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Cyclin E2 induces genomic instability by mechanisms distinct from cyclin E1
Cell Cycle
Abstract
Cyclin E1 drives the initiation of DNA replication, and deregulation of its periodic expression leads to mitotic delay associated with genomic instability. Since it is not known whether the closely related protein cyclin E2 shares these properties, we overexpressed cyclin E2 in breast cancer cells. This did not affect the duration of mitosis, nor did it cause an increase in p107 association with CDK2. In contrast, cyclin E1 overexpression led to inhibition of the APC complex, prolonged metaphase and increased p107 association with CDK2. Despite these different effects on the cell cycle, elevated levels of either cyclin E1 or E2 led to hallmarks of genomic instability, i.e., an increased proportion of abnormal mitoses, micronuclei and chromosomal aberrations. Cyclin E2 induction of genomic instability by a mechanism distinct from cyclin E1 indicates that these two proteins have unique functions in a cancer setting.
Supplementary resource (1)
Data
February 2016
... Cyclin E2 has greater S phase stability [17], and is independently transcribed and degraded [18]. Finally, while both cyclins E1 and E2 can induce genomic instability, cyclin E2 does not inhibit the APC Cdh1 complex [19], and the mechanistic basis for how it induces genomic instability is unknown. ...
... We examined T-47D breast cancer cells stably overexpressing cyclin E1-V5, cyclin E2-V5 or a vector control (pMIG -pMSCV-IRES-GFP) ( Figure 3A). Cells were previously sorted based on co-expression of the GFP protein and V5 expression into subpopulations of cells that had similar moderate overexpression of protein in each cell line [19]. ...
... We previously reported that cyclin E2 overexpression induces genomic instability in association with decreased chromosome condensation and failed nuclear envelope breakdown [19], both of which feature in endoreduplicating cells [35]. Cyclin E1 is also associated with genomic instability, but this is instead likely due to established roles in inhibiting preRC complex formation [25], causing focal genomic losses through replication stress [12] and by stabilising the APC Cdh1 complex [13,19]. ...
Genome doubling is an underlying cause of cancer cell aneuploidy and genomic instability, but few drivers have been identified for this process. Due to their physiological roles in the genome reduplication of normal cells, we hypothesised that the oncogenes cyclins E1 and E2 may be drivers of genome doubling in cancer. We show that both cyclin E1 (CCNE1) and cyclin E2 (CCNE2) mRNA are significantly associated with high genome ploidy in breast cancers. By live cell imaging and flow cytometry, we show that cyclin E2 overexpression promotes aberrant mitosis without causing mitotic slippage, and it increases ploidy with negative feedback on the replication licensing protein, Cdt1. We demonstrate that cyclin E2 localises with core preRC (pre-replication complex) proteins (MCM2, MCM7) on the chromatin of cancer cells. Low CCNE2 is associated with improved overall survival in breast cancers, and we demonstrate that low cyclin E2 protects from excess genome rereplication. This occurs regardless of p53 status, consistent with the association of high cyclin E2 with genome doubling in both p53 null/mutant and p53 wildtype cancers. In contrast, while cyclin E1 can localise to the preRC, its downregulation does not prevent rereplication, and overexpression promotes polyploidy via mitotic slippage. Thus, in breast cancer, cyclin E2 has a strong association with genome doubling, and likely contributes to highly proliferative and genomically unstable breast cancers.
... n = 4, mean + SEM, **P < 0.01, *P < 0.05, one-way ANOVA with Bonferonni correction. Cell cycle analysis and sample sizewas performed as described [66,67]. f Phase contrast photomicrographs of senescence-associated β-Galactosidase (β-Gal) staining in EsrCre or EsrCre-Hic1 lox/lox (Hic1KO) MEFs 5 days following tamoxifen treatment. ...
... Both EsrCre and p53KO cell lines were near tetraploid, resembling the stable karyotype of NIH-3T3 cells, a well-characterized MEF line generated by spontaneous immortalization (Fig. 3e, f) [40]. In keeping with the degree of anaphase bridge formation, numerical and segmental aneuploidy with large numbers of marker [66,67]. Cells were treated with vehicle or doxorubicin (Dox; 1 μM, 6 h). ...
... a Representative confocal photomicrographs showing anaphases in MEFs with the genotypes indicated. Cells were stained for tubulin (Red) and DNA (DAPI) (Blue) as described [66]. Scale bar = 5 μm. ...
Hypermethylated-in-Cancer 1 (Hic1) is a tumor suppressor gene frequently inactivated by epigenetic silencing and loss-of-heterozygosity in a broad range of cancers. Loss of HIC1, a sequence-specific zinc finger transcriptional repressor, results in deregulation of genes that promote a malignant phenotype in a lineage-specific manner. In particular, upregulation of the HIC1 target gene SIRT1, a histone deacetylase, can promote tumor growth by inactivating TP53. An alternate line of evidence suggests that HIC1 can promote the repair of DNA double strand breaks through an interaction with MTA1, a component of the nucleosome remodeling and deacetylase (NuRD) complex. Using a conditional knockout mouse model of tumor initiation, we now show that inactivation of Hic1 results in cell cycle arrest, premature senescence, chromosomal instability and spontaneous transformation in vitro. This phenocopies the effects of deleting Brca1, a component of the homologous recombination DNA repair pathway, in mouse embryonic fibroblasts. These effects did not appear to be mediated by deregulation of Hic1 target gene expression or loss of Tp53 function, and rather support a role for Hic1 in maintaining genome integrity during sustained replicative stress. Loss of Hic1 function also cooperated with activation of oncogenic KRas in the adult airway epithelium of mice, resulting in the formation of highly pleomorphic adenocarcinomas with a micropapillary phenotype in vivo. These results suggest that loss of Hic1 expression in the early stages of tumor formation may contribute to malignant transformation through the acquisition of chromosomal instability.
... Cyclin E2-CDK2 also causes genomic instability, but its mechanism is less well characterised (Caldon et al. 2013b). Unlike cyclin E1, genomic instability via cyclin E2 does not occur downstream of APC Cdh1 (Caldon et al. 2013b), but its overexpression causes the mis-segregation of chromosomes (Duffy et al. 2016). ...
... Cyclin E2-CDK2 also causes genomic instability, but its mechanism is less well characterised (Caldon et al. 2013b). Unlike cyclin E1, genomic instability via cyclin E2 does not occur downstream of APC Cdh1 (Caldon et al. 2013b), but its overexpression causes the mis-segregation of chromosomes (Duffy et al. 2016). ...
Cyclin E1 is one the most promising biomarkers in estrogen receptor positive (ER+) breast cancer for response to the new standard of care drug class, CDK4/6 inhibitors. Because of its strong predictive value, cyclin E1 expression may be used in the future to triage patients into potential responders and non-responders. Importantly, cyclin E1 is highly related to cyclin E2, and both cyclin E1 and cyclin E2 are estrogen target genes that can facilitate anti-estrogen resistance and can be highly expressed in breast cancer. However cyclin E1 and E2 are often expressed in different subsets of patients. This raises questions about whether the expression of cyclin E1 and cyclin E2 have different biological drivers, if high expressing subsets represent different clinical subtypes, and how to effectively develop a biomarker for E-cyclin expression. Finally, several pan-CDK inhibitors that target cyclin E-CDK2 activity have reached phase II clinical trials. In this review we outline the data identifying that different cohorts of patients have high expression of cyclins E1 and E2 in ER+ cancer, and address the implications for biomarker and therapeutic development.
... Lentiviral transduced Cyclin E shortened the delay by half an hour (Supplementary Figure 14c). These results correlate with published data demonstrating that aberrant expression of Cyclin E1 or its low molecular weight isoforms inhibited progression through mitosis due to Cyclin E1-Cdh1 binding, which resulted in inhibition of the APC complex and prolonged metaphase 46,47 . In addition, Cyclin E/Cdk2 phosphorylates Cdc25C. ...
... Overexpression of low molecular weight Cyclin E isoforms, on the other hand, led to premature inactivation of Cdc25C and faster mitotic exit 48 . Dysregulation of Cyclin E periodic expression led to mitotic delay associated with genomic instability, chromosome aberrations due to missegregation during metaphase, aberrant anaphase bridges, micronuclei and induction of multipolar anaphases leading to anaphase catastrophe 42,47,49 . Similar to ASPM Δ (Supplementary Figure 14a), deletion of the microcephaly disease gene Wdr62, resulted in a significant increased number of cortical PHH3 + cells associated with mitotic arrest 50 . ...
... In addition, cyclin E2 was previously shown to display high expression levels in lung cancer cells [11]. Cyclin E1 and E2 employ different mechanisms to induce chromosomal instability [12], and their degradation patterns were also different [13]. Am J Transl Res 2016;8(2):811-826 Further, in CHO cells, AKAP95 interacted with cyclin D and E during the G1/S phase of the cell cycle, and CDK2 inhibited the binding of AKAP95 to cyclin E [14]. ...
... The mechanism employed by cyclin E2 at inducing chromosomal instability differed from that employed by cyclin E1 [12]. Over-expression of cyclin E2 accelerated G1 progression in mammalian cells [11], but unlike cyclin E1, cyclin E2 was slightly, and in some cases, not even expressed in non-transformed cells [11]. ...
AKAP95 in lung cancer tissues showed higher expression than in paracancerous tissues. AKAP95 can bind with cyclin D and cyclin E during G1/S cell cycle transition, but its molecular mechanisms remain unclear. To identify the mechanism of AKAP95 in cell cycle progression, we performed AKAP95 transfection and silencing in A549 cells, examined AKAP95, cyclin E1 and cyclin E2 expression, and the interactions of AKAP95 with cyclins E1 and E2. Results showed that over-expression of AKAP95 promoted cell growth and AKAP95 bound cyclin E1 and E2, low molecular weight cyclin E1 (LWM-E1) and LWM-E2. Additionally AKAP95 bound cyclin E1 and LMW-E2 in the nucleus during G1/S transition, bound LMW-E1 during G1, S and G2/M, and bound cyclin E2 mainly on the nuclear membrane during interphase. Cyclin E2 and LMW-E2 were also detected. AKAP95 over-expression increased cyclin E1 and LMW-E2 expression but decreased cyclin E2 levels. Unlike cyclin E1 and LMW-E2 that were nuclear located during the G1, S and G1/S phases, cyclin E2 and LMW-E1 were expressed in all cell cycle phases, with cyclin E2 present in the cytoplasm and nuclear membrane, with traces in the nucleus. LMW-E1 was present in both the cytoplasm and nucleus. The 20 kDa form of LMW-E1 showed only cytoplasmic expression, while the 40 kDa form was nuclear expressed. The expression of AKAP95, cyclin E1, LMW-E1 and -E2, might be regulated by cAMP. We conclude that AKAP95 might promote cell cycle progression by interacting with cyclin E1 and LMW-E2. LMW-E2, but not cyclin E2, might be involved in G1/S transition. The binding of AKAP95 and LMW-E1 was found throughout cell cycle.
... Cyclin E1 and E2 have differences in tissue expression, transcription and post-transcriptional regulation, and have distinct affinities for other proteins, e.g. p107 [1,3]. In this study we examined the localisation of cyclin E1 and E2 and report unique sites of localisation in breast cancer cells. ...
... Data regulation in breast cancer via its unique interaction with NPAT. Cyclin E2 has a strong prognostic role in breast cancer [15], and induces genomic instability that is associated with defects in chromosome condensation [3]. This could be in part due to excessive histone production, as disruption of histone equilibrium is a predicted cause of genomic instability [17]. ...
The cyclin E oncogene activates CDK2 to drive cells from G1 to S phase of the cell cycle to commence DNA replication. It coordinates essential cellular functions with the cell cycle including histone biogenesis, splicing, centrosome duplication and origin firing for DNA replication. The two E-cyclins, E1 and E2, are assumed to act interchangeably in these functions. However recent reports have identified unique functions for cyclins E1 and E2 in different tissues, and particularly in breast cancer.
Cyclins E1 and E2 localise to distinct foci in breast cancer cells as well as co-localising within the cell. Both E-cyclins are found in complex with CDK2, at centrosomes and with the splicing machinery in nuclear speckles. However cyclin E2 uniquely co-localises with NPAT, the main activator of cell-cycle regulated histone transcription. Increased cyclin E2, but not cyclin E1, expression is associated with high expression of replication-dependent histones in breast cancers.
The preferential localisation of cyclin E1 or cyclin E2 to distinct foci indicates that each E-cyclin has unique roles. Cyclin E2 uniquely interacts with NPAT in breast cancer cells, and is associated with higher levels of histones in breast cancer. This could explain the unique correlations of high cyclin E2 expression with poor outcome and genomic instability in breast cancer.
... In several tumors, cyclin E overproduction has also been reported through CCNE1 gene amplification, the transcriptional activation of CCNE1 by c-MYC or E2F transcription factors, or the inactivation of the FBXW7 ubiquitin ligase adapter (reviewed in [52]). While cyclin E-CDK2 activity protects pluripotency in ESCs, it causes replication stress in the S phase by inducing incomplete DNA replication and abnormal mitotic progression in cancers [53,54]. ...
Simple Summary
The cell division cycle is tightly regulated to ensure faithful and complete DNA replication. A critical cell cycle phase is G1 in which cells prepare DNA for replication in S phase. Interestingly, stem cells and cancer cells have both similarities and differences in their cell cycle regulatory mechanisms. In this review, we address the role of various cell cycle regulators in controlling the dynamics of G1 phase in stem cells and cancer cells. We also discuss recent advances in understanding how core pluripotency factors regulate the cell cycle and play dual roles in stem cell pluripotency and in cancers where they are aberrantly expressed. A better understanding of these common regulatory networks could offer potential new therapeutic avenues for cancer.
Abstract
G1 cell cycle phase dynamics are regulated by intricate networks involving cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors, which control G1 progression and ensure proper cell cycle transitions. Moreover, adequate origin licensing in G1 phase, the first committed step of DNA replication in the subsequent S phase, is essential to maintain genome integrity. In this review, we highlight the intriguing parallels and disparities in G1 dynamics between stem cells and cancer cells, focusing on their regulatory mechanisms and functional outcomes. Notably, SOX2, OCT4, KLF4, and the pluripotency reprogramming facilitator c-MYC, known for their role in establishing and maintaining stem cell pluripotency, are also aberrantly expressed in certain cancer cells. In this review, we discuss recent advances in understanding the regulatory role of these pluripotency factors in G1 dynamics in the context of stem cells and cancer cells, which may offer new insights into the interconnections between pluripotency and tumorigenesis.
... In addition, high expression of CCNE1 and CCNE2 leads to genomic instability, which affects the normal progression of the M phase [104]. This finding is consistent with the restriction on the expression of these two genes in our rules to distinguish normal G2/M phase cells [105,106]. ...
