Cyclin E Overexpression in Human Mammary Epithelial Cells Promotes Epithelial Cancer-Specific Copy Number Alterations.

Cyclin E, a key cell cycle regulatory protein, has been linked to oncogenesis when dysregulated. We have previously shown that overexpression of cyclin E causes replication stress, leading to failure to complete replication at specific chromosomal loci during S phase of the cell cycle. This in turn promotes chromosomal damage during anaphase. Here we show that non-transformed human mammary epithelial cell clones that survive such aberrant mitoses have a specific and reproducible pattern of chromosomal Copy Number Alterations (CNAs) that we have characterized and termed the cyclin E CNA signature. Using a number of computational approaches, we show that this signature resembles one specific CNA pattern enriched in differentiated epithelial-like tumors of the breast and ovary. Analysis of the CNA profile of these clones provides a potential mechanism for cyclin E-mediated oncogenesis.

SCF(FBXW7)-mediated degradation of p53 promotes cell recovery after UV-induced DNA damage.

Eukaryotic cells have developed sophisticated mechanisms to ensure the integrity of the genome and prevent the transmission of altered genetic information to daughter cells. If this control system fails, accumulation of mutations would increase risk of diseases such as cancer. Ubiquitylation, an essential process for protein degradation and signal transduction, is critical for ensuring genome integrity as well as almost all cellular functions. Here, we investigated the role of the SKP1-Cullin-1-F-box protein (SCF)-[F-box and tryptophan-aspartic acid (WD) repeat domain containing 7 (FBXW7)] ubiquitin ligase in cell proliferation by searching for targets implicated in this process. We identified a hitherto-unknown FBXW7-interacting protein, p53, which is phosphorylated by glycogen synthase kinase 3 at serine 33 and then ubiquitylated by SCF(FBXW7) and degraded. This ubiquitylation is carried out in normally growing cells but primarily after DNA damage. Specifically, we found that SCF(FBXW7)-specific targeting of p53 is crucial for the recovery of cell proliferation after UV-induced DNA damage. Furthermore, we observed that amplification of FBXW7 in wild-type p53 tumors reduced the survival of patients with breast cancer. These results provide a rationale for using SCF(FBXW7) inhibitors in the treatment of this subset of tumors.-Galindo-Moreno, M., Giráldez, S., Limón-Mortés, M. C., Belmonte-Fernández, A., Reed, S. I., Sáez, C., Japón, M. Á., Tortolero, M., Romero, F. SCF(FBXW7)-mediated degradation of p53 promotes cell recovery after UV-induced DNA damage.

Reducing Mcl-1 gene dosage induces dopaminergic neuronal loss and motor impairments in Park2 knockout mice.

Mutations in the PARK2 gene are associated with early onset Parkinsonism. The Park2 -/- mouse, however, does not exhibit neurodegeneration or other Parkinson's disease (PD) phenotypes. Previously, we discovered that translation of Mcl-1, a pro-survival factor, is upregulated in the Park2 -/- mouse, suggesting a compensatory mechanism during development. Here we generated the Park2 -/- Mcl-1 +/- mouse and show that by reducing Mcl-1 gene dosage by 50%, the Park2 -/- genotype is sensitized, conferring both dopaminergic neuron loss and motor impairments. We propose that this murine model could be a useful tool for dissecting PD etiology and developing treatment strategies against this neurodegenerative disease.

CKS Proteins Promote Checkpoint Recovery by Stimulating Phosphorylation of Treslin.

CKS proteins are small (9-kDa) polypeptides that bind to a subset of the cyclin-dependent kinases. The two paralogs expressed in mammals, Cks1 and Cks2, share an overlapping function that is essential for early development. However, both proteins are frequently overexpressed in human malignancy. It has been shown that CKS protein overexpression overrides the replication stress checkpoint, promoting continued origin firing. This finding has led to the proposal that CKS protein-dependent checkpoint override allows premalignant cells to evade oncogene stress barriers, providing a causal link to oncogenesis. Here, we provide mechanistic insight into how overexpression of CKS proteins promotes override of the replication stress checkpoint. We show that CKS proteins greatly enhance the ability of Cdk2 to phosphorylate the key replication initiation protein treslin in vitro Furthermore, stimulation of treslin phosphorylation does not occur by the canonical adapter mechanism demonstrated for other substrates, as cyclin-dependent kinase (CDK) binding-defective mutants are capable of stimulating treslin phosphorylation. This effect is recapitulated in vivo, where silencing of Cks1 and Cks2 decreases treslin phosphorylation, and overexpression of wild-type or CDK binding-defective Cks2 prevents checkpoint-dependent dephosphorylation of treslin. Finally, we provide evidence that the role of CKS protein-dependent checkpoint override involves recovery from checkpoint-mediated arrest of DNA replication.

