Simvastatin activates the spindle assembly checkpoint and causes abnormal cell division by modifying small GTPases.

Simvastatin is an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which is a rate-limiting enzyme of the cholesterol synthesis pathway. It has been used clinically as a lipid-lowering agent to reduce low-density lipoprotein (LDL) cholesterol levels. In addition, antitumor activity has been demonstrated. Although simvastatin attenuates the prenylation of small GTPases, its effects on cell division in which small GTPases play an important role, have not been examined as a mechanism underlying its cytostatic effects. In this study, we determined its effect on cell division. Cell cycle synchronization experiments revealed a delay in mitotic progression in simvastatin-treated cells at concentrations lower than the IC50. Time-lapse imaging analysis indicated that the duration of mitosis, especially from mitotic entry to anaphase onset, was prolonged. In addition, simvastatin increased the number of cells exhibiting misoriented anaphase/telophase and bleb formation. Inhibition of the spindle assembly checkpoint (SAC) kinase Mps1 canceled the mitotic delay. Additionally, the number of cells exhibiting kinetochore localization of BubR1, an essential component of SAC, was increased, suggesting an involvement of SAC in the mitotic delay. Enhancement of F-actin formation and cell rounding at mitotic entry indicates that cortical actin dynamics were affected by simvastatin. The cholesterol removal agent methyl-ß-cyclodextrin (MßCD) accelerated mitotic progression differently from simvastatin, suggesting that cholesterol loss from the plasma membrane is not involved in the mitotic delay. Of note, the small GTPase RhoA, which is a critical factor for cortical actin dynamics, exhibited upregulated expression. In addition, Rap1 was likely not geranylgeranylated. Our results demonstrate that simvastatin affects actin dynamics by modifying small GTPases, thereby activating the spindle assembly checkpoint and causing abnormal cell division.

The Arabidopsis BUB1/MAD3 family protein BMF3 requires BUB3.3 to recruit CDC20 to kinetochores in spindle assembly checkpoint signaling.

The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation during cell division by monitoring kinetochore-microtubule attachment. Plants produce both sequence-conserved and diverged SAC components, and it has been largely unknown how SAC activation leads to the assembly of these proteins at unattached kinetochores to prevent cells from entering anaphase. In Arabidopsis thaliana, the noncanonical BUB3.3 protein was detected at kinetochores throughout mitosis, unlike MAD1 and the plant-specific BUB1/MAD3 family protein BMF3 that associated with unattached chromosomes only. When BUB3.3 was lost by a genetic mutation, mitotic cells often entered anaphase with misaligned chromosomes and presented lagging chromosomes after they were challenged by low doses of the microtubule depolymerizing agent oryzalin, resulting in the formation of micronuclei. Surprisingly, BUB3.3 was not required for the kinetochore localization of other SAC proteins or vice versa. Instead, BUB3.3 specifically bound to BMF3 through two internal repeat motifs that were not required for BMF3 kinetochore localization. This interaction enabled BMF3 to recruit CDC20, a downstream SAC target, to unattached kinetochores. Taken together, our findings demonstrate that plant SAC utilizes unconventional protein interactions for arresting mitosis, with BUB3.3 directing BMF3's role in CDC20 recruitment, rather than the recruitment of BUB1/MAD3 proteins observed in fungi and animals. This distinct mechanism highlights how plants adapted divergent versions of conserved cell cycle machinery to achieve specialized SAC control.

Spindle checkpoint activation by fungal orthologs of the S. cerevisiae Mps1 kinase.

