The mitotic spindle checkpoint is a critical determinant for topoisomerase-based chemotherapy

A novel strategy in cancer therapy is the induction of mitotic cell death by the pharmacological abrogation of cell cycle checkpoints. UCN-01 is such a compound that overrides the G2 cell cycle arrest induced by DNA damage and forces cells into a deleterious mitosis. The molecular pathways leading to mitotic cell death are largely unknown although recent evidence indicates that mitotic cell death represents a special case of apoptosis. Here, we demonstrate that the mitotic spindle checkpoint is activated upon chemotherapeutic treatment with topoisomerase II poisons and UCN-01. Cells that are forced to enter mitosis in the presence of topoisomerase inhibition arrest transiently in a prometaphase like state. By using a novel pharmacological inhibitor of the spindle checkpoint and spindle checkpoint-deficient cells we show that the spindle checkpoint function is required for the mitotic arrest and, most importantly, for efficient induction of mitotic cell death. Thus, our results demonstrate that the mitotic spindle checkpoint is an important determinant for the outcome of a chemotherapy based on the induction of mitotic cell death. Its frequent inactivation in human cancer might contribute to the observed resistance of tumor cells to these chemotherapeutic drugs.     

Requirement of a functional spindle checkpoint for arsenite-induced apoptosis

To understand the potential influence of spindle checkpoint function in response to arsenic trioxide (ATO)-induced apoptosis observed in cancer cell lines, we examined the correlation between activation of the spindle checkpoint and susceptibility to ATO-induced apoptosis in 10 cancer cell lines lacking functional p53. The ability to functionally activate the spindle checkpoint in each cancer cell line was assessed by the induction of mitotic arrest after Taxol treatment. Bromodeoxyuridine (BrdU) pulse-chase analysis of Taxol-treated cell lines with low mitotic arrest showed that they were not arrested at mitosis but divided abnormally, confirming that spindle checkpoint activation was impaired in these cell lines. Our results demonstrate that apoptosis was significantly induced by ATO in cancer cell lines with functional activation of the spindle checkpoint and substantial induction of mitotic arrest. Cell lines with negligible mitotic arrest exhibited little ATO-induced apoptosis. However, no such correlation was observed following treatment of cells with camptothecin, a topoisomerase I inhibitor. Furthermore, attenuation of the spindle checkpoint function by small interfering RNA-mediated silencing of BubR1 and Mad2 in cancer cells that were susceptible to ATO-induced mitotic arrest and apoptosis greatly reduced the induction of mitotic arrest and apoptosis by ATO and increased the formation of micronuclei or multinuclei in survived cells. The marked correlation between ATO-induced mitotic arrest and apoptosis indicates that the induction of apoptosis by ATO was highly dependent on the functional activation of the spindle checkpoint in cancer cells lacking normal p53 function.     

Genome‐wide siRNA screen reveals coupling between mitotic apoptosis and adaptation

The antimitotic anti‐cancer drugs, including taxol, perturb spindle dynamics, and induce prolonged, spindle checkpoint‐dependent mitotic arrest in cancer cells. These cells then either undergo apoptosis triggered by the intrinsic mitochondrial pathway or exit mitosis without proper cell division in an adaptation pathway. Using a genome‐wide small interfering RNA (siRNA) screen in taxol‐treated HeLa cells, we systematically identify components of the mitotic apoptosis and adaptation pathways. We show that the Mad2 inhibitor p31^comet actively promotes mitotic adaptation through cyclin B1 degradation and has a minor separate function in suppressing apoptosis. Conversely, the pro‐apoptotic Bcl2 family member, Noxa, is a critical initiator of mitotic cell death. Unexpectedly, the upstream components of the mitochondrial apoptosis pathway and the mitochondrial fission protein Drp1 contribute to mitotic adaption. Our results reveal crosstalk between the apoptosis and adaptation pathways during mitotic arrest.     

