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.
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