DEC1 Regulates Breast Cancer Cell Proliferation By Stabilizing Cyclin E Protein

Every year, millions of people die of cancers. Cancers, also known as malignant tumors, are a large family of diseases that involve abnormal cell growth with the potential to invade or spread to other parts of the body. The cells in the tumors have undergone unregulated and endless growth, which may be caused by disordered cell cycle. Evidence has shown that breast cancer that is accompanied by a high level of cyclin E expression or gene amplification always exhibits poor prognosis and clinical outcome. Several factors are known to regulate the level of cyclin E during the cell cycle progression.Differentiated embryo-chondrocyte expressed gene 1 (DEC1, also called STRA13 and SHARP2) belongs to a subfamily of basic helix-loop-helix (bHLH) transcription factors that are involved in a number of cell processes, including proliferation, apoptosis, and circadian rhythms and so on, aiming at regulating the tumor occurrence and development. Several studies which used protein overexpression and knockdown strategies have revealed the roles of DEC1 in cell cycle arrest, cell senescence and cell survival. Nevertheless, the mechanism of its role in cell proliferation is poorly understood. The cell cycle-dependent timing of DEC1 overexpression could affect the progression of the cell cycle through regulating the level of cyclin E protein. The main works of the paper are follows:1. In order to study the function of DEC1 in breast cancer cells, MTT and colony formation assays were used to detect the proliferation in DEC1 overexpressed MCF-7 and T47D cells. Overexpression of DEC 1 inhibited the proliferation and colony formation of both MCF-7 and T47D cells. The results showed that knockdown of DEC 1 in MCF-7 cells increased colony formation numbers and sizes compared to the control cells.2. To explore whether DEC1 protein levels are differently regulated at the various cell cycle stages, WB assay was used to measure the levels of DEC1 protein in MCF-7 cell extracts after synchronization by nocodazole. The expression of endogenous DEC1 was increased during late Gi-phase, and peaked at the Gi/S boundary before decreasing during the following S-phase, which almost precisely overlapped with the expression dynamics of cyclin E. Increase in the expression of cyclin E was induced by the overexpression of DEC1 in MCF-7 and T47D and in a dose-dependent manner. In addition, DEC1 could regulate the level of cyclin E protein independent of its transcriptional activity.3. Since DEC1 could stabilize cyclin E protein independent of its transcriptional activity, DEC1 might regulate the degradation of cyclin E. DEC1 markedly extended the half-life of cyclin E in MCF-7 using the half-life assays. The effect of DEC 1 on the stability of cyclin E depended on Fbw7a. Co-immunoprecipitation experiment showed that overexpression of DEC1 repressed the interaction between cyclin E and its E3 ligase Fbw7a, consequently reducing the level of polyunbiquitinated cyclin E.4. Co-immunoprecipitation experiment and Mammalian Two-hybrid System assay showed that DECl specifically interacted with cyclin E in MCF-7 cells whether the cells were under serum starvation or not. The interaction between endogenous DECl and cyclin E was also verified in MCF-7 cells. DECl and cyclin E exhibited a pronounced nuclear co-localization in Immunofluorescence staining assay. Co-immunoprecipitaion experiment was used to measure the dynamics of the interaction between DECl and cyclin E at different stage of the cell cycle. The interaction of these two protein displayed cell cycle-dependent dynamics was mainly detected at the Gi/S phase 8 hours after release from nocodazole treatment.5. Next to investigate the effect of DECl on the function of cyclin E and the progression of cell cycle, the WB assay, FACS assay and a mouse xenograft model was used. DECl promoted the formation and the activity of cyclin E/Cdk2 complex, which inhibited the subsequent association of cyclin A with Cdk2, and this had the effect of prolonging the S phase and suppressing the growth of breast cancer in a mouse xenograft model.Collectively, our data identified the connection of DECl and cyclin E, and found that DECl could stabilize cyclin E by blocking the proteasome pathway and hence, repressed the proliferation of breast cancer cells. The findings of this study would provide a better insight for conceiving a way to combat cancers, especially those mediated by cyclin E dysregulation, and may even provide a unique therapeutic strategy to control the growth of these cancer cells.