The role of AFAP1-AS1 in mitotic catastrophe and metastasis of triple-negative breast cancer cells by activating the PLK1 signaling pathway

Triple-negative breast cancer(TNBC)is characterized by fast growth,high metastasis,high invasion,and a lack of therapeutic targets.Mitosis and metastasis of TNBC cells are two important biological behaviors in TNBC malignant progression.It is well known that the long noncoding RNA AFAP1-AS1 plays a crucial role in various tumors,but whether AFAP1-AS1 is involved in the mitosis of TNBC cells remains unknown.In this study,we investigated the functional mechanism of AFAP1-AS1 in targeting Polo-like Kinase 1(PLK1)activation and participating in mitosis of TNBC cells.We detected the expression of AFAP1-AS1 in the TNBC patient cohort and primary cells by in situ hybridization(ISH),northern blot,fluorescent in situ hybridization(FISH)and cell nucleus/cytoplasm RNA fraction isolation.High AFAP1-AS1 expression was negatively correlated with overall survival(OS),disease-free survival(DFS),metastasis-free survival(MFS)and recurrence-free survival(RFS)in TNBC patients.We explored the function of AFAP1-AS1 by transwell,apoptosis,immunofluorescence(IF)and patient-derived xenograft(PDX)models in vitro and in vivo.We found that AFAP1-AS1 promoted TNBC primary cell survival by inhibiting mitotic catastrophe and increased TNBC primary cell growth,migration and invasion.Mechanistically,AFAP1-AS1 activated phosphorylation of the mitosis-associated kinase PLK1 protein.Elevated levels of AFAP1-AS1 in TNBC primary cells increased PLK1 pathway downstream gene expression,such as CDC25C,CDK1,BUB1 and TTK.More importantly,AFAP1-AS1 increased lung metastases in a mouse metastasis model.Taken together,AFAP1-AS1 functions as an oncogene that activates the PLK1 signaling pathway.AFAP1-AS1 could be used as a potential prognostic marker and therapeutic target for TNBC.

Mitotic Catastrophe的研究进展

细胞死亡是多细胞生物生命过程中重要的生理或病理现象,可分为坏死和程序性细胞死亡,而后者根据死亡细胞的形态学和发生机制的不同又可分为凋亡、自吞噬和mitotic catastrophe,其中mitotic catastrophe是近年来才被揭示报道,是指细胞在有丝分裂过程中死亡的现象,是一种发生在细胞有丝分裂期由于异常的细胞分裂而导致的细胞死亡,它常常伴随着细胞有丝分裂检查点的异常和基因或纺锤体结构的损伤而发生。现对mitotic catastrophe及相关的调控机制进行综述。

Genomic Instability in Cancer I: DNA-Repair Triggering Primitive Hereditary 4n-Skewed, Amitotic Division-System, the Culprit in EMT/MET/Metaplasia Cancer-Concepts

The objective was to gain proof of genome damage-repair induced mitotic slippage process (MSP) to 4n-diplochromosome skewed division-system, earlier suggested to have “cancer-deciding” consequences. Our damage-model showed two succeeding phases: molecular mutations for initiation of fitness-gained cells, and large chromosomal changes to aneuploidy from inherited DNA-breakage-repair inaccuracies. The mutations were gained while DNA-repair and DNA-replication, co-existed in the route to tetraploidy, a phenomenon also expressed for some existing unicellular organisms. These organisms also showed genome reductive, amitotic, meioticlike division, and was the origin of human genome conserved, self-inflicted 90° reorientation of the 4n nucleus relative to the cytoskeleton axis. In the in vitro DNA-damage model, this remarkable 4n-event deciding “flat-upright” cell-growth characteristics showed several consequences, for example, cancer-important, E-cadherin-β-catenin cell-to-cell adherence destruction, which gave diploid progeny cells, mobility freedom from cell contact inhibition, likely in renewal tissues. This 4n-skewed division-system with inheritance in progeny cells for repeat occurrences as mentioned for flat-up-right growth patterns is similar to claimed concepts of metaplasia-EMT/MET embryogenesis events in cancer evolution. A scrutiny of this literature, proof-wise invalidated this embryological concept by tetraploid 8C cells occurring in MET events and, was noted for small cell occurrence, i.e., diploidy from 4n-8C reductive division, an also event for tumor relapse cells, derived from genome damaging therapy agents. Pre-cancer hyperplasia reported MSP, cadherincatenin destruction and 90° perpendicularity to basal cell membrane. The DNA-damage-repair model can weed-out therapy-agents triggering 4n-skewed division. Cancer-control, beginning-information, is likely from mutational identity of the 4n derived fitness-gained cells.

