Spindle Defects Research Articles

Mitotic spindle defects and DNA damage induced by dimethoxycurcumin lead to an intrinsic apoptosis pathway in HepG2/C3A cells

Dimethoxycurcumin (DiMC), a synthetic analog of curcumin, was shown to have antiproliferative activity in human tumor cell lines. Therefore, we investigated its cytotoxic, antiproliferative, genotoxic, and apoptotic effect and correlated these evaluations with the expression of transcripts and proteins in the human hepatocellular carcinoma cell line (HepG2/C3A). Treatment with DiMC resulted in increased CYP2E1, CYP2C19 and CYP1A2 transcripts levels and was cytotoxic (≥10 μM). DiMC caused mitotic arrest by inducing monopolar spindle formation and was genotoxic increasing expression of the CDKN1A, GADD45A and PARP1 gene, key effectors in the cell cycle arrest and DNA repair pathways, respectively. This genotoxicity was caused by generation of reactive oxygen species and reduction of antioxidant proteins levels. Furthermore, we observed a decrease in important proteins involved in DNA repair. In addition to the observed apoptotic morphology and the presence of annexin labeling, we observed increased expression of BAK1 and CASP7 genes and caspase 3/7 protein activity, showing that these effects caused apoptosis through the intrinsic pathway in HepG2/C3A cells. Our results indicate that DiMC modulates important molecular targets leading to cell death even in metabolic competent cells models has considerable potential in anticancer therapy.

SIRT2 functions in aging, autophagy, and apoptosis in post-maturation bovine oocytes

AimsSirtuins have been implicated in the aging process, however, the functions of SIRT2 in post-maturation aging of oocytes are not fully understood. The purpose of the present investigation was to assess the roles of SIRT2 in aged oocytes and mechanisms involved. Main methodsThe fresh MII oocytes were aging in vitro, and treated with SIRT2 inhibitor (SirReal2), autophagy activator (Rapamycin), and autophagy inhibitor (3-Ma) for 24 h, respectively. Oocyte activation, cytoplasmic fragmentation, and spindle defects, mitochondrial distribution, ROS levels, ATP production, mitochondrial membrane potential, and early apoptosis were investigated. Western blotting was performed to determine LC3-II accumulation, SQSTM1 degradation, and caspase-3 activity. Key findingsSIRT2 expression gradually decreased in a time-dependent manner during oocyte aging. Treatment with SirReal2 significantly increased the rates of oocyte activation, cytoplasmic fragmentation, and spindle defects. In particular, the high ROS levels, abnormal mitochondrial distribution, low ATP production, and lost ΔΨm were observed in SirReal2-exposed oocytes. Further analysis revealed that LC3-II accumulation and SQSTM1 degradation were induced by SIRT2 inhibition. By performing early apoptosis analysis showed that oocyte aging was accompanied with cellular apoptosis, and SIRT2 inhibition increased apoptosis rates of aged oocytes. Importantly, upregulating autophagy with Rapamycin could mimic the effects of SIRT2 inhibition on apoptosis by increasing caspase-3 activation, whereas downregulating autophagy with 3-MA could abolish those effects by blocking caspase-3 activation. SignificanceOur results suggest that SIRT2 inactivation is a key mechanism underlying of cellular aging, and SIRT2 inhibition contributes to autophagy-dependent cellular apoptosis in post-maturation oocytes.

