Loss of APC induces polyploidy as a result of a combination of defects in mitosis and apoptosis

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene initiate a majority of colorectal cancers. Acquisition of chromosomal instability is an early event in these tumors. We provide evidence that the loss of APC leads to a partial loss of interkinetochore tension at metaphase and alters mitotic progression. Furthermore, we show that inhibition of APC in U2OS cells compromises the mitotic spindle checkpoint. This is accompanied by a decrease in the association of the checkpoint proteins Bub1 and BubR1 with kinetochores. Additionally, APC depletion reduced apoptosis. As expected from this combination of defects, tetraploidy and polyploidy are consequences of APC inhibition in vitro and in vivo. The removal of APC produced the same defects in HCT116 cells that have constitutively active beta-catenin. These data show that the loss of APC immediately induces chromosomal instability as a result of a combination of mitotic and apoptotic defects. We suggest that these defects amplify each other to increase the incidence of tetra- and polyploidy in early stages of tumorigenesis.     

Gain of function properties of mutant p53 proteins at the mitotic spindle cell cycle checkpoint

Mutations in the p53 tumor suppressor gene locus predispose human cells to chromosomal instability. This is due in part to interference of mutant p53 proteins with the activity of the mitotic spindle and postmitotic cell cycle checkpoints. Recent data demonstrates that wild type p53 is required for postmitotic checkpoint activity, but plays no role at the mitotic spindle checkpoint. Likewise, structural dominant p53 mutants demonstrate gain-of-function properties at the mitotic spindle checkpoint and dominant negative properties at the postmitotic checkpoint. At mitosis, mutant p53 proteins interfere with the control of the metaphase-to-anaphase progression by up-regulating the expression of CKs1, a protein that mediates activatory phosphorylation of the anaphase promoting complex (APC) by Cdc2. Cells that carry mutant p53 proteins overexpress CKs1 and are unable to sustain APC inactivation and mitotic arrest. Thus, mutant p53 gain-of-function at mitosis constitutes a key component to the origin of chromosomal instability in mutant p53 cells.     

The impact of p53 and p73 on aneuploidy and cancer

Initiation, progression and evasion are sequential steps in cancer formation, with autonomous cell proliferation as a final outcome. Genetic or epigenetic alterations of key regulatory genes of the cell cycle are frequently associated with these phenomena. Recently, chromosomal instability, a long-supposed driving force of tumorigenesis, was associated with dysregulation of mitotic genes, providing advantages to tumor cells. Numerous molecules thus provide a key link in the chain of relationships between chromosomal instability and cancer. Here, we discuss emerging evidence revealing that two p53 family members, p53 and p73, might be key regulatory genes at the heart of the relationship between chromosomal instability and cancer. We argue that the role of members of the p53 family as tumor suppressor proteins, their impact on the control of cellular ploidy, and their newly emerging connection with mitotic checkpoint regulatory genes support the suggestion that p73 and p53 could be two of the missing links among chromosomal instability, the mitotic checkpoint and cancer.     

Killing cells by targeting mitosis

Cell cycle deregulation is a common feature of human cancer. Tumor cells accumulate mutations that result in unscheduled proliferation, genomic instability and chromosomal instability. Several therapeutic strategies have been proposed for targeting the cell division cycle in cancer. Whereas inhibiting the initial phases of the cell cycle is likely to generate viable quiescent cells, targeting mitosis offers several possibilities for killing cancer cells. Microtubule poisons have proved efficacy in the clinic against a broad range of malignancies, and novel targeted strategies are now evaluating the inhibition of critical activities, such as cyclin-dependent kinase 1, Aurora or Polo kinases or spindle kinesins. Abrogation of the mitotic checkpoint or targeting the energetic or proteotoxic stress of aneuploid or chromosomally instable cells may also provide further benefits by inducing lethal levels of instability. Although cancer cells may display different responses to these treatments, recent data suggest that targeting mitotic exit by inhibiting the anaphase-promoting complex generates metaphase cells that invariably die in mitosis. As the efficacy of cell-cycle targeting approaches has been limited so far, further understanding of the molecular pathways modulating mitotic cell death will be required to move forward these new proposals to the clinic.     