Mitotic crossover- an evolutionary rudiment which promotes carcinogenesis of colorectal carcinoma

Mitotic crossover is a natural mechanism that is a main source of the genetic variability of primitive organisms.In complex organisms such as mammals,it represents an evolutionary rudiment which persisted as one of the numerous DNA repair mechanisms,and results in the production of homozygous allele combinations in all heterozygous genes located on the chromosome arm distal to the crossover.This event is familiar as loss of heterozygosity,which is one of the key mechanisms responsible for the development and progression of almost all cancers.We propose the hypothesis in which mitotic crossover is a principal source of the increased loss of heterozygosity that leads to the initiation and progression of colorectal carcinoma.The hypothesis could be tested by in vitro inhibition of Rad51 protein,orthotopic grafting of human colon cancer tissue into the gut of mice,and treatment with potential inhibitors.After these procedures,the frequency of mitotic crossover would be estimated.The development of selective inhibitors of mitotic crossover could stop further carcinogenesis of colorectal carcinoma,as well as many other neoplastic events.Loss of heterozygosity is an event responsible for carcinogenesis,its reduction by selective inhibitors of mitotic crossover could have a positive effect on cancer chemoprevention,as well as on growth reduction and a cessation in the progression of earlier developed tumors.

Transcriptional regulation of human polo-like kinases and early mitotic inhibitors

Human polo-like kinases （PLK1-PLK4） have been implicated in mitotic regulation and carcinogenesis. PLK1 phosphorylates early mitotic inhibitor 1 （Emil） to ensure mitosis entry, whereas Emi2 plays a key role during the meiotic cell cycle. Transcription factor E2F is primarily considered to regulate the G1/S transition of the cell cycle but its involvement in the regulation of mitosis has also been recently suggested. A gap still exists between the molecular basis of E2F and mitotic regulation. The present study was designed to characterize the transcriptional regulation of human PLK and Emi genes. Adenoviral overexpression of E2F1 increased PLK1 and PLK3 mRNA levels in A549 cells. A reporter gene assay revealed that the putative promoter regions of PLK1, PLK3, and PLK4 genes were responsive to activators E2F, E2F1-E2F3. We further characterized the putative promoter regions of Emil and Emi2 genes, and these could be regulated by activators E2F and E2F1-E2F4, respectively. Finally, PLK1-PLK4, Emil, and Emi2 mRNA expression levels in human adult, fetal tissues, and several cell lines indicated that each gene has a unique expression pattern but is uniquely expressed in common tissues and cells such as the testes and thymus. Collectively, these results indicate that E2F can integrate G1/S and G2/M to oscillate the cell cycle by regulating mitotic genes PLK and Emi, leading to determination of the cell fate.

Single-cell RNA sequencing of mitoticarrested prospermatogonia with DAZL::GFP chickens and revealing unique epigenetic reprogramming of chickens