Abstract 3871: A novel TACC3 inhibitor as an anti-cancer agent in breast cancer

Abstract Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3) is a microtubule-associated protein that is localized on mitotic spindles and ensures proper chromosomal segregation and microtubule stability. TACC3 is frequently amplified or mutated, and was demonstrated to be a prognostic factor in a broad spectrum of cancers which makes it a highly attractive therapeutic target. TACC3 knockdown was demonstrated to cause mitotic spindle defects and prevent proper mitotic progression causing apoptotic cell death. Inhibition of TACC3 with the currently available small molecule inhibitors, KHS101 and SPL-B, has been shown to reduce the growth of glioblastoma xenografts, and suppress tumor growth in ovarian cancer xenografts, respectively. However, none of these TACC3 inhibitors is being tested in clinics potentially due to low systemic stability or high IC50 (inhibitory concentration 50) values. Here, by combining rational drug design and screening approaches, we aimed to identify a novel and more potent TACC3 inhibitor that is effective in in vitro and in vivo systems and that can be used as a mitotic blocker in breast cancer (BC). A library of test compounds was generated by replacing certain functional groups of already available TACC3 inhibitors with their isosteric equivalents to improve potency and drug-like properties. Then, the generated compounds were tested in vitro in terms of growth inhibition in a panel of breast cancer cell lines of different breast cancer subtypes. The compounds were also compared based on their effect on the cellular processes that TACC3 is involved in, and were further compared with genomic TACC3 inhibition by siRNAs. Finally, the most promising agent was tested in vivo in terms of its anti-tumorigenic effects. BO-264 was identified as the most potent TACC3 inhibitor that effectively kills breast cancer cells of different subtypes when administered at nanomolar dose. Moreover, it demonstrated superior inhibitory effects on mitotic progression, DNA repair, and cellular viability as compared to currently available inhibitors. Oral administration of BO-264 suppressed tumor growth in breast cancer xenografts at a dose that caused no apparent toxicity. Specificity of BO-264 to TACC3 protein was tested by kinome profiling and further validated by state-of-art binding assays. Currently, we are testing the pharmacokinetics and pharmacodynamics properties and organ toxicity of our inhibitor and performing the detailed molecular characterization of BO-264-mediated anti-tumor effects. Overall, our preclinical findings suggest that our novel compound (BO-264) is potentially a specific TACC3 inhibitor, causing growth inhibition in breast cancer cell lines and xenograft models by inducing mitotic arrest, DNA damage and apoptosis. Considering the critical importance of TACC3 as a cancer biomarker and driver of disease progression, BO-264 has great potential to be used as a novel therapeutic strategy in BC. Citation Format: Ozge Akbulut, Deniz Lengerli, Ozge Saatci, Elif Duman, Urartu Seker, Burcu Caliskan, Erden Banoglu, Ozgur Sahin. A novel TACC3 inhibitor as an anti-cancer agent in 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 3871.

Microtubule-interfering agents, spindle defects, and interkinetochore tension.

Microtubule-interfering agents have been very useful both as biological tools in studying mitosis and as chemotherapeutic agents against cancer. It remains poorly understood how these agents converge on the spindle assembly checkpoint (SAC) to halt mitotic progression, while inhibiting microtubule dynamics by different mechanisms. Cells arrested at mitosis by various microtubule-interfering agents exhibit strikingly different defects in the mitotic spindle. However, all the arrested cells possess the 3F3/2 phosphoepitope at the sister kinetochores of chromosomes, indicating the decrease of tension across the paired kinetochores. In addition, microtubule-interfering agents result in a comparable reduction in the distance between sister kinetochores, suggesting that these agents decrease interkinetochore tension to similar degrees. Here, we discuss recent progress that suggests impairment of kinetochore-microtubule attachment and reduction of interkinetochore tension as common mechanisms underlying the persistent SAC activation in response to diverse microtubule-interfering agents.

Haploinsufficiency of GCP4 induces autophagy and leads to photoreceptor degeneration due to defective spindle assembly in retina

Retinopathy, owing to damage to the retina, often causes vision impairment, and the underlying molecular mechanisms are largely unknown. Using a gene targeting strategy, we generated mice with the essential gene Tubgcp4 knocked out. Homozygous mutation of Tubgcp4 resulted in early embryonic lethality due to abnormal spindle assembly caused by GCP4 (gamma-tubulin complex protein 4, encoded by Tubgcp4) depletion. Heterozygotes were viable through dosage compensation of one wild-type allele. However, haploinsufficiency of GCP4 affected the assembly of γ-TuRCs (γ-tubulin ring complexes) and disrupted autophagy homeostasis in retina, thus leading to photoreceptor degeneration and retinopathy. Notably, GCP4 exerted autophagy inhibition by competing with ATG3 for interaction with ATG7, thus interfering with lipidation of LC3B. Our findings justify dosage effects of essential genes that compensate for null alleles in viability of mutant mice and uncover dosage-dependent roles of GCP4 in embryo development and retinal homeostasis. These data have also clinical implications in genetic counseling on embryonic lethality and in development of potential therapeutic targets associated with retinopathy.

Combined Aurora Kinase A (AURKA) and WEE1 Inhibition Demonstrates Synergistic Antitumor Effect in Squamous Cell Carcinoma of the Head and Neck.

