Spindle Checkpoint Regulation Research Articles

First person – Shang-yang Chen

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Shang-yang Chen is first author on ‘ C. elegans spermatocyte divisions show a weak spindle checkpoint response’, published in JCS. Shang-yang is a PhD student in the lab of Jui-ching Wu at National Taiwan University National Taiwan University Hospital, Taipei, Taiwan, investigating spindle checkpoint regulation in male meiotic division.

Adrenaline blocks key cell cycle genes and exhibits antifibrotic and vasoconstrictor effects in glaucoma surgery

Adrenaline is a sympathomimetic drug used to maintain pupil dilation and to decrease the risk of bleeding. The aim of this study was to demonstrate if adrenaline could exert antifibrotic effects in glaucoma surgery. Adrenaline was tested in fibroblast-populated collagen contraction assays and there was a dose-response decrease in fibroblast contractility: matrices decreased to 47.4% (P = 0.0002) and 86.6% (P = 0.0036) with adrenaline 0.0005% and 0.01%, respectively. There was no significant decrease in cell viability even at high concentrations. Human Tenon's fibroblasts were also treated with adrenaline (0%, 0.0005%, 0.01%) for 24 h and RNA-Sequencing was performed on the Illumina NextSeq 2000. We carried out detailed gene ontology, pathway, disease and drug enrichment analyses. Adrenaline 0.01% upregulated 26 G1/S and 11 S-phase genes, and downregulated 23 G2 and 17 M-phase genes (P < 0.05). Adrenaline demonstrated similar pathway enrichment to mitosis and spindle checkpoint regulation. Adrenaline 0.05% was also injected subconjunctivally during trabeculectomy, PreserFlo Microshunt and Baerveldt 350 tube surgeries, and patients did not experience any adverse effects. Adrenaline is a safe and cheap antifibrotic drug that significantly blocks key cell cycle genes when used at high concentrations. Unless contraindicated, we recommend subconjunctival injections of adrenaline (0.05%) in all glaucoma bleb-forming surgeries.

Spindle Checkpoint Regulators in Insulin Signaling

The spindle checkpoint ensures accurate chromosome segregation during mitosis and guards against aneuploidy. Insulin signaling governs metabolic homeostasis and cell growth, and its dysregulation leads to metabolic disorders, such as diabetes. These critical pathways have been extensively investigated, but a link between the two has not been established until recently. Our recent study reveals a critical role of spindle checkpoint regulators in insulin signaling and metabolic homeostasis through regulating endocytosis of the insulin receptor (IR). These findings have linked spindle checkpoint proteins to metabolic regulation, expanding the connection between cell division and metabolism. Here, we briefly review the unexpected roles of spindle checkpoint regulators in vesicle trafficking and insulin signaling.

Pellino 1 inactivates mitotic spindle checkpoint by targeting BubR1 for ubiquitinational degradation.

Aberrant constitutive activation of receptor-mediated downstream signalling plays an active role in the deregulation of cell cycle control. The mitotic spindle checkpoint is important in preventing abnormal mitotic cell cycle with chromosome missegregation from achieving neoplastic aneuploidy. However, mechanisms coupling receptor-mediated signalling to mitotic spindle checkpoint regulation remain unclear. Pellino 1 is a receptor signal-responsive E3 ubiquitin ligase, and the application of certain receptor-mediated signalling regulates the expression and activity of Pellino 1. In the present study, Pellino 1 expression induced extensive chromosome aneuploidy and allowed abnormal mitotic cells to adapt and become aneuploid in vitro and in vivo. Pellino 1 directly interacted with BubR1, a key component of mitotic spindle checkpoint, in a mitotic cell-cycle dependent manner, and down-regulated the stability of BubR1 by ubiquitination-mediated degradation and induced mitotic dysfunction. In summary, Pellino 1 expression acts as an inhibitory signal of the homeostatic regulation of mitotic cell cycle and checkpoint, and thus contributes to the initiation and progression of neoplastic chromosome aneuploidy.

