BUB1 and CENP-U, Primed by CDK1, Are the Main PLK1 Kinetochore Receptors in Mitosis.

Reflecting its pleiotropic functions, Polo-like kinase 1 (PLK1) localizes to various sub-cellular structures during mitosis. At kinetochores, PLK1 contributes to microtubule attachments and mitotic checkpoint signaling. Previous studies identified a wealth of potential PLK1 receptors at kinetochores, as well as requirements for various mitotic kinases, including BUB1, Aurora B, and PLK1 itself. Here, we combine ectopic localization, in vitro reconstitution, and kinetochore localization studies to demonstrate that most and likely all of the PLK1 is recruited through BUB1 in the outer kinetochore and centromeric protein U (CENP-U) in the inner kinetochore. BUB1 and CENP-U share a constellation of sequence motifs consisting of a putative PP2A-docking motif and two neighboring PLK1-docking sites, which, contingent on priming phosphorylation by cyclin-dependent kinase 1 and PLK1 itself, bind PLK1 and promote its dimerization. Our results rationalize previous observations and describe a unifying mechanism for recruitment of PLK1 to human kinetochores.

RIPK1 and Caspase-8 Ensure Chromosome Stability Independently of Their Role in Cell Death and Inflammation.

Receptor-interacting protein kinase (RIPK) 1 functions as a key mediator of tissue homeostasis via formation of Caspase-8 activating ripoptosome complexes, positively and negatively regulating apoptosis, necroptosis, and inflammation. Here, we report an unanticipated cell-death- and inflammation-independent function of RIPK1 and Caspase-8, promoting faithful chromosome alignment in mitosis and thereby ensuring genome stability. We find that ripoptosome complexes progressively form as cells enter mitosis, peaking at metaphase and disassembling as cells exit mitosis. Genetic deletion and mitosis-specific inhibition of Ripk1 or Caspase-8 results in chromosome alignment defects independently of MLKL. We found that Polo-like kinase 1 (PLK1) is recruited into mitotic ripoptosomes, where PLK1's activity is controlled via RIPK1-dependent recruitment and Caspase-8-mediated cleavage. A fine balance of ripoptosome assembly is required as deregulated ripoptosome activity modulates PLK1-dependent phosphorylation of downstream effectors, such as BUBR1. Our data suggest that ripoptosome-mediated regulation of PLK1 contributes to faithful chromosome segregation during mitosis.

Molecular contribution to embryonic aneuploidy and karyotypic complexity in initial cleavage divisions of mammalian development.

Embryonic aneuploidy is highly complex, often leading to developmental arrest, implantation failure or spontaneous miscarriage in both natural and assisted reproduction. Despite our knowledge of mitotic mis-segregation in somatic cells, the molecular pathways regulating chromosome fidelity during the error-prone cleavage-stage of mammalian embryogenesis remain largely undefined. Using bovine embryos and live-cell fluorescent imaging, we observed frequent micro-/multi-nucleation of mis-segregated chromosomes in initial mitotic divisions that underwent unilateral inheritance, re-fused with the primary nucleus or formed a chromatin bridge with neighboring cells. A correlation between a lack of syngamy, multipolar divisions and asymmetric genome partitioning was also revealed, and single-cell DNA-seq showed propagation of primarily non-reciprocal mitotic errors. Depletion of the mitotic checkpoint protein BUB1B (also known as BUBR1) resulted in similarly abnormal nuclear structures and cell divisions, as well as chaotic aneuploidy and dysregulation of the kinase-substrate network that mediates mitotic progression, all before zygotic genome activation. This demonstrates that embryonic micronuclei sustain multiple fates, provides an explanation for blastomeres with uniparental origins, and substantiates defective checkpoints and likely other maternally derived factors as major contributors to the karyotypic complexity afflicting mammalian preimplantation development.

Hyper-active RAS/MAPK introduces cancer-specific mitotic vulnerabilities.