The cell cycle is composed of a series of ordered, highly regulated processes through which a cell grows and duplicates its genome and eventually divides into two daughter cells. According to the complex changes in cell structure and biosynthesis, the cell cycle is divided into four phases: gap 1 (G1), DNA synthesis (S), gap 2 (G2), and mitosis (M). Determining which cell cycle phases a cell is in is critical to the research of cancer development and pharmacy for targeting cell cycle. However, current detection methods have the following problems: (1) they are complicated and time consuming to perform, and (2) they cannot detect the cell cycle on a large scale. Rapid developments in single-cell technology have made dissecting cells on a large scale possible with unprecedented resolution. In the present research, we construct efficient classifiers and identify essential gene biomarkers based on single-cell RNA sequencing data through Boruta and three feature ranking algorithms (e.g., mRMR, MCFS, and SHAP by LightGBM) by utilizing four advanced classification algorithms. Meanwhile, we mine a series of classification rules that can distinguish different cell cycle phases. Collectively, we have provided a novel method for determining the cell cycle and identified new potential cell cycle-related genes, thereby contributing to the understanding of the processes that regulate the cell cycle.
... Cyclin E1 was mutated by site-directed mutagenesis as described 41 . MDA-MB-468 cells expressing the ecotropic receptor 42 were infected with pMSCV-IRES-GFP retrovirus expressing cyclin E1 wildtype and mutants as described 43 . Subpopulations with graded expression of GFP and cyclin proteins were separated by sterile flow cytometry and matched populations selected based on GFP expression. ...
Basal-like breast cancers (BLBC) are aggressive breast cancers that respond poorly to targeted therapies and chemotherapies. In order to define therapeutically targetable subsets of BLBC we examined two markers: cyclin E1 and BRCA1 loss. In high grade serous ovarian cancer (HGSOC) these markers are mutually exclusive, and define therapeutic subsets. We tested the same hypothesis for BLBC. Using a BLBC cohort enriched for BRCA1 loss, we identified convergence between BRCA1 loss and high cyclin E1 protein expression, in contrast to HGSOC in which CCNE1 amplification drives increased cyclin E1. In cell lines, BRCA1 loss was associated with stabilized cyclin E1 during the cell cycle, and BRCA1 siRNA led to increased cyclin E1 in association with reduced phospho-cyclin E1 T62. Mutation of cyclin E1 T62 to alanine increased cyclin E1 stability. We showed that tumors with high cyclin E1/ BRCA1 mutation in the BLBC cohort also had decreased phospho-T62, supporting this hypothesis. Since cyclin E1/CDK2 protects cells from DNA damage and cyclin E1 is elevated in BRCA1 mutant cancers, we hypothesized that CDK2 inhibition would sensitize these cancers to PARP inhibition. CDK2 inhibition induced DNA damage and synergized with PARP inhibitors to reduce cell viability in cell lines with homologous recombination deficiency, including BRCA1 mutated cell lines. Treatment of BRCA1 mutant BLBC patient-derived xenograft models with combination PARP and CDK2 inhibition led to tumor regression and increased survival. We conclude that BRCA1 status and high cyclin E1 have potential as predictive biomarkers to dictate the therapeutic use of combination CDK inhibitors/PARP inhibitors in BLBC.
... Gain in 19q12 included cell cycle regulator CyclinE1 (CCNE1). It had been reported that amplification and overexpression of CCNE1 were associated with chromosomal instability in GC and other cancers [23,40,41]. Previous TCGA study showed that patients with chromosomal instability (CIN, one of four molecular subtypes of GC identified by TCGA) exhibited the greatest benefit from adjuvant chemotherapy, as evidenced by significantly increased DFS rates [42]. ...
Background
Neoadjuvant chemotherapy (NACT) before radical gastrectomy is preferred for locally advanced gastric cancer (GC). However, clinical practices demonstrate that a considerable proportion of GC patients do not benefit from NACT, largely due to the lack of biomarkers for patient selection and prognosis prediction. A recent study revealed that patients with microsatellite instability-high (MSI-H) may be resistant to NACT, however, most tumors in Chinese GC patients (~ 95%) are characterized by microsatellite stability (MSS). Here, we aimed to discover new molecular biomarkers for this larger population.Methods
We performed whole-exome sequencing on 46 clinical samples (pre- and post-treatment) from 30 stage II/III MSS GC patients whose response to NACT was rigorously defined. Serum tumor markers (TMs), including AFP, CEA, CA199, CA724 and CA242 were measured during the course.ResultsHigh tumor mutation burden (TMB-H) and 19q12 amplification (19q12 +) were positively associated with the NACT response. When TMB and 19q12 amplification were jointly analyzed, those with TMB-H or 19q12 + showed favorable response to NACT (p = 0.035). Further, TMB-H was negatively correlated with ypN stage, lymph node metastasis, and macrophage infiltration. Patients with TMB-H showed better disease-free survival (DFS) than those with TMB-L (P = 0.025, HR = 0.1331), and this was further validated using two larger GC datasets: TCGA-STAD (p = 0.004) and ICGC-CN (p = 0.045).Conclusion
The combination of TMB-H and 19q12 + can serve as an early indicator of response to NACT. Superior to traditional clinical indicators, TMB-H is a robust and easily accessible candidate biomarker associated with better DFS, and can be evaluated at the time of diagnosis.
... Other studies implicated overexpressed cyclin E in inhibiting APC CDH1 activity, resulting in the abnormal accumulation of APC CDH1 substrates as cells enter mitosis. This, in turn, impairs progression through mitosis, thereby contributing to polyploidy (Caldon et al., 2013). Overexpression of cyclin E was also implicated in erroneous MCM loading, interference with nucleotide biosynthesis, induction of DNA damage in stalled forks, uncontrolled DNA replication origin firing, and replication-transcription collisions, and all these mechanisms may contribute to genome instability (Mazumder et al., 2007;Teixeira and Reed, 2017). ...
Abnormal activity of the core cell-cycle machinery is seen in essentially all tumor types and represents a driving force of tumorigenesis. Recent studies revealed that cell-cycle proteins regulate a wide range of cellular functions, in addition to promoting cell division. With the clinical success of CDK4/6 inhibitors, it is becoming increasingly clear that targeting individual cell-cycle components may represent an effective anti-cancer strategy. Here, we discuss the potential of inhibiting different cell-cycle proteins for cancer therapy.
... Evidence was provided showing that CDK inhibitors, used at concentrations that did not significantly affect cell proliferation, were able to reduce motility and invasiveness of BC cells [182]. Until now, CDK inhibitors have been primarily used for their action in the regulation of the cell cycle and cell proliferation [183], but the results reported above further support a role of CDK molecules in regulating pathways involved in cell migration control [184,185], suggesting the use of CDK inhibitors in metastatic BC to block the metastasis process. ...
Introduction:
Triple-negative breast cancer (TNBC) is the most difficult breast cancer subtype to treat because of its heterogeneity and lack of specific therapeutic targets. High Mobility Group A (HMGA) proteins are chromatin architectural factors that have multiple oncogenic functions in breast cancer, and they represent promising molecular therapeutic targets for this disease.
Areas covered:
We offer an overview of the strategies that have been exploited to counteract HMGA oncoprotein activities at the transcriptional and post-transcriptional levels. We also present the possibility of targeting cancer-associated factors that lie downstream of HMGA proteins and discuss the contribution of HMGA proteins to chemoresistance.
Expert opinion:
Different strategies have been exploited to counteract HMGA protein activities; these involve interfering with their nucleic acid binding properties and the blocking of HMGA expression. Some approaches have provided promising results. However, some unique characteristics of the HMGA proteins have not been exploited; these include their extensive protein-protein interaction network and their intrinsically disordered status that present the possibility that HMGA proteins could be involved in the formation of proteinaceous membrane-less organelles (PMLO) by liquid-liquid phase separation. These unexplored characteristics could open new pharmacological avenues to counteract the oncogenic contributions of HMGA proteins.
... Downregulation of miR-130a-3p could lead to stem cell dysfunction via increased KLF7 expression (Schuettpelz et al., 2012), whereas downregulation of miR-143-3p could decrease PAC survival by increasing the level of the proapoptotic factor AIFM1 (Bano and Prehn, 2018). On the other hand, upregulation of miR-30d and miR-23b could promote cell senescence and oxidative stress levels in PACs by decreasing the levels of CCNE2 (a promitotic factor) (Caldon et al., 2013) and GSH (an antioxidant enzyme) (Forman et al., 2009), respectively. The upregulation of miR-361 and miR-501 could decrease the angiogenic paracrine activity of PACs by targeting VEGF (Dal Monte et al., 2013) and MMP-13 (Kudo et al., 2012), respectively. ...
Background
Classical cardiovascular risk factors (CRFs) are associated with impaired angiogenic activities of bone marrow–derived proangiogenic cells (PACs) related to peripheral artery diseases (PADs) and ischemia-induced neovascularization. MicroRNAs (miRs) are key regulators of gene expression, and they are involved in the modulation of PAC function and PAC paracrine activity. However, the effects of CRFs on the modulation of miR expression in PACs are unknown.
Aims and Methods
We used a model of hindlimb ischemia and next-generation sequencing to perform a complete profiling of miRs in PACs isolated from the bone marrow of mice subjected to three models of CRFs: aging, smoking (SMK) and hypercholesterolemia (HC).
Results
Approximately 570 miRs were detected in PACs in the different CRF models. When excluding miRs with a very low expression level (<100 RPM), 40 to 61 miRs were found to be significantly modulated by aging, SMK, or HC. In each CRF condition, we identified downregulated proangiogenic miRs and upregulated antiangiogenic miRs that could contribute to explain PAC dysfunction. Interestingly, several miRs were similarly downregulated (e.g., miR-542-3p, miR-29) or upregulated (e.g., miR-501, miR-92a) in all CRF conditions. In silico approaches including Kyoto Encyclopedia of Genes and Genomes and cluster dendogram analyses identified predictive effects of these miRs on pathways having key roles in the modulation of angiogenesis and PAC function, including vascular endothelial growth factor signaling, extracellular matrix remodeling, PI3K/AKT/MAPK signaling, transforming growth factor beta (TGFb) pathway, p53, and cell cycle progression.
Conclusion
This study describes for the first time the effects of CRFs on the modulation of miR profile in PACs related to PAD and ischemia-induced neovascularization. We found that several angiogenesis-modulating miRs are similarly altered in different CRF conditions. Our findings constitute a solid framework for the identification of miRs that could be targeted in PACs in order to improve their angiogenic function and for the future development of novel therapies to improve neovascularization and reduce tissue damage in patients with severe PAD.
... gene was specifically expressed in male mice testis and restricted to type B spermatogonia and pre-meiotic germ cells. Although STRA8-deficient testis initiated meiosis, we discovered aberrant expressions of Cyclin A2 28-31 and Cyclin E2. [31][32][33][34] Both of these cycle factors, which began to express in late G1 phase, peaked in S phase and began to decline in G2 phase. The expression of Cyclin A2 and Cyclin E2 played a role in the initiation of DNA synthesis to prevent DNA replication. ...
STRA8 (Stimulated By Retinoic Acid Gene 8) is a retinoic acid (RA) induced gene that plays vital roles in spermatogonial proliferation, differentiation and meiosis. The SETD8 and STRA8 protein interaction was discovered using the yeast two‐hybrid technique using a mouse spermatogonial stem cell (SSC) cDNA library. The interaction of these two proteins was confirmed using co‐immunoprecipitation and identification of key domains governing the protein: protein complex. STRA8 and SETD8 showed a mutual transcriptional regulation pattern that provided evidence that SETD8 negatively regulated transcriptional activity of the STRA8 promoter. The SETD8 protein directly bound to the proximal promoter of the STRA8 gene. STRA8 increased the transcriptional activity of SETD8 promoter in a dose‐dependent manner. For the first time, we have discovered that STRA8 and SETD8 display a cell cycle‐dependent expression pattern in germline cells. Expression levels of SETD8 and H4K20me1 in S phase of STRA8 overexpression GC1 cells were different from that previously observed in tumour cell lines. In wild‐type mice testis, SETD8, H4K20me1 and PCNA co‐localized with STRA8 in spermatogonia. Further, our studies quantitated abnormal expression levels of cell cycle and ubiquitination‐related factors in STRA8 dynamic models. STRA8 and SETD8 may regulate spermatogenesis via Cdl4‐Clu4A‐Ddb1 ubiquitinated degradation axis in a PCNA‐dependent manner.
... The cross-reactivity in the immune response that was noted for cyclin E1 and cyclin E2 (Figs. 3 and 4) may be of great value in cyclin E-based therapy. Because the expression of cyclin E1 and cyclin E2 varies among tumor types and at different stages of the cell cycle [40][41][42], eliciting an immune response against either cyclin E1 or cyclin E2 would broaden the applicability of CCNE-based therapy. Furthermore, such therapy can be applied to solid tumors (i.e., breast and gastrointestinal malignancies) in addition to leukemia. ...
Immunotherapy targeting leukemia-associated antigens has shown promising results. Because of the heterogeneity of leukemia, vaccines with a single peptide have elicited only a limited immune response. Targeting several peptides together elicited peptide-specific cytotoxic T lymphocytes (CTLs) in leukemia patients, and this was associated with clinical responses. Thus, the discovery of novel antigens is essential. In the current study, we investigated cyclin E as a novel target for immunotherapy. Cyclin E1 and cyclin E2 were found to be highly expressed in hematologic malignancies, according to reverse transcription polymerase chain reaction and western blot analysis. We identified two HLA-A*0201 binding nonameric peptides, CCNE1M from cyclin E1 and CCNE2L from cyclin E2, which both elicited the peptide-specific CTLs. The peptide-specific CTLs specifically kill leukemia cells. Furthermore, CCNE1M and CCNE2L CTLs were increased in leukemia patients who underwent allogeneic hematopoietic stem cell transplantation, and this was associated with desired clinical outcomes. Our findings suggest that cyclin E1 and cyclin E2 are potential targets for immunotherapy in leukemia.
... All these genes play a key role in mitotic cell division. CCNE2 has been linked to endoreplication and genomic instability [24,25]. C21orf45, also known as MIS18A, is a kinetocore protein belonging to the Mis18 complex assembly that is crucial for CENPA deposition at the centromere [26,27]. ...
Platinum-based chemotherapy is the therapy of choice for epithelial ovarian cancer (EOC). Acquired resistance to platinum (PT) is a frequent event that leads to disease progression and predicts poor prognosis. To understand possible mechanisms underlying acquired PT-resistance, we have recently generated and characterized three PT-resistant isogenic EOC cell lines. Here, we more deeply characterize several PT-resistant clones derived from MDAH-2774 cells. We show that, in these cells, the increased PT resistance was accompanied by the presence of a subpopulation of multinucleated giant cells. This phenotype was likely due to an altered progression through the M phase of the cell cycle and accompanied by the deregulated expression of genes involved in M phase progression known to be target of mutant TP53. Interestingly, we found that PT-resistant MDAH cells acquired in the TP53 gene a novel secondary mutation (i.e., S185G) that accompanied the R273H typical of MDAH cells. The double p53S185G/R273H mutant increases the resistance to PT in a TP53 null EOC cellular model. Overall, we show how the selective pressure of PT is able to induce additional mutation in an already mutant TP53 gene in EOC and how this event could contribute to the acquisition of novel cellular phenotypes.