Mechanism of ubiquitin chain synthesis employed by a HECT domain ubiquitin ligase.

Homologous to E6AP C-terminal (HECT) ubiquitin (Ub) ligases (E3s) are a large class of enzymes that bind to their substrates and catalyze ubiquitination through the formation of a Ub thioester intermediate. The mechanisms by which these E3s assemble polyubiquitin chains on their substrates remain poorly defined. We report here that the Nedd4 family HECT E3, WWP1, assembles substrate-linked Ub chains containing Lys-63, Lys-48, and Lys-11 linkages (Lys-63 > Lys-48 > Lys-11). Our results demonstrate that WWP1 catalyzes the formation of Ub chains through a sequential addition mechanism, in which Ub monomers are transferred in a successive fashion to the substrate, and that ubiquitination by WWP1 requires the presence of a low-affinity, noncovalent Ub-binding site within the HECT domain. Unexpectedly, we find that the formation of Ub chains by WWP1 occurs in two distinct phases. In the first phase, chains are synthesized in a unidirectional manner and are linked exclusively through Lys-63 of Ub. In the second phase, chains are elongated in a multidirectional fashion characterized by the formation of mixed Ub linkages and branched structures. Our results provide new insight into the mechanism of Ub chain formation employed by Nedd4 family HECT E3s and suggest a framework for understanding how this family of E3s generates Ub signals that function in proteasome-independent and proteasome-dependent pathways.

CKS protein overexpression renders tumors susceptible to a chemotherapeutic strategy that protects normal tissues.

The cyclin-dependent kinase-interacting proteins Cyclin-dependent Kinase Subunit 1 and 2 (CKS1 and 2) are frequently overexpressed in cancer and linked to increased aggressiveness and poor prognoses. We previously showed that CKS protein overexpression overrides the replication stress checkpoint activated by oncoproteins. Since CKS overexpression and oncoprotein activation/overexpression are often observed in the same tumors, we have hypothesized that CKS-mediated checkpoint override could enhance the ability of premalignant cells experiencing oncoprotein-induced replication stress to expand. This tumor advantage, however, could represent a vulnerability to exploit therapeutically. Here, we first show in vitro that CKS protein overexpression selectively sensitizes tumor-derived cell lines to nucleoside analog-mediated toxicity under replication stress conditions. A treatment combination of the nucleoside analog gemcitabine and an agent that induces replication stress (thymidine or methotrexate) resulted in selective targeting of CKS protein-overexpressing tumor-derived cells while protecting proliferative cells with low CKS protein levels from gemcitabine toxicity. We validated this strategy in vivo and observed that Cks2-overexpressing mammary tumors in nude mice were selectively sensitized to gemcitabine under conditions of methotrexate-induced replication stress. These results suggest that high CKS expression might be useful as a biomarker to identify subgroups of cancer patients who might benefit from the described therapeutic approach.

Cyclin E Deregulation and Genomic Instability.

Precise replication of genetic material and its equal distribution to daughter cells are essential to maintain genome stability. In eukaryotes, chromosome replication and segregation are temporally uncoupled, occurring in distinct intervals of the cell cycle, S and M phases, respectively. Cyclin E accumulates at the G1/S transition, where it promotes S phase entry and progression by binding to and activating CDK2. Several lines of evidence from different models indicate that cyclin E/CDK2 deregulation causes replication stress in S phase and chromosome segregation errors in M phase, leading to genomic instability and cancer. In this chapter, we will discuss the main findings that link cyclin E/CDK2 deregulation to genomic instability and the molecular mechanisms by which cyclin E/CDK2 induces replication stress and chromosome aberrations during carcinogenesis.

Cyclin E deregulation promotes loss of specific genomic regions.

Cell-cycle progression is regulated by the cyclin-dependent kinase (Cdk) family of protein kinases, so named because their activation depends on association with regulatory subunits known as cyclins. Cyclin E normally accumulates at the G1/S boundary, where it promotes S phase entry and progression by activating Cdk2. In normal cells, cyclin E/Cdk2 activity is associated with DNA replication-related functions. However, deregulation of cyclin E leads to inefficient assembly of pre-replication complexes, replication stress, and chromosome instability. In malignant cells, cyclin E is frequently overexpressed, correlating with decreased survival in breast cancer patients. Transgenic mice deregulated for cyclin E in the mammary epithelia develop carcinoma, confirming that cyclin E is an oncoprotein. However, it remains unknown how cyclin E-mediated replication stress promotes genomic instability during carcinogenesis. Here, we show that deregulation of cyclin E causes human mammary epithelial cells to enter into mitosis with short unreplicated genomic segments at a small number of specific loci, leading to anaphase anomalies and ultimately deletions. Incompletely replicated regions are preferentially located at late-replicating domains, fragile sites, and breakpoints, including the mixed-lineage leukemia breakpoint cluster region (MLL BCR). Furthermore, these regions are characterized by a paucity of replication origins or unusual DNA structures. Analysis of a large set of breast tumors shows a significant correlation between cyclin E amplification and deletions at a number of the genomic loci identified in our study. Our results demonstrate how oncogene-induced replication stress contributes to genomic instability in human cancer.