Aneuploidy, the incorrect number of whole chromosomes, is a common feature of tumors that contributes to their initiation and evolution. Preventing aneuploidy requires properly functioning kinetochores, which are large protein complexes assembled on centromeric DNA that link mitotic chromosomes to dynamic spindle microtubules and facilitate chromosome segregation. The kinetochore leverages at least two mechanisms to prevent aneuploidy: error correction and the spindle assembly checkpoint (SAC). BubR1, a factor involved in both processes, was identified as a cancer dependency and therapeutic target in multiple tumor types; however, it remains unclear what specific oncogenic pressures drive this enhanced dependency on BubR1 and whether it arises from BubR1's regulation of the SAC or error-correction pathways. Here, we use a genetically controlled transformation model and glioblastoma tumor isolates to show that constitutive signaling by RAS or MAPK is necessary for cancer-specific BubR1 vulnerability. The MAPK pathway enzymatically hyperstimulates a network of kinetochore kinases that compromises chromosome segregation, rendering cells more dependent on two BubR1 activities: counteracting excessive kinetochore-microtubule turnover for error correction and maintaining the SAC. This work expands our understanding of how chromosome segregation adapts to different cellular states and reveals an oncogenic trigger of a cancer-specific defect.

BUB1B monoallelic germline variants contribute to prostate cancer predisposition by triggering chromosomal instability.

BACKGROUND: Prostate cancer (PrCa) is the most frequently diagnosed cancer in men. Variants in known moderate- to high-penetrance genes explain less than 5% of the cases arising at early-onset (< 56 years) and/or with familial aggregation of the disease. Considering that BubR1 is an essential component of the mitotic spindle assembly checkpoint, we hypothesized that monoallelic BUB1B variants could be sufficient to fuel chromosomal instability (CIN), potentially triggering (prostate) carcinogenesis. METHODS: To unveil BUB1B as a new PrCa predisposing gene, we performed targeted next-generation sequencing in germline DNA from 462 early-onset/familial PrCa patients and 1,416 cancer patients fulfilling criteria for genetic testing for other hereditary cancer syndromes. To explore the pan-cancer role of BUB1B, we used in silico BubR1 molecular modeling, in vitro gene-editing, and ex vivo patients' tumors and peripheral blood lymphocytes. RESULTS: Rare BUB1B variants were found in ~ 1.9% of the early-onset/familial PrCa cases and in ~ 0.6% of other cancer patients fulfilling criteria for hereditary disease. We further show that BUB1B variants lead to decreased BubR1 expression and/or stability, which promotes increased premature chromatid separation and, consequently, triggers CIN, driving resistance to Taxol-based therapies. CONCLUSIONS: Our study shows that different BUB1B variants may uncover a trigger for CIN-driven carcinogenesis, supporting the role of BUB1B as a (pan)-cancer predisposing gene with potential impact on genetic counseling and treatment decision-making.

The Mitotic Checkpoint Complex Requires an Evolutionary Conserved Cassette to Bind and Inhibit Active APC/C.

The Spindle Assembly Checkpoint (SAC) ensures genomic stability by preventing sister chromatid separation until all chromosomes are attached to the spindle. It catalyzes the production of the Mitotic Checkpoint Complex (MCC), which inhibits Cdc20 to inactivate the Anaphase Promoting Complex/Cyclosome (APC/C). Here we show that two Cdc20-binding motifs in BubR1 of the recently identified ABBA motif class are crucial for the MCC to recognize active APC/C-Cdc20. Mutating these motifs eliminates MCC binding to the APC/C, thereby abolishing the SAC and preventing cells from arresting in response to microtubule poisons. These ABBA motifs flank a KEN box to form a cassette that is highly conserved through evolution, both in the arrangement and spacing of the ABBA-KEN-ABBA motifs, and association with the amino-terminal KEN box required to form the MCC. We propose that the ABBA-KEN-ABBA cassette holds the MCC onto the APC/C by binding the two Cdc20 molecules in the MCC-APC/C complex.

E3-ubiquitin ligase, FBXW7 regulates mitotic progression by targeting BubR1 for ubiquitin-mediated degradation.