The inhibition of Aurora A abrogates the mitotic delay induced by microtubule perturbing agents

The spindle assembly checkpoint functions during mitosis to ensure that chromosomes are properly aligned in mitotic cells prior to the onset of anaphase, thereby ensuring an equal segregation of genetic material to each daughter cell. Defects in the function of this checkpoint lead to aneuploidy, and eventually to cell death or senescence. The Aurora-related kinases, and in particular Aurora B, have been shown to play a role in regulating the spindle assembly checkpoint. In this study, we demonstrate that Aurora A activity is required for maintainance of the spindle assembly checkpoint mediated-mitotic delay induced by microtubule perturbing agents. Inhibition of Aurora A using MLN8054, a selective small-molecule inhibitor of Aurora A, in paclitaxel- or nocodazole-treated cells induces cells to become multinucleated. Using time-lapse microscopy, we demonstrate that the multinucleation phenotype arises via mitotic slippage, which is significantly accelerated upon Aurora A inhibition. Under these conditions, the spindle assembly checkpoint protein BubR1 remains localized to kinetochores prior to mitotic slippage. Moreover, we demonstrate that Aurora B remains active in these mitotic cells, indicating that the mitotic slippage induced by MLN8054 is most likely due to the inhibition of Aurora A. This finding was corroborated by demonstrating that Aurora A depletion using RNA interference in paclitaxel-treated cells also induces multinucleation. Taken together, these results suggest that Aurora A is necessary for the maintenance of the mitotic delay induced in response to microtubule-perturbing agents.     

Effects of chemical manipulation of mitotic arrest and slippage on cancer cell survival and proliferation

Microtubule-targeting cancer therapies interfere with mitotic spindle dynamics and block cells in mitosis by activating the mitotic checkpoint. Cells arrested in mitosis may remain arrested for extended periods of time or undergo mitotic slippage and enter interphase without having separated their chromosomes. How extended mitotic arrest and mitotic slippage contribute to subsequent cell death or survival is incompletely understood. To address this question, automated fluorescence microscopy assays were designed and used to screen chemical libraries for modulators of mitotic slippage. Chlorpromazine and triflupromazine were identified as drugs that inhibit mitotic slippage and SU6656 and geraldol as chemicals that stimulate mitotic slippage. Using the drugs to extend mitotic arrest imposed by low concentrations of paclitaxel led to increased cell survival and proliferation after drug removal. Cells arrested at mitosis with paclitaxel or vinblastine and chemically induced to undergo mitotic slippage underwent several rounds of DNA replication without cell division and exhibited signs of senescence but eventually all died. By contrast, cells arrested at mitosis with the KSP/Eg5 inhibitor S-trityl-L-cysteine and induced to undergo mitotic slippage were able to successfully divide and continued to proliferate after drug removal. These results show that reinforcing mitotic arrest with drugs that inhibit mitotic slippage can lead to increased cell survival and proliferation, while inducing mitotic slippage in cells treated with microtubule-targeting drugs seems to invariably lead to protracted cell death.     

Mitotic checkpoints and the maintenance of the chromosome karyotype

The goal of the mitotic cell division is the faithful transmission of chromosomes to the daughter cells. To fulfil a correct separation of sister chromatids, kinetochores of all chromosomes should be correctly attached to spindle microtubules of opposite poles and should all be under tension. These events are monitored by the spindle checkpoint, which delays mitotic progression allowing time for corrections when errors occur in the dynamic interactions between chromosomes and microtubules. The G(1) post-mitotic checkpoint constitutes an additional checkpoint preventing further proliferation of cells that have undergone massive spindle damage. This review concentrates on the key structural and protein components which are pivotal for an accurate segregation of chromosomes during anaphase: the chromosome scaffold, sister chromatid cohesion and segregation and the kinetochores in higher eukaryotes. Furthermore, recent advances in understanding spindle and G(1) post-mitotic checkpoint and how they prevent aneuploidization and polyploidization are presented. In a last part the impact of aneuploidy and polyploidy on human health and in particular on cancer development is highlighted.     

