Prevent Chromosome Missegregation Research Articles

Aurora B kinase erases monopolar microtubule-kinetochore arrays at the meiosis I-II transition

During meiosis, faithful chromosome segregation requires monopolar spindle microtubule-kinetochore arrays in MI to segregate homologous chromosomes, but bipolar in MII to segregate sister chromatids. Using fission yeasts, we found that the universal Aurora B kinase localizes to kinetochores in metaphase I and in the mid-spindle during anaphase I, as in mitosis; but in the absence of an intervening S phase, the importin α Imp1 propitiates its release from the spindle midzone to re-localize at kinetochores during meiotic interkinesis. We show that "error-correction" activity of kinetochore re-localized Aurora B becomes essential to erase monopolar arrangements from anaphase I, a prerequisite to satisfy the spindle assembly checkpoint (SAC) and to generate proper bipolar arrays at the onset of MII. This microtubule-kinetochore resetting activity of Aurora B at the MI-MII transition is required to prevent chromosome missegregation in meiosis II, a type of error often associated with birth defects and infertility in humans.

Mitotic DNA Synthesis in Untransformed Human Cells Preserves Common Fragile Site Stability via a FANCD2-Driven Mechanism That Requires HELQ

Faithful genome duplication is a challenging task for dividing mammalian cells, particularly under replication stress where timely resolution of late replication intermediates (LRIs) becomes crucial prior to cell division. In human cancer cells, mitotic DNA repair synthesis (MiDAS) is described as a final mechanism for the resolution of LRIs to avoid lethal chromosome mis-segregation. RAD52-driven MiDAS achieves this mission in part by generating gaps/breaks on metaphase chromosomes, which preferentially occur at common fragile sites (CFS). We previously demonstrated that a MiDAS mechanism also exists in untransformed and primary human cells, which is RAD52 independent but requires FANCD2. However, the properties of this form of MiDAS are not well understood. Here, we report that FANCD2-driven MiDAS in untransformed human cells: 1) requires a prerequisite step of FANCD2 mono-ubiquitination by a subset of Fanconi anemia (FA) proteins, 2) primarily acts to preserve CFS stability but not to prevent chromosome mis-segregation, and 3) depends on HELQ, which potentially functions at an early step. Hence, FANCD2-driven MiDAS in untransformed cells is built to protect CFS stability, whereas RAD52-driven MiDAS in cancer cells is likely adapted to prevent chromosome mis-segregation at the cost of CFS expression. Notably, we also identified a novel form of MiDAS, which surfaces to function when FANCD2 is absent in untransformed cells. Our findings substantiate the complex nature of MiDAS and a link between its deficiencies and the pathogenesis of FA, a human genetic disease.

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.

Meiotic cells escape prolonged spindle checkpoint activity through kinetochore silencing and slippage.

To prevent chromosome mis-segregation, a surveillance mechanism known as the spindle checkpoint delays the cell cycle if kinetochores are not attached to spindle microtubules, allowing the cell additional time to correct improper attachments. During spindle checkpoint activation, checkpoint proteins bind the unattached kinetochore and send a diffusible signal to inhibit the anaphase promoting complex/cyclosome (APC/C). Previous work has shown that mitotic cells with depolymerized microtubules can escape prolonged spindle checkpoint activation in a process called mitotic slippage. During slippage, spindle checkpoint proteins bind unattached kinetochores, but the cells cannot maintain the checkpoint arrest. We asked if meiotic cells had as robust of a spindle checkpoint response as mitotic cells and whether they also undergo slippage after prolonged spindle checkpoint activity. We performed a direct comparison between mitotic and meiotic budding yeast cells that signal the spindle checkpoint through two different assays. We find that the spindle checkpoint delay is shorter in meiosis I or meiosis II compared to mitosis, overcoming a checkpoint arrest approximately 150 minutes earlier in meiosis than in mitosis. In addition, cells in meiosis I escape spindle checkpoint signaling using two mechanisms, silencing the checkpoint at the kinetochore and through slippage. We propose that meiotic cells undertake developmentally-regulated mechanisms to prevent persistent spindle checkpoint activity to ensure the production of gametes.

