Targeting mitotic exit for cancer treatment

For ordered mitotic progression, various proteins have to be regulated by an ubiquitin ligase, the anaphase-promoting complex or cyclosome (APC/C) with appropriate timing. Recent studies have implied that the activity of APC/C also contributes to release of mitotic checkpoint complexes (MCCs) from its target Cdc20 in the process of silencing the spindle assembly checkpoint (SAC). Here we describe a temperature-sensitive mutant (ubc11-P93L) in which cell cycle progression is arrested at mitosis. The mutant grows normally at the restrictive temperature when SAC is inactivated, suggesting that the arrest is not due to abnormal spindle assembly, but rather due to prolonged activation of SAC. Supporting this notion, MCCs remain bound to APC/C even when SAC is satisfied. The ubc11+ gene encodes one of the two E2 enzymes required for progression through mitosis in fission yeast. Remarkably, Slp1 (a fission yeast homolog of Cdc20), which is degraded in an APC/C-dependent manner, stays stable throughout the cell cycle in the ubc11-P93L mutant lacking the functional SAC. Other APC/C substrates, in contrast, were degraded on schedule. We have also found that a loss of Ubc4, the other E2 required for progression through mitosis, does not affect the stability of Slp1. We propose that each of the two E2 enzymes is responsible for collaborating with APC/C for a specific set of substrates, and that Ubc11 is responsible for regulating Slp1 with APC/C for silencing the SAC.

The ABBA Motif Binds APC/C Activators and Is Shared by APC/C Substrates and Regulators

Mammalian oocytes execute a unique meiotic programme involving 2 arrest stages and an unusually protracted preamble to chromosome segregation during the first meiotic division (meiosis I). How mammalian oocytes successfully navigate their exceptional meiotic journey has long been a question of immense interest. Understanding the minutiae of female mammalian meiosis I is not merely of academic interest as 80–90% of human aneuploidy is the consequence of errors arising at this particular stage of oocyte maturation, a stage with a peculiar vulnerability to aging. Recent evidence indicates that oocytes employ many of the same cast of proteins during meiosis I as somatic cells do during mitosis, often to execute similar tasks, but intriguingly, occasionally delegate them to unexpected and unprecedented roles. This is epitomised by the master cell-cycle regulon, the anaphase-promoting complex or cyclosome (APC/C), acting in concert with a critical APC/C-targeted surveillance mechanism, the spindle assembly checkpoint (SAC). Together, the APC/C and the SAC are among the most influential entities overseeing the fidelity of cell-cycle progression and the precision of chromosome segregation. Here I review the current status of pivotal elements underpinning homologue disjunction in mammalian oocytes including spindle assembly, critical biochemical anaphase-initiating events, APC/C activity and SAC signalling along with contemporary findings relevant to progressive oocyte SAC dysfunction as a model for age-related human aneuploidy.

The anaphase-promoting complex or cyclosome (APC/C) is a large ubiquitin ligase essential for the mitotic and meiotic cell cycle. It ligates chains of ubiquitin to its target proteins marking them for proteolysis by the 26S proteasome. APC/C activity and thereby stage-specific proteolysis are precisely regulated during the cell cycle to maintain the chronology of key events. This work characterized some of the mechanisms involved in APC/C regulation.In the first part, the requirement of post-translational modification with the small ubiquitin-like modifier SUMO on efficient APC/C-mediated protein degradation was tested. The anaphase inhibitor protein, Pds1, was found to be stabilized in cells defective for SUMOylation and due to this altered degradation, cells displayed defects in chromosome segregation. Furthermore also proteolysis of other APC/C target proteins, such as the mitotic cyclins Clb2, Clb3 and the polo-like kinase Cdc5, was delayed whereas degradation of unstable proteins that are not APC/C targets was unaffected. SUMOylation does not seem to be required for proteolysis in general but specifically for APC/C-mediated degradation.In the second part, cyclin Clb5 degradation and its importance for the exit from mitosis was analyzed. Clb5, which was previously identified as a target of the APC/C, was found to be degraded by a second, alternative mechanism independently of APC/C. It could be shown that the SCF ubiquitin-ligase is not involved in this process. Indeed, Clb5 is unstable even in mutant cells defective in both ubiquitin ligases, APC/C and SCF. The APC/C-independent mechanism remains to be identified, but under physiological conditions, this mechanism is obviously sufficient for Clb5 degradation in the cell cycle. Only under abnormal conditions, such as CLB5 overexpression, APC/C becomes essential for Clb5 degradation. In the third part of this work the regulation of the APC/C in meiosis by the meiosis-specific kinase Ime2 was investigated. Ime2, a negative APC/C regulator, is a highly unstable protein, and thus proteolysis could bear a regulatory mechanism. To investigate this, a truncated Ime2 protein was analyzed with regard to its functionality to induce a mitotic cell cycle arrest and to phosphorylate Cdh1. We showed that this truncated Ime2DC, lacking 241 amino acids at its C-terminus, is despite the truncation highly active and stable throughout meiosis. Furthermore, we found that homozygous strains for the truncated IME2 allele display a severe defect in meiosis II. These results indicate that either high Ime2 levels or loss of an unknown function causes the defect in meiosis II.