Checkpoint-independent stabilization of kinetochore-microtubule attachments by Mad2 in human cells

Faithful chromosome segregation is required for cell and organism viability and relies on both the mitotic checkpoint and the machinery that corrects kinetochore-microtubule (k-MT) attachment errors. Most solid tumors have aneuploid karyotypes and many missegregate chromosomes at high rates in a phenomenon called chromosomal instability (CIN). Mad2 is essential for mitotic checkpoint function and is frequently overexpressed in human tumors that are CIN. For unknown reasons, cells overexpressing Mad2 display high rates of lagging chromosomes. Here, we explore this phenomenon and show that k-MT attachments are hyperstabilized by Mad2 overexpression and that this undermines the efficiency of correction of k-MT attachment errors. Mad2 affects k-MT attachment stability independently of the mitotic checkpoint because k-MT attachments are unaltered upon Mad1 depletion and Mad2 overexpression hyperstabilizes k-MT attachments in Mad1-deficient cells. Mad2 mediates these effects with Cdc20 by altering the centromeric localization and activity of Aurora B kinase, a known regulator of k-MT attachment stability. These data reveal a new function for Mad2 to stabilize k-MT attachments independent of the checkpoint and explain why Mad2 overexpression increases chromosome missegregation to cause chromosomal instability in human tumors.     

A small-molecule inhibitor of Mps1 blocks the spindle-checkpoint response to a lack of tension on mitotic chromosomes

The spindle checkpoint prevents chromosome loss by preventing chromosome segregation in cells with improperly attached chromosomes [1, 2 and 3]. The checkpoint senses defects in the attachment of chromosomes to the mitotic spindle [4] and the tension exerted on chromosomes by spindle forces in mitosis [5, 6 and 7]. Because many cancers have defects in chromosome segregation, this checkpoint may be required for survival of tumor cells and may be a target for chemotherapy. We performed a phenotype-based chemical-genetic screen in budding yeast and identified an inhibitor of the spindle checkpoint, called cincreasin. We used a genome-wide collection of yeast gene-deletion strains and traditional genetic and biochemical analysis to show that the target of cincreasin is Mps1, a protein kinase required for checkpoint function [8]. Despite the requirement for Mps1 for sensing both the lack of microtubule attachment and tension at kinetochores, we find concentrations of cincreasin that selectively inhibit the tension-sensitive branch of the spindle checkpoint. At these concentrations, cincreasin causes lethal chromosome missegregation in mutants that display chromosomal instability. Our results demonstrate that Mps1 can be exploited as a target and that inhibiting the tension-sensitive branch of the spindle checkpoint may be a way of selectively killing cancer cells that display chromosomal instability.     

Chk1-Mad2 interaction

Chk1 is implicated in several checkpoints of the cell cycle acting as a key player in the signal transduction pathway activated in response to DNA damage and crucial for the maintenance of genomic stability. Chk1 also plays a role in the mitotic spindle checkpoint, which ensures the fidelity of mitotic segregation during mitosis, preventing chromosomal instability and aneuploidy. Mad2 is one of the main mitotic checkpoint components and also exerts a role in the cellular response to DNA damage. To investigate a possible crosslink existing between Chk1 and Mad2, we studied Mad2 protein levels after Chk1 inhibition either by specific siRNAs or by a specific and selective Chk1 inhibitor (PF-00477736), and we found that after Chk1 inhibition, Mad2 protein levels decrease only in tumor cells sensitive to Chk1 depletion. We then mapped six Chk1’s phosphorylatable sites on Mad2 protein, and found that Chk1 is able to phosphorylate Mad2 in vitro on more than one site, while it is incapable of phoshorylating the Mad2 form mutated on all six phosphorylatable sites. Moreover our studies demonstrate that Chk1 co-localizes and physically associates with Mad2 in cells both under unstressed conditions and after DNA damage, thus providing new and interesting evidence on Chk1 and Mad2 crosstalk in the DNA damage checkpoint and in the mitotic spindle checkpoint.     