There is an ongoing need for antifungal agents to treat humans. Identification of new antifungal agents can be based on screening compounds using whole cell assays. Screening compounds that target a particular molecule is possible in budding yeast wherein sophisticated strain engineering allows for controlled expression of endogenous or heterologous genes. We have considered the yeast Mps1 protein kinase as a reasonable target for antifungal agents because mutant or druggable forms of the protein, upon inactivation, cause rapid loss of cell viability. Furthermore, extensive analysis of the Mps1 in budding yeast has offered potential tactics for identifying inhibitors of its enzymatic activity. One such tactic is based on the finding that overexpression of Mps1 leads to cell cycle arrest via activation of the spindle assembly checkpoint. We have endeavored to adapt this assay to be based on the overexpression of Mps1 orthologs from pathogenic yeast in hopes of having a whole-cell assay system to test the activity of these orthologs. Mps1 orthologous genes from seven pathogenic yeast or other pathogenic fungal species were isolated and expressed in budding yeast. Two orthologs clearly produced phenotypes similar to those produced by the overexpression of budding yeast Mps1, indicating that this system for heterologous Mps1 expression has potential as a platform for identifying prospective antifungal agents.

Loss of UBE2S causes meiosis I arrest with normal spindle assembly checkpoint dynamics in mouse oocytes.

The timely degradation of proteins that regulate the cell cycle is essential for oocyte maturation. Oocytes are equipped to degrade proteins via the ubiquitin-proteasome system. In meiosis, anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin-ligase, is responsible for the degradation of proteins. Ubiquitin-conjugating enzyme E2 S (UBE2S), an E2 ubiquitin-conjugating enzyme, delivers ubiquitin to APC/C. APC/C has been extensively studied, but the functions of UBE2S in oocyte maturation and mouse fertility are not clear. In this study, we used Ube2s knockout mice to explore the role of UBE2S in mouse oocytes. Ube2s-deleted oocytes were characterized by meiosis I arrest with normal spindle assembly and spindle assembly checkpoint dynamics. However, the absence of UBE2S affected the activity of APC/C. Cyclin B1 and securin are two substrates of APC/C, and their levels were consistently high, resulting in the failure of homologous chromosome separation. Unexpectedly, the oocytes arrested in meiosis I could be fertilized and the embryos could become implanted normally, but died before embryonic day 10.5. In conclusion, our findings reveal an indispensable regulatory role of UBE2S in mouse oocyte meiosis and female fertility.

A novel HDAC6 inhibitor interferes microtubule dynamics and spindle assembly checkpoint and sensitizes cisplatin-induced apoptosis in castration-resistant prostate cancer.

BACKGROUND: Metastatic castration-resistant prostate cancer (CRPC), the most refractory prostate cancer, inevitably progresses and becomes unresponsive to hormone therapy, revealing a pressing unmet need for this disease. Novel agents targeting HDAC6 and microtubule dynamics can be a potential anti-CRPC strategy. METHODS: Cell proliferation was examined in CRPC PC-3 and DU-145 cells using sulforhodamine B assay and anchorage-dependent colony formation assay. Flow cytometric analysis of propidium iodide staining was used to determine cell-cycle progression. Cell-based tubulin polymerization assay and confocal immunofluorescence microscopic examination determine microtubule assembly/disassembly status. Protein expressions were determined using Western blot analysis. RESULTS: A total of 82 novel derivatives targeting HDAC6 were designed and synthesized, and Compound 25202 stood out, showing the highest efficacy in blocking HDAC6 (IC50, 3.5 nM in enzyme assay; IC50, 1.0 µM in antiproliferative assay in CRPC cells), superior to tubastatin A (IC50, 5.4 µM in antiproliferative assay). The selectivity and superiority of 25202 were validated by examining the acetylation of both α-tubulin and histone H3, detecting cell apoptosis and HDACs enzyme activity assessment. Notably, 25202 but not tubastatin A significantly decreased HDAC6 protein expression. 25202 prolonged mitotic arrest through the detection of cyclin B1 upregulation, Cdk1 activation, mitotic phosphoprotein levels, and Bcl-2 phosphorylation. Compound 25202 did not mimic docetaxel in inducing tubulin polymerization but disrupted microtubule organization. Compound 25202 also increased the phosphorylation of CDC20, BUB1, and BUBR1, indicating the activation of the spindle assembly checkpoint (SAC). Moreover, 25202 profoundly sensitized cisplatin-induced cell death through impairment of cisplatin-evoked DNA damage response and DNA repair in both ATR-Chk1 and ATM-Chk2 pathways. CONCLUSION: The data suggest that 25202 is a novel selective and potent HDAC6 inhibitor. Compound 25202 blocks HDAC6 activity and interferes microtubule dynamics, leading to SAC activation and mitotic arrest prolongation that eventually cause apoptosis of CRPC cells. Furthermore, 25202 sensitizes cisplatin-induced cell apoptosis through impeding DNA damage repair pathways.