The spindle checkpoint is a mitotic surveillance system which ensures equal segregation of sister chromatids. It delays anaphase onset by inhibiting the action of the E3 ubiquitin ligase known as the anaphase promoting complex or cyclosome (APC/C). Mad3/BubR1 is a key component of the mitotic checkpoint complex (MCC) which binds and inhibits the APC/C early in mitosis. Mps1Mph1 kinase is critical for checkpoint signalling and MCC-APC/C inhibition, yet few substrates have been identified. Here we identify Mad3 as a substrate of fission yeast Mps1Mph1 kinase. We map and mutate phosphorylation sites in Mad3, producing mutants that are targeted to kinetochores and assembled into MCC, yet display reduced APC/C binding and are unable to maintain checkpoint arrests. We show biochemically that Mad3 phospho-mimics are potent APC/C inhibitors in vitro, demonstrating that Mad3p modification can directly influence Cdc20Slp1-APC/C activity. This genetic dissection of APC/C inhibition demonstrates that Mps1Mph1 kinase-dependent modifications of Mad3 and Mad2 act in a concerted manner to maintain spindle checkpoint arrests.

The Four Canonical TPR Subunits of Human APC/C Form Related Homo-Dimeric Structures and Stack in Parallel to Form a TPR Suprahelix

The existence of two forms of the chromosome passenger complex (CPC) in the mammalian oocyte has meant that its role in female meiosis has remained unclear. Here we use loss- and gain-of function approaches to assess the meiotic functions of one of the shared components of these complexes, INCENP, and of the variable kinase subunits, Aurora B or Aurora C. We show that either the depletion of INCENP or the combined inhibition of Aurora kinases B and C activates the anaphase-promoting complex or cyclosome (APC/C) before chromosomes have properly congressed in meiosis I and also prevents cytokinesis and hence extrusion of the first polar body. Overexpression of Aurora C also advances APC/C activation and results in cytokinesis failure in a high proportion of oocytes, indicative of a dominant effect on CPC function. Together, this points to roles for the meiotic CPC in functions similar to the mitotic roles of the complex: correcting chromosome attachment to microtubules, facilitating the spindle-assembly checkpoint (SAC) function and enabling cytokinesis. Surprisingly, overexpression of Aurora B leads to a failure of APC/C activation, stabilization of securin and consequently a failure of chiasmate chromosomes to resolve - a dominant phenotype that is completely suppressed by depletion of INCENP. Taken together with the differential distribution of Aurora proteins B and C on chiasmate chromosomes, this points to differential functions of the two forms of CPC in regulating the separation of homologous chromosomes in meiosis I.

There were several errors in this article. In the Cdc5 experiment in Fig. 3A, the anti-G6PD immunoblot image from the Spo13 experiment in Fig. 3B was accidentally duplicated and flipped horizontally. Also in Fig. 3A, the experiment labeled Nrm1 actually shows the results from the Mps1 experiment. In Fig. 4C, the Spo12 stability profile in the wild-type CDH1 strain is identical to that shown in Fig. 3B. In Fig. 4, B and C, a black bar is now used to indicate excision of lanes from the same exposure of the same immunoblot.

BackgroundThe spindle assembly checkpoint (SAC) inhibits anaphase progression in the presence of insufficient kinetochore-microtubule attachments, but cells can eventually override mitotic arrest by a process known as mitotic slippage or adaptation. This is a problem for cancer chemotherapy using microtubule poisons.ResultsHere we describe mitotic slippage in yeast bub2Δ mutant cells that are defective in the repression of precocious telophase onset (mitotic exit). Precocious activation of anaphase promoting complex/cyclosome (APC/C)-Cdh1 caused mitotic slippage in the presence of nocodazole, while the SAC was still active. APC/C-Cdh1, but not APC/C-Cdc20, triggered anaphase progression (securin degradation, separase-mediated cohesin cleavage, sister-chromatid separation and chromosome missegregation), in addition to telophase onset (mitotic exit), during mitotic slippage. This demonstrates that an inhibitory system not only of APC/C-Cdc20 but also of APC/C-Cdh1 is critical for accurate chromosome segregation in the presence of insufficient kinetochore-microtubule attachments.ConclusionsThe sequential activation of APC/C-Cdc20 to APC/C-Cdh1 during mitosis is central to accurate mitosis. Precocious activation of APC/C-Cdh1 in metaphase (pre-anaphase) causes mitotic slippage in SAC-activated cells. For the prevention of mitotic slippage, concomitant inhibition of APC/C-Cdh1 may be effective for tumor therapy with mitotic spindle poisons in humans.