Faithful chromosome segregation requires correct attachment of kinetochores with the spindle microtubules. Erroneously-attached kinetochores recruit proteins to activate Spindle assembly checkpoint (SAC), which senses the errors and signals cells to delay anaphase progression for error correction. Temporal control of the levels of SAC activating-proteins is critical for checkpoint activation and silencing, but its mechanism is not fully understood. Here, we show that E3 ubiquitin ligase, SCF-FBXW7 targets BubR1 for ubiquitin-mediated degradation and thereby controls SAC in human cells. Depletion of FBXW7 results in prolonged metaphase arrest with increased stabilization of BubR1 at kinetochores. Similar kinetochore stabilization is also observed for BubR1-interacting protein, CENP-E. FBXW7 induced ubiquitination of both BubR1 and the BubR1-interacting kinetochore-targeting domain of CENP-E, but CENP-E domain degradation is dependent on BubR1. Interestingly, Cdk1 inhibition disrupts FBXW7-mediated BubR1 targeting and further, phospho-resistant mutation of Cdk1-targeted phosphorylation site, Thr 620 impairs BubR1-FBXW7 interaction and FBXW7-mediated BubR1 ubiquitination, supporting its role as a phosphodegron for FBXW7. The results demonstrate SCF-FBXW7 as a key regulator of spindle assembly checkpoint that controls stability of BubR1 and its associated CENP-E at kinetochores. They also support that upstream Cdk1 specific BubR1 phosphorylation signals the ligase to activate the process.

The Mre11-Rad50-Nbs1 complex mediates the robust recruitment of Polo to DNA lesions during mitosis in Drosophila.

The DNA damage sensor Mre11-Rad50-Nbs1 complex and Polo kinase are recruited to DNA lesions during mitosis. However, their mechanism of recruitment is elusive. Here, using live-cell imaging combined with micro-irradiation of single chromosomes, we analyze the dynamics of Polo and Mre11 at DNA lesions during mitosis in Drosophila These two proteins display distinct kinetics. Whereas Polo kinetics at double-strand breaks (DSBs) are Cdk1-driven, Mre11 promptly but briefly associates with DSBs regardless of the phase of mitosis and re-associates with DSBs in the proceeding interphase. Mechanistically, Polo kinase activity is required for its own recruitment and that of the mitotic proteins BubR1 and Bub3 to DSBs. Moreover, depletion of Rad50 severely impaired Polo kinetics at mitotic DSBs. Conversely, ectopic tethering of Mre11 to chromatin was sufficient to recruit Polo. Our study highlights a novel pathway that links the DSB sensor Mre11-Rad50-Nbs1 complex and Polo kinase to initiate a prompt, decisive response to the presence of DNA damage during mitosis.

SIRT1 regulates mitotic catastrophe via autophagy and BubR1 signaling.

Mitotic catastrophe (MC) is a suppressive mechanism that mediates the elimination of mitosis-deficient cells through apoptosis, necrosis or senescence after M phase block. SIRT1 is involved in the regulation of several cellular processes, including autophagy. However, the relationship between SIRT1 and MC has been largely obscure. Our study highlights that SIRT1 might be involved in the regulation of MC. We have shown that degradation of the SIRT1 protein via proteasome and lysosomal pathway was accompanied by MC induced via BMH-21. Overexpression of SIRT1 alleviated MC by decreasing the proportion of apoptotic and multinuclear cells induced by G2/M block and triggered autophagy whereas knockdown of SIRT1 aggravated MC and repressed autophagy. Furthermore, we found that serum starvation triggered autophagy evidently generated lower MC whereas siRNA of ATG5/7 suppressed autophagy leading to higher MC. ChIP analysis revealed that SIRT1 could bind to the promoter of BubR1, a component of spindle assembly checkpoint (SAC), to upregulate its expression. Overexpression of BubR1 decreased MC whereas knockdown of BubR1 increased it. These results reveal that SIRT1 regulates MC through autophagy and BubR1 signaling, and provide evidence for SIRT1, autophagy and BubR1 being the potential cancer therapeutic targets.

BuGZ facilitates loading of spindle assembly checkpoint proteins to kinetochores in early mitosis.