Sustained metaphase arrest in response to ionizing radiation in a non-small cell lung cancer cell line

Kodym, E., Kodym, R., Choy, H. and Saha, D. Sustained Metaphase Arrest in Response to Ionizing Radiation in a Non-small Cell Lung Cancer Cell Line. Radiat. Res. 169, 46-58 (2008). In solid tumors, non-apoptotic forms of tumor cell inactivation such as mitotic catastrophe appear to be predominant in the response to DNA-damaging agents. Despite its importance, the underlying molecular mechanisms of mitotic catastrophe have been only partially elucidated. We found that a large fraction of HCC2279 non-small cell lung cancer cells underwent mitotic catastrophe after irradiation. Cells were arrested in metaphase with chromosomal damage indicated by DNA fragments displaced from the metaphase plate and considerable numbers of residual gamma-H2AX foci. Although TP53 was nonfunctional, we detected a prompt radiation response on the level of checkpoint kinases. In contrast, CDC25A was the only checkpoint phosphatase that was responsive to radiation. CDC25B was not detectable, and CDC25C was constitutively phosphorylated at serine 216, leading to its cytoplasmic sequestration and functional inactivation. Therefore, radiation-induced mitotic catastrophe in HCC2279 cells appears to be induced by a combination of relative insufficiencies in the p53-mediated and checkpoint kinase-mediated pathways leading to premature entry into mitosis. Displaced chromosome fragments triggering an intra-M checkpoint in cells entering mitosis presumably result in a sustained metaphase arrest. The phenomenon found in these cells, which were derived directly from a human patient, might be responsible for therapy-induced genetic instability of tumors.     

WEE1 inhibition and genomic instability in cancer

One of the hallmarks of cancer is genomic instability controlled by cell cycle checkpoints. The G₁ and G₂ checkpoints allow DNA damage responses, whereas the mitotic checkpoint enables correct seggregation of the sister chromosomes to prevent aneuploidy. Cancer cells often lack a functional G₁ arrest and rely on G₂ arrest for DNA damage responses. WEE1 kinase is an important regulator of the G₂ checkpoint and is overexpressed in various cancer types. Inhibition of WEE1 is a promising strategy in cancer therapy in combination with DNA-damaging agents, especially when cancer cells harbor p53 mutations, as it causes mitotic catastrophy when DNA is not repaired during G₂ arrest. Cancer cell response to WEE1 inhibition monotherapy has also been demonstrated in various types of cancer, including p53 wild-type cancers. We postulate that chromosomal instability can explain tumor response to WEE1 monotherapy. Therefore, chromosomal instability may need to be taken into account when determining the most effective strategy for the use of WEE1 inhibitors in cancer therapy.     

Interplay between the DNA Damage Proteins MDC1 and ATM in the Regulation of the Spindle Assembly Checkpoint

To avoid genomic instability, cells have developed surveillance mechanisms such as the spindle assembly checkpoint (SAC) and the DNA damage response. ATM and MDC1 are central players of the cellular response to DNA double-strand breaks. Here, we identify a new role for these proteins in the regulation of mitotic progression and in SAC activation. MDC1 localizes at mitotic kinetochores following SAC activation in an ATM-dependent manner. ATM phosphorylates histone H2AX at mitotic kinetochores, and this phosphorylation is required for MDC1 localization at kinetochores. ATM and MDC1 are needed for kinetochore localization of the inhibitory mitotic checkpoint complex components, Mad2 and Cdc20, and for the maintenance of the mitotic checkpoint complex integrity. This probably relies on the interaction of MDC1 with the MCC. In this work, we have established that ATM and MDC1 maintain genomic stability not only by controlling the DNA damage response, but also by regulating SAC activation, providing an important link between these two essential biological processes.     

Unpicking neurodegeneration in a dish with human pluripotent stem cells

Eukaryotic cell division is an orderly and timely process involving the error-free segregation of chromosomes and cytoplasmic components to give rise to two separate daughter cells. Defects in genome maintenance mechanisms such as cell cycle checkpoints and DNA repair can impact the segregation of the genome during mitosis leading to multiple chromosomal imbalances. In mammals, the DNA damage checkpoint effector Checkpoint Kinase 1 (Chk1) is essential for responses to DNA replication errors, external DNA damage, and chromatin breaks. We reported recently that Chk1 also was essential for chromosome segregation and completion of cytokinesis to prevent genomic instability. Our studies demonstrated that Chk1 deficiency in mitotic cells causes chromosome mis-alignment, lagging chromosomes, chromosome mis- segregation, cytokinetic regression, and binucleation. In addition, abrogation of Chk1 resulted in aberrant localization of mitotic Aurora B kinase at the metaphase plate, anaphase spindle midzone, and cytokinetic midbody as studied both in various cell lines and in a mouse model. Therefore, inappropriate regulation of Chk1 levels during cell cycle progression will result in failed cell division and enhanced genomic instability.     