Functional analysis of Cdc20 reveals a critical role of CRY box in mitotic checkpoint signaling.

Accurate mitosis is coordinated by the spindle assembly checkpoint (SAC) through the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex or cyclosome (APC/C). As an essential regulator, Cdc20 promotes mitotic exit through activating APC/C and monitors kinetochore-microtubule attachment through activating SAC. Cdc20 requires multiple interactions with APC/C and MCC subunits to elicit these functions. Functionally assessing these interactions within cells requires efficient depletion of endogenous Cdc20, which is highly difficult to achieve by RNA interference (RNAi). Here we generated Cdc20 RNAi-sensitive cell lines which display a penetrant metaphase arrest by a single RNAi treatment. In this null background, we accurately measured the contribution of each known motif of Cdc20 on APC/C and SAC activation. The CRY box, a previously identified degron, was found critical for SAC by promoting MCC formation and its interaction with APC/C. These data reveal additional regulation within the SAC and establish a novel method to interrogate Cdc20.

Coregulation of NDC80 Complex Subunits Determines the Fidelity of the Spindle-Assembly Checkpoint and Mitosis.

NDC80 complex (NDC80C) is composed of four subunits (SPC24, SPC25, NDC80, and NUF2) and is vital for kinetochore-microtubule (KT-MT) attachment during mitosis. Paradoxically, NDC80C also functions in the activation of the spindle-assembly checkpoint (SAC). This raises an interesting question regarding how mitosis is regulated when NDC80C levels are compromised. Using a degron-mediated depletion system, we found that acute silencing of SPC24 triggered a transient mitotic arrest followed by mitotic slippage. SPC24-deficient cells were unable to sustain SAC activation despite the loss of KT-MT interaction. Intriguingly, our results revealed that other subunits of the NDC80C were co-downregulated with SPC24 at a posttranslational level. Silencing any individual subunit of NDC80C likewise reduced the expression of the entire complex. We found that the SPC24-SPC25 and NDC80-NUF2 subcomplexes could be individually stabilized using ectopically expressed subunits. The synergism of SPC24 downregulation with drugs that promote either mitotic arrest or mitotic slippage further underscored the dual roles of NDC80C in KT-MT interaction and SAC maintenance. The tight coordinated regulation of NDC80C subunits suggests that targeting individual subunits could disrupt mitotic progression and provide new avenues for therapeutic intervention. IMPLICATIONS: These results highlight the tight coordinated regulation of NDC80C subunits and their potential as targets for antimitotic therapies.

A coadapted KNL1 and spindle assembly checkpoint axis orchestrates precise mitosis in Arabidopsis.

The kinetochore scaffold 1 (KNL1) protein recruits spindle assembly checkpoint (SAC) proteins to ensure accurate chromosome segregation during mitosis. Despite such a conserved function among eukaryotic organisms, its molecular architectures have rapidly evolved so that the functional mode of plant KNL1 is largely unknown. To understand how SAC signaling is regulated at kinetochores, we characterized the function of the KNL1 gene in Arabidopsis thaliana. The KNL1 protein was detected at kinetochores throughout the mitotic cell cycle, and null knl1 mutants were viable and fertile but exhibited severe vegetative and reproductive defects. The mutant cells showed serious impairments of chromosome congression and segregation, that resulted in the formation of micronuclei. In the absence of KNL1, core SAC proteins were no longer detected at the kinetochores, and the SAC was not activated by unattached or misaligned chromosomes. Arabidopsis KNL1 interacted with SAC essential proteins BUB3.3 and BMF3 through specific regions that were not found in known KNL1 proteins of other species, and recruited them independently to kinetochores. Furthermore, we demonstrated that upon ectopic expression, the KNL1 homolog from the dicot tomato was able to functionally substitute KNL1 in A. thaliana, while others from the monocot rice or moss associated with kinetochores but were not functional, as reflected by sequence variations of the kinetochore proteins in different plant lineages. Our results brought insights into understanding the rapid evolution and lineage-specific connection between KNL1 and the SAC signaling molecules.