Abstract 3008: CEP55 is a determinant of genomic instability in aneuploid breast cancer cells and facilitates anti-mitotic drugs resistance by interacting directly with HSF1

Abstract Centrosomal proteins have long been known to act as a scaffolds, spindle checkpoint regulators as well as key regulating elements of microtubule organizing centre, hence as an essential component of cell cycle progression. CEP55 is a centrosomal protein originally identified as an indispensable regulator of cytokinesis, the final stage of cell division that divides cytoplasm equally amongst two daughter cells. Failure to faithfully complete cytokinesis due to loss of CEP55 resulted in primary genetic lesion leading to multinucleated cells, thus promotes tumourigenesis and aneuploidy. CEP55 has been to shown elevated in wide range of cancers including breast cancer, however the molecular mechanisms on how CEP55 mediated genomic instability in cancers are still not well understood. To decipher the functional role of CEP55 in regulating genomic instability in breast cancer, through a series of in-vitro and in-vivo experiments we found that high levels of CEP55 is significantly associates with basal-like breast cancer subtype with poor overall survival, relapse free survival and distant-metastasis free survival. Furthermore, we found that depletion of CEP55 decreased cell viability due to induction of apoptosis and hamper primary tumour growth due to clearance of aneuploid cells that are particularly important in oncogenic transformation. In addition, depletion of CEP55 hindered premature exit of mitotic arrested cells and this is partly mediated through CEP55-dependent interaction with HSF1 during mitosis to facilitate development of aneuploidy. Strikingly, we found that CEP55 mediates anti-mitotic drug resistance to docetaxel and PLK1 inhibition. Depletion of CEP55 increased sensitivity to anti-mitotic drugs in particular docetaxel or PLK1 inhibitor, providing a rationale to target CEP55 and its dependent-pathways in aggressive and aneuploid breast cancer. Citation Format: Murugan Kalimutho, Nicola Waddell, Jessie Jeffry, Sriganesh Srihari, Kum Kum Khanna. CEP55 is a determinant of genomic instability in aneuploid breast cancer cells and facilitates anti-mitotic drugs resistance by interacting directly with HSF1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3008. doi:10.1158/1538-7445.AM2015-3008

Depression of MAD2 inhibits apoptosis and increases proliferation and multidrug resistance in gastric cancer cells by regulating the activation of phosphorylated survivin

Mitotic arrest-deficient 2 (MAD2) is one of the essential mitotic spindle checkpoint regulators, and it can protect cells from aberrant chromosome segregation. The Mad2 gene is very rarely mutated in many kinds of human cancer, but aberrantly reduced expression of MAD2 has been correlated with defective mitotic checkpoints in several human cancers. We have previously found that the MAD2 expression level is also shown to be associated with the multidrug resistance of tumour cells. In this study, we constructed a small interfering RNA (siRNA) eukaryotic expression vector of MAD2 and downregulated MAD2 expression in the gastric cancer cell line SGC7901 by transfection of MAD2-siRNA. SGC7901 cells stably transfected with the MAD2-siRNA exhibited significantly increased expression of phosphorylated survivin protein and enhanced drug resistance. Furthermore, MAD2-siRNA suppressed the proliferation of SGC7901 cells and inhibited tumour formation in athymic nude mice. This study clearly reveals that downregulation of MAD2 could regulate the cell cycle, increase proliferation, and improve the drug resistance of gastric cancer cells by regulating the activation of phosphorylated survivin. It also suggests both that MAD2 might play an important role in the development of human gastric cancer and that silencing the MAD2 gene may help to deal with the multidrug resistance of gastric cancer cells.