Depletion of p31comet protein promotes sensitivity to antimitotic drugs

Antimitotic spindle poisons are among the most important chemotherapeutic agents available. However, precocious mitotic exit by mitotic slippage limits the cytotoxicity of spindle poisons. The MAD2-binding protein p31(comet) is implicated in silencing the spindle assembly checkpoint after all kinetochores are attached to spindles. In this study, we report that the levels of p31(comet) and MAD2 in different cell lines are closely linked with susceptibility to mitotic slippage. Down-regulation of p31(comet) increased the sensitivity of multiple cancer cell lines to spindle poisons, including nocodazole, vincristine, and Taxol. In the absence of p31(comet), lower concentrations of spindle poisons were required to induce mitotic block. The delay in checkpoint silencing was induced by an accumulation of mitotic checkpoint complexes. The increase in the duration of mitotic block after p31(comet) depletion resulted in a dramatic increase in mitotic cell death upon challenge with spindle poisons. Significantly, cells that are normally prone to mitotic slippage and resistant to spindle disruption-mediated mitotic death were also sensitized after p31(comet) depletion. These results highlight the importance of p31(comet) in checkpoint silencing and its potential as a target for antimitotic therapies.     

Combretastatin CA-4 and combretastatin derivative induce mitotic catastrophe dependent on spindle checkpoint and caspase-3 activation in non-small cell lung cancer cells

Combretastatin A-4 (CA-4), a natural stilbenoid isolated from Combretum caffrum, is a new vascular targeting agent (VTA) known for its antitumor activity due to its anti-tubulin properties. We investigated the molecular mechanisms leading to cell death in non-small cell lung cancer H460 cells induced by natural (CA-4) and synthetic stilbenoids (ST2151) structurally related to CA-4. We found that both compounds induced depolymerization and rearrangement of spindle microtubules, as well as an increasingly aberrant organization of metaphase chromosomes in a dose- and time-dependent manner. Prolonged exposition to ST2151 led cells to organize multiple sites of tubulin repolymerization, whereas tubulin repolymerization was observed only after CA-4 washout. H460 cells were arrested at a pro-metaphase stage, with condensed chromosomes and a triggered spindle assembly checkpoint, as evaluated by kinetochore localization of Bub1 and Mad1 antibodies. Persistent checkpoint activation led to mitochondrial membrane permeabilization (MMP) alterations, cytochrome c release, activation of caspase-9 and -3, PARP cleavage and DNA fragmentation. On the other hand, caspase-2, and -8 were not activated by the drug treatment. The ability of cells to reassemble tubulin in the presence of an activated checkpoint may be responsible for ST2151-induced multinucleation, a recognized sign of mitotic catastrophe. In conclusion, we believe that discovery of new agents able to trigger mitotic catastrophe cell death as a result of mitotic block and prolonged spindle checkpoint activation is particularly worthwhile, considering that tumor cells have a high proliferative rate and mitotic failure occurs irrespective of p53 status. Electronic Supplementary Material Supplementary material is available in the online version of this article at . Ilio Vitale and Antonio Antoccia contribuited equally to this work.     

The STARD9/Kif16a kinesin associates with mitotic microtubules and regulates spindle pole assembly

During cell division, cells form the microtubule-based mitotic spindle, a highly specialized and dynamic structure that mediates proper chromosome transmission to daughter cells. Cancer cells can show perturbed mitotic spindles and an approach in cancer treatment has been to trigger cell killing by targeting microtubule dynamics or spindle assembly. To identify and characterize proteins necessary for spindle assembly, and potential antimitotic targets, we performed a proteomic and genetic analysis of 592 mitotic microtubule copurifying proteins (MMCPs). Screening for regulators that affect both mitosis and apoptosis, we report the identification and characterization of STARD9, a kinesin-3 family member, which localizes to centrosomes and stabilizes the pericentriolar material (PCM). STARD9-depleted cells have fragmented PCM, form multipolar spindles, activate the spindle assembly checkpoint (SAC), arrest in mitosis, and undergo apoptosis. Interestingly, STARD9-depletion synergizes with the chemotherapeutic agent taxol to increase mitotic death, demonstrating that STARD9 is a mitotic kinesin and a potential antimitotic target.     