BuGZ is a kinetochore component that binds to and stabilizes Bub3, a key player in mitotic spindle assembly checkpoint signaling. Bub3 is required for kinetochore recruitment of Bub1 and BubR1, two proteins that have essential and distinct roles in the checkpoint. Both Bub1 and BubR1 localize to kinetochores through interactions with Bub3, which are mediated through conserved GLEBS domains in both Bub1 and BubR1. BuGZ also has a GLEBS domain, which is required for its kinetochore localization as well, presumably mediated through Bub3 binding. Although much is understood about the requirements for Bub1 and BubR1 interaction with Bub3 and kinetochores, much less is known regarding BuGZ's requirements. Here, we used a series of mutants to demonstrate that BuGZ kinetochore localization requires only its core GLEBS domain, which is distinct from the requirements for both Bub1 and BubR1. Furthermore, we found that the kinetics of Bub1, BubR1, and BuGZ loading to kinetochores differ, with BuGZ localizing prior to BubR1 and Bub1. To better understand how complexes containing Bub3 and its binding partners are loaded to kinetochores, we carried out size-exclusion chromatography and analyzed Bub3-containing complexes from cells under different spindle assembly checkpoint signaling conditions. We found that prior to kinetochore formation, Bub3 is complexed with BuGZ but not Bub1 or BubR1. Our results point to a model in which BuGZ stabilizes Bub3 and promotes Bub3 loading onto kinetochores in early mitosis, which, in turn, facilitates Bub1 and BubR1 kinetochore recruitment and spindle assembly checkpoint signaling.

Discovery of Novel Agents on Spindle Assembly Checkpoint to Sensitize Vinorelbine-Induced Mitotic Cell Death Against Human Non-Small Cell Lung Cancers.

Non-small cell lung cancer (NSCLC) accounts about 80% of all lung cancers. More than two-thirds of NSCLC patients have inoperable, locally advanced or metastatic tumors. Non-toxic agents that synergistically potentiate cancer-killing activities of chemotherapeutic drugs are in high demand. YL-9 was a novel and non-cytotoxic compound with the structure related to sildenafil but showing much less activity against phosphodiesterase type 5 (PDE5). NCI-H460, an NSCLC cell line with low PDE5 expression, was used as the cell model. YL-9 synergistically potentiated vinorelbine-induced anti-proliferative and apoptotic effects in NCI-H460 cells. Vinorelbine induced tubulin acetylation and Bub1-related kinase (BUBR1) phosphorylation, a necessary component in spindle assembly checkpoint. These effects, as well as BUBR1 cleavage, were substantially enhanced in co-treatment with YL-9. Several mitotic arrest signals were enhanced under combinatory treatment of vinorelbine and YL-9, including an increase of mitotic spindle abnormalities, increased cyclin B1 expression, B-cell lymphoma 2 (Bcl-2) phosphorylation and increased phosphoproteins. Moreover, YL-9 also displayed synergistic activity in combining with vinorelbine to induce apoptosis in A549 cells which express PDE5. In conclusion. the data suggest that YL-9 is a novel agent that synergistically amplifies vinorelbine-induced NSCLC apoptosis through activation of spindle assembly checkpoint and increased mitotic arrest of the cell cycle. YL-9 shows the potential for further development in combinatory treatment against NSCLC.

The kinetochore proteins CENP-E and CENP-F directly and specifically interact with distinct BUB mitotic checkpoint Ser/Thr kinases.

The segregation of chromosomes during cell division relies on the function of the kinetochores, protein complexes that physically connect chromosomes with microtubules of the spindle. The metazoan proteins, centromere protein E (CENP-E) and CENP-F, are components of a fibrous layer of mitotic kinetochores named the corona. Several of their features suggest that CENP-E and CENP-F are paralogs: they are very large (comprising ∼2700 and 3200 residues, respectively), contain abundant predicted coiled-coil structures, are C-terminally prenylated, and are endowed with microtubule-binding sites at their termini. Moreover, CENP-E contains an ATP-hydrolyzing motor domain that promotes microtubule plus end-directed motion. Here, we show that both CENP-E and CENP-F are recruited to mitotic kinetochores independently of the main corona constituent, the Rod/Zwilch/ZW10 (RZZ) complex. We identified specific interactions of CENP-F and CENP-E with budding uninhibited by benzimidazole 1 (BUB1) and BUB1-related (BUBR1) mitotic checkpoint Ser/Thr kinases, respectively, paralogous proteins involved in mitotic checkpoint control and chromosome alignment. Whereas BUBR1 was dispensable for kinetochore localization of CENP-E, BUB1 was stringently required for CENP-F localization. Through biochemical reconstitution, we demonstrated that the CENP-E/BUBR1 and CENP-F/BUB1 interactions are direct and require similar determinants, a dimeric coiled-coil in CENP-E or CENP-F and a kinase domain in BUBR1 or BUB1. Our findings are consistent with the existence of structurally similar BUB1/CENP-F and BUBR1/CENP-E complexes, supporting the notion that CENP-E and CENP-F are evolutionarily related.

lncRNA CCAT1 is a biomarker for the proliferation and drug resistance of esophageal cancer via the miR-143/PLK1/BUBR1 axis.