FANCD2 promotes mitotic rescue from transcription-mediated replication stress in SETX-deficient cancer cells

Replication stress (RS) is a leading cause of genome instability and cancer development. A substantial source of endogenous RS originates from the encounter between the transcription and replication machineries operating on the same DNA template. This occurs predominantly under specific contexts, such as oncogene activation, metabolic stress, or a deficiency in proteins that specifically act to prevent or resolve those transcription-replication conflicts (TRCs). One such protein is Senataxin (SETX), an RNA:DNA helicase involved in resolution of TRCs and R-loops. Here we identify a synthetic lethal interaction between SETX and proteins of the Fanconi anemia (FA) pathway. Depletion of SETX induces spontaneous under-replication and chromosome fragility due to active transcription and R-loops that persist in mitosis. These fragile loci are targeted by the Fanconi anemia protein, FANCD2, to facilitate the resolution of under-replicated DNA, thus preventing chromosome mis-segregation and allowing cells to proliferate. Mechanistically, we show that FANCD2 promotes mitotic DNA synthesis that is dependent on XPF and MUS81 endonucleases. Importantly, co-depleting FANCD2 together with SETX impairs cancer cell proliferation, without significantly affecting non-cancerous cells. Therefore, we uncovered a synthetic lethality between SETX and FA proteins for tolerance of transcription-mediated RS that may be exploited for cancer therapy.

Therapeutic Gene Editing of HSCs Ex Vivo without in Vitro Culture Avoids Genotoxicity, Simplifies Procedures, and Preserves Efficiency and Stemness

Therapeutic gene editing of HSCs promises to cure inherited blood disorders. However, current ex vivo gene editing protocols may be associated with genotoxic potential and complex in vitro cell manufacturing procedures. Base and prime editing reduce double strand breaks (DSBs) but are generally less efficient and might have unique genotoxic potential (e.g. untargeted deaminase activity). Here we test the hypothesis that therapeutic gene editing with targeted endonucleases disrupting HbF-repressing elements for b-hemoglobinopathies may be made safer, simpler, and at least as effective by focusing the editing on nondividing cells. Genotoxic risks of DSBs appear to be concentrated in cycling cells. For example, Cas9-induced DSBs can result in long deletions at the on-target site that appear to be mainly the result of unidirectional extensive resection selectively active in S/G2 cell cycle phases1. In addition, DSBs must be resolved prior to mitosis to prevent chromosome mis-segregation leading to copy number variation and cytoplasmic micronuclei2,3. We measured the cell cycle state of mobilized PB CD34+ HSPCs immediately after collection, or after 24 or 48 hours of culture with cytokines (SCF, TPO, and FLT3L each at 100 ng/ml). 100% of cells were in G0/G1 at 0 hours, 94.6%±1.5% at 24 hours, and 76.7%±2.6% at 48 hours. We electroporated CD34+ HSPCs with 0, 24, or 48 hours of cytokine culture before performing 3xNLS-HiFi-SpCas9:sgRNA ribonucleoprotein (RNP) electroporation targeting the BCL11A erythroid enhancer. We employed a flow cytometry assay to quantify micronuclei in edited HSPCs. The fraction of micronuclei was increased in cells electroporated with Cas9:sgRNA RNP following 24 or 48 hours of cytokine stimulation but not in unstimulated cells. We performed ddPCR on sorted nuclei and micronuclei using assays specific to distal chromosome 2p (telomeric to the BCL11A cleavage site) and to chr1 as a reference autosome. We found ~9-fold enrichment of chr2p in micronuclei after 24 or 48 hours of pre-stimulation cytokine culture prior to gene editing, but strikingly did not observe any enrichment of chr2p in micronuclei in cells edited without pre-stimulation culture, suggesting that chromosome mis-segregation and unwanted downstream consequences could be avoided by editing quiescent HSCs (Fig. 1A). In addition, we developed ddPCR assays to measure genome editing outcomes after editing the BCL11A enhancer with paired cleavages (at the +58 and +55 enhancers) separated by 3-kb. These assays measure: 1) programmed 3-kb deletions, 2) programmed 3-kb inversions, or 3) short indels or unedited alleles. Any missing signals (genomic copies fewer than found on a reference nontargeted autosome) may represent long deletions or rearrangements. Editing cells without pre-stimulation culture was associated with undetectable missing signals (inferred long deletions and rearrangements) as compared to editing after 24 or 48 hours of pre-stimulation culture. We compared our standard protocol of 24 hours of pre-stimulation cytokine culture and 24 hours of post-electroporation cytokine culture to a protocol in which CD34+ HSPCs were electroporated immediately after thawing and then cryopreserved 15 minutes after electroporation, without any cytokine culture, before infusion to NBSGW mice. After 16 weeks, HbF levels were potently induced to similar levels after gene editing in recipients of HSPCs without and with cytokine culture (Fig. 1B). Overall human engraftment was similar for all groups, as was multilineage repopulation. Gene edit frequencies were similar for recipients of HSPCs without and with cytokine culture. We observed comparable editing and engraftment potency of HSPCs without as compared to with cytokine culture across mobilization type (G-CSF or plerixafor), donor disease status (healthy and SCD), and sgRNA target sequence. Together, these results suggest that editing HSPCs ex vivo without in vitro culture: avoids unintended on-target genotoxicity potential, including production of micronuclei, long deletions, and rearrangements; simplifies protocols, eliminating time, cost, and complexity; and preserves gene editing efficiency and HbF induction and HSC repopulation potency. Editing nondividing cells selectively could be a straightforward modification to improve ex vivo editing as well as to advance future efforts for safe and potent in vivo editing of HSCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Lis1-dynein drives corona compaction and limits erroneous microtubule attachment at kinetochores.