Human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCC) commonly bear disruptive mutations in TP53, resulting in treatment resistance. In these patients, direct targeting of p53 has not been successful, but synthetic lethal approaches have promise. Although Aurora A kinase (AURKA) is overexpressed and an oncogenic driver, its inhibition has only modest clinical effects in HPV-negative HNSCC. We explored a novel combination of AURKA and WEE1 inhibition to overcome intrinsic resistance to AURKA inhibition.Experimental Design: AURKA protein expression was determined by fluorescence-based automated quantitative analysis of patient specimens and correlated with survival. We evaluated treatment with the AURKA inhibitor alisertib (MLN8237) and the WEE1 inhibitor adavosertib (AZD1775), alone or in combination, using in vitro and in vivo HNSCC models. Elevated nuclear AURKA correlated with worse survival among patients with p16(-) HNSCC. Alisertib caused spindle defects, G2-M arrest and inhibitory CDK1 phosphorylation, and cytostasis in TP53 mutant HNSCC FaDu and UNC7 cells. Addition of adavosertib to alisertib instead triggered mitotic entry and mitotic catastrophe. Moreover, in FaDu and Detroit 562 xenografts, this combination demonstrated synergistic effects on tumor growth and extended overall survival compared with either vehicle or single-agent treatment. Combinatorial treatment with adavosertib and alisertib leads to synergistic antitumor effects in in vitro and in vivo HNSCC models. These findings suggest a novel rational combination, providing a promising therapeutic avenue for TP53-mutated cancers.

Spatiotemporal control of spindle disassembly in fission yeast.

Maintenance of genomic stability during cell division is one of the most important cellular tasks, and it critically depends on the faithful replication of the genetic material and its equal partitioning into daughter cells, gametes, or spores in the case of yeasts. Defective mitotic spindle assembly and disassembly both result in changes in cellular ploidy that ultimately impinge proliferation fitness and might increase tumor malignancy. Although a great progress has been made in understanding how spindles are assembled to orchestrate chromosome segregation, much less is known about how they are disassembled once completed their function. Here, we review two recently uncovered mechanisms of spindle disassembly that operate at different stages of the fission yeast life cycle.

Exposure to aristolochic acid I compromises the maturational competency of porcine oocytes via oxidative stress-induced DNA damage.

Aristolochic acid (AA) is a class of carcinogenic and nephrotoxic nitrophenanthrene carboxylic acids naturally found in Aristolochia plants. These plants have been widely used as herbal medicines and also enter the human food chain as the persistent soil pollutants. It has been known that AA exposure is implicated in multiple cancer types, kidney failure and ovarian dysfunction. However, whether AA exposure would influence the oocyte quality has not yet determined. Here, we document that AAI has the negative effects on the competency of oocyte maturation and fertilization. We show that AAI exposure leads to the oocyte meiotic failure via impairing the meiotic apparatus, displaying a prominently defective spindle assembly, actin dynamics and mitochondrial integrity. AAI exposure also causes the abnormal distribution of cortical granules and ovastacin, which is consistent with the observation that fewer sperm bound to the zona pellucida surrounding the unfertilized AAI-exposed eggs, contributing to the fertilization failure. In addition, AAI exposure induces the increased levels of ROS, DNA damage and early apoptosis in porcine oocytes. Collectively, we demonstrate that AAI exposure perturbs the oocyte meiotic progression and fertilization capacity via disruption of both nuclear maturation and cytoplasmic maturation of oocyte, which might be caused by the excessive oxidative stress-induced DNA damage and apoptosis.

N-acetyl-L-cysteine (NAC) delays post-ovulatory oocyte aging in mouse.

The quality of post-ovulatory oocytes decreases with aging. In this study, we aimed to investigate the effects of N-acetyl-L-cysteine (NAC), a broadly used antioxidant, on oocyte quality in mouse post-ovulatory oocyte aging in vitro. NAC at 0.6mM concentration was added to culture medium (M2), and the quality of oocytes was analyzed at 6h, 12h, 18h and 24h of culture. We found that the frequency of spindle defects decreased in NAC-treated oocytes compared to those without NAC treatment. NAC treatment significantly decreased abnormal distribution of cortical granules (CGs) in oocytes during aging for 18h and 24h. Decreased intracellular reactive oxygen species (ROS) was also observed. Increased intracellular ATP levels and decreased abnormal distribution of mitochondria could be observed with NAC supplementation during post-ovulatory oocyte aging in vitro. These results indicate that NAC will maintain the quality of oocytes, and delay post-ovulatory oocyte aging as studied in the mouse.