... Exchanges that were obviously due to "flipping" at the centrosome were omitted from quantitation. Mitotic abnormalities were scored as described previously 68,69 . ...
The collapse of stalled replication forks is a major driver of genomic instability. Several committed mechanisms exist to resolve replication stress. These pathways are particularly pertinent at telomeres. Cancer cells that use Alternative Lengthening of Telomeres (ALT) display heightened levels of telomere-specific replication stress, and co-opt stalled replication forks as substrates for break-induced telomere synthesis. FANCM is a DNA translocase that can form independent functional interactions with the BLM-TOP3A-RMI (BTR) complex and the Fanconi anemia (FA) core complex. Here, we demonstrate that FANCM depletion provokes ALT activity, evident by increased break-induced telomere synthesis, and the induction of ALT biomarkers. FANCM-mediated attenuation of ALT requires its inherent DNA translocase activity and interaction with the BTR complex, but does not require the FA core complex, indicative of FANCM functioning to restrain excessive ALT activity by ameliorating replication stress at telomeres. Synthetic inhibition of FANCM-BTR complex formation is selectively toxic to ALT cancer cells.
... For example, the common candidate driver lncRNA PVT1 was associated with hallmark "Genome Instability and Mutation" in three cancer types (OV, LUAD and HNSC). In OV, PVT1 was mutually exclusive with CCNE1 ( Figure 6E-F), and deregulation of CCNE1 expression led to genomic instability via mitotic delay (Caldon et al., 2013). But in LUAD, PVT1 exhibited mutually exclusive with CDKN2A ( Figure 6E, 6G), which induced DNA replication stress and subsequently contributed to genomic instability. ...
Substantial cancer genome sequencing efforts have discovered many important driver genes contributing to tumorigenesis. However, very little is known about genetic alterations of long non-coding RNAs (lncRNAs) in cancer. There is thus a need for systematic surveys of driver lncRNAs. Through integrative analysis of 5,918 tumors across 11 cancer types, we revealed that lncRNAs have undergone dramatic genomic alterations, many of which are mutually exclusive with well-known cancer genes. Using the hypothesis of functional redundancy of mutual exclusivity, we developed a computational framework to identify driver lncRNAs associated with different cancer hallmarks. Applying it to pan-cancer data, we identified 378 candidate driver lncRNAs, whose genomic features highly resemble the known cancer driver genes (such as, high conservation and early replication). We further validated the candidate driver lncRNAs involved in "tissue invasion and metastasis" in lung adenocarcinoma and breast cancer, and highlighted their potential roles in improving clinical outcomes. In summary, we have generated a comprehensive landscape of cancer candidate driver lncRNAs, which could act as a starting point for future functional explorations, the identification of biomarkers, and lncRNA-based target therapy.
... CCNE2 is frequently upregulated in human pulmonary dysplasia and malignancy and is correlated with poor prognosis in lung cancer patients [15][16][17][18][19] . In several cancer types (including NSCLC and breast cancer), CCNE2 upregulation occurs as an early event 15,34 , and overexpression of this cyclin acts as an inducer of genomic instability and polyploidy, differently from cyclin E1, D, or A overexpression 35,36 . Although we did not determine the impact of uc.339-induced upregulation of CCNE2 on the genomic instability of the tested cell lines, this area warrants further investigation. ...
The transcribed ultraconserved regions (T-UCRs) encode long non-coding RNAs implicated in human carcinogenesis. Their mechanisms of action and the factors regulating their expression in cancers are poorly understood. Here we show that high expression of uc.339 correlates with lower survival in 210 non-small cell lung cancer (NSCLC) patients. We provide evidence from cell lines and primary samples that TP53 directly regulates uc.339. We find that transcribed uc.339 is upregulated in archival NSCLC samples, functioning as a decoy RNA for miR-339-3p, -663b-3p, and -95-5p. As a result, Cyclin E2, a direct target of all these microRNAs is upregulated, promoting cancer growth and migration. Finally, we find that modulation of uc.339 affects microRNA expression. However, overexpression or downregulation of these microRNAs causes no significant variations in uc.339 levels, suggesting a type of interaction for uc.339 that we call “entrapping”. Our results support a key role for uc.339 in lung cancer.
... Proteins upregulated in this cluster included DNA damage response proteins MutS Homolog 2 (MSH2), Proliferating Cell Nuclear Antigen (PCNA), and BRCA1 Associated Protein 1 (BAP1). Cyclin E1, which displays copy number gain in 36% of GBMs and whose overexpression results in chromosome instability 27 , was also upregulated in this cluster, as was NF-κB-p65 (pS536), an inflammatory and stress response protein. These results indicate that the PN region specifically expresses a variety of stress response proteins, possibly related to severe hypoxia, acidosis, immune cell infiltrate and tissue damage. ...
Glioblastoma (GBM) contains diverse microenvironments with uneven distributions of oncogenic alterations and signaling networks. The diffusely infiltrative properties of GBM result in residual tumor at neurosurgical resection margins, representing the source of relapse in nearly all cases and suggesting that therapeutic efforts should be focused there. To identify signaling networks and potential druggable targets across tumor microenvironments (TMEs), we utilized 5-ALA fluorescence-guided neurosurgical resection and sampling, followed by proteomic analysis of specific TMEs. Reverse phase protein array (RPPA) was performed on 205 proteins isolated from the tumor margin, tumor bulk, and perinecrotic regions of 13 previously untreated, clinically-annotated and genetically-defined high grade gliomas. Differential protein and pathway signatures were established and then validated using western blotting, immunohistochemistry, and comparable TCGA RPPA datasets. We identified 37 proteins differentially expressed across high-grade glioma TMEs. We demonstrate that tumor margins were characterized by pro-survival and anti-apoptotic proteins, whereas perinecrotic regions were enriched for pro-coagulant and DNA damage response proteins. In both our patient cohort and TCGA cases, the data suggest that TMEs possess distinct protein expression profiles that are biologically and therapeutically relevant.
... For example, cyclin E2 is the major E-cyclin within HLBs in breast cancer cells and has a strong prognostic role in breast cancer [114,115]. Cyclin E2 has a particular role in coordinating the cell cycle with histone transcription and can induce genomic instability that is associated with defects in chromosome condensation partly due to excessive histone production [114,116]. Analysis of the transcriptome profiles of breast cancers from TCGA showed that high cyclin E2 expression is associated with high levels of replicationdependent histones, which could explain the correlations of high cyclin E2 expression with poor outcome and genomic instability in breast cancer [114]. The expression of ATAD2, the human homolog of Yta7 correlates with clinical outcome of breast cancer patients [57]. ...
The expression of core histone genes is cell cycle regulated. Large amounts of histones are required to restore duplicated chromatin during S phase when DNA replication occurs. Over-expression and excess accumulation of histones outside S phase are toxic to cells and therefore cells need to restrict histone expression to S phase. Misregulation of histone gene expression leads to defects in cell cycle progression, genome stability, DNA damage response and transcriptional regulation. Here, we discussed the factors involved in histone gene regulation as well as the underlying mechanism. Understanding the histone regulation mechanism will shed lights on elucidating the side effects of certain cancer chemotherapeutic drugs and developing potential biomarkers for tumor cells.
... Proteins upregulated in this cluster included DNA damage response proteins MutS Homolog 2 (MSH2), Proliferating Cell Nuclear Antigen (PCNA), and BRCA1 Associated Protein 1 (BAP1). Cyclin E1, which displays copy number gain in 36% of GBMs and whose overexpression results in chromosome instability 27 , was also upregulated in this cluster, as was NF-κB-p65 (pS536), an inflammatory and stress response protein. These results indicate that the PN region specifically expresses a variety of stress response proteins, possibly related to severe hypoxia, acidosis, immune cell infiltrate and tissue damage. ...
Glioblastoma (GBM) is highly heterogeneous and micro-environmental diversity may necessitate therapeutically targeting specific tumor regions rather than treating the tumor as a homogenous entity. The diffusely infiltrative properties of GBM result in residual tumor at resection margins in nearly all cases, suggesting that targeting efforts should focus on this region. To identify potential druggable targets at the tumor margin, we utilized fluorescence-guided surgical resection based on 5-ALA, followed by proteomic analysis of individual tumor regions that were precisely defined during surgery. Reverse phase protein array (RPPA) was performed on 206 proteins isolated from the tumor margin, bulk, and perinecrotic regions of 13 genetically defined patient samples (10 GBM, 3AA) in order to identify differentially expressed perturbable molecular pathways or signaling networks. Using ANOVA and post-hoc Tukey-HSD, we identified 37 proteins that were differentially expressed among the three regions. We performed unsupervised hierarchical clustering on these 37 proteins and found that in general, samples from the perinecrotic and bulk regions clustered together and those from the margin clustered together. 25 of these 37 proteins were overexpressed and 12 underexpressed at the tumor margin. Among differentially expressed proteins, we focused further efforts on Akt, PAI-1, and Tyro3. In comparison to the perinecrotic region, Akt and Tyro3 were found to be overexpressed at the tumor margin while PAI-1 was underexpressed at the margin. A common signaling abnormality of GBM is upregulated Akt activity, typically downstream of EGFR, PDGFR or c-Met amplification, PTEN loss or activating mutations in PI3K subunits. Tyro3 is a promising target because of its activation of the PI3K/Akt/mTOR pathway and its role in coagulation. PAI-1 (plasminogen activator inhibitor 1) is a protein that promotes coagulation and could induce intratumoral thrombosis, a defining feature of GBMs. We present differential protein expression patterns among specific tumor microenvironments as potential therapeutic targets.
... CCNE2 (cyclin E2) specifically interacts with the CIP/KIP family of CDK inhibitors and plays a role in cell cycle G1/S transition. Elevated CCNE2 level can lead to genomic instability such as increased proportion of abnormal mitoses, micronuclei, and chromosomal aberrations [19]. Significantly increased expression levels of CCNE2 have been observed in various tumors such as those of the lung, breast, pancreas, and nasopharyngx, and have been shown to play important roles in the proliferation, invasion, metastasis, and poor prognosis of these cancers [20,21]. ...
Objectives
Lung cancer in Xuanwei (LCXW), China, is known throughout the world for its distinctive characteristics, but little is known about its pathogenesis. The purpose of this study was to screen potential novel “driver genes” in LCXW.
Methods
Genome-wide DNA copy number alterations (CNAs) were detected by array-based comparative genomic hybridization and differentially expressed genes (DEGs) by gene expression microarrays in 8 paired LCXW and non-cancerous lung tissues. Candidate driver genes were screened by integrated analysis of CNAs and DEGs. The candidate genes were further validated by real-time quantitative polymerase chain reaction.
Results
Large numbers of CNAs and DEGs were detected, respectively. Some of the most frequently occurring CNAs included gains at 5p15.33-p15.32, 5p15.1-p14.3, and 5p14.3-p14.2 and losses at 11q24.3, 21q21.1, 21q22.12-q22.13, and 21q22.2. Integrated analysis of CNAs and DEGs identified 24 candidate genes with frequent copy number gains and concordant upregulation, which were considered potential oncogenes, including CREB3L4, TRIP13, and CCNE2. In addition, the analysis identified 19 candidate genes with a negative association between copy number change and expression change, considered potential tumor suppressor genes, including AHRR, NKD2, and KLF10. One of the most studied oncogenes, MYC, may not play a carcinogenic role in LCXW.
Conclusions
This integrated analysis of CNAs and DEGs identified several potential novel LCXW-related genes, laying an important foundation for further research on the pathogenesis of LCXW and identification of novel biomarkers or therapeutic targets.
... We generated a list of human homologs of yeast dCIN genes to uncover amplified and/or overexpressed genes that may be relevant to tumorigenesis. Our list contains two previously characterized dCIN genes in humans (51,52,85) as well as eight genes whose overexpression had a clear role in cancer progression (86). Our candidate testing approach identified two additional genes, meaning that 20% (4/20) of yeast dCIN genes were phenotypically conserved in humans. ...
Significance
Chromosome instability (CIN) is well established as an enabling characteristic of cancer that contributes to cancer initiation and progression. Here, we identify 245 genes whose individual overexpression causes CIN in yeast, and we show that the overexpression of several of these dosage CIN (dCIN) genes also causes CIN in human cells. We demonstrate that genetic screens in yeast can be used to determine candidate target genes for specific killing of cancer cells overexpressing dCIN genes. Rhabdomyosarcoma cells with elevated levels of Tdp1 are one example where the histone deacetylase inhibitors valproic acid and trichostatin A can be used to specifically kill these cells. We have generated a list of dCIN candidate genes that can facilitate selective targeting of tumor cells.
... CCNE2, cyclin E2; miR, microRNA; UTR, untranslated region; NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. A B C significantly in tumor-derived cells (20), suggesting that mechanisms distinct from CCNE1 induce tumorigenesis and progression (21). Previous studies indicate that CCNE2 overexpression is associated with pathogenesis (22), endocrine resistance (23), metastasis and reduced survival (24) in breast cancer. ...
MicroRNAs (miRNAs) act as tumor promoters or tumor suppressors in different human malignancies. In the current study, using an Agilent miRNA microarray, miR‑30a was found to be a significantly downregulated miRNA in castration‑resistant prostate cancer (CRPC) tissues, compared with androgen‑dependent prostate cancer tissues. Aberrant expression of cyclin E2 (CCNE2) has been reported in a variety of types of cancer including prostate cancer, and correlates with clinical outcome. The purpose of the current study was to determine the functions of miR‑30a in CRPC cell lines and identify whether CCNE2 was regulated by miR‑30a. To analyze the associations between miR‑30a and CCNE2 expression levels, pathological specimens were collected, and reverse transcription‑quantitative polymerase chain reaction and immunohistochemical staining were conducted. The effect of miR‑30a overexpression on CRPC cell lines and the predicted target gene, CCNE2, were evaluated by MTT assay, flow cytometry, tumor formation, luciferase reporter assay and western blotting. miR‑30a overexpression resulted in a significant suppression of cell growth in vitro, and reduced tumorigenicity in vivo. miR‑30a repressed the expression of CCNE2 through binding to its 3'‑untranslated region. CCNE2 was observed to be overexpressed in patients with CRCP and had an approximately inverse correlation with the level of miR‑30a. The results suggest that miR‑30a may function as a novel tumor suppressor in CRPC. Its anti‑oncogenic activity may occur by the reduced expression of a distinct cell cycle protein, CCNE2.