Mitotic cell division requires that kinetochores form microtubule attachments that can segregate chromosomes and control mitotic progression via the spindle assembly checkpoint. During prometaphase, kinetochores shed a domain called the fibrous corona as microtubule attachments form. This shedding is mediated, in part, by the minus-end directed motor dynein, which 'strips' cargoes along K-fibre microtubules. Despite its essentiality, little is known about how dynein stripping is regulated and how it responds to attachment maturation. Lis1 (also known as PAFAH1B1) is a conserved dynein regulator that is mutated in the neurodevelopmental disease lissencephaly. Here, we have combined loss-of-function studies, high-resolution imaging and separation-of-function mutants to define how Lis1 contributes to dynein-mediated corona stripping in HeLa cells. Cells depleted of Lis1 fail to disassemble the corona and show a delay in metaphase as a result of persistent checkpoint activation. Furthermore, we find that although kinetochore-tethered Lis1-dynein is required for error-free microtubule attachment, the contribution of Lis1 to corona disassembly can be mediated by a cytoplasmic pool. These findings support the idea that Lis1 drives dynein function at kinetochores to ensure corona disassembly and prevent chromosome mis-segregation.

Mitotic Checkpoint Imbalances in Familial Cancer

Numerical chromosomal aberrations are highly frequent in cancer cells. However, tumor-associated mutations in regulators of the mitotic machinery that controls chromosome segregation are rather rare. By sequencing families with hereditary cancer, Chen and colleagues report two novel heterozygous mutations in CDC20, a coactivator of the anaphase-promoting complex (APC/C) and a target of the spindle assembly checkpoint (SAC) that prevents chromosome missegregation during mitosis. CDC20 mutations result in partial SAC functionality and segregate with tumor susceptibility in families with aneuploid ovarian cancers and other malignancies. The expression of these mutations in a knock-in mouse model accelerates the development of Myc-induced lymphomas and mortality, strongly supporting the notion that partial dysfunction of mitotic regulators may have profound implications in spontaneous and hereditary cancer. See related article by Chen et al., p. 3499.

BUB3, beyond the Simple Role of Partner.

