Hec1 overexpression hyperactivates the mitotic checkpoint and induces tumor formation in vivo.
Ontology highlight
ABSTRACT:
SUBMITTER: Diaz-Rodriguez E
PROVIDER: S-EPMC2570608 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Hec1 is a conserved mitotic regulator critical for spindle checkpoint control, kinetochore functionality, and cell survival. Overexpression of Hec1 has been detected in a variety of human cancers and is linked to poor prognosis of primary breast cancers. Through a chemical genetic screening, we have identified a small molecule, N-(4-[2,4-dimethyl-phenyl]-thiazol-2-yl)-benzamide (INH1), which specifically disrupts the Hec1/Nek2 interaction via direct Hec1 binding. Treating cells with INH1 triggered reduction of kinetochore-bound Hec1 as well as global Nek2 protein level, consequently leading to metaphase chromosome misalignment, spindle aberrancy, and eventual cell death. INH1 effectively inhibited the proliferation of multiple human breast cancer cell lines in culture (GI(50), 10-21 micromol/L). Furthermore, treatment with INH1 retarded tumor growth in a nude mouse model bearing xenografts derived from the human breast cancer line MDA-MB-468, with no apparent side effects. This study suggests that the Hec1/Nek2 pathway may serve as a novel mitotic target for cancer intervention by small compounds.
| S-EPMC2709694 | biostudies-literature
Project description:Microtubule inhibitors are widely used in cancer chemotherapy. These drugs characteristically induce mitotic arrest and cell death but the mechanisms linking the two are not firmly established. One of the problems is that cancer cells vary widely in their sensitivity to these agents, and thus comparison of data from different systems is difficult. To alleviate this problem we sought to molecularly induce mitotic death and study its mechanisms, by expressing non-degradable cyclin B (R42A) in HeLa cells. However, this approach failed to induce significant mitotic arrest, Cdk1 activation, or phosphorylation of anti-apoptotic Bcl-2 proteins, all characteristics of cells treated with microtubule inhibitors. Furthermore, cyclin B1-R42A induced rapid cell death, and when expressed in synchronized cells, cell death occurred in G1 phase. Decreasing the plasmid concentration reduced transfection efficiency but restored mitotic arrest and eliminated non-specific death. These results show that inappropriate overexpression of cyclin B1 causes non-specific cell death and suggest caution in its use for the study of mitotic events.
| S-EPMC4240608 | biostudies-literature
Project description:Most cancer cells show chromosomal instability, a condition where chromosome missegregation occurs frequently. We found that chromosome oscillation, an iterative chromosome motion during metaphase, is attenuated in cancer cell lines. We also found that metaphase phosphorylation of Hec1 at serine 55, which is mainly dependent on Aurora A on the spindle, is reduced in cancer cell lines. The Aurora A-dependent Hec1-S55 phosphorylation level was regulated by the chromosome oscillation amplitude and vice versa: Hec1-S55 and -S69 phosphorylation by Aurora A is required for efficient chromosome oscillation. Furthermore, enhancement of chromosome oscillation reduced the number of erroneous kinetochore-microtubule attachments and chromosome missegregation, whereas inhibition of Aurora A during metaphase increased such errors. We propose that Aurora A-mediated metaphase Hec1-S55 phosphorylation through chromosome oscillation, together with Hec1-S69 phosphorylation, ensures mitotic fidelity by eliminating erroneous kinetochore-microtubule attachments. Attenuated chromosome oscillation and the resulting reduced Hec1-S55 phosphorylation may be a cause of CIN in cancer cell lines.
| S-EPMC8129796 | biostudies-literature
Project description:Amitozyn (Am) is a semi-synthetic drug produced by the alkylation of major celandine (Chelidonium majus L.) alkaloids with the organophosphorous compound N,N'N'-triethylenethiophosphoramide (ThioTEPA). We show here that the treatment of living cells with Am reversibly perturbs the microtubule cytoskeleton, provoking a dose-dependent cell arrest in the M phase. Am changed the dynamics of tubulin polymerization in vitro, promoted the appearance of aberrant mitotic phenotypes in HeLa cells and induced apoptosis by the activation of caspase-9, caspase-3 and PARP, without inducing DNA breaks. Am treatment of HeLa cells induced changes in the phosphorylation of the growth suppressor pRb that coincided with maximum mitotic index. The dose-dependent and reversible anti-proliferative effect of Am was observed in several transformed cell lines. Importantly, the drug was also efficient against multidrug-resistant, paclitaxel-resistant or p53-deficient cells. Our results thus open the way to further pre-clinical evaluation of Am.