... CCNE2 has been detected in various prognostic gene expression profiles that predict a shorter metastasis-free survival or relapse-free interval in breast cancer patients [14][15][16]. CCNE2 overexpression in breast cancer cells induces genomic instability but does not affect mitotic progression [17,18]. Therefore, even though it appears that CCNE2 might play a role in cancer progression, its underlying molecular mechanism is unknown. ...
High Mobility Group A1 (HMGA1) is an architectural chromatin factor that promotes neoplastic transformation and progression. However, the mechanism by which HMGA1 exerts its oncogenic function is not fully understood. Here, we show that cyclin E2 (CCNE2) acts downstream of HMGA1 to regulate the motility and invasiveness of basal-like breast cancer cells by promoting the nuclear localization and activity of YAP, the downstream mediator of the Hippo pathway. Mechanistically, the activity of MST1/2 and LATS1/2, the core kinases of the Hippo pathway, are required for the HMGA1- and CCNE2-mediated regulation of YAP localization. In breast cancer patients, high levels of HMGA1 and CCNE2 expression are associated with the YAP/TAZ signature, supporting this connection. Moreover, we provide evidence that CDK inhibitors induce the translocation of YAP from the nucleus to the cytoplasm, resulting in a decrease in its activity. These findings reveal an association between HMGA1 and the Hippo pathway that is relevant to stem cell biology, tissue homeostasis, and cancer.
... Noya et al. [49] and Nguyen et al. [50] reported that HPV E7 increases the expression of cyclins E and A and by this promotes malignant trans-formation [51]. Furthermore, dysregulated Cyclin E expression induces chromosomal instability and initiates apoptosis [52,53]. Both cyclins as part of the cyclin-dependent-kinasecyclin (CDK-cyclin) complex represent key proteins in the transition from G1 to S phase (Cyclin E2), during S phase (Cyclin E2/A2) and from S to G2 phase (Cyclin A2). ...
Squamous cell carcinoma of the head and neck region (HNSCC), which is related to an infection with human papilloma virus (HPV), responds better to simultaneous radio-chemotherapy with Cisplatin based regimens than HPV-negative tumors. The underlying molecular mechanisms for this clinical observation are not fully understood. Therefore, the response of four HPV-positive (HPV+) (UM-SCC-47, UM-SCC-104, 93-VU-147T, UPCI:SCC152) and four HPV-negative (HPV-) (UD-SCC-1, UM-SCC-6, UM-SCC-11b, UT-SCC-33) HNSCC cell lines to x-irradiation ± Cisplatin incubation in terms of clonogenic survival, cell cycle progression, protein expression (cyclin A2, cyclin E2, E6, E7, p53) and induction of apoptosis, was investigated. HPV+ cells were more radio- and chemosensitive and were more effectively sensitized to x-irradiation by simultaneous Cisplatin incubation than HPV- cell lines. HPV+ cell lines revealed an increased and prolonged G2/M arrest after irradiation, whereas Cisplatin induced a blockage of cells in S phase. In comparison to irradiation only, addition of Cisplatin significantly enhanced apoptosis especially in HPV+ cell lines. While irradiation alone increased the amount of HPV E6 and E7 proteins, both were down-regulated by Cisplatin incubation either alone or in combination with x-rays, which however did not increase the expression of endogenous p53. Our results demonstrate that cell cycle deregulation together with downregulation of HPV E6 and E7 proteins facilitating apoptosis after Cisplatin incubation promote the enhanced sensitivity of HPV+ HNSCC cells to simultaneous radio-chemotherapy. Combined effects of irradiation and Cisplatin appear to be relevant in mediating the enhanced therapeutic response of HPV-related HNSCC and are indicative of the benefit of combined modality approaches in future treatment optimization strategies.
... CCNE2 (cyclin E2), which is encoded by the CCNE2 gene in humans, plays a critical role in the G1/S portion of cell cycle [28]. CCNE2 increased proportion of abnormal mitoses, micronuclei and chromosomal aberrations in cancer setting [29]. In the present study, the potential NPCrelated genes including MCM4 and CCNE2 were enriched in cell cycle and p53 signaling pathway which are both associated with the pathogenesis of NPC [30][31][32][33]. ...
To reveal the potential microRNAs (miRNAs), genes, pathways and regulatory network involved in the process of nasopharyngeal carcinoma (NPC) by using the method of bioinformatics.
Gene expression profiles GSE12452 (31 NPC and 10 normal samples) and GSE53819 (18 NPC and 18 normal samples), as well as miRNA expression profiles GSE32960 (312 NPC and 18 normal samples) and GSE36682 (62 NPC and 6 normal samples) were obtained from Gene Expression Omnibus database. The differentially expressed genes (DEGs) and miRNAs (DEmiRNAs) between NPC and normal samples were identified by using t-test based on MATLAB software (FDR < 0.01), followed by pathway enrichment analysis based on DAVID software (P-value < 0.1). Then, DEmiRNA-DEG regulatory network was constructed.
A total of 1254 DEGs and 107 DEmiRNAs were identified, respectively. Then, 16 pathways (including cell cycle) and 32 pathways (including pathways in cancer) were enriched by DEGs and target genes of DEmiRNAs, respectively. Furthermore, DEmiRNA-DEG regulatory network was constructed, containing 12 DEmiRNAs (including has-miR-615-3P) and 180 DEGs (including MCM4 and CCNE2).
has-miR-615-3p might take part in the pathogenetic process of NPC through regulating MCM4 which is enriched in cell cycle. The DEmiRNAs identified in the present study might serve as new biomarkers for NPC.
... It is noteworthy that overexpression of CCNE2 has been reported in mammary epithelial cells treated in vitro with zidovudine [21]. CCNE2 is known to be up-regulated in many tumors [22][23][24], which may contribute to chromosome instability and even tumorigenesis. SMC1A was down-regulated in CD3 þ T cells in the zidovudine-exposed group. ...
OBJECTIVES: Zidovudine and tenofovir are the two main nucleos(t)ide analogs used to prevent mother-to-child transmission of HIV. In vitro, both drugs bind to and integrate into human DNA and inhibit telomerase. The objective of the present study was to assess the genotoxic effects of either zidovudine or tenofovir-based combination therapies on cord blood cells in newborns exposed in utero.
DESIGN: We compared the aneuploid rate and the gene expression profiles in cord blood samples from newborns exposed either to zidovudine or tenofovir-based combination therapies during pregnancy and from unexposed controls (n = 8, 9, and 8, respectively).
METHODS: The aneuploidy rate was measured on the cord blood T-cell karyotype. Gene expression profiles of cord blood T cells and hematopoietic stem and progenitor cells were determined with microarrays, analyzed in a gene set enrichment analysis and confirmed by real-time quantitative PCRs.
RESULTS: Aneuploidy was more frequent in the zidovudine-exposed group (26.3%) than in the tenofovir-exposed group (14.2%) or in controls (13.3%; P < 0.05 for both). The transcription of genes involved in DNA repair, telomere maintenance, nucleotide metabolism, DNA/RNA synthesis, and the cell cycle was deregulated in samples from both the zidovudine and the tenofovir-exposed groups.
CONCLUSION: Although tenofovir has a lower clastogenic impact than zidovudine, gene expression profiling showed that both drugs alter the transcription of DNA repair and telomere maintenance genes.This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License, where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially. http://creativecommons.org/licenses/by-nc-nd/4.0.
... It is noteworthy that overexpression of CCNE2 has been reported in mammary epithelial cells treated in vitro with zidovudine [21]. CCNE2 is known to be up-regulated in many tumors [22][23][24], which may contribute to chromosome instability and even tumorigenesis. SMC1A was down-regulated in CD3 þ T cells in the zidovudine-exposed group. ...
Opioid use may affect HIV infection through altered expression of HIV co-receptors. This was examined in Indonesia among antiretroviral therapy (ART)-naive HIV patients, many of whom use drugs. CCR5 expression on CD4 cells was higher in heroin (P = 0.007), methadone (P = 0.024) and former opioid users (P = 0.003) compared to nonusers, whereas production of RANTES and other CCR5 ligands was similar or lower. This suggests that opioids can affect HIV susceptibility through up-regulation of CCR5 or down-regulation of its ligands.
... Nevertheless, Keyomarsi group have recently demonstrated that full length cyclin E1 protein, but not cyclin E2, can be cleaved [26] and that the low molecular weight form of cyclin E1 can play an important role in tumorigenesis. In addition, Musgrove group have lately reported that cyclin E2 influences genomic instability via mechanisms that are distinct from cyclin E1 [27]. In addition, the present work demonstrated that stimulation of hERG1 channel leads to activation of a degradation pathway for cyclin E2 that is distinct from cyclin E1. ...
Cyclin E2 gene amplification, but not cyclin E1, has been recently defined as marker for poor prognosis in breast cancer, and appears to play a major role in proliferation and therapeutic resistance in several breast cancer cells. Our laboratory has previously reported that stimulation of the hERG1 potassium channel with selective activators led to down-regulation of cyclin E2 in breast cancer cells. In this work, we demonstrate that stimulation of hERG1 promotes an ubiquitin-proteasome-dependent degradation of cyclin E2 in multiple breast cancer cell lines representing Luminal A, HER2+ and Trastuzumab-resistant breast cancer cells. In addition we have also reveal that hERG1 stimulation induces an increase in intracellular calcium that is required for cyclin E2 degradation. This novel function for hERG1 activity was specific for cyclin E2, as cyclins A, B, D E1 were unaltered by the treatment.
Our results reveal a novel mechanism by which hERG1 activation impacts the tumor marker cyclin E2 that is independent of cyclin E1, and suggest a potential therapeutic use for hERG1 channel activators.
... These functions of cyclin E are essential when nondividing cells exit the quiescent state and resume cell proliferation but may be redundant with the activities of other cell cycle regulators in continuously proliferating cells [28]. It was reported that overexpression of cyclin E2, a subtype of E-cyclins, also induced genomic instability by a typical mechanism [29]. XIAP ΔRING expression and its translocation into nucleus resulted in its binding and interaction with E2F1 protein, by which induced in E2F transactivation and subsequently led to an E2Fdependent cyclin E overexpression. ...
The inhibitor of apoptosis protein XIAP (X-linked inhibitor of apoptosis protein) is a well-documented protein that is located in cytoplasm acting as a potent regulator of cell apoptosis. Here, we showed that expressing XIAP with RING (Really Interesting New Gene) domain deletion (XIAP△RING) in cancer cells promoted cancer cell anchorage-independent growth and G1/S phase transition companied with increasing cyclin e transcription activity and protein expression. Further studies revealed that XIAP△RING was mainly localized in nuclear with increased binding with E2F1, whereas XIAP with BIR (Baculoviral IAP Repeat) domains deletion (XIAP△BIRs) was entirely presented in cytoplasma with losing its binding with E2F1, suggesting that RING domain was able to inhibit BIR domains nuclear localization, by which impaired BIRs binding with E2F1 in cellular nucleus in intact cells. These studies identified a new function of XIAP protein in cellular nucleus is to regulate E2F1 transcriptional activity by binding with E2F1 in cancer cells. Our current finding of an effect of XIAP△RING expression on cancer cell anchorage-independent growth suggests that overexpression of this protein may contribute to genetic instability associated with cell cycle and checkpoint perturbations, in addition to its impact on cellular apoptosis.
... For example, p53 can negatively regulate the activity of cyclin D. 36 CCND3, a cyclin D gene, and CCNE2, a cyclin E gene, activate cyclin-dependent kinases. 44,45 MEGF4 is involved in the induction of cell proliferation. 46 CREB regulates the cell cycle arrest in cancer cells. ...
Graphene oxide (GO) can be potentially used in biomedical and nonbiomedical products. The in vivo studies have demonstrated that GO is predominantly deposited in the lung. In the present study, we employed SOLiD sequencing technique to investigate the molecular control of in vitro GO toxicity in GLC-82 pulmonary adenocarcinoma cells by microRNAs (miRNAs), a large class of short noncoding RNAs acting to post-transcriptionally inhibit gene expression. In GLC-82 cells, GO exposure at concentrations more than 50 mg/L resulted in severe reduction in cell viability, induction of lactate dehydrogenase (LDH) leakage, reactive oxygen species (ROS) production and apoptosis, and dysregulation of cell cycle. GO was localized in cytosol, mitochondria, endoplasmic reticulum, and nucleus of cells. Based on SOLiD sequencing, we identified 628 up-regulated and 25 down-regulated miRNAs in GO exposed GLC-82 cells. Expression of some selected dysregulated miRNAs was concentration-dependent in GO exposed GLC-82 cells. The dysregulated miRNAs and their predicted targeted genes were involved in many biological processes. By combining both information on targeted genes for dysregulated miRNAs and known signaling pathways for apoptosis control, we hypothesize that the dysregulated miRNAs could activate both death receptor pathway by influencing functions of tumor necrosis factor α (TNFα) receptor and Caspase-3 and mitochondrial pathway by affecting functions of p53 and Bcl-2 in GO exposed GLC-82 cells. Our results provide an important molecular basis at the miRNAs level for explaining in vitro GO toxicity. Our data will be also useful for developing new strategies to reduce GO toxicity such as surface chemical modification.
Chromosomal instability (CIN) is a hallmark of cancer and comprises structural CIN (S-CIN) and numerical or whole chromosomal CIN (W-CIN). Recent work indicated that replication stress (RS), known to contribute to S-CIN, also affects mitotic chromosome segregation, possibly explaining the common co-existence of S-CIN and W-CIN in human cancer. Here, we show that RS-induced increased origin firing is sufficient to trigger W-CIN in human cancer cells. We discovered that overexpression of origin firing genes, including GINS1 and CDC45, correlates with W-CIN in human cancer specimens and causes W-CIN in otherwise chromosomally stable human cells. Furthermore, modulation of the ATR-CDK1-RIF1 axis increases the number of firing origins and leads to W-CIN. Importantly, chromosome missegregation upon additional origin firing is mediated by increased mitotic microtubule growth rates, a mitotic defect prevalent in chromosomally unstable cancer cells. Thus, our study identifies increased replication origin firing as a cancer-relevant trigger for chromosomal instability.