Oncogenic RAS induces accelerated transition through G2/M and promotes defects in the G2 DNA damage and mitotic spindle checkpoints

Activating mutations of RAS are prevalent in thyroid follicular neoplasms, which commonly have chromosomal losses and gains. In thyroid cells, acute expression of HRAS(V12) increases the frequency of chromosomal abnormalities within one or two cell cycles, suggesting that RAS oncoproteins may interfere with cell cycle checkpoints required for maintenance of a stable genome. To explore this, PCCL3 thyroid cells with conditional expression of HRAS(V12) or HRAS(V12) effector mutants were presynchronized at the G(1)/S boundary, followed by activation of expression of RAS mutants and release from the cell cycle block. Expression of HRAS(V12) accelerated the G(2)/M phase by approximately 4 h and promoted bypass of the G(2) DNA damage and mitotic spindle checkpoints. Accelerated passage through G(2)/M and bypass of the G(2) DNA damage checkpoint, but not bypass of the mitotic spindle checkpoint, required activation of mitogen-activated protein kinase (MAPK). However, selective activation of the MAPK pathway was not sufficient to disrupt the G(2) DNA damage checkpoint, because cells arrested appropriately in G(2) despite conditional expression of HRAS(V12,S35) or BRAF(V600E). By contrast to the MAPK requirement for radiation-induced G(2) arrest, RAS-induced bypass of the mitotic spindle checkpoint was not prevented by pretreatment with MEK inhibitors. These data support a direct role for the MAPK pathway in control of G(2) progression and regulation of the G(2) DNA damage checkpoint. We propose that oncogenic RAS activation may predispose cells to genomic instability through both MAPK-dependent and independent pathways that affect critical checkpoints in G(2)/M.     

ZW10 links mitotic checkpoint signaling to the structural kinetochore

The mitotic checkpoint ensures that chromosomes are divided equally between daughter cells and is a primary mechanism preventing the chromosome instability often seen in aneuploid human tumors. ZW10 and Rod play an essential role in this checkpoint. We show that in mitotic human cells ZW10 resides in a complex with Rod and Zwilch, whereas another ZW10 partner, Zwint-1, is part of a separate complex of structural kinetochore components including Mis12 and Ndc80-Hec1. Zwint-1 is critical for recruiting ZW10 to unattached kinetochores. Depletion from human cells or Xenopus egg extracts is used to demonstrate that the ZW10 complex is essential for stable binding of a Mad1-Mad2 complex to unattached kinetochores. Thus, ZW10 functions as a linker between the core structural elements of the outer kinetochore and components that catalyze generation of the mitotic checkpoint-derived "stop anaphase" inhibitor.     

Upregulated Op18/stathmin activity causes chromosomal instability through a mechanism that evades the spindle assembly checkpoint

Op18/stathmin (Op18) is a microtubule-destabilizing protein that is phosphorylation-inactivated during mitosis and its normal function is to govern tubulin subunit partitioning during interphase. Human tumors frequently overexpress Op18 and a tumor-associated Q18→E mutation has been identified that confers hyperactivity, destabilizes spindle microtubules, and causes mitotic aberrancies, polyploidization, and chromosome loss in K562 leukemia cells. Here we determined whether wild-type and mutant Op18 have the potential to cause chromosomal instability by some means other than interference with spindle assembly, and thereby bypassing the spindle assembly checkpoint. Our approach was based on Op18 derivatives with distinct temporal order of activity during mitosis, conferred either by differential phosphorylation inactivation or by anaphase-specific degradation through fusion with the destruction box of cyclin B1. We present evidence that excessive Op18 activity generates chromosomal instability through interference occurring subsequent to the metaphase-to-anaphase transition, which reduces the fidelity of chromosome segregation to spindle poles during anaphase. Similar to uncorrected merotelic attachment, this mechanism evades detection by the spindle assembly checkpoint and thus provides an additional route to chromosomal instability.     