Background:Germ cell mitotic arrest is conserved in many vertebrates,including birds,although the time of entry or exit into quiescence phase differs.Mitotic arrest is essential for the normal differentiation of male germ cells into spermatogonia and accompanies epigenetic reprogramming and meiosis inhibition from embryonic development to post-hatch.However,mitotic arrest was not well studied in chickens because of the difficulty in obtaining pure germ cells from relevant developmental stage.Results:We performed single-cell RNA sequencing to investigate transcriptional dynamics of male germ cells during mitotic arrest in DAZL::GFP chickens.Using differentially expressed gene analysis and K-means clustering to analyze cells at different developmental stages(E12,E16,and hatch),we found that metabolic and signaling pathways were regulated,and that the epigenome was reprogrammed during mitotic arrest.In particular,we found that histone H3K9 and H3K14 acetylation(by HDAC2)and DNA demethylation(by DNMT3B and HELLS)led to a transcriptionally permissive chromatin state.Furthermore,we found that global DNA demethylation occurred gradually after the onset of mitotic arrest,indicating that the epigenetic-reprogramming schedule of the chicken genome differs from that of the mammalian genome.DNA hypomethylation persisted after hatching,and methylation was slowly re-established 3 weeks later.Conclusions:We found a unique epigenetic-reprogramming schedule of mitotic-arrested chicken prospermatogonia and prolonged hypomethylation after hatching.This will provide a foundation for understanding the process of germ-cell epigenetic regulation in several species for which this process is not clearly described.Our findings on the biological processes related to sex-specific differentiation of prospermatogonia could help studying germline development in vitro more elaborately.

Cytological and transcriptional analysis reveal phosphatidylinositol signaling pathway plays key role in mitotic division of Pyropia yezoensis

The phosphatidylinositol(PI)signaling system,a central regulator of eukaryotic metabolism,is widely found in eukaryotes for regulating a variety of cell activities.Most of the genes in the PI signaling system were found conserved in Pyropia yezoensis.In this experiment,wortmannin was used as an inhibitor to inhibit the activity of phosphatidylinositol-3 kinase(PI3K),an important regulator of the PI signaling system.After wortmannin treatment,the mitotic division of P.yezoensis was signifi cantly inhibited in a dose-dependent manner,and the mitotic division percentage was reduced by 68.1%and 91.9%in the 5-and 10-μmol/L groups,respectively.When thalli were treated with wortmannin,the levels of reactive oxygen species(ROS)were signifi cantly decreased.Furthermore,the expression level of PI3K was inhibited and the expression levels of downstream genes regulated by PI3K was signifi cantly changed.In the PI3K-AGC signaling pathway,the expression levels of Serine/threonine protein kinase(AGC)and cyclindependent kinases A(CDKA)were downregulated,while WEE1 kinase gene(WEE1)was upregulated.Three nicotinamide adenine dinucleotide phosphate(NADPH)oxidase genes were downregulated after wortmannin treatment.These results indicate that the PI signaling system plays an important role in the regulation of cell activity in P.yezoensis.It was speculated that the growth and development of P.yezoensis might be regulated by P.yezoensis PI3K,which promoted the expression of the AGC gene and further regulates the expression of downstream WEE1 and CDKA genes to advance mitotic division,and also promoted the expression level of NADPH oxidase that regulates ROS homeostasis.

Mitotic Activity in Cowpea (<i>Vigna unguiculata</i>(L.) Land Race “Olaudi” Walp) in Nigeria

Cowpea, Vigna unguiculata is a major staple for millions of households in the tropics. A cowpea land race, “Olaudi” collected from South-Eastern Nigeria was investigated for mitotic indices during cell division. Root tips were pre-treated with 8-hydroxyquinoline for three hours, fixed in Carnoy’s solution (alcohol-acetic acid solution 3:1 v/v) for 24 hours, hydrolysed in 1 NHCl and squashed in aceto-orcein. Various stages of mitosis were recorded to determine mitotic activity in dividing cells. Results obtained showed that active cell division occurred between 7.00 AM and 2.00 PM but peaks between 11.00 AM and 1.00 PM. Although cells at the prophase stage were in a continuum, they became observable between 8.00 AM and 3.00 PM, but greater proportion of cells was observed at 12.00 PM. Cells at metaphase were generally observed during a six hours period between 8.00 AM and 1.00 PM, but the peak period was 11.00 AM. Mitotic activity at anaphase commenced around 10.00 AM late morning but plateaus for two hours early afternoon between 12.00 PM and 2.00 PM. Telophase activity was mostly observed at 11.00 AM within a five hour period between 9.00 AM and 2.00 PM. Mitotic index peaked at 1.00 PM with 65.3% of cells in active cell division, with a concomitant high mean germination percentage (63.5%). This study provides useful basic information to guide further cytogenetics research in the genetic improvement of this local cowpea landrace.