The BUB3 protein plays a key role in the activation of the spindle assembly checkpoint (SAC), a ubiquitous surveillance mechanism that ensures the fidelity of chromosome segregation in mitosis and, consequently, prevents chromosome mis-segregation and aneuploidy. Besides its role in SAC signaling, BUB3 regulates chromosome attachment to the spindle microtubules. It is also involved in telomere replication and maintenance. Deficiency of the BUB3 gene has been closely linked to premature aging. Upregulation of the BUB3 gene has been found in a variety of human cancers and is associated with poor prognoses. Here, we review the structure and functions of BUB3 in mitosis, its expression in cancer and association with survival prognoses, and its potential as an anticancer target.

Counteraction between Astrin-PP1 and Cyclin-B-CDK1 pathways protects chromosome-microtubule attachments independent of biorientation

Defects in chromosome-microtubule attachment can cause chromosomal instability (CIN), frequently associated with infertility and aggressive cancers. Chromosome-microtubule attachment is mediated by a large macromolecular structure, the kinetochore. Sister kinetochores of each chromosome are pulled by microtubules from opposing spindle-poles, a state called biorientation which prevents chromosome missegregation. Kinetochore-microtubule attachments that lack the opposing-pull are detached by Aurora-B/Ipl1. It is unclear how mono-oriented attachments that precede biorientation are spared despite the lack of opposing-pull. Using an RNAi-screen, we uncover a unique role for the Astrin-SKAP complex in protecting mono-oriented attachments. We provide evidence of domains in the microtubule-end associated protein that sense changes specific to end-on kinetochore-microtubule attachments and assemble an outer-kinetochore crescent to stabilise attachments. We find that Astrin-PP1 and Cyclin-B-CDK1 pathways counteract each other to preserve mono-oriented attachments. Thus, CIN prevention pathways are not only surveying attachment defects but also actively recognising and stabilising mature attachments independent of biorientation.

Mitotic Errors Promote Genomic Instability and Leukemia in a Novel Mouse Model of Fanconi Anemia.

Fanconi anemia (FA) is a disease of genomic instability and cancer. In addition to DNA damage repair, FA pathway proteins are now known to be critical for maintaining faithful chromosome segregation during mitosis. While impaired DNA damage repair has been studied extensively in FA-associated carcinogenesis in vivo, the oncogenic contribution of mitotic abnormalities secondary to FA pathway deficiency remains incompletely understood. To examine the role of mitotic dysregulation in FA pathway deficient malignancies, we genetically exacerbated the baseline mitotic defect in Fancc-/- mice by introducing heterozygosity of the key spindle assembly checkpoint regulator Mad2. Fancc-/-;Mad2+/- mice were viable, but died from acute myeloid leukemia (AML), thus recapitulating the high risk of myeloid malignancies in FA patients better than Fancc-/-mice. We utilized hematopoietic stem cell transplantation to propagate Fancc-/-; Mad2+/- AML in irradiated healthy mice to model FANCC-deficient AMLs arising in the non-FA population. Compared to cells from Fancc-/- mice, those from Fancc-/-;Mad2+/- mice demonstrated an increase in mitotic errors but equivalent DNA cross-linker hypersensitivity, indicating that the cancer phenotype of Fancc-/-;Mad2+/- mice results from error-prone cell division and not exacerbation of the DNA damage repair defect. We found that FANCC enhances targeting of endogenous MAD2 to prometaphase kinetochores, suggesting a mechanism for how FANCC-dependent regulation of the spindle assembly checkpoint prevents chromosome mis-segregation. Whole-exome sequencing revealed similarities between human FA-associated myelodysplastic syndrome (MDS)/AML and the AML that developed in Fancc-/-; Mad2+/- mice. Together, these data illuminate the role of mitotic dysregulation in FA-pathway deficient malignancies in vivo, show how FANCC adjusts the spindle assembly checkpoint rheostat by regulating MAD2 kinetochore targeting in cell cycle-dependent manner, and establish two new mouse models for preclinical studies of AML.

Localization of the ubiquitin ligase Dma1 to the fission yeast contractile ring is modulated by phosphorylation.