The Heritability of Pigment Dispersion Syndrome and Pigmentary Glaucoma

Pigment dispersion syndrome (PDS) and pigmentary glaucoma (PG) are presumed to be inherited in an autosomal dominant manner. We examine relatives of patients with PDS and PG in order to determine the heritability of these diseases. This was a prospective, cross-sectional study. One hundred and one patients with PDS were prospectively recruited over 11months. Four of the patients had PDS without ocular hypertension or glaucoma, 6 had PDS and ocular hypertension, and 91 had PG. Criteria for PDS were 2 of 3 signs: Krukenberg spindle, midperipheral iris transillumination defects, and/or heavy trabecular meshwork pigmentation. Criteria for PG were PDS and 2 of 3 signs: intraocular pressure >21mm Hg, glaucomatous optic nerve damage, and/or glaucomatous visual field loss. Ninety-nine first-degree relatives living within a 100-mile radius of Iowa City, Iowa were examined in the clinic to determine the probability of familial transmission. A total of 10 of 99 (10.10%) first-degree relatives were diagnosed with PDS (1 with PDS alone, 2 with PDS and ocular hypertension, and 7 with PG). Seven families with ≥2 affected members were identified. The majority of affected family members (8/10) showed moderate to heavy trabecular meshwork pigmentation and either Krukenberg spindle or transillumination defects. Most of the cases of PDS in our study were sporadic, and the risk to first-degree relatives is lower than previously reported. However, there are families with apparent autosomal dominant inheritance of PDS in which the risk to relatives may be high.

PKCβ1 regulates meiotic cell cycle in mouse oocyte

ABSTRACT PKCβI, a member of the classical protein kinase C family, plays key roles in regulating cell cycle transition. Here, we report the expression, localization and functions of PKCβI in mouse oocyte meiotic maturation. PKCβI and p-PKCβI (phosphor-PKCβI) were expressed from germinal vesicle (GV) stage to metaphase II (MII) stage. Confocal microscopy revealed that PKCβI was localized in the GV and evenly distributed in the cytoplasm after GV breakdown (GVBD), and it was concentrated at the midbody at telophase in meiotic oocytes. While, p-PKCβI was concentrated at the spindle poles at the metaphase stages and associated with midbody at telophase. Depletion of PKCβI by specific siRNA injection resulted in defective spindles, accompanied with spindle assembly checkpoint activation, metaphase I arrest and failure of first polar body (PB1) extrusion. Live cell imaging analysis also revealed that knockdown of PKCβI resulted in abnormal spindles, misaligned chromosomes, and meiotic arrest of oocytes arrest at the Pro-MI/MI stage. PKCβI depletion did not affect the G2/M transition, but its overexpression delayed the G2/M transition through regulating Cyclin B1 level and Cdc2 activity. Our findings reveal that PKCβI is a critical regulator of meiotic cell cycle progression in oocytes. Abbreviations: PKC, protein kinase C; COC, cumulus-oocyte complexes; GV, germinal vesicle; GVBD, germinal vesicle breakdown; Pro-MI, first pro-metaphase; MI, first metaphase; Tel I, telophase I; MII, second metaphase; PB1, first polar body; SAC, spindle assembly checkpoint

CDK1-CCNB1 creates a spindle checkpoint-permissive state by enabling MPS1 kinetochore localization.

Spindle checkpoint signaling is initiated by recruitment of the kinase MPS1 to unattached kinetochores during mitosis. We show that CDK1-CCNB1 and a counteracting phosphatase PP2A-B55 regulate the engagement of human MPS1 with unattached kinetochores by controlling the phosphorylation status of S281 in the kinetochore-binding domain. This regulation is essential for checkpoint signaling, since MPS1S281A is not recruited to unattached kinetochores and fails to support the recruitment of other checkpoint proteins. Directly tethering MPS1S281A to the kinetochore protein Mis12 bypasses this regulation and hence the requirement for S281 phosphorylation in checkpoint signaling. At the metaphase-anaphase transition, MPS1 S281 dephosphorylation is delayed because PP2A-B55 is negatively regulated by CDK1-CCNB1 and only becomes fully active once CCNB1 concentration falls below a characteristic threshold. This mechanism prolongs the checkpoint-responsive period when MPS1 can localize to kinetochores and enables a response to late-stage spindle defects. By acting together, CDK1-CCNB1 and PP2A-B55 thus create a spindle checkpoint-permissive state and ensure the fidelity of mitosis.