A Role for Mitogen-activated Protein Kinase in the Spindle Assembly Checkpoint in XTC Cells

The spindle assembly checkpoint prevents cells whose spindles are defective or chromosomes are misaligned from initiating anaphase and leaving mitosis. Studies of Xenopus egg extracts have implicated the Erk2 mitogen-activated protein kinase (MAP kinase) in this checkpoint. Other studies have suggested that MAP kinases might be important for normal mitotic progression. Here we have investigated whether MAP kinase function is required for mitotic progression or the spindle assembly checkpoint in vivo in Xenopus tadpole cells (XTC). We determined that Erk1 and/or Erk2 are present in the mitotic spindle during prometaphase and metaphase, consistent with the idea that MAP kinase might regulate or monitor the status of the spindle. Next, we microinjected purified recombinant XCL100, a Xenopus MAP kinase phosphatase, into XTC cells in various stages of mitosis to interfere with MAP kinase activation. We found that mitotic progression was unaffected by the phosphatase. However, XCL100 rendered the cells unable to remain arrested in mitosis after treatment with nocodazole. Cells injected with phosphatase at prometaphase or metaphase exited mitosis in the presence of nocodazole—the chromosomes decondensed and the nuclear envelope re-formed—whereas cells injected with buffer or a catalytically inactive XCL100 mutant protein remained arrested in mitosis. Coinjection of constitutively active MAP kinase kinase-1, which opposes XCL100's effects on MAP kinase, antagonized the effects of XCL100. Since the only known targets of MAP kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells.     

Stem cells, senescence, neosis and self-renewal in cancer

We describe the basic tenets of the current concepts of cancer biology, and review the recent advances on the suppressor role of senescence in tumor growth and the breakdown of this barrier during the origin of tumor growth. Senescence phenotype can be induced by (1) telomere attrition-induced senescence at the end of the cellular mitotic life span (MLS*) and (2) also by replication history-independent, accelerated senescence due to inadvertent activation of oncogenes or by exposure of cells to genotoxins. Tumor suppressor genes p53/pRB/p16INK4A and related senescence checkpoints are involved in effecting the onset of senescence. However, senescence as a tumor suppressor mechanism is a leaky process and senescent cells with mutations or epimutations in these genes escape mitotic catastrophe-induced cell death by becoming polyploid cells. These polyploid giant cells, before they die, give rise to several cells with viable genomes via nuclear budding and asymmetric cytokinesis. This mode of cell division has been termed neosis and the immediate neotic offspring the Raju cells. The latter inherit genomic instability and transiently display stem cell properties in that they differentiate into tumor cells and display extended, but, limited MLS, at the end of which they enter senescent phase and can undergo secondary/tertiary neosis to produce the next generation of Raju cells. Neosis is repeated several times during tumor growth in a non-synchronized fashion, is the mode of origin of resistant tumor growth and contributes to tumor cell heterogeneity and continuity. The main event during neosis appears to be the production of mitotically viable daughter genome after epigenetic modulation from the non-viable polyploid genome of neosis mother cell (NMC). This leads to the growth of resistant tumor cells. Since during neosis, spindle checkpoint is not activated, this may give rise to aneuploidy. Thus, tumor cells also are destined to die due to senescence, but may escape senescence due to mutations or epimutations in the senescent checkpoint pathway. A historical review of neosis-like events is presented and implications of neosis in relation to the current dogmas of cancer biology are discussed. Genesis and repetitive re-genesis of Raju cells with transient "stemness" via neosis are of vital importance to the origin and continuous growth of tumors, a process that appears to be common to all types of tumors. We suggest that unlike current anti-mitotic therapy of cancers, anti-neotic therapy would not cause undesirable side effects. We propose a rational hypothesis for the origin and progression of tumors in which neosis plays a major role in the multistep carcinogenesis in different types of cancers. We define cancers as a single disease of uncontrolled neosis due to failure of senescent checkpoint controls.     