Basal‐like breast cancer (BLBC) has a greater overlap in molecular features with high‐grade serous ovarian cancer (HGSOC) than with other breast cancer subtypes. Similarities include BRCA1 mutation, high frequency of TP53 mutation, and amplification of CCNE1 (encoding the cyclin E1 protein) in 6–34% of cases, and these features can be used to group patients for targeted therapies in clinical trials. In HGSOC, we previously reported two subsets with high levels of cyclin E1: those in which CCNE1 is amplified, have intact homologous recombination (HR), and very poor prognosis; and a CCNE1 non‐amplified subset, with more prevalent HR defects. Here, we investigate whether similar subsets are identifiable in BLBC that may allow alignment of patient grouping in clinical trials of agents targeting cyclin E1 overexpression. We examined cyclin E1 protein and CCNE1 amplification in a cohort of 76 BLBCs and validated the findings in additional breast cancer datasets. Compared to HGSOC, CCNE1 amplified BLBC had a lower level of amplification (3.5 versus 5.2 copies) and lower relative cyclin E1 protein, a lack of correlation of amplification with expression, and no association with polyploidy. BLBC with elevated cyclin E1 protein also had prevalent HR defects, and high‐level expression of the cyclin E1 deubiquitinase ubiquitin‐specific protease 28 (USP28). Using a meta‐analysis across multiple studies, we determined that cyclin E1 protein overexpression but not amplification is prognostic in BLBC, while both cyclin E1 overexpression and amplification are prognostic in HGSOC. Overall CCNE1 gene amplification is not equivalent between BLBC and HGSOC. However, high cyclin E1 protein expression can co‐occur with HR defects in both BLBC and HGSOC, and is associated with poor prognosis in BLBC.
Chromosomal instability (CIN) is a hallmark of cancer and comprises structural CIN (S-CIN) and whole chromosome instability (W-CIN). Replication stress (RS), a condition of slowed or stalled DNA replication during S phase, has been linked to S-CIN, whereas defects in mitosis leading to chromosome missegregation and aneuploidy can account for W-CIN. It is well established that RS can activate additional replication origin firing that is considered as a rescue mechanism to suppress chromosomal instability in the presence of RS. In contrast, we show here that an increase in replication origin firing during S phase can contribute to W-CIN in human cancer cells. Increased origin firing can be specifically triggered by overexpression of origin firing genes including GINS1 and CDC45, whose elevated expression significantly correlates with W-CIN in human cancer specimens. Moreover, endogenous mild RS present in cancer cells characterized by W-CIN or modulation of the origin firing regulating ATR-CDK1-RIF1 axis induces dormant origin firing, which is sufficient to trigger chromosome missegregation and W-CIN. Importantly, chromosome missegregation upon increased dormant origin firing is mediated by increased microtubule growth rates leading to the generation of lagging chromosomes in mitosis, a condition prevalent in chromosomally unstable cancer cells. Thus, our study identified increased or dormant replication origin firing as a hitherto unrecognized, but cancer-relevant trigger for chromosomal instability.
Estrogens, via induction of their specific receptors (e.g., ER-α), regulate cell proliferation, differentiation and morphogenesis in mammary epithelium. Cell cycle progression is driven by activation of complexes consisting of cyclin-dependent kinases (CDKs) and cyclins, which also modulate the activity of ER-α. Loss of control over the cell cycle results in accelerated cell division and malignant transformation. Thus, a reciprocal relation exists between estrogen signaling and cell proliferation. Based on these findings, a new concept was developed to reduce ER-α activity and bring the cell cycle in transformed cells to heel. Prevention of ER-α activation and control over the deregulated cell cycle was achieved by supplementation with pharmacological CDK inhibitors alone or in combination with selective antiestrogens.
Increasing evidence for the role of histone modifying enzymes on non-histone substrates, including those in the cytoplasm, has been emerging in recent years. Some chromatin modifying proteins, such as the histone methyl transferase EZH2, compartmentalize to both the nucleus and cytoplasm; whereby EZH2 can methylate histones and actin, respectively.1 Alternatively, the mammalian nuclear membrane, during mitosis, disintegrates and the structural boundary separating nuclear and cytoplasmic proteins breaks down. This allows nuclear proteins to diffuse into the cytoplasm and potentially catalyze non-nuclear substrates. Evidence suggests that the Gcn5 containing histone acetyl transferase complex ATAC controls mitotic progression through the modification on non-histone targets during mitosis.2 And more recently, studies have shown that the HIPK2 kinase controls cytokinesis through the phosphorylation of histone H2B localized at the midbody, the site of cell abscission at the end of mitosis.3 What these and other studies suggest is that coordination and crosstalk between the chromatin and cytoskeletal structures is much more intertwined than previously appreciated.
Comment on: Palii SS, et al. Cell Cycle 2013; 12: In this issue.
When the cell cycle is arrested, even though growth-promoting pathways such as mTOR are still active, then cells senesce. For example, induction of either p21 or p16 arrests the cell cycle without inhibiting mTOR, which, in turn, converts p21/p16-induced arrest into senescence (geroconversion). Here we show that geroconversion is accompanied by dramatic accumulation of cyclin D1 followed by cyclin E and replicative stress. When p21 was switched off, senescent cells (despite their loss of proliferative potential) progressed through S phase, and levels of cyclins D1 and E dropped. Most cells entered mitosis and then died, either during mitotic arrest or after mitotic slippage, or underwent endoreduplication. Next, we investigated whether inhibition of mTOR would prevent accumulation of cyclins and loss of mitotic competence in p21-arrested cells. Both nutlin-3, which inhibits mTOR in these cells, and rapamycin suppressed geroconversion during p21-induced arrest, decelerated accumulation of cyclins D1 and E and decreased replicative stress. When p21 was switched off, cells successfully progressed through both S phase and mitosis. Also, senescent mouse embryonic fibroblasts (MEFs) overexpressed cyclin D1. After release from cell cycle arrest, senescent MEFs entered S phase but could not undergo mitosis and did not proliferate. We conclude that cellular senescence is characterized by futile hyper-mitogenic drive associated with mTOR-dependent mitotic incompetence.
The ubiquitin–proteasome pathway plays an important role in control of the abundance of cell cycle regulators. Mice lacking Skp2, an F-box protein and substrate recognition component of an Skp1–Cullin–F-box protein (SCF) ubiquitin ligase, were generated. Although Skp2-/- animals are viable, cells in the mutant mice contain markedly enlarged nuclei with polyploidy and multiple centrosomes, and show a reduced growth rate and increased apoptosis. Skp2-/- cells also exhibit increased accumulation of both cyclin E and p27Kip1. The elimination of cyclin E during S and G2 phases is impaired in Skp2-/- cells, resulting in loss of cyclin E periodicity. Biochemical studies showed that Skp2 interacts specifically with cyclin E and thereby promotes its ubiquitylation and degradation both in vivo and in vitro. These results suggest that specific degradation of cyclin E and p27Kip1 is mediated by the SCFSkp2 ubiquitin ligase complex, and that Skp2 may control chromosome replication and centrosome duplication by determining the abundance of cell cycle regulators.
FGF signaling inhibits chondrocyte proliferation and requires the function of the p107 and p130 members of the Rb protein family to execute growth arrest. p107 dephosphorylation plays a critical role in the chondrocyte response to FGF, as overexpression of cyclin D1/CDK4 complexes (the major p107 kinase) in rat chondrosarcoma (RCS) cells overcomes FGF-induced p107 dephosphorylation and growth arrest. In cells overexpressing cyclin D1/CDK4, FGF-induced downregulation of cyclin E/CDK2 activity was absent. To examine the role of cyclin E/CDK2 complexes in mediating FGF-induced growth arrest, this kinase was overexpressed in RCS cells. FGF-induced dephosphorylation of either p107 or p130 was not prevented by overexpressing cyclin E/CDK2 complexes. Unexpectedly, however, FGF-treated cells exhibited sustained proliferation even in the presence of hypophosphorylated p107 and p130. Both pocket proteins were able to form repressive complexes with E2F4 and E2F5 but these repressors were not translocated into the nucleus and therefore were unable to occupy their respective target DNA sites. Overexpressed cyclin E/CDK2 molecules were stably associated with p107 and p130 in FGF-treated cells in the context of E2F repressive complexes. Taken together, our data suggest a novel mechanism by which cyclin E/CDK2 complexes can promote cell cycle progression in the presence of dephosphorylated Rb proteins and provide a novel insight into the key Retinoblastoma/E2F/cyclin E pathway. Our data also highlight the importance of E2F4/p130 complexes for FGF-mediated growth arrest in chondrocytes.
Recent identification of the modular CLS motifs responsible for cyclins A and E localization on centrosomes has revealed a tight linkage between the nuclear and centrosomal cycles. These G1/S cyclins must localize on the centrosome in order for DNA replication to occur in the nucleus, whereas essential DNA replication factors also function on the centrosome to prevent centrosome overduplication. Both events are dependent on the presence of an intact CLS within each cyclin. Here we compare the cyclins A and E CLSs at the structural and functional levels and identify a new cyclin A CLS mutant that disrupts all CLS functions and reduces the affinity of cyclin A for Cdk2. Analysis of interactions of the CLS motif within the cyclin molecules highlights the importance of the cyclin CBOX1 region for Cdk2 binding.
DNA interstrand crosslinks (ICLs) are highly toxic because they block the progression of replisomes. The Fanconi Anemia (FA) proteins, encoded by genes that are mutated in FA, are important for repair of ICLs. The FA core complex catalyzes the monoubiquitination of FANCD2, and this event is essential for several steps of ICL repair. However, how monoubiquitination of FANCD2 promotes ICL repair at the molecular level is unknown. Here, we describe a highly conserved protein, KIAA1018/MTMR15/FAN1, that interacts with, and is recruited to sites of DNA damage by, the monoubiquitinated form of FANCD2. FAN1 exhibits endonuclease activity toward 5' flaps and has 5' exonuclease activity, and these activities are mediated by an ancient VRR_nuc domain. Depletion of FAN1 from human cells causes hypersensitivity to ICLs, defects in ICL repair, and genome instability. These data at least partly explain how ubiquitination of FANCD2 promotes DNA repair.
Condensation and segregation of mitotic chromosomes is a critical process for cellular propagation, and, in mammals, mitotic errors can contribute to the pathogenesis of cancer. In this report, we demonstrate that the retinoblastoma protein (pRB), a well-known regulator of progression through the G1 phase of the cell cycle, plays a critical role in mitotic chromosome condensation that is independent of G1-to-S-phase regulation. Using gene targeted mutant mice, we studied this aspect of pRB function in isolation, and demonstrate that it is an essential part of pRB-mediated tumor suppression. Cancer-prone Trp53(-/-) mice succumb to more aggressive forms of cancer when pRB's ability to condense chromosomes is compromised. Furthermore, we demonstrate that defective mitotic chromosome structure caused by mutant pRB accelerates loss of heterozygosity, leading to earlier tumor formation in Trp53(+/-) mice. These data reveal a new mechanism of tumor suppression, facilitated by pRB, in which genome stability is maintained by proper condensation of mitotic chromosomes.
Loss of G1/S control is a hallmark of cancer, and is often caused by inactivation of the retinoblastoma pathway. However, mouse embryonic fibroblasts lacking the retinoblastoma genes RB1, p107, and p130 (TKO MEFs) are still subject to cell cycle control: Upon mitogen deprivation, they enter and complete S phase, but then firmly arrest in G2. We now show that G2-arrested TKO MEFs have accumulated DNA damage. Upon mitogen readdition, cells resume proliferation, although only part of the damage is repaired. As a result, mitotic cells show chromatid breaks and chromatid cohesion defects. These aberrations lead to aneuploidy in the descendent cell population. Thus, our results demonstrate that unfavorable growth conditions can cause genomic instability in cells lacking G1/S control. This mechanism may allow premalignant tumor cells to acquire additional genetic alterations that promote tumorigenesis.
Overexpression of the low molecular weight isoforms (LMW-E) of cyclin E induces chromosome instability; however, the degree to which these tumor-specific forms cause genomic instability differs from that of full-length cyclin E (EL), and the underlying mechanism(s) has yet to be elucidated. Here, we show that EL and LMW-E overexpression impairs the G(2)-M transition differently and leads to different degrees of chromosome instability in a breast cancer model system. First, the most significant difference is that EL overexpression prolongs cell cycle arrest in prometaphase, whereas LMW-E overexpression reduces the length of mitosis and accelerates mitotic exit. Second, LMW-E-overexpressing cells are binucleated or multinucleated with amplified centrosomes, whereas EL-overexpressing cells have the normal complement of centrosomes. Third, LMW-E overexpression causes mitotic defects, chromosome missegregation during metaphase, and anaphase bridges during anaphase, most of which are not detected on EL induction. LMW-E induces additional mitotic defects in cooperation with p53 loss in both normal and tumor cells. Fourth, LMW-E-overexpressing cells fail to arrest in the presence of nocodazole. Collectively, the mitotic defects mediated by LMW-E induction led to failed cytokinesis and polyploidy, suggesting that LMW-E expression primes cells to accrue chromosomal instability by shortening the length of mitosis. Lastly, LMW-E expression in human breast cancer tissues correlates with centrosome amplification and higher nuclear grade. These results suggest that LMW-E overexpression leads to higher centrosome numbers in breast cancer, which is a prerequisite for genomic instability.
The highly conserved E-type cyclins are core components of the cell cycle machinery, facilitating the transition into S phase through activation of the cyclin dependent kinases, and assembly of pre-replication complexes on DNA. Cyclin E1 and cyclin E2 are assumed to be functionally redundant, as cyclin E1-/- E2-/- mice are embryonic lethal while cyclin E1-/- and E2-/- single knockout mice have primarily normal phenotypes. However more detailed studies of the functions and regulation of the E-cyclins have unveiled potential additional roles for these proteins, such as in endoreplication and meiosis, which are more closely associated with either cyclin E1 or cyclin E2. Moreover, expression of each E-cyclin can be independently regulated by distinct transcription factors and microRNAs, allowing for context-specific expression. Furthermore, cyclins E1 and E2 are frequently expressed independently of one another in human cancer, with unique associations to signatures of poor prognosis. These data imply an absence of co-regulation of cyclins E1 and E2 during tumorigenesis and possibly different contributions to cancer progression. This is supported by in vitro data identifying divergent regulation of the two genes, as well as potentially different roles in vivo.
Metazoan replication-dependent histone mRNAs terminate in a conserved stem-loop structure rather than a polyA tail. Formation of this unique mRNA 3' end requires Stem-loop Binding Protein (SLBP), which directly binds histone pre-mRNA and stimulates 3' end processing. The 3' end stem-loop is necessary for all aspects of histone mRNA metabolism, including replication coupling, but its importance to organism fitness and genome maintenance in vivo have not been characterized.
In Drosophila, disruption of the Slbp gene prevents normal histone pre-mRNA processing and causes histone pre-mRNAs to utilize the canonical 3' end processing pathway, resulting in polyadenylated histone mRNAs that are no longer properly regulated. Here we show that Slbp mutants display genomic instability, including loss of heterozygosity (LOH), increased presence of chromosome breaks, tetraploidy, and changes in position effect variegation (PEV). During imaginal disc growth, Slbp mutant cells show defects in S phase and proliferate more slowly than control cells.
These data are consistent with a model in which changing the 3' end of histone mRNA disrupts normal replication-coupled histone mRNA biosynthesis and alters chromatin assembly, resulting in genomic instability, inhibition of cell proliferation, and impaired development.