Glucose Signaling Is Connected to Chromosome Segregation Through Protein Kinase A Phosphorylation of the Dam1 Kinetochore Subunit in Saccharomycescerevisiae.

The Dam1 complex is an essential component of the outer kinetochore that mediates attachments between spindle microtubules and chromosomes. Dam1p, a subunit of the Dam1 complex, binds to microtubules and is regulated by Aurora B/Ipl1p phosphorylation. We find that overexpression of cAMP-dependent protein kinase (PKA) catalytic subunits (i.e., TPK1, TPK2, TPK3) is lethal in DAM1 mutants and increases the rate of chromosome loss in wild-type cells. Replacing an evolutionarily conserved PKA site (S31) in Dam1p with a nonphosphorylatable alanine suppressed the high-copy PKA dosage lethality in dam1-1 Consistent with Dam1p as a target of PKA, we find that in vitro PKA can directly phosphorylate S31 in Dam1p and we observed phosphorylation of S31 in Dam1p purified from asynchronously growing yeast cells. Cells carrying high-copy TPK2 or a Dam1p phospho-mimetic S31D mutant displayed a reduction in Dam1p localization at the kinetochore, suggesting that PKA phosphorylation plays a role in assembly and/or stability of the Dam1 complex. Furthermore, we observed spindle defects associated with S31 phosphorylation. Finally, we find that phosphorylation of Dam1p on S31 is reduced when glucose is limiting as well as during α-factor arrest, conditions that inhibit PKA activity. These observations suggest that the PKA site of Dam1p participates in regulating kinetochore activity. While PKA is a well-established effector of glucose signaling, our work shows for the first time that glucose-dependent PKA activity has an important function in chromosome segregation.

18 Characterisation of early embryonic cellular defects after somatic cell nuclear transfer in fish

Cryopreservation of genetic resources is of major interest for the security of biodiversity and the sustainability of the agronomic industry. Cryopreservation of somatic cells is one means of preserving the genome of both parents, but regeneration of functional breeders requires the mastering of somatic cell NT (SCNT). In mammals, SCNT consists of injecting the somatic nuclei into enucleated recipient oocytes to obtain clones that bear the genome of the donor. Cloning in fish presents some advantages over cloning in mammals: oocytes are produced by the hundreds, and development is external. Moreover, embryonic genome activation takes place after 10 mitoses only. These features are favourable to a better understanding of cellular reprogramming of the somatic cell. Nonenucleated oocytes are often used for SCNT, which allows the production of 2n clones bearing only the donor genome when the maternal chromatin is spontaneously removed by an unknown mechanism (25% of cases). This ability can help us understand the fate of the maternal chromatin and the role of its surrounding factors on the cellular behaviour of the somatic cell during the first developmental steps of embryonic development: meiosis resumption (MR) and mitosis. In this study, fin somatic cells and mature oocytes were obtained from a 2-year-old goldfish (Carassius auratus). For SCNT, the whole fin cell was injected into the sperm entry site, and the oocyte was activated by water contact after 30min to begin the development. Somatic and maternal chromatin behaviour during MR and early mitosis were characterised by immunofluorescence (Hoechst/Vybrant labelling for chromatin identification and α-tubulin for spindle organisation). Variability in chromatin condensation was observed after the somatic cell injection before the activation. Clone analysis (n=16) revealed that some of them presented a condensed somatic nuclei (71%) and others presented a decondensed nuclei (29%). After activation, most clones underwent normal polar body extrusion of the maternal chromatin (66%, n=69/95 clones v. 96%, n=96/100 controls oocytes) without extrusion of a somatic polar body. Afterward, clones that presented a first symmetric division (n=14/35) were analysed at the 2-cells stage and compared with fertilized embryos (n=7). The maternal chromatin was observed as fragmented on the cleavage furrow (79%), where it cannot contribute to the development. Spindle defects were observed in 50% of clone cells (v. 0% in controls), such as multicentrosomal spindles, chromosome misalignment, abnormal segregation, and DNA fragmentation. To conclude, those clone (2n or 3n) defects are probably due to the chromatin condensation observed before activation. The fish oocyte volume did not allow the MR of the condensed somatic chromatin, and that may induce an abnormal anaphase and the following clone developmental defects.

