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. ? ? ?