The anaphase-promoting complex or cyclosome (APC/C) is an ubiquitin protein ligase that together with Cdc20 and Cdh1 targets mitotic proteins for degradation by the proteosome. APC-Cdc20 activity during mitosis triggers anaphase by destroying securin and cyclins. APC-Cdh1 promotes degradation of cyclins and other proteins during G(1). We show that loss of APC/C during embryogenesis is early lethal before embryonic day E6.5 (E6.5). To investigate the role of APC/C in quiescent cells, we conditionally inactivated the subunit Apc2 in mice. Deletion of Apc2 in quiescent hepatocytes caused re-entry into the cell cycle and arrest in metaphase, resulting in liver failure. Re-entry into the cell cycle either occurred without any proliferative stimulus or could be easily induced. We demonstrate that the APC has an additional function to prevent hepatocytes from unscheduled re-entry into the cell cycle.

Self-Renewal and Differentiation in Hematopoietic Stem and Progenitor Cells Is Controlled By the APC/C Coactivator Cdh1

The mitotic spindle assembly checkpoint (SAC) signaling has been emerging as an important promising mechanism in cancer therapy. A prolonged mitotic arrest in response to cancer chemotherapy induces apoptosis, mitotic catastrophe, mitotic slippage and senescence, depending on what cell line and/or inhibitor is studied. In consequence, a weakened or disturbed SAC signaling in human cancers might contribute to drug resistance both to spindle inhibitors and to DNA damaging agents. In several studies the mitotic arrest deficient 2 (MAD2α) has been implicated in the association between failure in the SAC signaling and chemotherapy response. In this study, we identified a novel splicing variant of MAD2, designated as MAD2γ. This isoform was ubiquitously expressed in several cancer cell lines and non-cancerous primary foreskin fibroblasts. When it was ectopically expressed, MAD2γ localized in the nucleus. Its overexpression in a fully functional SAC-competent cancer cell line, HCT116, reduced the mitotic index, suggesting SAC impairment. Furthermore, the endogenous overexpression of MAD2γ in testicular germ cell tumor patients was associated with a resistant response to cisplatin-based chemotherapy, whereas expression of MAD2α was not significantly different between resistant and sensitive patients. In addition, HCT116 raised significantly its endogenous expression of MAD2γ only in response to cisplatin, but not that of MAD2α. Our data suggest that MAD2γ may have an opposing role to MAD2α in the activation of the SAC signaling, and that its expression is associated to a resistant response to DNA-damaging agents, such as cisplatin-based chemotherapy. This highlight the importance to study alternative splicing that may compromise the SAC function, which has been regarded as a promise for cancer therapy. Citation Format: Alejandro Lopez-Saavedra, Rodrigo Caceres, Fernando Luna, Irwin Hernandez, Luis Alonso Herrera. MAD2γ, a new MAD2 isoform, is ubiquitously expressed in distinct cell lines, reduces mitotic arrest, and associates with resistance to cisplatin-based chemotherapy in testicular germ cell tumors. [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 5457. doi:10.1158/1538-7445.AM2015-5457

Mitotic spindle assembly is a critical step for cell division that happens once per cell cycle. The centrosome is a structural organelle that is important in spindle assembly as centrosomes act as the microtubule organizing centers at the spindle poles during mitosis. Deviation from normal spindle assembly can lead to activation of the spindle assembly checkpoint (SAC). Some of the most successful antimitotic drugs activate the SAC in order to arrest cancer cells in mitosis to induce subsequent apoptosis. Recently, kinesins and kinases implicated in spindle assembly have gained attention as possible therapeutic targets, as their functions rely on their ability to hydrolyze ATP. Our labs previous proteomic and genetic studies identified steroidogenic acute regulatory protein‐related lipid transfer domain containing 9 (STARD9) as a kinesin involved in spindle assembly. Its depletion invokes multipolar spindles in dividing cancer cells and most surprisingly, STARD9‐depleted cancer cells display increased Taxol sensitivity. STARD9 plays a functional role in the stabilization of pericentriolar material (PCM) during centriole maturation, but its mechanism of action and its regulation are not yet fully understood. To elucidate these mechanisms we have used multiple proteomic approaches to study each of STARD9's major domains. These approaches have identified putative interacting proteins that may play regulatory roles in PCM stabilization and we have validated some of these associations in vitro. We are currently focused on determining the importance of these STARD9 interactions for PCM stabilization. Our work looks to further the knowledge of STARD9's function within centrosome assembly and more broadly cancer cell division.Support or Funding InformationUCLA Tumor Cell Biology Training Program (USHHS Ruth L. Kirschstein Institutional National Research Service Award # T32 CA009056)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An Essential Function of the C. elegans Ortholog of TPX2 Is to Localize Activated Aurora A Kinase to Mitotic Spindles

How Do so Few Control so Many?