A Pan-Cancer Analysis of the Immunological and Prognostic Role of BUB1 Mitotic Checkpoint Serine/Threonine Kinase B (BUB1B) in Human Tumors.

BACKGROUND: BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) is a member of the spindle assembly checkpoint family and is related to cancer disease progression, invasion, metastasis, and functional promotion of angiogenesis. Several studies have noted that the BUB1B gene is frequently upregulated in various types of cancers. However, the expression patterns of BUB1B across different cancer types and its diagnostic and prognostic potential have not been investigated from a pan-cancer perspective. METHODS: The Cancer Genome Atlas (TCGA) data were used to explore the diagnostic and prognostic immunological potential of BUB1B in 33 cancer types. RESULTS: BUB1B was almost universally upregulated across all cancers, with increased protein expression in at least six cancer types and an enhanced phosphorylation level of S670 in two cancer types. Furthermore, BUB1B expression was negatively associated with clinical progression and prognosis in most cancers. BUB1B expression was positively associated with tumor mutational burden and microsatellite instability in 17 and 7 cancer types, respectively, and there was a correlation between BUB1B expression and DNA methylation at multiple probes in 30 cancer types. Additionally, a positive relationship existed between BUB1B expression and the infiltration levels of Th2, Tcm, and T helper cells, whereas BUB1B showed a negative correlation with the infiltration levels of other immune cells in multiple cancers. Moreover, functions associated with cell cycle progression and ubiquitin-mediated proteolysis were involved in the functional mechanism of BUB1B. CONCLUSIONS: Our pan-cancer study offers a comprehensive understanding of the role of BUB1B in tumorigenesis and tumor immunity across different types of cancer.

Genetic heterogeneity in p53-null leukemia increases transiently with spindle assembly checkpoint inhibition and is not rescued by p53.

Chromosome gains or losses often lead to copy number variations (CNV) and loss of heterozygosity (LOH). Both quantities are low in hematologic "liquid" cancers versus solid tumors in data of The Cancer Genome Atlas (TCGA) that also shows the fraction of a genome affected by LOH is ~ one-half of that with CNV. Suspension cultures of p53-null THP-1 leukemia-derived cells conform to these trends, despite novel evidence here of genetic heterogeneity and transiently elevated CNV after perturbation. Single-cell DNAseq indeed reveals at least 8 distinct THP-1 aneuploid clones with further intra-clonal variation, suggesting ongoing genetic evolution. Importantly, acute inhibition of the mitotic spindle assembly checkpoint (SAC) produces CNV levels that are typical of high-CNV solid tumors, with subsequent cell death and down-selection to novel CNV. Pan-cancer analyses show p53 inactivation associates with aneuploidy, but leukemias exhibit a weaker trend even though p53 inactivation correlates with poor survival. Overexpression of p53 in THP-1 does not rescue established aneuploidy or LOH but slightly increases cell death under oxidative or confinement stress, and triggers p21, a key p53 target, but without affecting net growth. Our results suggest that factors other than p53 exert stronger pressures against aneuploidy in liquid cancers, and identifying such CNV suppressors could be useful across liquid and solid tumor types.

TTK/MPS1 inhibitor OSU-13 targets the mitotic checkpoint and is a potential therapeutic strategy for myeloma.