Mitotic Studies on <i>Combretum</i>Loefl. from Nigeria

Cytological studies involving root-tip chromosomes of ten Combretum species belonging to the family Combretaceae from Nigeriawere carried out. The result showed that C. platypterum, C. racemosum, C. constrictum, C. bracteatum and a yet to be identified Combretum sp.2 have a chromosome number of 2n = 26. Also, C. racemosum, C. zenkeri and Combretum sp.3 (yet to be identified) have a chromosome number of 2n = 39. A basic chromosome number of x = 13 for the genus is therefore proposed.

Systems Approach to Mitotic Chromosome Motions

Recent experiments revealing possible nanoscale electrostatic interactions in force generation at kinetochores for chromosome motions have prompted speculation regarding possible models for interactions between positively charged molecules in kinetochores and negative charge on C-termini near the plus ends of microtubules. A clear picture of how kinetochores establish and maintain a dynamic coupling to microtubules for force generation during the complex motions of mitosis remains elusive. The molecular cell biology paradigm requires that specific molecules, or molecular geometries, for force generation be identified. However, it is possible to account for mitotic chromosome motions within a systems approach in terms of experimentally known cellular electric charge distributions interacting over nanometer distances.

Mitotic inheritance of DNA methylation: more than just copy and paste

Decades of investigation on DNA methylation have led to deeper insights into its metabolic mechanisms and biological functions.This understanding was fueled by the recent development of genome editing tools and our improved capacity for analyzing the global DNA methylome in mammalian cells.This review focuses on the maintenance of DNA methylation patterns during mitotic cell division.We discuss the latest discoveries of the mechanisms for the inheritance of DNA methylation as a stable epigenetic memory.We also highlight recent evidence showing the rapid turnover of DNA methylation as a dynamic gene regulatory mechanism.A body of work has shown that altered DNA methylomes are common features in aging and disease.We discuss the potential links between methylation maintenance mechanisms and diseaseassociated methylation changes.

The tumor suppressor CYLD controls epithelial morphogenesis and homeostasis by regulating mitotic spindle behavior and adherens junction assembly

Epithelial morphogenesis and homeostasis are essential for animal development and tissue regeneration, and epithelial disorganization is associated with developmental disorders and tumorigenesis. However, the molecular mechanisms that contribute to the morphogenesis and homeostasis of the epithelium remain elusive. Herein, we report a novel role for the cylindromatosis （CYLD） tumor suppressor in these events. Our results show that CYLD depletion disrupts epithelial organization in both Drosophila egg chambers and mouse skin and intestinal epithelia. Microscopic analysis of proliferating cells in mouse epithelial tissues and cultured organoids reveals that loss of CYLD synergizes with tumor-promoting agents to cause the misorientation of the mitotic spindle. Mechanistic studies show that CYLD accu- mulates at the cell cortex in epithelial tissues and cultured ceils, where it promotes the formation of epithelial adherens junctions through the modulation of microtuhule dynamics. These data suggest that CYLD controls epithelial morphogenesis and homeostasis by modulating the assembly of adherens junctions and ensuring proper orientation of the mitotic spindle. Our findings thus provide novel insight into the role of CYLD in development, tissue homeostasis, and tumorigenesis.