MAP kinase meets mitosis: A role for Raf Kinase Inhibitory Protein in spindle checkpoint regulation

Raf Kinase Inhibitory Protein (RKIP) is an evolutionarily conserved protein that functions as a modulator of signaling by the MAP kinase cascade. Implicated as a metastasis suppressor, Raf Kinase Inhibitory Protein depletion correlates with poor prognosis for breast, prostate and melanoma tumors but the mechanism is unknown. Recent evidence indicates that Raf Kinase Inhibitory Protein regulates the mitotic spindle assembly checkpoint by controlling Aurora B Kinase activity, and the mechanism involves Raf/MEK/ERK signaling. In contrast to elevated MAP kinase signaling during the G1, S or G2 phases of the cell cycle that activates checkpoints and induces arrest or senescence, loss of RKIP during M phase leads to bypass of the spindle assembly checkpoint and the generation of chromosomal abnormalities. These results reveal a role for Raf Kinase Inhibitory Protein and the MAP kinase cascade in ensuring the fidelity of chromosome segregation prior to cell division. Furthermore, these data highlight the need for precise titration of the MAP kinase signal to ensure the integrity of the spindle assembly process and provide a mechanism for generating genomic instability in tumors. Finally, these results raise the possibility that RKIP status in tumors could influence the efficacy of treatments such as poisons that stimulate the Aurora B-dependent spindle assembly checkpoint.

A census of mitotic cancer genes: new insights into tumor cell biology and cancer therapy

Tumor cell proliferation is frequently associated with genetic or epigenetic alterations in key regulators of the cell cycle. Most known oncogenes and tumor suppressors target entry into the cell cycle and control the G(1)/S transition. However, tumor-associated alterations in spindle formation or chromosome segregation are also frequent and may result in chromosomal instability. In fact, a few centrosomal or mitotic proteins such as aurora A, polo-like kinase 1 and PTTG1 (securin) have been reported to act as oncogenes. Some spindle checkpoint regulators such as the BUB kinases or MAD2 protect cells from aberrant chromosome segregation and may therefore function as suppressors of malignant transformation. However, few cancer-associated mutations in these or other mitotic regulators have been described thus far and many of these molecules do not fit into the classical definition of 'oncogenes' or 'tumor suppressor genes'. In some cases, both over-expression and decreased expression of these genes result in mitotic arrest. Moreover, some mitotic regulators such as MAD2 are either up- or down-regulated depending on the tumor types and, in both cases, these alterations result in chromosomal imbalances and tumor development. Minor changes in protein levels that do not compromise cell viability might therefore be sufficient to dysregulate the mitotic cycle and induce genomic instability. Despite the limited knowledge on the molecular basis of these processes, the clinical success of mitotic poisons such as taxanes reinforces the interest in these molecules, their involvement in human cancer and the therapeutic opportunities to modulate their function in cancer treatment.

ATM is activated by mitotic stress and suppresses centrosome amplification in primary but not in tumor cells

Centrosome amplification has been proposed to contribute to the development of aneuploidy and genome instability. Here, we show that Ataxia-Telangiectasia Mutated (ATM) is localized to the centrosome and co-purified with gamma-tubulin. The importance of ATM in centrosome duplication is demonstrated in Atm-deficient primary mouse embryonic fibroblasts that display centrosome amplification. Interestingly, centrosome amplification was not observed in tumor cell lines derived from Atm and p21 double deficient mouse. Our results also indicate that both p53 and p21 operate in the same pathway as ATM in regulating centrosome biogenesis. Finally, a potential role of ATM in spindle checkpoint regulation is demonstrated by which ATM protein is activated by mitotic stress. These results suggest a role of ATM in spindle checkpoint regulation and indicate that ATM suppresses genome instability and cellular transformation by regulating centrosome biogenesis.