Protein phosphorylation regulates a multitude of biological processes. However, the large number of protein kinases and their substrates generates an enormously complex phosphoproteome. The cyclin-dependent kinases--the CDKs--comprise a class of enzymes that regulate cell cycle progression and play important roles in tumorigenesis. However, despite intense study, only a limited number of mammalian CDK substrates are known. A comprehensive understanding of CDK function requires the identification of their substrate network.
We describe a simple and efficient approach to identify potential cyclin A-CDK2 targets in complex cell lysates. Using a kinase engineering strategy combined with chemical enrichment and mass spectrometry, we identified 180 potential cyclin A-CDK2 substrates and more than 200 phosphorylation sites. About 10% of these candidates function within pathways related to cell division, and the vast majority are involved in other fundamental cellular processes. We have validated several candidates as direct cyclin A-CDK2 substrates that are phosphorylated on the same sites that we identified by mass spectrometry, and we also found that one novel substrate, the ribosomal protein RL12, exhibits site-specific CDK2-dependent phosphorylation in vivo.
We used methods entailing engineered kinases and thiophosphate enrichment to identify a large number of candidate CDK2 substrates in cell lysates. These results are consistent with other recent proteomic studies, and suggest that CDKs regulate cell division via large networks of cellular substrates. These methods are general and can be easily adapted to identify direct substrates of many other protein kinases.
Eukaryotic cells become committed to proliferate during the G1 phase of the cell cycle. In budding yeast, commitment occurs when the catalytic subunit of a protein kinase, encoded by the CDC28 gene (the homolog of the fission yeast cdc2+ gene), binds to a positively acting regulatory subunit, a cyclin. Related kinases are also required for progression through the G1 phase in higher eukaryotes. The role of cyclins in controlling G1 progression in mammalian cells was tested by construction of fibroblasts that constitutively overexpress human cyclin E. This was found to shorten the duration of G1, decrease cell size, and diminish the serum requirement for the transition from G1 to S phase. These observations show that cyclin levels can be rate-limiting for G1 progression in mammalian cells and suggest that cyclin synthesis may be the target of physiological signals that control cell proliferation.
p107 is a retinoblastoma protein-related phosphoprotein that, when overproduced, displays a growth inhibitory function. It interacts with and modulates the activity of the transcription factor, E2F-4. In addition, p107 physically associates with cyclin E-CDK2 and cyclin A-CDK2 complexes in late G1 and at G1/S, respectively, an indication that cyclin-dependent kinase complexes may regulate, contribute to, and/or benefit from p107 function during the cell cycle. Our results show that p107 phosphorylation begins in mid G1 and proceeds through late G1 and S and that cyclin D-associated kinase(s) contributes to this process. In addition, E2F-4 binds selectively to hypophosphorylated p107, and G1 cyclin-dependent p107 phosphorylation leads to the dissociation of p107-E2F-4 complexes as well as inactivation of p107 G1 blocking function.
G1 cyclin E controls the initiation of DNA synthesis by activating CDK2, and abnormally high levels of cyclin E expression have frequently been observed in human cancers. We have isolated a novel human cyclin, cyclin E2, that contains significant homology to cyclin E. Cyclin E2 specifically interacts with CDK inhibitors of the CIP/KIP family and activates both CDK2 and CDK3. The expression of cyclin E2 mRNA oscillates periodically throughout the cell cycle, peaking at the G1/S transition, and exhibits a pattern of tissue specificity distinct from that of cyclin E1. Cyclin E2 encodes a short lived protein whose turnover is most likely governed by the proteasome pathway and is regulated by phosphorylation on a conserved Thr-392 residue. Expression of the viral E6 oncoprotein in normal human fibroblasts increases the steady state level of cyclin E2, but not cyclin E1, while expression of the E7 oncoprotein upregulates both. These data suggest that the expression of these two G1 E-type cyclins may be similarly regulated by the pRb function, but distinctly by the p53 activity.
We report here the cloning and characterization of human and mouse cyclin E2, which define a new subfamily within the vertebrate E-type cyclins, while all previously identified family-members belong to the cyclin El subfamily. Cyclin E2/CKD2 and cyclin E/CDK2 complexes phosphorylate histone H1 in vitro with similar specific activities and both are inhibited by p27Kip1. Cyclin E2 mRNA levels in human cells oscillate throughout the cell cycle and peak at the G1/S boundary, in parallel with the cyclin E mRNA. In cells, cyclin E2 is complexed with CDK2, p27 and p21. Like cyclin E, cyclin E2 is an unstable protein in vivo and is stabilized by proteasome inhibitors. Cyclin E2-associated kinase activity rises in late G1 and peaks very close to cyclin E activity. In two malignantly transformed cell lines, cyclin E2 activity is sustained throughout S phase, while cyclin E activity has already declined and cyclin A activity is only beginning to rise. We speculate that cyclin E2 is not simply redundant with cyclin E, but may regulate distinct rate-limiting pathway(s) in G1-S control.
A novel cyclin gene was discovered by searching an expressed sequence tag database with a cyclin box profile. The human cyclin E2 gene encodes a 404-amino-acid protein that is most closely related to cyclin E. Cyclin E2 associates with Cdk2 in a functional kinase complex that is inhibited by both p27
Kip1
and p21
Cip1
. The catalytic activity associated with cyclin E2 complexes is cell cycle regulated and peaks at the G
1
/S transition. Overexpression of cyclin E2 in mammalian cells accelerates G
1
, demonstrating that cyclin E2 may be rate limiting for G
1
progression. Unlike cyclin E1, which is expressed in most proliferating normal and tumor cells, cyclin E2 levels were low to undetectable in nontransformed cells and increased significantly in tumor-derived cells. The discovery of a novel second cyclin E family member suggests that multiple unique cyclin E-CDK complexes regulate cell cycle progression.
Phosphorylation of histone H3 at serine 10 occurs during mitosis in diverse eukaryotes and correlates closely with mitotic and meiotic chromosome condensation. To better understand the function of H3 phosphorylation in vivo, we created strains of Tetrahymena in which a mutant H3 gene (S10A) was the only gene encoding the major H3 protein. Although both micronuclei and macronuclei contain H3 in typical nucleosomal structures, defects in nuclear divisions were restricted to mitotically dividing micronuclei; macronuclei, which are amitotic, showed no defects. Strains lacking phosphorylated H3 showed abnormal chromosome segregation, resulting in extensive chromosome loss during mitosis. During meiosis, micronuclei underwent abnormal chromosome condensation and failed to faithfully transmit chromosomes. These results demonstrate that H3 serine 10 phosphorylation is causally linked to chromosome condensation and segregation in vivo and is required for proper chromosome dynamics.
Cyclin E/Cdk2 acts at the G1/S-phase transition to promote the E2F transcriptional program and the initiation of DNA synthesis. To explore further how cyclin E/Cdk2 controls S-phase events, we examined the subcellular localization of the cyclin E/Cdk2 interacting protein p220(NPAT) and its regulation by phosphorylation. p220 is localized to discrete nuclear foci. Diploid fibroblasts in Go and G1 contain two p220 foci, whereas S- and G2-phase cells contain primarily four p220 foci. Cells in metaphase and telophase have no detectable focus. p220 foci contain cyclin E and are coincident with Cajal bodies (CBs), subnuclear organelles that associate with histone gene clusters on chromosomes 1 and 6. Interestingly, p220 foci associate with chromosome 6 throughout the cell cycle and with chromosome 1 during S phase. Five cyclin E/Cdk2 phosphorylation sites in p220 were identified. Phospho-specific antibodies against two of these sites react with p220 within CBs in a cell cycle-specific manner. The timing of p220 phosphorylation correlates with the appearance of cyclin E in CBs at the G1/S boundary, and this phosphorylation is maintained until prophase. Expression of p220 activates transcription of the histone H2B promoter. Importantly, mutation of Cdk2 phosphorylation sites to alanine abrogates the ability of p220 to activate the histone H2B promoter. Collectively, these results strongly suggest that p220(NPAT) links cyclical cyclin E/Cdk2 kinase activity to replication-dependent histone gene transcription.
Substrates of cyclin-cdk2 kinases contain two distinct primary sequence motifs: a cyclin-binding RXL motif and one or more
phosphoacceptor sites (consensus S/TPXK/R or S/TP). To identify novel cyclin-cdk2 substrates, we searched the database for
proteins containing both of these motifs. One such protein is human HIRA, the homologue of two cell cycle-regulated repressors
of histone gene expression in Saccharomyces cerevisiae, Hir1p and Hir2p. Here we demonstrate that human HIRA is an in vivo substrate of a cyclin-cdk2 kinase. First, HIRA bound
to and was phosphorylated by cyclin A- and E-cdk2 in vitro in an RXL-dependent manner. Second, HIRA was phosphorylated in
vivo on two consensus cyclin-cdk2 phosphoacceptor sites and at least one of these, threonine 555, was phosphorylated by cyclin
A-cdk2 in vitro. Third, phosphorylation of HIRA in vivo was blocked by cyclin-cdk2 inhibitor p21cip1. Fourth, HIRA became phosphorylated on threonine 555 in S phase when cyclin-cdk2 kinases are active. Fifth, HIRA was localized
preferentially to the nucleus, where active cyclin A- and E-cdk2 are located. Finally, ectopic expression of HIRA in cells
caused arrest in S phase and this is consistent with the notion that it is a cyclin-cdk2 substrate that has a role in control
of the cell cycle.
Cell cycle arrests in the G1, S, and G2phases occur in mammalian cells after ionizing irradiation and appear to protect cells from permanent genetic damage and transformation.
Though Brca1 clearly participates in cellular responses to ionizing radiation (IR), conflicting conclusions have been drawn
about whether Brca1 plays a direct role in cell cycle checkpoints. Normal Nbs1 function is required for the IR-induced S-phase
checkpoint, but whether Nbs1 has a definitive role in the G2/M checkpoint has not been established. Here we show that Atm and Brca1 are required for both the S-phase and G2 arrests induced by ionizing irradiation while Nbs1 is required only for the S-phase arrest. We also found that mutation of
serine 1423 in Brca1, a target for phosphorylation by Atm, abolished the ability of Brca1 to mediate the G2/M checkpoint but did not affect its S-phase function. These results clarify the checkpoint roles for each of these three
gene products, demonstrate that control of cell cycle arrests must now be included among the important functions of Brca1
in cellular responses to DNA damage, and suggest that Atm phosphorylation of Brca1 is required for the G2/M checkpoint.
Cyclin E is a G(1) 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. Here we report that the LMW forms of cyclin E are hyperactive biochemically and they can more readily induce G(1)-to-S progression in transfected normal cells than the full-length form of the protein can. Through biochemical and mutational analyses we have identified two proteolytically sensitive sites in the amino terminus of human cyclin E that are cleaved to generate the LMW isoforms found in tumor cells. Not only are the LMW forms of cyclin E functional, as they phosphorylate substrates such as histone H1 and GST-Rb, but also their activities are higher than the full-length cyclin E. These nuclear localized LMW forms of cyclin E are also biologically functional, as their overexpression in normal cells increases the ability of these cells to enter S and G(2)/M. Lastly, we show that cyclin E is selectively cleaved in vitro by the elastase class of serine proteases to generate LMW forms similar to those observed in tumor cells. These studies suggest that the defective entry into and exit from S phase by tumor cells is in part due to the proteolytic processing of cyclin E, which generates hyperactive LMW isoforms whose activities have been modified from that of the full-length protein.
The results presented describe the effects of various spectator ligands, attached to a platinum 1,2-intrastand d(GpG) cross-link
in duplex DNA, on the binding of high mobility group box (HMGB) domains and the TATA-binding protein (TBP). In addition to
cisplatin-modified DNA, 15-base pair DNA probes modified by {Pt(1R,2R-diaminocyclohexane)}2+,cis-{Pt(NH3)(cyclohexylamine)}2+, {Pt(ethylenediamine)}2+,cis-{Pt(NH3)(cyclobutylamine)}2+, and cis-{Pt(NH3)(2-picoline)}2+were examined. Electrophoretic mobility shift assays show that both the A and B domains of HMGB1 as well as TBP discriminate
between different platinum-DNA adducts. HMGB1 domain A is the most sensitive to the nature of the spectator ligands on platinum.
The effect of the spectator ligands on protein binding also depends highly on the base pairs flanking the platinated d(GpG)
site. Double-stranded oligonucleotides containing the AG*G*C sequence, where the asterisks denote the sites of platination,
with different spectator ligands are only moderately discriminated by the HMGB proteins and TBP, but the recognition of dsTG*G*A
is highly dependent on the ligands. The effects of HMGB1 overexpression in a BG-1 ovarian cancer cell line, induced by steroid
hormones, on the sensitivity of cells treated with [Pt(1R,2R-diaminocyclohexane)Cl2] andcis-[Pt(NH3)(cyclohexylamine)Cl2] were also examined. The results suggest that HMGB1 protein levels influence the cellular processing ofcis-{Pt(NH3)- (cyclohexylamine)}2+, but not {Pt((1R,2R)-diaminocyclohexane)}2+, DNA lesions. This result is consistent with the observed binding of HMGB1a to platinum-modified dsTG*G*A probes but not
with the binding affinity of HMGB1a and HMGB1 to platinum-damaged dsAG*G*C oligonucleotides. These experiments reinforce the
importance of sequence context in platinum-DNA lesion recognition by cellular proteins.
Long-term growth inhibition, arrest in G1 phase and reduced activity of both cyclin D1-Cdk4 and cyclin E-Cdk2 are elicited by progestin treatment of breast cancer
cells in culture. Decreased cyclin expression, induction of p18INK4c and increased association of the CDK inhibitors p21WAF1/Cip1 and p27Kip1 with cyclin E-Cdk2 have been implicated in these responses. To determine the role of decreased cyclin expression, T-47D human
breast cancer cells constitutively expressing cyclin D1 or cyclin E were treated with the progestin ORG 2058. Overexpression
of cyclin E had only a modest effect on growth inhibition. Although cyclin E expression was maintained during progestin treatment,
cyclin E-Cdk2 activity decreased by ∼60%. This was accompanied by p27Kip1 association with cyclin E-Cdk2, indicating that both cyclin E down-regulation and p27Kip1 recruitment contribute to the decrease in activity. In contrast, overexpression of cyclin D1 induced progestin resistance
and cell proliferation continued despite decreased cyclin E-Cdk2 activity. Progestin treatment of cyclin D1-overexpressing
cells was associated with increased p27Kip1 association with cyclin E-Cdk2. Thus the ability of cyclin D1 to confer progestin resistance does not depend on sequestration
of p27Kip1 away from cyclin E-Cdk2, providing evidence for a critical function of cyclin D1 other than as a high-capacity “sink” for
p27Kip1. These data indicate that regulation of cyclin D1 is a critical element of progestin inhibition in breast cancer cells and
suggest that breast cancers overexpressing cyclin D1 may respond poorly to progestin therapy.