| S-EPMC3591406 | biostudies-literature
Project description:Microtubules are among the most successful targets for anticancer therapy because they play important roles in cell proliferation as they constitute the mitotic spindle, which is critical for chromosome segregation during mitosis. Hence, identifying new therapeutic targets encoding proteins that regulate microtubule assembly and function specifically in cancer cells is critical. In the present study, we identified a candidate gene that promotes tumor progression, ribonucleic acid export 1 (RAE1), a mitotic checkpoint regulator, on chromosome 20q through a bioinformatics approach using datasets of colorectal cancer (CRC), including The Cancer Genome Atlas (TCGA). RAE1 was ubiquitously amplified and overexpressed in tumor cells. High expression of RAE1 in tumor tissues was positively associated with distant metastasis and was an independent poor prognostic factor in CRC. In vitro and in vivo analysis showed that RAE1 promoted tumor growth, inhibited apoptosis, and promoted cell cycle progression, possibly with a decreased proportion of multipolar spindle cells in CRC. Furthermore, RAE1 induced chemoresistance through its anti-apoptotic effect. In addition, overexpression of RAE1 and significant effects on survival were observed in various types of cancer, including CRC. In conclusion, we identified RAE1 as a novel gene that facilitates tumor growth in part by inhibiting apoptosis and promoting cell cycle progression through stabilizing spindle bipolarity and facilitating tumor growth. We suggest that it is a potential therapeutic target to overcome therapeutic resistance of CRC.
| S-EPMC8353924 | biostudies-literature
Project description:Aided by translocated virulence proteins, Agrobacterium tumefaciens transforms plant cells with oncogenic T-DNA. In the host cells the virulence protein VirD5 moves to the nucleus, where it becomes localized at the kinetochores, and disturbs faithful chromosome segregation, but the molecular mechanism underlying this remains unknown. To gain more insight, we screened amongst the kinetochore proteins for VirD5 interactors using bimolecular fluorescence complementation assays, and tested chromosome segregation in yeast cells. We found that VirD5 interacts with the conserved mitotic Aurora kinase Ipl1 in yeast and likewise with plant Aurora kinases. In vitro VirD5 was found to stimulate the activity of Ipl1. Phosphorylation of substrates by Ipl1 in vivo is known to result in the detachment between kinetochore and spindle microtubule. This is necessary for error correction, but increased Ipl1/Aurora kinase activity is known to cause spindle instability, explaining enhanced chromosome mis-segregation seen in the presence of VirD5. That activation of the Ipl1/Aurora kinase at least partially underlies the toxicity of VirD5 became apparent by artificial boosting the activity of the specific counteracting phosphatase Glc7 in vivo, which relieved the toxicity. These findings reveal a novel mechanism by which a pathogenic bacterium manipulates host cells.
| S-EPMC6667905 | biostudies-other
Project description:BackgroundAneuploidy and chromosomal instability (CIN) are common abnormalities in human cancer. Alterations of the mitotic spindle checkpoint are likely to contribute to these phenotypes, but little is known about somatic alterations of mitotic spindle checkpoint genes in breast cancer.MethodsTo obtain further insight into the molecular mechanisms underlying aneuploidy in breast cancer, we used real-time quantitative RT-PCR to quantify the mRNA expression of 76 selected mitotic spindle checkpoint genes in a large panel of breast tumor samples.ResultsThe expression of 49 (64.5%) of the 76 genes was significantly dysregulated in breast tumors compared to normal breast tissues: 40 genes were upregulated and 9 were downregulated. Most of these changes in gene expression during malignant transformation were observed in epithelial cells.Alterations of nine of these genes, and particularly NDC80, were also detected in benign breast tumors, indicating that they may be involved in pre-neoplastic processes.We also identified a two-gene expression signature (PLK1 + AURKA) which discriminated between DNA aneuploid and DNA diploid breast tumor samples. Interestingly, some DNA tetraploid tumor samples failed to cluster with DNA aneuploid breast tumors.ConclusionThis study confirms the importance of previously characterized genes and identifies novel candidate genes that could be activated for aneuploidy to occur. Further functional analyses are required to clearly confirm the role of these new identified genes in the molecular mechanisms involved in breast cancer aneuploidy. The novel genes identified here, and/or the two-gene expression signature, might serve as diagnostic or prognostic markers and form the basis for novel therapeutic strategies.