Perspectives and mechanisms for targeting mitotic catastrophe in cancer treatment

Mitotic catastrophe is distinct from other cell death modes due to unique nuclear alterations characterized as multi and/or micronucleation. Mitotic catastrophe is a common and virtually unavoidable consequence during cancer therapy. However, a comprehensive understanding of mitotic catastrophe remains lacking. Herein, we summarize the anticancer drugs that induce mitotic catastrophe, including microtubule-targeting agents, spindle assembly checkpoint kinase inhibitors, DNA damage agents and DNA damage response inhibitors. Based on the relationships between mitotic catastrophe and other cell death modes, we thoroughly evaluated the roles played by mitotic catastrophe in cancer treatment as well as its advantages and disadvantages. Some strategies for overcoming its shortcomings while fully utilizing its advantages are summarized and proposed in this review. We also review how mitotic catastrophe regulates cancer immunotherapy. These summarized findings suggest that the induction of mitotic catastrophe can serve as a promising new therapeutic approach for overcoming apoptosis resistance and strengthening cancer immunotherapy.     

Characterization of the cytotoxic mechanism of Mana-Hox, an analog of manzamine alkaloids

Mana-Hox is a synthetic analog of manzamines, which are beta-carboline alkaloids isolated from marine sponges. Mana-Hox exhibited cytotoxicity against various tumor cell lines with the IC(50) range from 1 to 5 microM. Cell cycle synchronization and flow cytometric analysis showed that Mana-Hox delayed cell cycle progression at mitosis. At the concentration that delayed mitotic progression, bipolar spindle with lagged chromosomes and multipolar spindle with disorganized chromosomes were detected. The presence of such aberrant mitotic cells accompanied by the activation of spindle checkpoint that delayed cells exit from mitosis. However, after a short delay, lagged chromosomes were able to display in the abnormal metaphase plates, and subsequent cell division resulting in chromosome missegregation. Furthermore, the aberrant mitotic cells showed lower viability, indicating that Mana-Hox-induced cell death resulting from chromosome missegregation. This study is the first to explore cytotoxic mechanism of a manzamine-related compound and understand its potential as a lead compound for the development of future anticancer agents.     

Microtubules,microtubule-interfering agents and apoptosis

Microtubules are dynamic polymers that play crucial roles in a large number of cellular functions. Their pivotal role in mitosis makes them a target for the development of anticancer drugs. Microtubule-damaging agents suppress microtubule dynamics, leading to disruption of the mitotic spindle in dividing cells, cell cycle arrest at M phase, and late apoptosis. A better understanding of the processes coupling microtubule damage to the onset of apoptosis will reveal sites of potential intervention in cancer chemotherapy. Inhibition of microtubule dynamics induces persistent modification of biological processes (M arrest) and signaling pathways (mitotic spindle assembly checkpoint activation, Bcl-2 phosphorylation, c-Jun NH₂-terminal kinase activation), which ultimately lead to apoptosis through the accumulation of signals that finally reach the threshold for the onset of apoptosis or through diminishing the threshold for engagement of cell death. Microtubules serve also as scaffolds for signaling molecules that regulate apoptosis, such as Bim and survivin, and their release from microtubules affect the activities of these apoptosis regulators. Thus, sustained modification of signaling routes and changes in the scaffolding properties of microtubules seem to constitute two major processes in the apoptotic response induced by microtubule-interfering agents.     