The timing of cytokinesis relative to other mitotic events in the fission yeast Schizosaccharomyces pombe is controlled by the septation initiation network (SIN). During a mitotic checkpoint, the SIN is inhibited by the E3 ubiquitin ligase Dma1 to prevent chromosome mis-segregation. Dma1 dynamically localizes to spindle pole bodies (SPBs) and the contractile ring (CR) during mitosis, though its role at the CR is unknown. Here, we examined whether Dma1 phosphorylation affects its localization or function. We found that preventing Dma1 phosphorylation by substituting the six phosphosites with alanines diminished its CR localization but did not affect its mitotic checkpoint function. These studies reinforce the conclusion that Dma1 localization to the SPB is key to its role in the mitotic checkpoint.

Bub1 and CENP-U redundantly recruit Plk1 to stabilize kinetochore-microtubule attachments and ensure accurate chromosome segregation.

Bub1 is required for the kinetochore/centromere localization of two essential mitotic kinases Plk1 and Aurora B. Surprisingly, stable depletion of Bub1 by ∼95% in human cells marginally affects whole chromosome segregation fidelity. We show that CENP-U, which is recruited to kinetochores by the CENP-P and CENP-Q subunits of the CENP-O complex, is required to prevent chromosome mis-segregation in Bub1-depleted cells. Mechanistically, Bub1 and CENP-U redundantly recruit Plk1 to kinetochores to stabilize kinetochore-microtubule attachments, thereby ensuring accurate chromosome segregation. Furthermore, unlike its budding yeast homolog, the CENP-O complex does not regulate centromeric localization of Aurora B. Consistently, depletion of Bub1 or CENP-U sensitizes cells to the inhibition of Plk1 but not Aurora B kinase activity. Taken together, our findings provide mechanistic insight into the regulation of kinetochore function, which may have implications for targeted treatment of cancer cells with mutations perturbing kinetochore recruitment of Plk1 by Bub1 or the CENP-O complex.

A CTDNEP 1-Lipin 1-mTOR Regulatory Network Restricts ER Membrane Biogenesis to Enable Chromosome Motions Necessary for Mitotic Fidelity

The endoplasmic reticulum (ER) dramatically restructures in open mitosis to become excluded from the mitotic spindle; however, the significance of ER reorganization to mitotic progression is not known. Here, we demonstrate that limiting ER membrane biogenesis enables mitotic chromosome movements necessary for chromosome biorientation and prevention of micronuclei formation. Aberrantly expanded ER membranes increase the effective viscosity of the mitotic cytoplasm to physically restrict chromosome dynamics – slowed chromosome motions impede correction of mitotic errors induced by transient disassembly, leading to severe micronucleation. We define the mechanistic link between regulation of ER membrane biogenesis and mitotic fidelity by demonstrating that a CTDNEP1-lipin 1-mTOR regulatory network limits ER lipid synthesis to prevent chromosome missegregation. Together, this work shows that ER membranes reorganize in mitosis to enable chromosome movements necessary for mitotic error correction and reveal dysregulated lipid metabolism as a potential source of aneuploidy in cancer cells. 

First meiotic anaphase requires Cep55-dependent inhibitory cyclin-dependent kinase 1 phosphorylation.

During mitosis, anaphase is triggered by anaphase-promoting complex (APC)-mediated destruction of securin and cyclin B1, which leads to inactivation of cyclin-dependent kinase 1 (Cdk1). By regulating APC activity, the mitotic spindle assembly checkpoint (SAC) therefore has robust control over anaphase timing to prevent chromosome mis-segregation. Mammalian oocytes are prone to aneuploidy, the reasons for which remain obscure. In mitosis, Cep55 is required post-anaphase for the final steps of cytokinesis. We found that Cep55-depleted mouse oocytes progress normally through early meiosis I, but that anaphase I fails as a result of persistent Cdk1 activity. Unexpectedly, Cdk1 inactivation was compromised following Cep55 depletion, despite on-time SAC silencing and intact APC-mediated proteolysis. We found that impaired Cdk1 inactivation was caused by inadequate inhibitory Cdk1 phosphorylation consequent upon failure to suppress Cdc25 phosphatase, identifying a proteolysis-independent step necessary for anaphase I. Thus, the SAC in oocytes does not exert exclusive control over anaphase I initiation, providing new insight into vulnerability to error.

