Cyclin-dependent kinase subunit (Cks) 1 or Cks2 overexpression overrides the DNA damage response barrier triggered by activated oncoproteins.

Cyclin-dependent kinase subunit (Cks) proteins are small cyclin-dependent kinase-interacting proteins that are frequently overexpressed in breast cancer, as well as in a broad spectrum of other human malignancies. However, the mechanistic link between Cks protein overexpression and oncogenesis is still unknown. In this work, we show that overexpression of Cks1 or Cks2 in human mammary epithelial and breast cancer-derived cells, as well as in other cell types, leads to override of the intra-S-phase checkpoint that blocks DNA replication in response to replication stress. Specifically, binding of Cks1 or Cks2 to cyclin-dependent kinase 2 confers partial resistance to the effects of inhibitory tyrosine phosphorylation mediated by the intra-S-phase checkpoint, allowing cells to continue replicating DNA even under conditions of replicative stress. Because many activated oncoproteins trigger a DNA damage checkpoint response, which serves as a barrier to proliferation and clonal expansion, Cks protein overexpression likely constitutes one mechanism whereby premalignant cells can circumvent this DNA damage response barrier, conferring a proliferative advantage under stress conditions, and therefore contributing to tumor development.

Cyclin-dependent kinase-associated proteins Cks1 and Cks2 are essential during early embryogenesis and for cell cycle progression in somatic cells.

Cks proteins associate with cyclin-dependent kinases and have therefore been assumed to play a direct role in cell cycle regulation. Mammals have two paralogs, Cks1 and Cks2, and individually deleting the gene encoding either in the mouse has previously been shown not to impact viability. In this study we show that simultaneously disrupting CKS1 and CKS2 leads to embryonic lethality, with embryos dying at or before the morula stage after only two to four cell division cycles. RNA interference (RNAi)-mediated silencing of CKS genes in mouse embryonic fibroblasts (MEFs) or HeLa cells causes cessation of proliferation. In MEFs CKS silencing leads to cell cycle arrest in G(2), followed by rereplication and polyploidy. This phenotype can be attributed to impaired transcription of the CCNB1, CCNA2, and CDK1 genes, encoding cyclin B1, cyclin A, and Cdk1, respectively. Restoration of cyclin B1 expression rescues the cell cycle arrest phenotype conferred by RNAi-mediated Cks protein depletion. Consistent with a direct role in transcription, Cks2 is recruited to chromatin in general and to the promoter regions and open reading frames of genes requiring Cks function with a cell cycle periodicity that correlates with their transcription.

Cyclin E dysregulation and chromosomal instability in endometrial cancer.

Deregulation of cyclin E, an activator of cyclin-dependent kinase 2 (Cdk2), has been associated with a broad spectrum of human malignancies. Yet the mechanism linking abnormal cyclin E expression to carcinogenesis is largely unknown. The gene encoding the F-box protein hCdc4, a key component of the molecular machinery that targets cyclin E for degradation, is frequently mutated in endometrial cancer, leading to deregulation of cyclin E expression. Here we show that hCDC4 gene mutation and hyperphosphorylation of cyclin E, a parameter that usually correlates with hCDC4 mutation, have a strong statistically significant association with polypoidy and aneuploidy in endometrial cancer. On the contrary, elevated expression of cyclin E by itself was not significantly correlated with polyploidy or aneuploidy when tumors of similar grade are evaluated. These data suggest that impairment of cell cycle regulated proteolysis of cyclin E may be linked to carcinogenesis by promoting genomic instability.

Mutation of hCDC4 leads to cell cycle deregulation of cyclin E in cancer.

hCDC4, the gene that encodes the F-box protein responsible for targeting cyclin E for ubiquitin-mediated proteolysis, has been found to be mutated in a number of primary cancers and cancer-derived cell lines. We have observed that functional inactivation of hCDC4 does not necessarily correlate with elevated levels of cyclin E in tumors. Here we show, however, that hCDC4 mutation in primary tumors correlates strongly with loss of cell cycle regulation of cyclin E. Similarly, a breast carcinoma-derived cell line mutated for hCDC4 exhibits cell cycle deregulation of cyclin E, but periodic expression is restored by reintroducing hCDC4 via retroviral transduction. Conversely, small interfering RNA-mediated silencing of hCdc4 deregulates cyclin E with respect to the cell cycle. These results indicate that hCdc4 function is an absolute prerequisite for cell cycle regulation of cyclin E levels, and loss of hCdc4 function is sufficient to deregulate cyclin E.

Requirement of Cks2 for the first metaphase/anaphase transition of mammalian meiosis.

We generated mice lacking Cks2, one of two mammalian homologs of the yeast Cdk1-binding proteins, Suc1 and Cks1, and found them to be viable but sterile in both sexes. Sterility is due to failure of both male and female germ cells to progress past the first meiotic metaphase. The chromosomal events up through the end of prophase I are normal in both CKS2-/- males and females, suggesting that the phenotype is due directly to failure to enter anaphase and not a consequence of a checkpoint-mediated metaphase I arrest.

Seek and destroy: SCF ubiquitin ligases in mammalian cell cycle control.

The eukaryotic cell cycle consists of a series of sequential phases, the order of which is highly regulated to ensure the faithful transmission of intact genome equivalents to daughter cells. Progression through the cell cycle depends on the activity of cyclin-dependent kinases (Cdks), which drive the transitions between phases by targeting numerous, but largely unknown, substrates for phosphorylation. The activity of Cdks is subject to both positive and negative regulation by their temporal association with cyclins and Cdk inhibitors, respectively. Whereas Cdks are constitutively expressed throughout the cell cycle, the levels of cyclins and Cdk inhibitors are regulated by both transcriptional and post-transcriptional processes. The discovery that many cyclins and Cdk inhibitors are unstable proteins has implicated regulated protein degradation as a critical mechanism in cell cycle control. Proteolysis allows for the rapid removal of cell cycle regulators promoting irreversible transitions between cell cycle phases. The rapid removal of positive regulators prevents them from interfering with regulation of subsequent cell cycle events. In this review, we highlight the recent advances of our understanding of how a recently discovered ubiquitin ligase, designated SCF, contributes to mammalian cell cycle control.

hCDC4 gene mutations in endometrial cancer.

Cyclin-dependent kinase 2 activated by cyclin E is involved in the initiation of DNA replication and other S phase functions. Consistent with this role, cyclin E protein accumulates at the G1-S phase transition and declines during early S phase. This profile of expression is the result of periodic transcription and ubiquitin-mediated proteolysis directed by SCF(hCdc4). However, in many types of human tumors cyclin E protein is elevated and deregulated relative to the cell cycle by an unknown mechanism. Here, we show that the F-box protein hCdc4 that targets cyclin E to the SCF (Skp1-Cull-F-box) protein ubiquitin ligase is mutated in at least 16% of human endometrial tumors. Mutations were found either in the substrate-binding domain of the protein or at the amino terminus, suggesting a critical role for the region of hCdc4 upstream of the F-box. hCDC4 gene mutations were accompanied by loss of heterozygosity and correlated with aggressive disease. The hCDC4 gene is localized to chromosome region 4q32, which is deleted in over 30% of human tumors. Our results show that the hCDC4 gene is mutated in primary human tumors and suggest that it may function as a tumor suppressor in the genesis of many human cancers.