Cell-based expression cloning for identification of polypeptides that hypersensitize mammalian cells to mitotic arrest

Microtubule inhibitors such as Vinblastine and Paclitaxel are chemotherapy agents that activate the mitotic spindle checkpoint, arresting cells in mitosis and leading to cell death. The pathways that connect mitotic arrest to cell death are not well characterized. We developed a mammalian cell-based cDNA cloning method to isolate proteins and protein fragments whose expression inhibits colony formation in the presence of microtubule inhibitors. Understanding how these proteins impact cellular responses to microtubule drugs will lead to better understanding of the biochemical pathways connecting mitotic arrest and cell death in mammalian cells and may provide novel targets that can enhance microtubule inhibitor-mediated chemotherapy.     

Stabilization of P/CAF,as a ubiquitin ligase toward MDM2, suppresses mitotic cell death through p53-p21 activation in HCT116 cells with SIRT2 suppression

We previously reported that the suppression of SIRT2, an NAD + -dependent protein deacetylases, induces p53 accumulation via degradation of p300 and the subsequent MDM2 degradation, eventually leading to apoptosis in HeLa cells. The present study identified a novel pathway of p53 accumulation by SIRT2 suppression in HCT116(p53+/+) cells in which SIRT2 suppression led to escape from mitotic cell death caused by spindle assembly checkpoint activation induced by microtubule inhibitors such as nocodazole but not apoptosis or G1 or G2 arrest. We found that SIRT2 interacts with P/CAF, a histone acetyltransferase, which also acts as a ubiquitin ligase against MDM2. SIRT2 suppression led to an increase of P/CAF acetylation and its stabilization followed by a decrease in MDM2 and activation of the p53-p21 pathway. Depression of mitotic cell death in HCT116(p53+/+) cells with SIRT2 suppression was released by suppression of P/CAF or p21. Thus, the P/CAF-MDM2-p53-p21 axis enables the escape from mitotic cell death and confers resistance to nocodazole in HCT116(p53+/+) cells with SIRT2 suppression. As SIRT2 has attracted attention as a potential target for cancer therapeutics for p53 regulation, the present study provides a molecular basis for the efficacy of SIRT2 for future cancer therapy based on p53 regulation. These findings also suggest an undesirable function of the SIRT2 suppression associated with activation of the p53-p21 pathway in the suppression of mitotic cell death caused by spindle assembly checkpoint activation.     

A spindle checkpoint functions during mitosis in the early Caenorhabditis elegans embryo

During mitosis, chromosome segregation is regulated by a spindle checkpoint mechanism. This checkpoint delays anaphase until all kinetochores are captured by microtubules from both spindle poles, chromosomes congress to the metaphase plate, and the tension between kinetochores and their attached microtubules is properly sensed. Although the spindle checkpoint can be activated in many different cell types, the role of this regulatory mechanism in rapidly dividing embryonic animal cells has remained controversial. Here, using time-lapse imaging of live embryonic cells, we show that chemical or mutational disruption of the mitotic spindle in early Caenorhabditis elegans embryos delays progression through mitosis. By reducing the function of conserved checkpoint genes in mutant embryos with defective mitotic spindles, we show that these delays require the spindle checkpoint. In the absence of a functional checkpoint, more severe defects in chromosome segregation are observed in mutants with abnormal mitotic spindles. We also show that the conserved kinesin CeMCAK, the CENP-F-related proteins HCP-1 and HCP-2, and the core kinetochore protein CeCENP-C all are required for this checkpoint. Our analysis indicates that spindle checkpoint mechanisms are functional in the rapidly dividing cells of an early animal embryo and that this checkpoint can prevent chromosome segregation defects during mitosis.     