Abstract 2008: CENP-E kinesin facilitates chemokine ligand CCL18 translocation to promote migration in triple negative breast cancer

Abstract Our research is focused on the molecular mechanisms responsible for enhanced cellular proliferation and migration as it relates to triple negative breast cancer (TNBC). Advances in visualization techniques and technologies work to provide an in-depth view of various cellular mechanisms, allowing us to capture molecular level protein-protein interactions previously unknown. There are more than 40 kinesin motor proteins found within the spindle assembly checkpoint, classified into 14 families, which each play essential roles in microtubule-dependent intracellular trafficking. This assembly is a critical component to the cell cycle control complex that acts during mitosis to prevent chromosome missegregation and aneuploidy. Centromere associated protein E (CENP-E) is a kinesin motor protein that shows aberrant overexpression in TNBC which facilitates exponential cell growth and eventual metastasis that occurs in TNBC. Growing awareness through scientific discoveries sheds light on the fact that it is a rare occurrence for biological functions to be attributed to a single mutated macromolecule. Most physiological processes arise from a complex orchestration of cellular events occurring between multiple molecules acting in concert. The nature of protein-protein transactions that drive gene activation appear to be dynamic in TNBC cells thus enabling uncontrolled proliferation. We believe that a pleiotropic network of small molecules may be working cooperatively with CENP-E to facilitate the exponential cell growth and eventual metastasis that occurs in TNBC cells. Currently, we are carrying out spectral imaging and biophysical force measurements to delineate CCL18-elicited cell mechanics and breast cancer metastasis. To address this, we have incorporated syntelin, a small inhibitory molecule targeted to CENP-E in an effort to analyze the resultant effect it has on this network of proteins we believe may be involved in the CENP-E signaling cascade. Taken together, these results indicate that syntelin may comprise a novel targeted chemotherapeutic intervention for TNBC. Citation Format: McKay M. Mullen. CENP-E kinesin facilitates chemokine ligand CCL18 translocation to promote migration in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2008.

Cellular response upon proliferation in the presence of an active mitotic checkpoint.

Eukaryotic cells treated with microtubule-targeting agents activate the spindle assembly checkpoint to arrest in mitosis and prevent chromosome mis-segregation. A fraction of mitotically arrested cells overcomes the block and proliferates even under persistent checkpoint-activating conditions. Here, we asked what allows proliferation in such unfavourable conditions. We report that yeast cells are delayed in mitosis at each division, implying that their spindle assembly checkpoint remains responsive. The arrest causes their cell cycle to be elongated and results in a size increase. Growth saturates at mitosis and correlates with the repression of various factors involved in translation. Contrary to unperturbed cells, growth of cells with an active checkpoint requires Cdh1. This peculiar cell cycle correlates with global changes in protein expression whose signatures partly overlap with the environmental stress response. Hence, cells dividing with an active checkpoint develop recognisable specific traits that allow them to successfully complete cell division notwithstanding a constant mitotic checkpoint arrest. These properties distinguish them from unperturbed cells. Our observation may have implications for the identification of new therapeutic windows and targets in tumors.

Overexpression of Fbxo6 inactivates spindle checkpoint by interacting with Mad2 and BubR1

ABSTRACT The spindle assembly checkpoint prevents chromosome mis-segregation during mitosis by delaying sister chromatid separation. Several F-box protein members play critical roles in maintaining genome stability and regulating cell cycle progress via ubiquitin-mediated protein degradation. Here, we showed that Fbxo6 critically regulated spindle checkpoint and chromosome segregation. Fbxo6 was phosphorylated during mitosis. Overexpression of Fbxo6 lead to faster exit from nocodazole-induced mitosis arrest through premature sister chromatid separation. Moreover, we found substantially more binuclear and multilobed nuclei cells accompanied with impaired cell viability in Fbxo6-overexpressed HeLa cells. Mechanistically, Fbxo6 interacted with spindle checkpoint proteins including Mad2 and BubR1 leading to the premature exit from mitosis. Overall, we revealed a novel role of Fbxo6 in regulating spindle checkpoint, which may shed light on the regulation of genome instability of cancer cells.