Cks1 enhances transcription efficiency at the GAL1 locus by linking the Paf1 complex to the 19S proteasome.

Cks1 was originally identified based on genetic interactions with CDC28, the gene that encodes Cdk1 in the budding yeast Saccharomyces cerevisiae. Subsequent work has shown that Cks1 binds Cdc28 and modulates its activity against certain substrates. However, the Cks1/Cdc28 complex also has a role in transcriptional chromatin remodeling not related to kinase activity. In order to elucidate protein networks associated with Cks1 transcriptional functions, proteomic analysis was performed on immunoaffinity-purified Cks1, identifying a physical interaction with the Paf1 complex. Specifically, we found that the Paf1 complex component Rtf1 interacts directly with Cks1 and that this interaction is essential for efficient recruitment of Cks1 to chromatin in the context of GAL1 gene induction. We further found that Cks1 in this capacity serves as an adaptor allowing Rtf1 to recruit 19S proteasome particles, shown to be required for efficient RNA production from some rapidly inducible genes such as GAL1.

Parkin-dependent degradation of the F-box protein Fbw7ß promotes neuronal survival in response to oxidative stress by stabilizing Mcl-1.

Parkinson's disease (PD) is characterized by progressive loss of midbrain dopaminergic neurons resulting in motor dysfunction. While most PD is sporadic in nature, a significant subset can be linked to either dominant or recessive germ line mutations. PARK2, encoding the ubiquitin ligase parkin, is the most frequently mutated gene in hereditary Parkinson's disease. Here, we present evidence for a neuronal ubiquitin ligase cascade involving parkin and the multisubunit ubiquitin ligase SCF(Fbw7ß). Specifically, parkin targets the SCF substrate adapter Fbw7ß for proteasomal degradation. Furthermore, we show that the physiological role of parkin-mediated regulation of Fbw7ß levels is the stabilization of the mitochondrial prosurvival factor Mcl-1, an SCF(Fbw7ß) target in neurons. We show that neurons depleted of parkin become acutely sensitive to oxidative stress due to an inability to maintain adequate levels of Mcl-1. Therefore, loss of parkin function through biallelic mutation of PARK2 may lead to death of dopaminergic neurons through unregulated SCF(Fbw7ß)-mediated ubiquitylation-dependent proteolysis of Mcl-1.

Ubiquitin ligases and cell cycle control.

The ubiquitin-proteasome system plays a pivotal role in the sequence of events leading to cell division known as the cell cycle. Not only does ubiquitin-mediated proteolysis constitute a critical component of the core oscillator that drives the cell cycle in all eukaryotes, it is also central to the mechanisms that ensure that the integrity of the genome is maintained. These functions are primarily carried out by two families of E3 ubiquitin ligases, the Skp/cullin/F-box-containing and anaphase-promoting complex/cyclosome complexes. However, beyond those functions associated with regulation of central cell cycle events, many peripheral cell cycle-related processes rely on ubiquitylation for signaling, homeostasis, and dynamicity, involving additional types of ubiquitin ligases and regulators. We are only beginning to understand the diversity and complexity of this regulation.

Fbw7α and Fbw7γ collaborate to shuttle cyclin E1 into the nucleolus for multiubiquitylation.

Cyclin E1, an activator of cyclin-dependent kinase 2 (Cdk2) that promotes replicative functions, is normally expressed periodically within the mammalian cell cycle, peaking at the G(1)-S-phase transition. This periodicity is achieved by E2F-dependent transcription in late G(1) and early S phases and by ubiquitin-mediated proteolysis. The ubiquitin ligase that targets phosphorylated cyclin E is SCF(Fbw7) (also known as SCF(Cdc4)), a member of the cullin ring ligase (CRL) family. Fbw7, a substrate adaptor subunit, is expressed as three splice-variant isoforms with different subcellular distributions: Fbw7α is nucleoplasmic but excluded from the nucleolus, Fbw7ß is cytoplasmic, and Fbw7γ is nucleolar. Degradation of cyclin E in vivo requires SCF complexes containing Fbw7α and Fbw7γ, respectively. In vitro reconstitution showed that the role of SCF(Fbw7α) in cyclin E degradation, rather than ubiquitylation, is to serve as a cofactor of the prolyl cis-trans isomerase Pin1 in the isomerization of a noncanonical proline-proline bond in the cyclin E phosphodegron. This isomerization is required for subsequent binding and ubiquitylation by SCF(Fbw7γ). Here we show that Pin1-mediated isomerization of the cyclin E phosphodegron and subsequent binding to Fbw7γ drive nucleolar localization of cyclin E, where it is ubiquitylated by SCF(Fbw7γ) prior to its degradation by the proteasome. It is possible that this constitutes a mechanism for rapid inactivation of phosphorylated cyclin E by nucleolar sequestration prior to its multiubiquitylation and degradation.