TheDrosophilaγ-Tubulin Small Complex Subunit Dgrip84 Is Required for Structural and Functional Integrity of the Spindle Apparatus

Gamma-tubulin, a protein critical for microtubule assembly, functions within multiprotein complexes. However, little is known about the respective role of gamma-tubulin partners in metazoans. For the first time in a multicellular organism, we have investigated the function of Dgrip84, the Drosophila orthologue of the Saccharomyces cerevisiae gamma-tubulin-associated protein Spc97p. Mutant analysis shows that Dgrip84 is essential for viability. Its depletion promotes a moderate increase in the mitotic index, correlated with the appearance of monopolar or unpolarized spindles, impairment of centrosome maturation, and increase of polyploid nuclei. This in vivo study is strengthened by an RNA interference approach in cultured S2 cells. Electron microscopy analysis suggests that monopolar spindles might result from a failure of centrosome separation and an unusual microtubule assembly pathway via centriolar triplets. Moreover, we point to an involvement of Dgrip84 in the spindle checkpoint regulation and in the maintenance of interphase microtubule dynamics. Dgrip84 also seems essential for male meiosis, ensuring spindle bipolarity and correct completion of cytokinesis. These data sustain that Dgrip84 is required in some aspects of microtubule dynamics and organization both in interphase and mitosis. The nature of a minimal gamma-tubulin complex necessary for proper microtubule organization in the metazoans is discussed.

Analysis ofAurora-A andhMPS1 mitotic kinases in mantle cell lymphoma

Aurora-A and hMPS1 are kinases involved in spindle checkpoint and centrosome duplication regulation and whose alterations have been associated with cell transformation and chromosome instability in different tumor models. In this study, we have examined the possible alterations of these genes in 58 mantle cell lymphomas (MCLs) and 4 MCL-related cell lines. Aurora-A was also examined in 46 diffuse large B-cell lymphomas (DLBCLs). Aurora-A and hMPS1 mRNA expression levels were related to tumor proliferative activity. Interestingly, a MCL case with the highest number or chromosomal imbalances also showed an extremely high value of Aurora-A mRNA expression. No Aurora-A gene amplifications were detected in any tumor or cell line, whereas hemizygous hMPS1 gene deletions were observed in 23% of MCLs and 3 of the 4 cell lines. However, no expression alterations or gene mutations were detected in these cases. The Aurora-A proposed cancer susceptibility polymorphic variant (P31I) was observed with a similar frequency in MCL, DLBCL, chronic lymphocytic leukemia and in the 431 healthy controls. However, the 3 MCLs and 4 DLBCLs with the homozygous variant of this polymorphism had particular clinical characteristics with an unusual early-age presentation and second epithelial malignancies in MCL and extranodal origin in DLBCL. These findings indicate that Aurora-A and hMPS1 aberrations are uncommon in aggressive lymphomas but Aurora-A overexpression may contribute to numerical chromosomal alterations in occasional MCL. Although the Aurora-A P31I polymorphic variant is not directly involved in a genetic predisposition to these lymphomas, it may modulate the clinical presentation of these tumors.

Identification of an overlapping binding domain on Cdc20 for Mad2 and anaphase-promoting complex: model for spindle checkpoint regulation.

Activation of the anaphase-promoting complex (APC) is required for anaphase initiation and for exit from mitosis in mammalian cells. Cdc20, which specifically recognizes APC substrates involved in the metaphase-to-anaphase transition, plays a pivotal role in APC activation through direct interaction with the APC. The activation of the APC by Cdc20 is prevented by the interaction of Cdc20 with Mad2 when the spindle checkpoint is activated. Using deletion mutagenesis and peptide mapping, we have identified the sequences in Cdc20 that target it to Mad2 and the APC, respectively. These sequences are distinct but overlapping, providing a possible structural explanation for the internal modulation of the APC-Cdc20 complex by Mad2. In the course of these studies, a truncation mutant of Cdc20 (1-153) that constitutively binds Mad2 but fails to bind the APC was identified. Overexpression of this mutant induces the formation of multinucleated cells and increases their susceptibility to undergoing apoptosis when treated with microtubule-inhibiting drugs. Our experiments demonstrate that disruption of the Mad2-Cdc20 interaction perturbs the mitotic checkpoint, leading to premature activation of the APC, sensitizing the cells to the cytotoxic effects of microtubule-inhibiting drugs.