Recent evidence indicates that long noncoding RNA colon cancer-associated transcript-1 (lncRNA CCAT1) is abundantly expressed in esophageal cancer and is closely related to the occurrence, development, invasion, metastasis, and drug resistance of this disease. However, the role and molecular mechanisms of CCAT1 in the cell proliferation and chemoresistance of esophageal cancer are largely unknown. The correlation between CCAT1 expression and drug resistance to cisplatin (CDDP) in esophageal squamous cell carcinoma (ESCC) cells was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and quantitative real-time polymerase chain reaction (qRT-PCR) assays. CCAT1 knockdown and miR-143 overexpression or inhibition were used to verify the effects on proliferation and drug resistance via MTT, western blotting, flow cytometry, and immunofluorescence assays. qRT-PCR and western blotting were applied to detect the potential regulatory relationship among CCAT1, miR-143, PLK1, and BUBR1. A xenograft tumor assay was performed to validate the role of CCAT1 in vivo. The expression of CCAT1 was positively correlated with drug resistance in several ESCC cell lines. CCAT1 knockdown and miR-143 overexpression inhibited cell proliferation and CDDP drug resistance. Moreover, the downstream target of CCAT1 was found to be miR-143, which can regulate the expression of PLK1 and BUBR1. In vivo assays showed that CCAT1 knockdown suppressed tumor growth and enhanced the sensitivity of tumors to CDDP in nude mice. Taken together, we discovered a novel mechanism by which CCAT1 promotes cell proliferation and enhances drug resistance by regulating the miR-143/PLK1/BUBR1 signaling axis both in vitro and in vivo. Our findings further suggest that lncRNA CCAT1 may be a potential therapeutic target for overcoming chemoresistance in esophageal cancer.

BubR1 Is Essential for Thio-Dimethylarsinic Acid-Induced Spindle Assembly Checkpoint and Mitotic Cell Death for Preventing the Accumulation of Abnormal Cells.

Thio-dimethylarsinic acid (thio-DMA) was detected in human urine after exposure to inorganic arsenic and arsenosugars consumed by marine algae. Our previous studies have shown that thio-DMA disturbed the cell cycle progression and arrested cells in mitosis, though the biological significance or the mechanism by which thio-DMA-induced mitotic phase accumulation occurs is yet to be understood. In this study, we showed that thio-DMA promotes the phosphorylation of BubR1 protein, which is one of the constituents of the spindle assembly checkpoint (SAC) complex and accumulates in the cell in mitotic phase. Binding of Mad2 to CDC20, also known as the marker of the mitotic checkpoint complex (MCC) formation during the activation of SAC, was enhanced and mitotic associated cell death by apoptosis was promoted in HeLa cells but not in HepG2 cells. Basal BubR1 protein level in HepG2 was 10-times lower than that of HeLa cells. Consequently, BubR1 knockdown HeLa cells were generated by small interfering RNA (siRNA) technique. The MCC formation and mitotic arrest induced by thio-DMA were completely inhibited in BubR1 knockdown cells. Moreover, BubR1 knockdown cells could survive in the medium containing higher concentrations of thio-DMA with some abnormalities such as larger cell size, huge nucleus, multiple nuclei, and abnormal DNA contents. Especially, cyclin B1 negative tetraploid cells, which signify interphase cells with tetraploid, increased and survived after 48-72 h treatment with thio-DMA. Thus, these results suggest that BubR1-mediated SAC activation and MCC formation are one of the defense systems for preventing the accumulation and survival of abnormal cells induced by thio-DMA.

Downregulation of Mad2 and BubR1 increase the malignant potential and nocodazole resistance by compromising spindle assembly checkpoint signaling pathway in cervical carcinogenesis.