Despite substantial recent advances in treatment, multiple myeloma (MM) remains an incurable disease, with a shortage of treatment options for patients with high-risk disease, warranting the need for novel therapeutic targets and treatment approaches. Threonine and tyrosine kinase (TTK), also known as monopolar spindle 1 (MPS1), is a kinase essential for the mitotic spindle checkpoint whose expression correlates to unfavorable prognosis in several cancers. Here, we report the importance of TTK in MM, and the effects of the TTK inhibitor OSU-13. Elevated TTK expression correlated with amplification/ gain of 1q21 and decreased overall and event-free survival in MM. Treatment with OSU-13 inhibited TTK activity efficiently and selectively at a similar concentration range to other TTK inhibitor clinical candidates. OSU-13 reduced proliferation and viability of primary human MM cells and cell lines, especially those with high 1q21 copy numbers, and triggered apoptosis through caspase 3 and 7 activation. In addition, OSU-13 induced DNA damage and severe defects in chromosome alignment and segregation, generating aneuploidy. In vivo, OSU-13 decreased tumor growth in mice with NCI-H929 xenografts. Collectively, our findings reveal that inhibiting TTK with OSU-13 is a potential therapeutic strategy for MM, particularly for a subset of high-risk patients with poor outcome.

PP2A inhibition causes synthetic lethality in BRCA2-mutated prostate cancer models via spindle assembly checkpoint reactivation.

Mutations in the BRCA2 tumor suppressor gene have been associated with an increased risk of developing prostate cancer. One of the paradoxes concerning BRCA2 is the fact that its inactivation affects genetic stability and is deleterious for cellular and organismal survival, while BRCA2-mutated cancer cells adapt to this detriment and malignantly proliferate. Therapeutic strategies for tumors arising from BRCA2 mutations may be discovered by understanding these adaptive mechanisms. In this study, we conducted forward genetic synthetic viability screenings in Caenorhabditis elegans brc-2 (Cebrc-2) mutants and found that Ceubxn-2 inactivation rescued the viability of Cebrc-2 mutants. Moreover, loss of NSFL1C, the mammalian ortholog of CeUBXN-2, suppressed the spindle assembly checkpoint (SAC) activation and promoted the survival of BRCA2-deficient cells. Mechanistically, NSFL1C recruited USP9X to inhibit the polyubiquitination of AURKB and reduce the removal of AURKB from the centromeres by VCP, which is essential for SAC activation. SAC inactivation is common in BRCA2-deficient prostate cancer patients, but PP2A inhibitors could reactivate the SAC and achieve BRCA2-deficient prostate tumor synthetic lethality. Our research reveals the survival adaptation mechanism of BRCA2-deficient prostate tumor cells and provides different angles for exploring synthetic lethal inhibitors in addition to targeting DNA damage repair pathways.

The mitotic checkpoint kinase BUB1 is a direct and actionable target of MYB in adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is a head and neck cancer that frequently originates in salivary glands, but can also strike other exocrine glands such as the breast. A key molecular alteration found in the majority of ACC cases is MYB gene rearrangements, leading to activation of the oncogenic transcription factor MYB. In this study, we used immortalised breast epithelial cells and an inducible MYB transgene as a model of ACC. Molecular profiling confirmed that MYB-driven gene expression causes a transition into an ACC-like state. Using this new cell model, we identified BUB1 as a targetable kinase directly controlled by MYB, whose pharmacological inhibition caused MYB-dependent synthetic lethality, growth arrest and apoptosis of patient-derived cells and organoids.

Small-molecule inhibition of kinesin KIF18A reveals a mitotic vulnerability enriched in chromosomally unstable cancers.

Chromosomal instability (CIN) is a hallmark of cancer, caused by persistent errors in chromosome segregation during mitosis. Aggressive cancers like high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have a high frequency of CIN and TP53 mutations. Here, we show that inhibitors of the KIF18A motor protein activate the mitotic checkpoint and selectively kill chromosomally unstable cancer cells. Sensitivity to KIF18A inhibition is enriched in TP53-mutant HGSOC and TNBC cell lines with CIN features, including in a subset of CCNE1-amplified, CDK4-CDK6-inhibitor-resistant and BRCA1-altered cell line models. Our KIF18A inhibitors have minimal detrimental effects on human bone marrow cells in culture, distinct from other anti-mitotic agents. In mice, inhibition of KIF18A leads to robust anti-cancer effects with tumor regression observed in human HGSOC and TNBC models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via KIF18A inhibition.