Cyclin E1 (formerly called cyclin E) and the recently described cyclin E2 belong to the family of E-type cyclins that operate during the G(1)/S phase progression in mammalian cells. The two E-cyclins share a catalytic partner, cyclin-dependent kinase 2 (CDK2), and activate their associated kinase activities at similar times during cell cycle progression. Despite these similarities, it is unknown whether the two proteins perform distinct functions, or, alternatively, they control S-phase entry of different cell types in a tissue-specific fashion. To start addressing in vivo functions of E-cyclins, we determined the expression pattern of cyclins E1 and E2 during normal mouse development. We found that the two E-cyclins showed very similar patterns of expression; both were expressed within the proliferating compartment during embryo development. Analyses of cells and tissues lacking members of the retinoblastoma (pRB) family of proteins revealed that the expression of both cyclins is controlled in a pRB-dependent, but p107- and p130-independent fashion, likely through the pRB-dependent E2F transcription factors. We also found that cyclins E1 and E2 are expressed at high levels in mouse breast tumors driven by the Myc oncogene. Last, we found that cyclin E2 is overexpressed in approximately 24% of analyzed human mammary carcinomas. Collectively these findings suggest that the expression of cyclins E1 and E2 is governed by similar molecular circuitry.
Breast cancer patients with the same stage of disease can have markedly different treatment responses and overall outcome. The strongest predictors for metastases (for example, lymph node status and histological grade) fail to classify accurately breast tumours according to their clinical behaviour. Chemotherapy or hormonal therapy reduces the risk of distant metastases by approximately one-third; however, 70-80% of patients receiving this treatment would have survived without it. None of the signatures of breast cancer gene expression reported to date allow for patient-tailored therapy strategies. Here we used DNA microarray analysis on primary breast tumours of 117 young patients, and applied supervised classification to identify a gene expression signature strongly predictive of a short interval to distant metastases ('poor prognosis' signature) in patients without tumour cells in local lymph nodes at diagnosis (lymph node negative). In addition, we established a signature that identifies tumours of BRCA1 carriers. The poor prognosis signature consists of genes regulating cell cycle, invasion, metastasis and angiogenesis. This gene expression profile will outperform all currently used clinical parameters in predicting disease outcome. Our findings provide a strategy to select patients who would benefit from adjuvant therapy.
Estrogens stimulate proliferation of estrogen receptor positive MCF7 breast cancer cells while antiestrogens signal a G0/G1 growth arrest. In MCF7 cells, arrest is mediated through the CDK inhibitors p21 and p27 and through a decrease in cyclin E/CDK2 kinase activity. We found that in MCF7 cells, overexpression of cyclin E partially abrogates a tamoxifen mediated growth arrest. Overexpression of cyclin E is accompanied by a decrease in the levels of RB and CDK inhibitor p21 but an increase in CDK inhibitor p27. Cyclin E overexpression also alters the composition of E2F transcription factor complexes. The E2F4/p107/cyclin E/CDK2 complex, a minor component in proliferating control cells that is absent in growth-arrested cells, is more abundant in both proliferating and tamoxifen treated cyclin E overexpressing cells. Conversely, levels of the quiescence associated E2F/p130 complex is not detected in these cells. Expression from the E2F dependant promoter is elevated in proliferating and tamoxifen treated cyclin E overexpressing cells. This study suggests that a modest overexpression of cyclin E abrogates the tamoxifen mediated growth arrest through modification of the RB/E2F pathway. Moreover, these results provide one explanation of why some cells that express the estrogen receptor may be unresponsive to antiestrogens.
To study the regulation of cyclin-dependent kinase (CDK) activity during mitotic exit in mammalian cells, we constructed murine cell lines that constitutively express a stabilized mutant of cyclin A (cyclin A47). Even though cyclin A47 was expressed throughout mitosis and in G1 cells, its associated CDK activity was inactivated after the transition from metaphase to anaphase. Cyclin A47 associated with both p21 and p27 during mitotic exit, implicating these proteins in CDK inactivation. However, cyclin A47 was fully inhibited during the M-to-G1 transition in p21(-/-) p27(-/-) fibroblasts. Also, the CDKs associated with cyclin A47 were not inactivated by phosphorylation at tyrosines. The protein responsible for CDK inactivation during mitotic exit in p21/p27 null cells was the Rb family member, p107. p107 bound to cyclin A47 when p21 and p27 were absent, and cyclin A47-CDK activity was not inactivated during the M-to-G1 transition in p21(-/-) p27(-/-) p107(-/-) null fibroblasts. Enforced expression of cyclin A in cells lacking all three CDK inhibitors induced rapid tetraploidization, indicative of mitotic failure/endoreduplication. We concluded that cyclin proteolysis and CDK inhibitors constitute redundant pathways that control cyclin A-CDK activity during mitotic exit in mammalian cells and that loss of these pathways can cause genetic instability.
The deregulated expression of cyclin E as measured by the overexpression of its low molecular weight (LMW) isoforms is a powerful predictor of poor outcome in patients with breast cancer. The mechanism by which these LMW forms give tumor cells a growth advantage is not known and is the subject of this article. In this article, we provide the pathobiological mechanisms of how these LMW forms are involved in disease progression. Specifically, we show that overexpression of the LMW forms of cyclin E but not the full-length form in MCF-7 results in (a) their hyperactivity because of increased affinity for cdk2 and resistance to inhibition by the cyclin-dependent kinase inhibitors p21 and p27, (b) resistance to the growth inhibiting effects of antiestrogens, and (c) chromosomal instability. Lastly, tumors from breast cancer patients overexpressing the LMW forms of cyclin E are polyploid in nature and are resistant to endocrine therapy. Collectively, the biochemical and functional differences between the full-length and the LMW isoforms of cyclin E provide a molecular mechanism for the poor clinical outcome observed in breast cancer patients harboring tumors expressing high levels of the LMW forms of cyclin E. These properties of the LMW forms cyclin E suggest that they are not just surrogate markers of poor outcome but bona fide mediators of aggressive disease and potential therapeutic targets for patients whose tumors overexpress these forms.
Deregulation of cyclin E expression has been associated with a broad spectrum of human malignancies. Analysis of DNA replication in cells constitutively expressing cyclin E at levels similar to those observed in a subset of tumor-derived cell lines indicates that initiation of replication and possibly fork movement are severely impaired. Such cells show a specific defect in loading of initiator proteins Mcm4, Mcm7, and to a lesser degree, Mcm2 onto chromatin during telophase and early G1 when Mcm2-7 are normally recruited to license origins of replication. Because minichromosome maintenance complex proteins are thought to function as a heterohexamer, loading of Mcm2-, Mcm4-, and Mcm7-depleted complexes is likely to underlie the S phase defects observed in cyclin E-deregulated cells, consistent with a role for minichromosome maintenance complex proteins in initiation of replication and fork movement. Cyclin E-mediated impairment of DNA replication provides a potential mechanism for chromosome instability observed as a consequence of cyclin E deregulation.
Nucleostemin (NS) was identified as a stem cell- and cancer cell-enriched nucleolar protein that controls the proliferation of these cells. Here, we report the mechanism that regulates its dynamic shuttling between the nucleolus and nucleoplasm. The nucleolar residence of nucleostemin involves a transient and a long-term binding by the basic and GTP-binding domains, and a dissociation mechanism mediated by the COOH-terminal region. This cycle is propelled by the GTP binding state of nucleostemin. We propose that a rapid nucleostemin cycle is designed to translate extra- and intra-cellular signals into the amount of nucleostemin in the nucleolus in a bidirectional and fast manner.
Cdc45 is required for initiation of DNA replication and fork progression, but its function in these processes remains unknown. We show that targeting Cdc45 to specific chromosomal sites in mammalian cells results in large-scale chromatin decondensation that strongly correlates with histone H1 phosphorylation. Cdk2 is recruited to sites of Cdc45 decondensation, and Cdk2 inhibitors reduce the level of decondensation. Targeting wild-type Cdk2, but not kinase-defective Cdk2, to chromatin is also effective at inducing decondensation involving phospho-H1. Cdc45, Cdk2, Cyclin A, and phospho-H1 associate with chromatin during S-phase, and Cdc45, Cdk2, and an active H1 kinase physically interact. Replicating DNA and phospho-H1 foci colocalize in vivo, and S-phase progression and H1 phosphorylation are directly related and Cdk2 dependent. Because Cdk2 colocalizes with replication foci and H1 regulates higher-order chromatin, we suggest a model in which Cdc45 recruits Cdk2 to replication foci, resulting in H1 phosphorylation, chromatin decondensation, and facilitation of fork progression.
The deregulated expression of cyclin E as measured by the overexpression of its low molecular weight (LMW) isoforms is a powerful predictor of poor outcome in patients with breast cancer. The mechanism by which these LMW forms give tumor cells a growth advantage is not known and is the subject of this article. In this article, we provide the pathobiological mechanisms of how these LMW forms are involved in disease progression. Specifically, we show that overexpression of the LMW forms of cyclin E but not the full-length form in MCF-7 results in (a) their hyperactivity because of increased affinity for cdk2 and resistance to inhibition by the cyclin-dependent kinase inhibitors p21 and p27, (b) resistance to the growth inhibiting effects of antiestrogens, and (c) chromosomal instability. Lastly, tumors from breast cancer patients overexpressing the LMW forms of cyclin E are polyploid in nature and are resistant to endocrine therapy. Collectively, the biochemical and functional differences between the full-length and the LMW isoforms of cyclin E provide a molecular mechanism for the poor clinical outcome observed in breast cancer patients harboring tumors expressing high levels of the LMW forms of cyclin E. These properties of the LMW forms cyclin E suggest that they are not just surrogate markers of poor outcome but mediators of aggressive disease and potential therapeutic targets for patients whose tumors overexpress these forms.
Cyclin E2, but not cyclin E1, is included in several gene signatures that predict disease progression in either tamoxifen-resistant or metastatic breast cancer. We therefore examined the role of cyclin E2 in antiestrogen resistance in vitro and its potential for therapeutic targeting through cyclin-dependent kinase (CDK) inhibition. High expression of CCNE2, but not CCNE1, was characteristic of the luminal B and HER2 subtypes of breast cancer and was strongly predictive of shorter distant metastasis-free survival following endocrine therapy. After antiestrogen treatment of MCF-7 breast cancer cells, cyclin E2 mRNA and protein were downregulated and cyclin E2-CDK2 activity decreased. However, this regulation was lost in tamoxifen-resistant (MCF-7 TAMR) cells, which overexpressed cyclin E2. Expression of either cyclin E1 or E2 in T-47D breast cancer cells conferred acute antiestrogen resistance, suggesting that cyclin E overexpression contributes to the antiestrogen resistance of tamoxifen-resistant cells. Ectopic expression of cyclin E1 or E2 also reduced sensitivity to CDK4, but not CDK2, inhibition. Proliferation of tamoxifen-resistant cells was inhibited by RNAi-mediated knockdown of cyclin E1, cyclin E2, or CDK2. Furthermore, CDK2 inhibition of E-cyclin overexpressing cells and tamoxifen-resistant cells restored sensitivity to tamoxifen or CDK4 inhibition. Cyclin E2 overexpression is therefore a potential mechanism of resistance to both endocrine therapy and CDK4 inhibition. CDK2 inhibitors hold promise as a component of combination therapies in endocrine-resistant disease as they effectively inhibit cyclin E1 and E2 overexpressing cells and enhance the efficacy of other therapeutics.
Chromosomal instability in early cancer stages is caused by stress on DNA replication. The molecular basis for replication perturbation in this context is currently unknown. We studied the replication dynamics in cells in which a regulator of S phase entry and cell proliferation, the Rb-E2F pathway, is aberrantly activated. Aberrant activation of this pathway by HPV-16 E6/E7 or cyclin E oncogenes significantly decreased the cellular nucleotide levels in the newly transformed cells. Exogenously supplied nucleosides rescued the replication stress and DNA damage and dramatically decreased oncogene-induced transformation. Increased transcription of nucleotide biosynthesis genes, mediated by expressing the transcription factor c-myc, increased the nucleotide pool and also rescued the replication-induced DNA damage. Our results suggest a model for early oncogenesis in which uncoordinated activation of factors regulating cell proliferation leads to insufficient nucleotides that fail to support normal replication and genome stability.
Chromosome instability (CIN) is a common feature of tumor cells. By monitoring chromosome segregation, we show that depletion of the retinoblastoma protein (pRB) causes rates of missegregation comparable with those seen in CIN tumor cells. The retinoblastoma tumor suppressor is frequently inactivated in human cancers and is best known for its regulation of the G1/S-phase transition. Recent studies have shown that pRB inactivation also slows mitotic progression and promotes aneuploidy, but reasons for these phenotypes are not well understood. Here we describe the underlying mitotic defects of pRB-deficient cells that cause chromosome missegregation. Analysis of mitotic cells reveals that pRB depletion compromises centromeric localization of CAP-D3/condensin II and chromosome cohesion, leading to an increase in intercentromeric distance and deformation of centromeric structure. These defects promote merotelic attachment, resulting in failure of chromosome congression and an increased propensity for lagging chromosomes following mitotic delay. While complete loss of centromere function or chromosome cohesion would have catastrophic consequences, these more moderate defects allow pRB-deficient cells to proliferate but undermine the fidelity of mitosis, leading to whole-chromosome gains and losses. These observations explain an important consequence of RB1 inactivation, and suggest that subtle defects in centromere function are a frequent source of merotely and CIN in cancer.
During estrogen-induced proliferation, c-Myc and cyclin D1 initiate independent pathways that activate cyclin E1-Cdk2 by sequestration
and/or downregulation of the CDK inhibitor p21Waf1/Cip1, without significant increases in cyclin E1 protein levels. In contrast, cyclin E2 undergoes a marked increase in expression,
which occurs within 9 to 12 h of estrogen treatment of antiestrogen-pretreated MCF-7 breast cancer cells. Both E cyclins are
important to estrogen action, as small interfering RNA (siRNA)-mediated knockdown of either cyclin E1 or cyclin E2 attenuated
estrogen-mediated proliferation. Inducible expression of cyclin D1 upregulated cyclin E2, while siRNA-mediated knockdown of
cyclin D1 attenuated estrogen effects on cyclin E2. However, manipulation of c-Myc levels did not profoundly affect cyclin
E2. Cyclin E2 induction by estrogen was accompanied by recruitment of E2F1 to the cyclin E1 and E2 promoters, and cyclin D1 induction was sufficient for E2F1 recruitment. siRNA-mediated knockdown of the chromatin remodelling
factor CHD8 prevented cyclin E2 upregulation. Together, these data indicate that cyclin E2-Cdk2 activation by estrogen occurs
via E2F- and CHD8-mediated transcription of cyclin E2 downstream of cyclin D1. This contrasts with the predominant regulation
of cyclin E1-Cdk2 activity via CDK inhibitor association downstream of both c-Myc and cyclin D1 and indicates that cyclins
E1 and E2 are not always coordinately regulated.