PO-224 consequences of FOXP1-mediated transcriptional repression of the mitotic checkpoint gene MAD2 in breast cancer

IntroductionThe mitotic checkpoint, or spindle assembly checkpoint (SAC), is a safeguard mechanism that prevents chromosome missegregation during mitosis. It is well established that overexpression of the mitotic checkpoint component MAD2 leads to chromosome instability (CIN) and tumorigenesis. MAD2 expression is controlled by transcriptional activator and repressor E2Fs, as well as by cell cycle dependent element/cell cycle homology region (CDE/CHR) sequences in its promoter. However, precisely how MAD2 transcription is regulated has remained elusive. Focusing on breast cancer, we therefore aimed to identify novel MAD2 transcriptional regulators and to characterise how these might contribute to CIN and tumour formation.Material and methodsPotential MAD2 transcriptional regulators were identified using a DNA-protein pull-down assay and mass spectrometry. In-depth characterisation of protein-DNA interactions and transcriptional effects were studied using chromatin immunoprecipitation, promoter mutagenesis and reporter assays. Cell lines in which identified repressors were stably knocked-down by shRNAs were used to investigate changes in MAD2 protein levels, cell cycle progression and chromosome segregation fidelity. In silico analyses and immunohistochemistry on tissue microarrays were used to study associations between expression of the regulators and MAD2 at the mRNA and protein levels in breast cancer. Repressor expression levels were also assessed in the context of breast cancer patient prognosis and survival.Results and discussionsWe identified the forkhead box transcription factor FOXP1 as a main transcriptional repressor of MAD2 expression. FOXP1 reduces MAD2 promoter activity via direct binding to CDE/CHR elements. Furthermore, knockdown of FOXP1 expression increases the proportion of cells in G2/M phase. In addition, low FOXP1 expression strongly correlates with high MAD2 expression at the mRNA and protein levels, and with poor patient prognosis in breast cancer, especially triple-negative breast cancer.ConclusionWe identified FOXP1 as a novel transcriptional repressor of MAD2 expression. Our results suggest that reduced FOXP1 levels in breast cancer affect both MAD2 expression and cell cycle progression. This may promote CIN and tumour formation via upregulation of MAD2. Our results may have important implications for breast cancer diagnostics and, potentially, therapeutic targeting.

C‐terminal domain of Mad1 interacts with Mad2 to catalyze O‐C Mad2 conversion in coordination with mitotic checkpoints

Aneuploidy occurs when chromosomes are unequally separated. It is important that all chromosomes' kinetochores attach to spindle microtubules before the cell starts to divide; if not, the chromosomes will be distributed unevenly between the two daughter cells. In order to prevent chromosome missegregation, the mitotic checkpoint complex (MCC) prevents chromosomes that are unattached to spindles from division. Human MCC is composed of multiple proteins: Bub3, BubR1, Cdc20, and Mad2. Kinetochores that are unattached to spindles give out the signal that triggers the MCC to assemble. Once formed, the MCC is able to inhibit anaphase‐promoting complex/cyclosome (APC/C). Without the activation of APC/C, the anaphase progression is temporarily stopped. In our study, we investigated Mad2, which is a critical part of the MCC. There are two functional conformations of Mad2, closed and opened, also known as C‐Mad2 and O‐Mad2, respectively. In the MCC, Mad2 exists in closed form (C‐Mad2). Therefore, to assemble the MCC, O‐Mad2 needs to be converted to C‐Mad2. Our research focuses on the pathway where Mad1 recruits O‐Mad2 and converts it to C‐Mad2. Previous research was concentrated on the middle part of Mad1 (Mad1‐MIM), and it was posited that Mad1 could not independently recruit O‐Mad2 without forming a complex with C‐Mad2. However, our research on the C‐terminal domain (CTD) of Mad1 indicates that Mad1‐CTD binds to both O‐ and C‐Mad2. This result suggests that CTD‐Mad1 could potentially recruit O‐Mad2, and convert it to C‐Mad2 which then assembles into the MCC.Support or Funding InformationThe work was carried out during the summer of 2017 in the Department of Biological Sciences at the University of Toledo in Dr. Song‐Tao Liu's lab with support from the Department of Chemistry at Wabash College.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.