Cyclin-dependent kinases are regulators and effectors of oscillations driven by a transcription factor network.

During embryonic cell cycles, B-cyclin-CDKs function as the core component of an autonomous oscillator. Current models for the cell-cycle oscillator in nonembryonic cells are slightly more complex, incorporating multiple G1, S phase, and mitotic cyclin-CDK complexes. However, periodic events persist in yeast cells lacking all S phase and mitotic B-cyclin genes, challenging the assertion that cyclin-CDK complexes are essential for oscillations. These and other results led to the proposal that a network of sequentially activated transcription factors functions as an underlying cell-cycle oscillator. Here we examine the individual contributions of a transcription factor network and cyclin-CDKs to the maintenance of cell-cycle oscillations. Our findings suggest that while cyclin-CDKs are not required for oscillations, they do contribute to oscillation robustness. A model emerges in which cyclin expression (thereby, CDK activity) is entrained to an autonomous transcriptional oscillator. CDKs then modulate oscillator function and serve as effectors of the oscillator.

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.

Reduced spermatogonial proliferation and decreased fertility in mice overexpressing cyclin E in spermatogonia.

Cyclin E is a key component of the cell cycle regulatory machinery, contributing to the activation of Cdk2 and the control of cell cycle progression at several stages. Cyclin E expression is tightly regulated, by periodic transcription and ubiquitin-mediated degradation. Overexpression of cyclin E has been associated with tumor development and poor prognosis in several tumor types, including germ cell tumors and both cyclin E and its partner Cdk2 are required for normal spermatogenesis. Here we have generated and characterized transgenic mice overexpressing a cyclin E mutant protein, resistant to ubiquitin-mediated proteolysis, in testicular germ cells, under the control of the human EF-1alpha promoter. The transgenic mice develop normally and live a normal life span, with no signs of testicular tumor development. The transgenic mice display however reduced fertility and testicular atrophy, due to reduced spermatogonial proliferation as a consequence of deregulated cyclin E levels. Overall our results show that deregulation of cyclin E expression contribute to infertility, due to inability of the spermatogonial cells to start the mitotic cycles prior to entering meiosis.

Cks1, Cdk1, and the 19S proteasome collaborate to regulate gene induction-dependent nucleosome eviction in yeast.

Cks1, Cdk1 (Cdc28), and the proteasome are required for efficient transcriptional induction of GAL1 and other genes in Saccharomyces cerevisiae. We show here that one function of these proteins is to reduce nucleosome density on chromatin in a gene induction-specific manner. The transcriptional requirement for Cks1 can be bypassed if nucleosome density is reduced by an alternative pathway, indicating that this is the primary function of Cks1 in the context of gene induction. We further show that Cks1, Cdk1, and the 19S subunit of the proteasome are recruited to chromatin by binding directly to the histone H4 amino-terminal tail. However, this activity of the proteasome does not require the protease activity associated with the 20S subunit. These data suggest a model where binding of a complex consisting of Cks1, Cdk1, and the 19S proteasome to histone H4 leads to removal of nucleosomes via a nonproteolytic activity of the proteasome.

Phosphorylation of Mcm2 by Cdc7 promotes pre-replication complex assembly during cell-cycle re-entry.

Cyclin E has been shown to have a role in pre-replication complex (Pre-RC) assembly in cells re-entering the cell cycle from quiescence. The assembly of the pre-RC, which involves the loading of six MCM subunits (Mcm2-7), is a prerequisite for DNA replication. We found that cyclin E, through activation of Cdk2, promotes Mcm2 loading onto chromatin. This function is mediated in part by promoting the accumulation of Cdc7 messenger RNA and protein, which then phosphorylates Mcm2. Consistent with this, a phosphomimetic mutant of Mcm2 can bypass the requirement for Cdc7 in terms of Mcm2 loading. Furthermore, ectopic expression of both Cdc6 and Cdc7 can rescue the MCM loading defect associated with expression of dominant-negative Cdk2. These results are consistent with a role for cyclin E-Cdk2 in promoting the accumulation of Cdc6 and Cdc7, which is required for Mcm2 loading when cells re-enter the cell cycle from quiescence.

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.