AIM: To explore the involvement of Mad2 and BubR1 in cervical carcinogenesis. METHODS: The expressions of Mad2 and BubR1 in tissues of high-grade squamous intraepithelial lesions (HSIL), low-grade squamous intraepithelial lesions (LSIL) and chronic cervicitis were analyzed immunohistochemistrily and compared with those of p16INK4A . PEGFP-Mad2 and pEGFP-BubR1 were transfected into SiHa cells to overexpress Mad2 and BubR1 and Si-RNAs to knockdown. Cell viability was measured by cell counting kit-8 (CCK-8) assay. Migration and invasion capabilities were detected by Transwell. Propidium iodide staining with flow cytometry was used for cell cycle analysis and apoptosis was detected using Annexin V/7-AAD staining after nocodazole treatment. RESULTS: The expression of Mad2 was significantly lower in HSIL than those in chronic cervicitis and LSIL, however, the expression of BubR1 showed no significant differences. To detect HSIL in cervical lesions, Mad2 had a sensitivity of 88.44% and a specificity of 87.23%, Mad2 was less sensitive and more specific than p16INK4a . In SiHa cells, knockdown of Mad2 and BubR1 increased cell growth, reinforced invasion capacity and migration potency, inhibited apoptosis and decreased G2-phase distribution after nocodazole treatment. Oppositely, the overexpression strategies made cells show decreased malignant behaviors, raised apoptosis and increased G2-phase distribution. CONCLUSION: Mad2 negativity was specific to identify HSIL immunohistochemistrily. Downregulation of Mad2 and BubR1 increase the malignant behavior and nocodazole resistance of SiHa cells via causing spindle assembly checkpoint defect. This mechanism may contribute to cervical carcinogenesis and resistance to microtubule-targeting drugs.

SIRT2 induces the checkpoint kinase BubR1 to increase lifespan.

Mice overexpressing the mitotic checkpoint kinase gene BubR1 live longer, whereas mice hypomorphic for BubR1 (BubR1(H/H)) live shorter and show signs of accelerated aging. As wild-type mice age, BubR1 levels decline in many tissues, a process that is proposed to underlie normal aging and age-related diseases. Understanding why BubR1 declines with age and how to slow this process is therefore of considerable interest. The sirtuins (SIRT1-7) are a family of NAD(+)-dependent deacetylases that can delay age-related diseases. Here, we show that the loss of BubR1 levels with age is due to a decline in NAD(+) and the ability of SIRT2 to maintain lysine-668 of BubR1 in a deacetylated state, which is counteracted by the acetyltransferase CBP. Overexpression of SIRT2 or treatment of mice with the NAD(+) precursor nicotinamide mononucleotide (NMN) increases BubR1 abundance in vivo. Overexpression of SIRT2 in BubR1(H/H) animals increases median lifespan, with a greater effect in male mice. Together, these data indicate that further exploration of the potential of SIRT2 and NAD(+) to delay diseases of aging in mammals is warranted.

Functional and Structural Characterization of Bub3·BubR1 Interactions Required for Spindle Assembly Checkpoint Signaling in Human Cells.

The spindle assembly checkpoint (SAC) is an essential safeguarding mechanism devised to ensure equal chromosome distribution in daughter cells upon mitosis. The proteins Bub3 and BubR1 are key components of the mitotic checkpoint complex, an essential part of the molecular machinery on which the SAC relies. In the present work we have performed a detailed functional and biochemical characterization of the interaction between human Bub3 and BubR1 in cells and in vitro Our results demonstrate that genetic knockdown of Bub3 abrogates the SAC, promotes apoptosis, and inhibits the proliferation of human cancer cells. We also show that the integrity of the human mitotic checkpoint complex depends on the specific recognition between BubR1 and Bub3, for which the BubR1 Gle2 binding sequence motif is essential. This 1:1 binding event is high affinity, enthalpy-driven and with slow dissociation kinetics. The affinity, kinetics, and thermodynamic parameters of the interaction are differentially modulated by small regions in the N and C termini of the Gle2 binding domain sequence, suggesting the existence of "hotspots" for this protein-protein interaction. Furthermore, we show that specific disruption of endogenous BubR1·Bub3 complexes in human cancer cells phenocopies the effects observed in gene targeting experiments. Our work enhances the current understanding of key members of the SAC and paves the road for the pursuit of novel targeted cancer therapies based on SAC inhibition.