Intersectin‐Cdc42 interaction is required for orderly meiosis in porcine oocytes

Intersectins (ITSNs) have been shown to act as adaptor proteins that govern multiple cellular events via regulating Cdc42 activity. However, it remains to be determined whether the ITSN-Cdc42 pathway is functional in porcine oocytes. To address this question, we used a small molecule, ZCL278, to selectively disrupt the ITSN2-Cdc42 interaction. In the presentstudy, we find that porcine oocytes exposed to ZCL278 are unable to completely progress through meiosis. Meanwhile, the spindle defects and chromosomal congression failure are frequently detected in these oocytes. In support of this, we observed the accumulated distribution of vesicle-like ITSN2 signals around the chromosome/spindle region during porcine oocyte maturation. In addition, our results also showed that inhibition of the ITSN-Cdc42 interaction impairs the actin polymerization in porcine oocytes. In summary, the findings support a model where ITSNs, through the interaction with Cdc42, modulates the assembly of meiotic apparatus and actin polymerization, consequently ensuring the orderly meiotic progression during porcine oocyte maturation.

Alterations in oocyte mitochondrial number and function are related to spindle defects and occur with maternal aging in mice and humans†

The objective of this work was to determine the role of mitochondria in the loss of oocyte quality with maternal aging. Our results show that mitochondrial DNA (mtDNA) copy number and function are reduced in eggs from aged mice after both in vivo and in vitro maturation. Higher incidences of spindle abnormalities were observed in old eggs. However, no correlation with egg ATP content was found. In vitro matured eggs from aged mice did not have a normal cortical distribution of active mitochondria and were subject to increased oxidative stress due to higher levels of reactive oxygen species and lower expression of glutamate-cysteine ligase, catalytic subunit (Gclc). Supplementation of antioxidants during in vitro maturation of old eggs mitigated this affect, resulting in increased mtDNA copy number and mitochondrial function, a mitochondria distribution pattern similar to young eggs, and improved chromosomal alignment. Eggs from women of advanced maternal age (AMA) had lower mitochondrial function than eggs from young women, although both age groups displayed a cortical distribution pattern of active mitochondria. In contrast to the mouse, human eggs from AMA women had higher mtDNA copy number than eggs from young women following in vitro maturation. In summary, oocytes of older females are more susceptible to perturbations in mitochondrial number and function, which are associated with increased spindle abnormalities and oxidative stress during in vitro maturation. These results demonstrate that oocyte mitochondria play a critical role in age-related infertility.

MiR-584-5p potentiates vincristine and radiation response by inducing spindle defects and DNA damage in medulloblastoma

Despite improvements in overall survival, only a modest percentage of patients survives high-risk medulloblastoma. The devastating side effects of radiation and chemotherapy substantially reduce quality of life for surviving patients. Here, using genomic screens, we identified miR-584-5p as a potent therapeutic adjuvant that potentiates medulloblastoma to radiation and vincristine. MiR-584-5p inhibited medulloblastoma growth and prolonged survival of mice in pre-clinical tumor models. MiR-584-5p overexpression caused cell cycle arrest, DNA damage, and spindle defects in medulloblastoma cells. MiR-584-5p mediated its tumor suppressor and therapy-sensitizing effects by targeting HDAC1 and eIF4E3. MiR-584-5p overexpression or HDAC1/eIF4E3 silencing inhibited medulloblastoma stem cell self-renewal without affecting neural stem cell growth. In medulloblastoma patients, reduced expression of miR-584-5p correlated with increased levels of HDAC1/eIF4E3. These findings identify a previously undefined role for miR-584-5p/HDAC1/eIF4E3 in regulating DNA repair, microtubule dynamics, and stemness in medulloblastoma and set the stage for a new way to treat medulloblastoma using miR-584-5p.

HDAC3 inhibition disrupts the assembly of meiotic apparatus during porcine oocyte maturation.