| S-EPMC3058099 | biostudies-literature
Project description:The basic helix-loop-helix transcription factor, Id1, was shown to induce tetraploidy in telomerase-immortalized nasopharyngeal epithelial cells in this study. Using both transient and stable Id1-expressing cell models, multiple mitotic aberrations were detected, including centrosome amplification, binucleation, spindle defects, and microtubule perturbation. Many of these abnormal phenotypes have previously been reported in cells overexpressing Aurora A. Further experiments showed that Id1 could stabilize Aurora A, whereas knocking down Aurora A expression in Id1-expressing cells could rescue some of the mitotic defects. The mechanisms by which Aurora A could be modulated by Id1 were explored. DNA amplification of the Aurora A locus was not involved. Id1 could only weakly activate the transcriptional activity of the Aurora A promoter. We found that Id1 overexpression could affect Aurora A degradation, leading to its stabilization. Aurora A is normally degraded from mitosis exit by the APC/C(Cdh1)-mediated proteasomal proteolysis pathway. Our results revealed that Id1 and Cdh1 are binding partners. The association of Id1 and Cdh1 was found to be dependent on the canonical destruction box motif of Id1, the increased binding of which may compete with the interaction between Cdh1 and Aurora A, leading to stabilization of Aurora A in Id1-overexpressing cells.
| S-EPMC2397319 | biostudies-literature
Project description:Mechanisms of spindle pole formation rely on minus-end-directed motor proteins. gamma-Tubulin is present at the centre of poles, but its function during pole formation is completely unknown. To address the role of gamma-tubulin in spindle pole formation, we overexpressed GFP (green fluorescent protein)-fused gamma-tubulin (gamma-Tu-GFP) in Xenopus oocytes and produced self-assembled mitotic asters in the oocyte extracts. gamma-Tu-GFP associated with endogenous alpha-, beta- and gamma-tubulin, suggesting that it acts in the same manner as that of endogenous gamma-tubulin. During the process of aster formation, gamma-Tu-GFP aggregated as dots on microtubules, and then the dots were translocated to the centre of the aster along microtubules in a manner dependent on cytoplasmic dynein activity. Inhibition of the function of gamma-tubulin by an anti-gamma-tubulin antibody resulted in failure of microtubule organization into asters. This defect was restored by overexpression of gamma-Tu-GFP, confirming the necessity of gamma-tubulin in microtubule recruitment for aster formation. We also examined the effects of truncated gamma-tubulin mutants, which are difficult to solubly express in other systems, on aster formation. The middle part of gamma-tubulin caused abnormal organization of microtubules in which minus ends of microtubules were not tethered, but dispersed. An N-terminus-deleted mutant prevented recruitment of microtubules into asters, similar to the effect of the anti-gamma-tubulin antibody. The results indicate possible roles of gamma-tubulin in spindle pole formation and show that the system developed in the present study could be useful for analysing roles of many proteins that are difficult to solubly express.
| S-EPMC1180710 | biostudies-literature
Project description:Chromosomal instability (CIN) is a driving force for cancer development. The most common causes of CIN include the dysregulation of the spindle assembly checkpoint (SAC), which is a surveillance mechanism that prevents premature chromosome separation during mitosis by targeting anaphase-promoting complex/cyclosome (APC/C). DAB2IP is frequently silenced in advanced prostate cancer (PCa) and is associated with aggressive phenotypes of PCa. Our previous study showed that DAB2IP activates PLK1 and functions in mitotic regulation. Here, we report the novel mitotic phosphorylation of DAB2IP by Cdks, which mediates DAB2IP's interaction with PLK1 and the activation of the PLK1-Mps1 pathway. DAB2IP interacts with Cdc20 in a phosphorylation-independent manner. However, the phosphorylation of DAB2IP inhibits the ubiquitylation of Cdc20 in response to SAC, and blocks the premature release of the APC/C-MCC. The PLK1-Mps1 pathway plays an important role in mitotic checkpoint complex (MCC) assembly. It is likely that DAB2IP acts as a scaffold to aid PLK1-Mps1 in targeting Cdc20. Depletion or loss of the Cdks-mediated phosphorylation of DAB2IP destabilizes the MCC, impairs the SAC, and increases chromosome missegregation and subsequent CIN, thus contributing to tumorigenesis. Collectively, these results demonstrate the mechanism of DAB2IP in SAC regulation and provide a rationale for targeting the SAC to cause lethal CIN against DAB2IP-deficient aggressive PCa, which exhibits a weak SAC.
| S-EPMC8782720 | biostudies-literature