Emerging links among Chromosome Instability (CIN), cancer, and aging.

Aneuploidy was predicted to cause cancer. To test the prediction, various Chromosome Instability (CIN) mice models that carry transgenic mutations in mitotic regulators have been created. The availability of these mice has aided researchers in discovering connections between CIN, cancer, and aging. This review will focus on recent interdisciplinary findings regarding how CIN and aneuploidy affect carcinogenesis, immune dysfunction, and aging. High CIN can be generated in vivo by various intrinsic alterations (e.g., gene mutation, epigenetic modification) and extrinsic/environmental challenges (e.g., biological, chemical, biophysical), while immune surveillance, cell death, and natural turnover can remove cells with CIN. CIN itself is mutagenic and may cause further cellular mutations, which can be carcinogenic. Mitotically damaged cells can activate senescence-related tumor suppressors (e.g., p21WAF1 , p27KIP1 , p16INK4A ), which may lead to tissue-level senescence/aging through inflammatory paracrine mechanisms called Senescence-Associated Secretory Phenotype (SASP) and Senescence Inflammatory Response (SIR). Organs with high CIN show altered gene expressions in both organ-specific and non-specific manners. Organ-specific gene expression signatures include activation of oncogenic pathways. Non-organ-specific gene expression signatures include metabolic changes and downregulations in immune functions. Immune surveillance normally targets senescent cells and tetraploid cells, a form of aneuploidy, for elimination. However, with partial immune dysfunction, immune surveillance is weakened with systemic CIN. In this case, more senescent cells and aneuploid cells survive, which further leads to an inflammatory, pro-tumorigenic, and senescent/aging microenvironment. We also discuss how we may intervene in this sequence of events to prevent CIN- or age-related carcinogenesis and/or some aspects of tissue aging. © 2016 Wiley Periodicals, Inc.

Oncoprotein YAP regulates the spindle checkpoint activation in a mitotic phosphorylation-dependent manner through up-regulation of BubR1.

The transcriptional co-activator YAP (Yes-associated protein) functions as an oncogene; however, it is largely unclear how YAP exerts its oncogenic role. In this study, we further explored the functional significance of YAP and its mitotic phosphorylation in the spindle checkpoint. We found that the dynamic mitotic phosphorylation of YAP was CDC14-dependent. We also showed that YAP was required for the spindle checkpoint activation induced by spindle poisons. Mitotic phosphorylation of YAP was required for activation of the spindle checkpoint. Furthermore, enhanced expression of active YAP hyperactivated the spindle checkpoint and induced mitotic defects in a mitotic phosphorylation-dependent manner. Mechanistically, we documented that mitotic phosphorylation of YAP controlled transcription of genes associated with the spindle checkpoint. YAP constitutively associated with BubR1 (BUB1-related protein kinase), and knockdown of BubR1 relieved YAP-driven hyperactivation of the spindle checkpoint. Finally, we demonstrated that YAP promoted epithelial cell invasion via both mitotic phosphorylation and BubR1-dependent mechanisms. Together, our results reveal a novel link between YAP and the spindle checkpoint and indicate a potential mechanism underlying the oncogenic function of YAP through dysregulation of the spindle checkpoint.

EDD, a ubiquitin-protein ligase of the N-end rule pathway, associates with spindle assembly checkpoint components and regulates the mitotic response to nocodazole.

In this work, we identify physical and genetic interactions that implicate E3 identified by differential display (EDD) in promoting spindle assembly checkpoint (SAC) function. During mitosis, the SAC initiates a mitotic checkpoint in response to chromosomes with kinetochores unattached to spindle pole microtubules. Similar to Budding uninhibited by benzimidazoles-related 1 (BUBR1) siRNA, a bona fide SAC component, EDD siRNA abrogated G2/M accumulation in response to the mitotic destabilizing agent nocodazole. Furthermore, EDD siRNA reduced mitotic cell viability and, in nocodazole-treated cells, increased expression of the promitotic progression protein cell division cycle 20 (CDC20). Copurification studies also identified physical interactions with CDC20, BUBR1, and other components of the SAC. Taken together, these observations highlight the potential role of EDD in regulating mitotic progression and the cellular response to perturbed mitosis.