Spindle assembly checkpoint insensitivity allows meiosis-II despite chromosomal defects in aged eggs.

Chromosome segregation errors in mammalian oocyte meiosis lead to developmentally compromised aneuploid embryos and become more common with advancing maternal age. Known contributors include age-related chromosome cohesion loss and spindle assembly checkpoint (SAC) fallibility in meiosis-I. But how effective the SAC is in meiosis-II and how this might contribute to age-related aneuploidy is unknown. Here, we developed genetic and pharmacological approaches to directly address the function of the SAC in meiosis-II. We show that the SAC is insensitive in meiosis-II oocytes and that as a result misaligned chromosomes are randomly segregated. Whilst SAC ineffectiveness in meiosis-II is not age-related, it becomes most prejudicial in oocytes from older females because chromosomes that prematurely separate by age-related cohesion loss become misaligned in meiosis-II. We show that in the absence of a robust SAC in meiosis-II these age-related misaligned chromatids are missegregated and lead to aneuploidy. Our data demonstrate that the SAC fails to prevent cell division in the presence of misaligned chromosomes in oocyte meiosis-II, which explains how age-related cohesion loss can give rise to aneuploid embryos.

The signalling lipid PI3,5P2 is essential for timely mitotic exit.

Coordination of mitotic exit with chromosome segregation is key for successful mitosis. Mitotic exit in budding yeast is executed by the mitotic exit network (MEN), which is negatively regulated by the spindle position checkpoint (SPOC). SPOC kinase Kin4 is crucial for SPOC activation in response to spindle positioning defects. Here, we report that the lysosomal signalling lipid phosphatidylinositol-3,5-bisphosphate (PI3,5P2) has an unanticipated role in the timely execution of mitotic exit. We show that the lack of PI3,5P2 causes a delay in mitotic exit, whereas elevated levels of PI3,5P2 accelerates mitotic exit in mitotic exit defective cells. Our data indicate that PI3,5P2 promotes mitotic exit in part through impairment of Kin4. This process is largely dependent on the known PI3,5P2 effector protein Atg18. Our work thus uncovers a novel link between PI3,5P2 and mitotic exit.

A bifunctional kinase-phosphatase module balances mitotic checkpoint strength and kinetochore-microtubule attachment stability.

Two major mechanisms safeguard genome stability during mitosis: the mitotic checkpoint delays mitosis until all chromosomes have attached to microtubules, and the kinetochore-microtubule error-correction pathway keeps this attachment process free from errors. We demonstrate here that the optimal strength and dynamics of these processes are set by a kinase-phosphatase pair (PLK1-PP2A) that engage in negative feedback from adjacent phospho-binding motifs on the BUB complex. Uncoupling this feedback to skew the balance towards PLK1 produces a strong checkpoint, hypostable microtubule attachments and mitotic delays. Conversely, skewing the balance towards PP2A causes a weak checkpoint, hyperstable microtubule attachments and chromosome segregation errors. These phenotypes are associated with altered BUB complex recruitment to KNL1-MELT motifs, implicating PLK1-PP2A in controlling auto-amplification of MELT phosphorylation. In support, KNL1-BUB disassembly becomes contingent on PLK1 inhibition when KNL1 is engineered to contain excess MELT motifs. This elevates BUB-PLK1/PP2A complex levels on metaphase kinetochores, stabilises kinetochore-microtubule attachments, induces chromosome segregation defects and prevents KNL1-BUB disassembly at anaphase. Together, these data demonstrate how a bifunctional PLK1/PP2A module has evolved together with the MELT motifs to optimise BUB complex dynamics and ensure accurate chromosome segregation.

Signaling protein abundance modulates the strength of the spindle assembly checkpoint.

