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与正常细胞的明显特征。因此，了解调控细胞周期蛋白的机制对于开发专门针对肿瘤细胞的治疗策略是不可或缺的。Cyclin E是一种细胞周期蛋白，介导GO释放和G1向S的相变，显示出一种独特的肿瘤特异性去调节与乳腺癌、卵巢癌和结直肠癌的恶性相关。特别是，在许多类型的癌症中，全长野生型细胞周期蛋白E被加工成低分子量异构体。在永生化乳腺上皮细胞和乳腺癌细胞系及转基因小鼠中，低分子量细胞周期蛋白E亚型由蛋白水解性裂解或全长细胞周期蛋白E交替翻译而成，可诱导细胞早期G1期向S期转变，延长S期，延长基因组不稳定性，增加细胞增殖和转移，并改变细胞形态和发育。LMW Cyclin 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，相关的高活性激酶活性增强，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，恢复细胞周期调节功能，有可能减轻多种肿瘤的恶性程度。E3泛素连接酶特异性因子Fbw7是一种F-box蛋白，能结合并介导正常细胞中细胞周期蛋白E的降解，使其成为抑制肿瘤细胞中低分子细胞周期蛋白E活性的候选蛋白。Fbw7基因突变与一些乳腺、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细胞周期蛋白E的调节。然而，Fbw7对肿瘤细胞中全长和LMW细胞周期蛋白E亚型的调节机制仍有待阐明。我们假设LMW Cyclin E亚型改变了与包括Fbw7在内的Cyclin E结合蛋白的相互作用，并与新的蛋白质相互作用，从而使LMW Cyclin E亚型致癌。我们建议通过免疫组织化学、体外酶分析和细胞学来确定Fbw7对全长和LMW细胞周期蛋白E亚型的调节机制，包括对细胞周期蛋白E稳定性、激酶活性和细胞周期调节的影响。我们还建议进行一种新颖的高通量筛选，以确定全长和低分子周期蛋白E相互作用因子，并表征这些周期蛋白E复合体的细胞和生化特性。阐明调控细胞周期蛋白E稳定性和功能的信号分子或信号通路可以为肿瘤细胞中细胞周期蛋白E的异常表达提供合理的靶点。

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.