Cyclin E, one of the activators of the cyclin-dependent kinase Cdk2, is expressed near the G1-S phase transition and is thought to be critical for the initiation of DNA replication and other S-phase functions. Accumulation of cyclin E at the G1-S boundary is achieved by periodic transcription coupled with regulated proteolysis linked to autophosphorylation of cyclin E. The proper timing and amplitude of cyclin E expression seem to be important, because elevated levels of cyclin E have been associated with a variety of malignancies and constitutive expression of cyclin E leads to genomic instability. Here we show that turnover of phosphorylated cyclin E depends on an SCF-type protein-ubiquitin ligase that contains the human homologue of yeast Cdc4, which is an F-box protein containing repeated sequences of WD40 (a unit containing about 40 residues with tryptophan (W) and aspartic acid (D) at defined positions). The gene encoding hCdc4 was found to be mutated in a cell line derived from breast cancer that expressed extremely high levels of cyclin E.

Cyclin E, a regulatory subunit of cyclin-dependent kinase 2 (Cdk2), is an important regulator of entry into S phase in the mammalian cell cycle. In normal dividing cells, cyclin E accumulates at the G1/S-phase boundary and is degraded as cells progress through S phase. However, in many human tumours cyclin E is overexpressed and the levels of protein and kinase activity are often deregulated relative to the cell cycle. It is not understood how alterations in expression of cyclin E contribute to tumorigenesis. Here we show that constitutive cyclin-E overexpression in both immortalized rat embryo fibroblasts and human breast epithelial cells results in chromosome instability (CIN). In contrast, analogous expression of cyclin D1 or A does not increase the frequency of CIN. Cyclin-E-expressing cells that exhibit CIN have normal centrosome numbers. However, constitutive overexpression of cyclin E impairs S-phase progression, indicating that aberrant regulation of this process may be responsible for the CIN observed. These results indicate that downregulation of cyclin-E/Cdk2 kinase activity following the G1/S-phase transition may be necessary for the maintenance of karyotypic stability.

A yeast screen was developed to identify mutations in human cyclin E that lead to stabilization of the protein in order to identify determinants important for cyclin E turnover. Both C‐terminal truncations and missense mutations near the C‐terminus of cyclin E conferred hyperstability in vivo, suggesting that sequences in this region were critical for turnover. The following observations indicate that autophosphorylation of CDK2/cyclin E on Thr380 of the cyclin regulates cyclin E destruction: (i) mutation of Thr380 to Ala stabilizes cyclin E in yeast and mammalian cells; (ii) cyclin E/CDK2 autophosphorylates on cyclin E in vitro and cyclin E is a phosphoprotein in vivo in mammalian cells; (iii) the T380A mutation eliminates phosphorylation on the same site in mammalian cells and in vitro; (iv) inhibiting CDK2 activity in vivo stabilizes cyclin E; (v) the T380A mutation prevents ubiquitination of cyclin E. These results suggest a model where activation of cyclinE/CDK2 is coupled to cyclin E turnover via site‐specific phosphorylation, which acts as a signal for ubiquitination and proteasome processing.

The Met receptor tyrosine kinase and its ligand,

Triple-negative breast cancer (TNBC) accounts for 10-15% of all breast cancer cases. TNBCs lack estrogen and progesterone receptors and express low levels of HER2, and therefore do not respond to hormonal or anti-HER2 therapies. TNBC is a particularly aggressive form of breast cancer that generally displays poorer prognosis compared to other breast cancer subtypes. TNBC is chemotherapy sensitive, and this treatment remains the standard of care despite its limited benefit. Recent advances with novel agents have been made for specific subgroups with PD-L1 + tumors or germline Brca-mutated tumors. However, only a fraction of these patients responds to immune checkpoint or PARP inhibitors and even those who do respond often develop resistance and relapse. Various new agents and combination strategies have been explored to further understand molecular and immunological aspects of TNBC. In this review, we discuss clinical trials in the management of TNBC as well as perspectives for potential future treatments. Contents 1. Introduction 2. Current treatment paradigm 3. Investigational drugs 4. New potential therapeutic strategies 5. Conclusion KWANG-AI WON 1,2 and CHARLES SPRUCK 3