Histone deacetylases (HDACs) are involved in a wide array of biological processes. However, the role of HDAC3 in porcine oocytes remains unclear. In the current study, we examine the effects of HDAC3 inhibition on porcine oocyte maturation using RGFP966, a selective HDAC3 inhibitor. We find that suppression of HDAC3 activity prevents not only the expansion of cumulus cells but also the meiotic progression of oocytes. It is interesting to note that HDAC3 displays a spindle-like distribution pattern as the porcine oocytes enter meiosis. In line with this, confocal microscopy reveals the high frequency of spindle defects and chromosomal congression failure in metaphase oocytes exposed to RGFP966. Moreover, HDAC3 inhibition results in the hyperacetylation of α-tubulin during oocyte meiosis. These findings indicate that HDAC3 activity might control the microtubule stability via the deacetylation of tubulin, which is critical for maintaining the proper spindle assembly, accurate chromosome separation, and orderly meiotic progression during porcine oocyte maturation.

Spindlin1 alters the metaphase to anaphase transition in meiosis I through regulation of BUB3 expression in porcine oocytes.

Spindlin 1 (SPIN1), which contains Tudor-like domains, regulates maternal transcripts via interaction with a messenger RNA (mRNA)-binding protein. SPIN1 is involved in tumorigenesis in somatic cells and is highly expressed in cancer cells. Nevertheless, the role of SPIN1 in porcine oocyte maturation remains totally unknown. To explore the function of SPIN1 in porcine oocyte maturation, knockdown, and overexpression techniques were used. SPIN1 mRNA was identified in maternal stages ranging from GV to MII. SPIN1 was localized in the cytoplasm and to chromosomes during meiosis. SPIN1 knockdown accelerated first polar body extrusion. Oocytes with overexpressed SPIN1 were arrested at the MI stage. SPIN1 depletion caused meiotic spindle defects and chromosome instability. The BUB3 signal was investigated, confirming that SPIN1 affects the stability of Bub3 mRNA as well as BUB3 expression. Further, overexpression of SPIN1 inhibited the degradation and regulation of G2/mitotic-specific cyclin-B1. In summation, SPIN1 regulates the meiotic cell cycle by modulating the activation of the spindle assembly checkpoint.

Abstract A45: Tumor suppressor miR-584-5p regulates MYC and sensitizes MYC-amplified medulloblastoma to vincristine and ionizing radiation

Abstract Despite recent improvements in the overall survival of medulloblastoma (MB), only a modest percentage of patients survive high-risk disease. Furthermore, the toxicity associated with current therapies inflicts debilitating lifelong side effects on surviving MB patients. Using an unbiased microRNA screen, we identified miR-584-5p as a potent tumor suppressor that uniquely sensitizes MB to both ionizing radiation (IR) and vincristine (VCR), which are commonly used in combination to treat MB. Our studies revealed that miR-584-5p inhibited MB stem cell proliferation and consequently growth in vivo. Notably, we discovered that miR-584-5p imparts its tumor-suppressor and therapy-sensitizing effects by inhibiting histone deacetylase 1 (HDAC1), eukaryotic translation initiation factor 4e family member 3 (eIF4E3), and C-MYC. We demonstrated that miR-584-5p overexpression or the knockdown of its target genes including eIF4E3 and HDAC1 resulted in G2-M arrest, DNA damage, spindle defects, and apoptosis in MB cells. Meta-analysis of normal brain and MB tumor samples showed that while miR-584-5p is highly expressed in normal brain and cerebellum, it is highly downregulated across all MB subtypes. miR-584-5p targets, on the other hand, are highly overexpressed in MB tumors compared to normal cerebellum. Taken together, this study highlights a previously unidentified role for miR-584-5p, eIF4E3, and HDAC1 in regulating microtubule dynamics and DNA repair pathways. In addition, our study identifies novel targets that can affect the therapeutic efficacy of VCR and IR in MB and sets the stage for a new paradigm of treating medulloblastoma using microRNA-based therapeutics. Citation Format: Nourhan Abdelfattah, Subapriya Rajamanickam, Santosh Timilsina, Panneerdoss Subbarayalu, Benjamin Onyeagucha, Manjeet Rao. Tumor suppressor miR-584-5p regulates MYC and sensitizes MYC-amplified medulloblastoma to vincristine and ionizing radiation [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A45.