MCDB: A comprehensive curated mitotic catastrophe database for retrieval, protein sequence alignment, and target prediction

Mitotic catastrophe(MC)is a form of programmed cell death induced by mitotic process disorders,which is very important in tumor prevention,development,and drug resistance.Because rapidly increased data for MC is vigorously promoting the tumor-related biomedical and clinical study,it is urgent for us to develop a professional and comprehensive database to curate MC-related data.Mitotic Catastrophe Database(MCDB)consists of 1214 genes/proteins and 5014 compounds collected and organized from more than 8000 research articles.Also,MCDB defines the confidence level,classification criteria,and uniform naming rules for MC-related data,which greatly improves data reliability and retrieval convenience.Moreover,MCDB develops protein sequence alignment and target prediction functions.The former can be used to predict new potential MC-related genes and proteins,and the latter can facilitate the identification of potential target proteins of unknown MC-related compounds.In short,MCDB is such a proprietary,standard,and comprehensive database for MC-relate data that will facilitate the exploration of MC from chemists to biologists in the fields of medicinal chemistry,molecular biology,bioinformatics,oncology and so on.The MCDB is distributed on http://www.combio-lezhang.online/MCDB/indexhtml/.

Down-regulation of mitotic checkpoint in transformed human embryo lung fibroblasts induced by N-methyl-N'-nitro-N-nitrosoguaridine

Background Mutations in mitotic checkpoint genes have been detected in several human cancers, which exhibit chromosome instability. We wanted to know whether mutation of hBub1 could occur in transformed human embryo lung fibroblasts (HELF) cells induced by a chemical carcinogen.Methods HELF cells were transformed by N-methyl-N’-nitro-N- nitrosoguaridine (MNNG), and three flasks of transformed HELF cells (named as T1, T2, and T3) were selected as amplifiers, and mutations of hBub1 in these transformed cells were analyzed by PCR-SSCP and sequencing.Results It was found that any one of three transformed cell lines exhibited aneuploidy with a low mitotic checkpoint function. Subsequent PCR-SSCP and sequence analysis showed an AGT to CGT or ATT mutation at codon 80 in hBub1 gene in T1 cells with a resultant change in amino acid sequence.Conclusion Our study demonstrated that the mitotic checkpoint genes could be targets of MNNG.

Flavonoids kaempferide and 4,2’-dihydroxy-4’,5’,6’-trimethoxychalcone inhibit mitotic clonal expansion and induce apoptosis during the early phase of adipogenesis in 3T3-L1 cells

Objective: Kaempferide and 4,2'-dihydroxy-4',5',6'-trimethoxychalcone (DTMC) are two major flavonoids found in Chromolaena odoraia Linn, leaf extract. The aim of this study was to elucidate the mechanism by which these two flavonoids exerted their effect on adipogenesis. The inhibitory effect of kaempferide and DTMC on adipocyte differentiation and their mechanisms involving mitotic clonal expansion (MCE) and apoptosis during the early stage of adipogenesis were investigated. Methods: Confluent 3T3-L1 preadipocytes were induced to differentiate and exposed to the flavonoids during various phases of difTerentiaiion. Intracellular lipid accumulation, cell density and expression of the transcription factors peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding pro-teins a were assessed using AdipoRed, Oil red 0 and Western blot assays. Effects of both flavonoids on cell proliferation and apoptosis were also determined by carboxyfluorescein diacetate succinimidyl ester and annexin V-fluorescein isothiocyanate/propidium iodide-staining assays, respectively. Results: Kaempferide and DTMC showed significant, concentration-dependent anti-adipogenic activity and effect on cell density in the early phase of adipogenesis. The expression of the transcription factors seemed to be reduced when the treatment was prolonged or in the early phase of adipogenesis. These flavonoids interrupted MCE via inhibition of preadipocyte proliferation and induction of apoptosis. DTMC was nearly three times more potent than kaempferide in inducing apoptosis. Conclusion: Kaempferide and DTMC exerted their anti-adipogenic activity through inhibition of MCE, either by suppressing cell proliferation or by inducing apoptosis during the early phase of differentiation.