Caffeine promotes apoptosis in mitotic spindle checkpoint-arrested cells

The spindle assembly checkpoint arrests cells in mitosis when defects in mitotic spindle assembly or partitioning of the replicated genome are detected. This checkpoint blocks exit from mitosis until the defect is rectified or the cell initiates apoptosis. In this study we have used caffeine to identify components of the mechanism that signals apoptosis in mitotic checkpoint-arrested cells. Addition of caffeine to spindle checkpoint-arrested cells induced >40% apoptosis within 5 h. It also caused proteasome-mediated destruction of cyclin B1, a corresponding reduction in cyclin B1/cdk1 activity, and reduction in MPM-2 reactivity. However, cells retained MAD2 staining at the kinetochores, an indication of continued spindle checkpoint function. Blocking proteasome activity did not block apoptosis, but continued spindle checkpoint function was essential for apoptosis. After systematically eliminating all known targets, we have identified p21-activated kinase PAK1, which has an anti-apoptotic function in spindle checkpoint-arrested cells, as a target for caffeine inhibition. Knockdown of PAK1 also increased apoptosis in spindle checkpoint-arrested cells. This study demonstrates that the spindle checkpoint not only regulates mitotic exit but apoptosis in mitosis through the activity of PAK1.     

The transforming acidic coiled coil 3 protein is essential for spindle-dependent chromosome alignment and mitotic survival

Cancer-associated centrosomal transforming acidic coiled coil (TACC) proteins are involved in mitotic spindle function. By employing gene targeting, we have recently described a nonredundant and essential role of TACC3 in regulating cell proliferation. In this study, we used an inducible RNA interference approach to characterize the molecular function of TACC3 and its role in mitotic progression and cell survival. Our data demonstrate that a TACC3 knockdown arrests G(1) checkpoint-compromised HeLa cells prior to anaphase with aberrant spindle morphology and severely misaligned chromosomes. Interestingly, TACC3-depleted cells fail to accumulate the mitotic kinase Aurora B and the checkpoint protein BubR1 to normal levels at kinetochores. Moreover, localization of the structural protein Ndc80 at outer kinetochores is reduced, indicating a defective kinetochore-microtubule attachment in TACC3-deficient cells. As a consequence of prolonged TACC3 depletion, cells undergo caspase-dependent cell death that relies on a spindle checkpoint-dependent mitotic arrest. TACC3 knockdown cells that escape from this arrest by mitotic slippage become highly polyploid and accumulate supernumerary centrosomes. Similarly, deficiency of the post-mitotic cell cycle inhibitor p21(WAF) exacerbates the effects of TACC3 depletion. Our findings therefore point to an essential role of TACC3 in spindle assembly and cellular survival and identify TACC3 as a potential therapeutic target in cancer cells.     

Unattached kinetochores rather than intrakinetochore tension arrest mitosis in taxol-treated cells

Kinetochores attach chromosomes to the spindle microtubules and signal the spindle assembly checkpoint to delay mitotic exit until all chromosomes are attached. Light microscopy approaches aimed to indirectly determine distances between various proteins within the kinetochore (termed Delta) suggest that kinetochores become stretched by spindle forces and compact elastically when the force is suppressed. Low Delta is believed to arrest mitotic progression in taxol-treated cells. However, the structural basis of Delta remains unknown. By integrating same-kinetochore light microscopy and electron microscopy, we demonstrate that the value of Delta is affected by the variability in the shape and size of outer kinetochore domains. The outer kinetochore compacts when spindle forces are maximal during metaphase. When the forces are weakened by taxol treatment, the outer kinetochore expands radially and some kinetochores completely lose microtubule attachment, a condition known to arrest mitotic progression. These observations offer an alternative interpretation of intrakinetochore tension and question whether Delta plays a direct role in the control of mitotic progression.     

Spindle poisons and cell fate: a tale of two pathways

Spindle poisons, such as paclitaxel and vinblastine, exert their potent anti-neoplastic effects through activation of the spindle assembly checkpoint (SAC), thereby arresting cells in mitosis. Unfortunately, only certain cancers are susceptible to these drugs, and many patients fail to respond to treatment. We review the pathways that are triggered by spindle poisons and highlight recent studies that describe the great variability of tumor cells in responding to these drugs. We also describe the recent identification of an apoptotic pathway that is activated by mitotic arrest in response to spindle poisons. Emerging from these studies is not only a greater understanding of how these classic antimitotic agents bring about cell death, but also a wealth of potential new targets of anticancer therapeutics.