E-cyclins control the transition of quiescent cells into the cell cycle. Two E-cyclins, CcnE1 and CcnE2, have been described, but their specific contributions to cell cycle reentry in vivo are poorly understood. Liver regeneration following partial hepatectomy is an excellent in vivo model for the study of cell cycle reentry of quiescent cells. We investigated the relevance of E-cyclins in directing resting hepatocytes into the cell cycle after partial hepatectomy using CcnE1 and CcnE2 knockout mice.
Partial hepatectomy (70%) was performed in CcnE1 (E1(-/-)) and CcnE2 (E2(-/-)) knockout and wild-type mice. Liver regeneration was monitored by cell cycle markers for G(1)/S phase, S phase, and M phase as well as by determining the liver/body weight ratio after partial hepatectomy. Ploidy of hepatocytes was determined by fluorescence-activated cell sorting and fluorescent in situ hybridization.
CcnE1 deletion resulted in normal liver regeneration with a slight delay of the G(1)/S-phase transition and a defect in endoreplication of otherwise polyploid hepatocytes. Surprisingly, E2(-/-) mice displayed accelerated and sustained DNA synthesis after partial hepatectomy, excessive endoreplication in hepatocytes, and a liver mass that was 45% greater than that of wild-type mice after termination of the regeneration process. CcnE2 depletion induced overexpression of CcnE1 and prolonged cdk2 kinase activity after partial hepatectomy.
CcnE2 has an unexpected role in repressing CcnE1; the phenotype of E2(-/-) mice appears to result from CcnE1 overexpression and cdk2 hyperactivation. CcnE1 and CcnE2 therefore have nonredundant functions for S-phase entry and endoreplication during liver regeneration.
E-type cyclins (E1 and E2) regulate the S phase program in the mammalian cell division cycle. Expression of cyclin E1 and E2 is frequently deregulated in a variety of cancer types and a wealth of experimental evidence supports an oncogenic role of these proteins in human tumorigenesis. Although the molecular mechanisms responsible for cyclin E1 deregulation in cancer are well defined, little is known regarding cyclin E2. Here we report that cyclin E2 is targeted for ubiquitin-dependent proteolysis by the ubiquitin ligase SCF(Fbxw7/hCdc4). Ubiquitylation is triggered by phosphorylation of cyclin E2 on residues Thr392 and Ser396, and to a lesser extent Thr74, contained in two consensus Cdc4-phosphodegrons. Furthermore, we found that ectopic expression of cyclin E1 enhances the ubiquitin-dependent proteolysis of cyclin E2 in vivo, suggesting a potential cross-talk in the regulation of E-type cyclin activity. Since SCF(Fbxw7/hCdc4) is functionally inactivated in several human cancer types, alteration of this molecular pathway could contribute to the deregulation of cyclin E2 in tumorigenesis.
Conditional overexpression of human cyclins B1, D1, and E was accomplished by using a synthetic cDNA expression system based on the Escherichia coli tetracycline repressor. After induction of these cyclins in asynchronous Rat-1 fibroblasts, a decrease in the length of the G1 interval was observed for cyclins D1 and E, consistent with an acceleration of the G1/S phase transition. We observed, in addition, a compensatory lengthening of S phase and G2 so that the mean cell cycle length in populations constitutively expressing these cyclins was unchanged relative to those of their uninduced counterparts. We found that expression of cyclin B1 had no effect on cell cycle dynamics, despite elevated levels of cyclin B-associated histone H1 kinase activity. Induction of cyclins D1 and E also accelerated entry into S phase for synchronized cultures emerging from quiescence. However, whereas cyclin E exerted a greater effect than cyclin D1 in asynchronous cycling cells, cyclin D1 conferred a greater effect upon stimulation from quiescence, suggesting a specific role for cyclin D1 in the G0-to-G1 transition. Overexpression of cyclins did not prevent cells from entering into quiescence upon serum starvation, although a slight delay in attainment of quiescence was observed for cells expressing either cyclin D1 or cyclin E. These results suggest that cyclins D1 and E are rate-limiting activators of the G1-to-S phase transition and that cyclin D1 might play a specialized role in facilitating emergence from quiescence.
Deregulated expression of several cell cycle regulatory genes has been demonstrated to be associated with cancer. In particular, a strong correlation has been established between inappropriate cyclin E expression and human breast cancer. To determine the ability of cyclin E to play a causative role in mammary tumorigenesis, regulatory sequences from the ovine beta-lactoglobulin gene were utilized to specifically target expression of human cyclin E to the mammary glands of pregnant and lactating mice. Lactating mammary glands of transgenic mice expressing cyclin E contained areas of hyperplasia, primarily papillary projections of hyperplastic cells, which were rarely observed in lactating glands of control mice. Over 10% of female cyclin E transgenic mice have developed mammary carcinomas, with latencies ranging from 8 to 13 months. Tumor analysis revealed the presence of transgene-specific cyclin E RNA and protein, as well as cyclin E- and cdk2-associated kinase activity, suggesting that cyclin E is likely a contributing component of tumorigenic progression in this model system.
Estrogen-induced progression through G
1
phase of the cell cycle is preceded by increased expression of the G
1
-phase regulatory proteins c-Myc and cyclin D1. To investigate the potential contribution of these proteins to estrogen action, we derived clonal MCF-7 breast cancer cell lines in which c-Myc or cyclin D1 was expressed under the control of the metal-inducible metallothionein promoter. Inducible expression of either c-Myc or cyclin D1 was sufficient for S-phase entry in cells previously arrested in G
1
phase by pretreatment with ICI 182780, a potent estrogen antagonist. c-Myc expression was not accompanied by increased cyclin D1 expression or Cdk4 activation, nor was cyclin D1 induction accompanied by increases in c-Myc. Expression of c-Myc or cyclin D1 was sufficient to activate cyclin E-Cdk2 by promoting the formation of high-molecular-weight complexes lacking the cyclin-dependent kinase inhibitor p21, as has been described, following estrogen treatment. Interestingly, this was accompanied by an association between active cyclin E-Cdk2 complexes and hyperphosphorylated p130, identifying a previously undefined role for p130 in estrogen action. These data provide evidence for distinct c-Myc and cyclin D1 pathways in estrogen-induced mitogenesis which converge on or prior to the formation of active cyclin E-Cdk2-p130 complexes and loss of inactive cyclin E-Cdk2-p21 complexes, indicating a physiologically relevant role for the cyclin E binding motifs shared by p130 and p21.
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.
Cyclin E/cyclin-dependent kinase 2 complexes are essential during the cell cycle for entrance into S phase. Cyclin E expression starts in mid-G1, reaches a maximum at S-phase entrance, and undergoes proteolysis mediated by the ubiquitin pathway as the cell progresses through S phase. Laser scanning cytometry, a microscope-based cytofluorometer combining the advantages of both flow and image analysis, allowed the determination of subcellular localization of cyclin E, p27, and retinoblastoma protein during cell cycle progression in normal human fibroblasts and nine bladder cancer cell lines. We observed that in normal fibroblasts and most tumor cell lines, cyclin E localizes in the nucleoplasm during mid-G1, and is translocated to the nucleolus during G1-S-phase transition, and its levels are undetectable in G2-M phase. Neither levels nor subcellular localization of p27 and retinoblastoma protein was cell cycle dependent in normal or tumor cells. However, four of nine bladder cancer cell lines continued to express cyclin E in all phases of the cycle, and image analysis revealed that it was localized to nucleoli. These observations suggest that the nucleolus mediates a cyclin E "shuttling" between the nucleus and the cytoplasm that is probably involved in its regulation and that this mechanism could be disrupted in bladder cancer.
Cyclin A1 is tissue-specifically expressed during spermatogenesis, but it is also highly expressed in acute myeloid leukemia (AML). Its pathogenetic role in AML and in the cell cycle of leukemic blasts is unknown. B-myb is essential for G1/S transition and has been shown to be phosphorylated by the cyclin A2/cdk2 complex. Here it is demonstrated that cyclin A1 interacts with the C-terminal portion of B-myb as shown by glutathione S-transferase (GST) precipitation. This interaction is confined to cyclin A1 because binding could not be detected between cyclin A2 and B-myb. Also, cdk2 was not pulled down by GST-B-myb from U937 lysates. In addition, co-immunoprecipitation of cyclin A1 and B-myb in leukemic cells evidenced protein interaction in vivo. Baculovirus-expressed cyclin A1/cdk2 complexes were able to phosphorylate human as well as murine B-myb in vitro. Tryptic phosphopeptide mapping revealed that cyclin A1/cdk2 complexes phosphorylated the C-terminal part of B-myb at several sites including threonine 447, 490, and 497 and serine 581. These phosphorylation sites have been demonstrated to be important for the enhancement of B-myb transcriptional activity. Further studies showed that cyclin A1 cooperated with B-myb to transactivate myb binding site containing promoters including the promoter of the human cyclin A1 gene. Taken together, the data suggest that cyclin A1 is a tissue-specific regulator of B-myb function and activates B-myb in leukemic blasts. (Blood. 2001;97:2091-2097)
Cyclin E binds and activates the cyclin-dependent kinase Cdk2 and catalyzes the transition from the G1 phase to the S phase of the cell cycle. The amount of cyclin E protein present in the cell is tightly controlled by ubiquitin-mediated
proteolysis. Here we identify the ubiquitin ligase responsible for cyclin E ubiquitination as SCFFbw7 and demonstrate that it is functionally conserved in yeast, flies, and mammals. Fbw7 associates specifically with phosphorylated
cyclin E, and SCFFbw7catalyzes cyclin E ubiquitination in vitro. Depletion of Fbw7 leads to accumulation and stabilization of cyclin E in vivo
in human andDrosophila melanogaster cells. Multiple F-box proteins contribute to cyclin E stability in yeast, suggesting an overlap in SCF E3 ligase specificity
that allows combinatorial control of cyclin E degradation.
Cyclin E, in conjunction with its catalytic partner cdk2, is rate limiting for entry into the S phase of the cell cycle. Cancer cells frequently contain mutations within the cyclin D-Retinoblastoma protein pathway that lead to inappropriate cyclin E-cdk2 activation. Although deregulated cyclin E-cdk2 activity is believed to directly contribute to the neoplastic progression of these cancers, the mechanism of cyclin E-induced neoplasia is unknown.
We studied the consequences of deregulated cyclin E expression in primary cells and found that cyclin E initiated a p53-dependent response that prevented excess cdk2 activity by inducing expression of the p21Cip1 cdk inhibitor. The increased p53 activity was not associated with increased expression of the p14ARF tumor suppressor. Instead, cyclin E led to increased p53 serine15 phosphorylation that was sensitive to inhibitors of the ATM/ATR family. When either p53 or p21cip1 was rendered nonfunctional, then the excess cyclin E became catalytically active and caused defects in S phase progression, increased ploidy, and genetic instability.
We conclude that p53 and p21 form an inducible barrier that protects cells against the deleterious consequences of cyclin E-cdk2 deregulation. A response that restrains cyclin E deregulation is likely to be a general protective mechanism against neoplastic transformation. Loss of this response may thus be required before deregulated cyclin E can become fully oncogenic in cancer cells. Furthermore, the combination of excess cyclin E and p53 loss may be particularly genotoxic, because cells cannot appropriately respond to the cell cycle anomalies caused by excess cyclin E-cdk2 activity.
E type cyclins (E1 and E2) are believed to drive cell entry into the S phase. It is widely assumed that the two E type cyclins are critically required for proliferation of all cell types. Here, we demonstrate that E type cyclins are largely dispensable for mouse development. However, endoreplication of trophoblast giant cells and megakaryocytes is severely impaired in the absence of cyclin E. Cyclin E-deficient cells proliferate actively under conditions of continuous cell cycling but are unable to reenter the cell cycle from the quiescent G(0) state. Molecular analyses revealed that cells lacking cyclin E fail to normally incorporate MCM proteins into DNA replication origins during G(0)-->S progression. We also found that cyclin E-deficient cells are relatively resistant to oncogenic transformation. These findings define a molecular function for E type cyclins in cell cycle reentry and reveal a differential requirement for cyclin E in normal versus oncogenic proliferation.
Metazoan replication-dependent histone mRNAs accumulate to high levels during S phase as a result of an increase in the rate of histone gene transcription, pre-mRNA processing, and mRNA stability at the G1-S transition. However, relatively little is known about the contribution of these processes to histone expression in the cell cycles of early development, which often lack a G1 phase. In post-blastoderm Drosophila embryos, zygotic expression of the stg(cdc25) phosphatase in G2 activates cyclin/cdc2 kinases and triggers mitosis. Here we show that histone transcription initiates in late G2 of cycle 14 in response to stg(cdc25) and in anticipation of S phase of the next cycle, which occurs immediately following mitosis. Mutation of stg(cdc25) arrests cells in G2 and prevents histone transcription. Expression of a mutant form of Cdc2 that bypasses the requirement for stg(cdc25) activates histone transcription during G2 in stg(cdc25) mutant embryos. Thus, in these embryonic cycles, histone transcription is controlled by the principal G2-M regulators, string(cdc25), and cdc2 kinase, rather than solely by regulators of the G1-S transition. After the introduction of G1-S control midway through embryogenesis, histone expression depends on DNA replication and the function of cyclin E, and no longer requires stg(cdc25). Thus, during the altered cell cycles of early animal development, different cell cycle mechanisms are employed to ensure that the production of histones accompanies DNA synthesis.
Genome-wide measures of gene expression can identify patterns of gene activity that subclassify tumours and might provide a better means than is currently available for individual risk assessment in patients with lymph-node-negative breast cancer.
We analysed, with Affymetrix Human U133a GeneChips, the expression of 22000 transcripts from total RNA of frozen tumour samples from 286 lymph-node-negative patients who had not received adjuvant systemic treatment.
In a training set of 115 tumours, we identified a 76-gene signature consisting of 60 genes for patients positive for oestrogen receptors (ER) and 16 genes for ER-negative patients. This signature showed 93% sensitivity and 48% specificity in a subsequent independent testing set of 171 lymph-node-negative patients. The gene profile was highly informative in identifying patients who developed distant metastases within 5 years (hazard ratio 5.67 [95% CI 2.59-12.4]), even when corrected for traditional prognostic factors in multivariate analysis (5.55 [2.46-12.5]). The 76-gene profile also represented a strong prognostic factor for the development of metastasis in the subgroups of 84 premenopausal patients (9.60 [2.28-40.5]), 87 postmenopausal patients (4.04 [1.57-10.4]), and 79 patients with tumours of 10-20 mm (14.1 [3.34-59.2]), a group of patients for whom prediction of prognosis is especially difficult.
The identified signature provides a powerful tool for identification of patients at high risk of distant recurrence. The ability to identify patients who have a favourable prognosis could, after independent confirmation, allow clinicians to avoid adjuvant systemic therapy or to choose less aggressive therapeutic options.