The Short Isoform of Nuclear Mitotic Apparatus Protein 1 Functions as a Putative Tumor Suppressor

Background：Nuclear mitotic apparatus protein 1 （NuMA 1） had been reported to produce three groups of isoforms categorized as long,middle,and short groups,of which short NuMA displayed distinct localization patterns compared to long and middle isoforms.However,the function of short NuMA was not clear in the progress of cancer formation.This study aimed to unveil the role of short NuMA in cancer pathogenesis.Methods：The expression levels of short isoforms were explored in paired gastric carcinoma （GC） samples and different cell lines.Furthermore,the short isoform behaved as a putative tumor suppressor based on cell proliferation and cell colony formation assays.Pull-down assay and whole-genome gene expression analysis were carried out to search candidate interaction partners of short NuMA.Results：The expression of short NuMA was highly expressed in S and G2 phases of the cell cycle;compared with nontumor tissues,short NuMA downregulated in nine GCs （GC 1 [0.131,P =5 × 10^-4];GC2 [0.316,P =3 × 10^-5];GC3 [0.111,P =6 × 10^-4];GC4 [0.456,P =0.011];GC5 [0.474,P=0.001];GC6 [0.311,P=0.004];GC7 [0.28,P=3 × 10^-5];GC8 [0.298,P=0.007];and GC9 [0.344,P=0.002]）.Besides,high expression of short NuMA significantly inhibits cell growth （2.43 × 10^-5 vs.2.97 × 10^-5,P =0.0029） and cell clone information in vitro （70 vs.2,P =1.67 × 10^-45）.Short NuMA could bind with alpha-actinin-4 （ACTN4）,a putative tumor promoting gene.Overexpression of short NuMA could tremendously decrease the expression of MYB proto-oncogene like 2 （MYBL2） of about 92-fold,which played an important role in the cell cycles.Conclusions：Short isoform of NuMA might be functioned as a putative role of tumor suppressor.Further studies should be made to illuminate the relationship between ACTN4,MYBL2,and tumor progression.

A Long Type of TBCK Is a Novel Cytoplasmic and Mitotic Apparatus-Associated Protein Likely Suppressing Cell Proliferation

Autoantibodies from patients with various connective tissue diseases have been shown to be specific probes that can detect cellular structures, including centrosome, centromere/kineto- chore, spliceosome, Golgi complex and the rough endoplasmic reticulum （Louvard et al., 1982; Rattner et al., 1998;

AMPK-mediated activation of MCU stimulates mitochondrial Ca2+entry to promote mitotic progression

With the support by the National Natural Science Foundation of China,the research team directed by Prof.Pan Xin(潘欣)and Prof.Zhang XueMin(张学敏)at the State Key Laboratory of Toxicology and Medical Countermeasures,Institute of Pharmacology and Toxicology,National Center of Biomedical Analysis,recently reported that a mitochondrial Ca2+transient is essential for mitotic progression,which was published as a cover story in Nature Cell Biology(2019,21:476—486).

Distinctive phosphorylation pattern during mitotic exit network(MEN)regulation is important for the development and pathogenicity of Magnaporthe oryzae

The mitotic exit network(MEN)pathway is a vital kinase cascade regulating the timely and correct progress of cell division.In the rice blast fungus Magnaporthe oryzae,the MEN pathway,consisting of conserved protein kinases MoSep1 and MoMob1-MoDbf2,is important in the development and pathogenicity of the fungus.We found that deletion of MoSEP1 affects the phosphorylation of MoMob1,but not MoDbf2,in contrast to what was found in the buddy yeast Saccharomyces cerevisiae,and verified this finding by in vitro phosphorylation assay and mass spectrom-etry(MS)analysis.We also found that S43 residue is the critical phosphor-site of MoMob1 by MoSep1,and proved that MoSep1-dependent MoMob1 phosphorylation is essential for cell division during the development of M.oryzae.We further provided evidence demonstrating that MoSep1 phosphorylates MoMob1 to maintain the cell cycle during vegetative growth and infection.Taken together,our results revealed that the MEN pathway has both distinct and conservative functions in regulating the cell cycle during the development and pathogenesis of M.oryzae.

Mitotic chromosome mis-segregation mediated by Separase phosphosite mutation leads to aneuploidy and impaired oogenesis

Aneuploidy is the leading genetic cause of human fertility failure. It is considered results from errors in meiotic and post-zygotic mitotic chromosome segregation. Using a