During mitosis, unattached kinetochores in a dividing cell signal to the spindle assembly checkpoint (SAC) to delay anaphase onset and prevent chromosome missegregation.1,2,3,4 The signaling activity of these kinetochores and the likelihood of chromosome missegregation depend on the amount of SAC signaling proteins each kinetochore recruits.5,6,7,8 Therefore, factors that control SAC protein recruitment must be thoroughly understood. Phosphoregulation of kinetochore and SAC signaling proteins due to the concerted action of many kinases and phosphatases is a significant determinant of the SAC protein recruitment to signaling kinetochores.9 Whether the abundance of SAC proteins also influences the recruitment and signaling activity of human kinetochores has not been studied.8,10 Here, we reveal that the low cellular abundance of the SAC signaling protein Bub1 limits its own recruitment and that of BubR1 and restricts the SAC signaling activity of the kinetochore. Conversely, Bub1 overexpression results in higher recruitment of SAC proteins, producing longer delays in anaphase onset. We also find that the number of SAC proteins recruited by a signaling kinetochore is inversely correlated with the total number of signaling kinetochores in the cell. This correlation likely arises from the competition among the signaling kinetochores to recruit from a limited pool of signaling proteins, including Bub1. The inverse correlation may allow the dividing cell to prevent a large number of signaling kinetochores in early prophase from generating an overly large signal while enabling the last unaligned kinetochore in late prometaphase to signal at the maximum strength.

Lycorine inhibits the proliferation of neuroblastoma neuro-2a cells by inducing G2/M phase cell cycle arrest and apoptosis.

Lycorine, a benzylphenanthridine-type alkaloid extracted form Amarillidaceae genera, exhibits an efficacy against various types of cancer. Nonetheless, the impact of lycorine treatment on neuroblastoma has not yet been investigated. Here we utilized a combinatorial strategy to explore and to understand the effect of lycorine on neuroblastoma Neuro-2a cells. Our results indicated that lycorine inhibits the Neuro-2a cells proliferation by promoting cell apoptosis. In addition, wound healing assay revealed that lycorine inhibits the Neuo-2a cells migration. Comparative transcriptome analysis showed that lycorine has the potential to affect cycle pathway. Flow cytometry analysis confirmed that lycorine arrested the Neuro-2a cell cycle at G2/M phase. Furthermore, we detected that the protein expression of Cyclin A, Cyclin B1 and Cyclin E were decreased, whereas protein of p53, Tgfß3, Gadd45ß, Gadd45γ, p21 and p27 were increased after treatment with lycorine. Collectively, we propose that lycorine might be a valuable candidate therapeutic agent in combating neuroblastoma.

MTA1 localizes to the mitotic spindle apparatus and interacts with TPR in spindle assembly checkpoint regulation.

We previously identified a cell cycle-dependent periodic subcellular distribution of cancer metastasis-associated antigen 1 (MTA1) and unraveled a novel role of MTA1 in inhibiting spindle damage-induced spindle assembly checkpoint (SAC) activation in cancer cells. However, the more detailed subcellular localization of MTA1 in mitotic cells and its copartner in SAC regulation in cancer cells are still poorly understood. Here, through immunofluorescent colocalization analysis of MTA1 and alpha-tubulin in mitotic cancer cells, we reveal that MTA1 is dynamically localized to the spindle apparatus throughout the entire mitotic process. We also demonstrated a reversible upregulation of MTA1 expression upon spindle damage-induced SAC activation, and time-lapse imaging assays indicated that MTA1 silencing delayed the mitotic metaphase-anaphase transition in cancer cells. Further investigation revealed that MTA1 interacts and colocalizes with Translocated Promoter Region (TPR) on spindle microtubules in mitotic cells, and this interaction is attenuated on SAC activation. TPR is well-implicated in SAC regulation via binding the MAD1-MAD2 complex, however, no interactions between MTA1 and MAD1 or MAD2 were detected in our coimmunoprecipitation (co-IP) assays, suggesting that the MTA1-TPR may represent a distinct SAC-associated complex separate from the previously reported TPR-MAD1/MAD2 complex. Our data provide new insights into the subcellular localization and molecular function of MTA1 in SAC regulation in cancer, and indicate that intervention of the MTA1-TPR interaction may be effective to modulate SAC and hence chromosomal instability (CIN) in tumorigenesis.