选方法来鉴定全长和低分子量周期蛋白E相互作用物，并表征这些周期蛋白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，可以找到针对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.