The IKK Inhibitor BMS-345541 Affects Multiple Mitotic Cell Cycle Transitions

The IκB kinase (IKK) complex controls processes such as inflammation, immune responses, cell survival and the proliferation of both normal and tumor cells. By activating NFκB, the IKK complex contributes to G1/S transition and first evidence has been presented that IKKα also regulates entry into mitosis. At what stage IKK is required and whether IKK also contributes to progression through mitosis and cytokinesis, however, has not yet been determined. In this study, we use BMS-345541, a potent allosteric small molecule inhibitor of IKK, to inhibit IKK specifically during G2 and during mitosis. We show that BMS-345541 affects several mitotic cell-cycle transitions, including mitotic entry, prometaphase to anaphase progression and cytokinesis. Adding BMS-345541 to the cells released from arrest in S-phase blocked the activation of aurora A, B and C, Cdk1 activation and histone H3 phosphorylation. Additionally, treatment of the mitotic cells with BMS-345541 resulted in precocious cyclin B1 and securin degradation, defective chromosome separation and improper cytokinesis. BMS-345541 was also found to override the spindle checkpoint in nocodazole-arrested cells. In vitro kinase assays using BMS-345541 indicate that these effects are not primarily due to a direct inhibitory effect of BMS-345541 on mitotic kinases such as Cdk1, Aurora A or B, Plk1 or NEK2. This study points towards a new potential role of IKK in cell cycle progression. Since deregulation of the cell-cycle is one of the hallmarks of tumor formation and progression, the newly discovered level of BMS 345541 function could be useful for cell-cycle control studies and may provide valuable clues for the design of future therapeutics.     

The Tumor Cell and Telomerase

Imbalanced activity of the mechanism that controls cell division is a prerequisite for malignant transformation of a normal cell. The present review considers this multi-step mechanism, which is usually called the G1-S checkpoint. Besides, tumor cells are characterized by the presence of telomerase, an enzyme responsible for restoration of chromosome ends after replication and thus providing for unlimited cell division. The main point of the present article is to find out whether the activation of telomerase is controlled by the G1-S checkpoint or does not depend on it. The principal components of the G1-S checkpoint, such as cyclin-dependent kinases, retinoblastoma and E2F proteins, control the activity of telomerase. In their turn, they accumulate and transmit signals from various sources inside and outside the cell. Thus, various changes in tumor cells can activate telomerase through the G1-S checkpoint. Such are the suggested effects on telomerase of Myc, p53, Waf1, protein kinases B and C, Wnt5A, TGFbeta, WT1, and estrogens. However, Myc, p53, WT1, estrogens, protein kinases B and C, and TGFbeta can also directly influence telomerase independently of the G1-S checkpoint mechanism. Moreover, in 30% of human tumors the gene of the key subunit of telomerase (hTERT) is amplified, possibly due to chromosomal rearrangements unassociated with the activity of the G1-S checkpoint. Thus, telomerase seems to be activated not by a single agent but due to combined action of various factors, both with involvement of the G1-S checkpoint mechanism and independently of it.     

Association of MAD2 expression with mitotic checkpoint competence in hepatoma cells

Chromosomal instability (CIN) refers to high rates of chromosomal gains and losses and is a major cause of genomic instability of cells. It is thought that CIN caused by loss of mitotic checkpoint contributes to carcinogenesis. In this study, we evaluated the competence of mitotic checkpoint in hepatoma cells and investigated the cause of mitotic checkpoint defects. We found that 6 (54.5%) of the 11 hepatoma cell lines were defective in mitotic checkpoint control as monitored by mitotic indices and flow-cytometric analysis after treatment with microtubule toxins. Interestingly, all 6 hepatoma cell lines with defective mitotic checkpoint showed significant underexpression of mitotic arrest deficient 2 (MAD2), a key mitotic checkpoint protein. The level of MAD2 underexpression was significantly associated with defective mitotic checkpoint response (p<0.001). In addition, no mutations were found in the coding sequences of MAD2 in all 11 hepatoma cell lines. Our findings suggest that MAD2 deficiency may cause a mitotic checkpoint defect in hepatoma cells.