RESUMO
During mitosis, chromatin condensation shapes chromosomes as separate, rigid, and compact sister chromatids to facilitate their segregation. Here, we show that, unlike wild-type yeast chromosomes, non-chromosomal DNA circles and chromosomes lacking a centromere fail to condense during mitosis. The centromere promotes chromosome condensation strictly in cis through recruiting the kinases Aurora B and Bub1, which trigger the autonomous condensation of the entire chromosome. Shugoshin and the deacetylase Hst2 facilitated spreading the condensation signal to the chromosome arms. Targeting Aurora B to DNA circles or centromere-ablated chromosomes or releasing Shugoshin from PP2A-dependent inhibition bypassed the centromere requirement for condensation and enhanced the mitotic stability of DNA circles. Our data indicate that yeast cells license the chromosome-autonomous condensation of their chromatin in a centromere-dependent manner, excluding from this process non-centromeric DNA and thereby inhibiting their propagation.
Assuntos
Centrômero/genética , Cromossomos Fúngicos/genética , Mitose , Saccharomyces cerevisiae/genética , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/genética , Sirtuína 2/metabolismoRESUMO
Liquid-liquid phase separation (LLPS) of putative assembly scaffolds has been proposed to drive the biogenesis of membraneless compartments. LLPS scaffolds are usually identified through in vitro LLPS assays with single macromolecules (homotypic), but the predictive value of these assays remains poorly characterized. Here, we apply a strategy to evaluate the robustness of homotypic LLPS assays. When applied to the chromosomal passenger complex (CPC), which undergoes LLPS in vitro and localizes to centromeres to promote chromosome biorientation, LLPS propensity in vitro emerged as an unreliable predictor of subcellular localization. In vitro CPC LLPS in aqueous buffers was enhanced by commonly used crowding agents. Conversely, diluted cytomimetic media dissolved condensates of the CPC and of several other proteins. We also show that centromeres do not seem to nucleate LLPS, nor do they promote local, spatially restrained LLPS of the CPC. Our strategy can be adapted to purported LLPS scaffolds of other membraneless compartments.
Assuntos
Centrômero , Humanos , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , Substâncias Macromoleculares/metabolismo , Substâncias Macromoleculares/química , Separação de FasesRESUMO
Reflecting its pleiotropic functions, Polo-like kinase 1 (PLK1) localizes to various sub-cellular structures during mitosis. At kinetochores, PLK1 contributes to microtubule attachments and mitotic checkpoint signaling. Previous studies identified a wealth of potential PLK1 receptors at kinetochores, as well as requirements for various mitotic kinases, including BUB1, Aurora B, and PLK1 itself. Here, we combine ectopic localization, in vitro reconstitution, and kinetochore localization studies to demonstrate that most and likely all of the PLK1 is recruited through BUB1 in the outer kinetochore and centromeric protein U (CENP-U) in the inner kinetochore. BUB1 and CENP-U share a constellation of sequence motifs consisting of a putative PP2A-docking motif and two neighboring PLK1-docking sites, which, contingent on priming phosphorylation by cyclin-dependent kinase 1 and PLK1 itself, bind PLK1 and promote its dimerization. Our results rationalize previous observations and describe a unifying mechanism for recruitment of PLK1 to human kinetochores.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Histonas/metabolismo , Cinetocoros/metabolismo , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Células HeLa , Histonas/genética , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Quinase 1 Polo-LikeRESUMO
The kinetochore complex is a conserved machinery that connects chromosomes to spindle microtubules. During meiosis, the kinetochore is restructured to accommodate a specialized chromosome segregation pattern. In budding yeast, meiotic kinetochore remodeling is mediated by the temporal changes in the abundance of a single subunit called Ndc80. We previously described the regulatory events that control the timely synthesis of Ndc80. Here, we report that Ndc80 turnover is also tightly regulated in meiosis: Ndc80 degradation is active in meiotic prophase, but not in metaphase I. Ndc80 degradation depends on the ubiquitin ligase APCAma1 and is mediated by the proteasome. Importantly, Aurora B-dependent Ndc80 phosphorylation, a mark that has been previously implicated in correcting erroneous microtubule-kinetochore attachments, is essential for Ndc80 degradation in a microtubule-independent manner. The N terminus of Ndc80, including a 27-residue sequence and Aurora B phosphorylation sites, is both necessary and sufficient for kinetochore protein degradation. Finally, defects in Ndc80 turnover predispose meiotic cells to chromosome mis-segregation. Our study elucidates the mechanism by which meiotic cells modulate their kinetochore composition through regulated Ndc80 degradation, and demonstrates that Aurora B-dependent regulation of kinetochores extends beyond altering microtubule attachments.
Assuntos
Aurora Quinase B/metabolismo , Cinetocoros/metabolismo , Meiose/fisiologia , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Microtúbulos/metabolismo , ProteóliseRESUMO
The distinctive segregation patterns of chromosomes in mitosis and meiosis are dictated in part by the kinetochores, the structures on chromosomes that attach them to the microtubules of the spindle. Inappropriate mitosis-like chromosome segregation in meiosis leads to gametes with incorrect chromosome numbers. New findings by Chen and colleagues (pp. 209-225) in this issue of Genes & Development reveal how cells restructure their kinetochores when they enter meiosis. Their results describe an interconnected set of mechanisms that provides multiple layers of protection from the carryover of mitotic chromosome segregation patterns into meiotic cells.
Assuntos
Cinetocoros , Meiose , Segregação de Cromossomos , Microtúbulos , MitoseRESUMO
Both an increased frequency of chromosome missegregation (chromosomal instability, CIN) and the presence of an abnormal complement of chromosomes (aneuploidy) are hallmarks of cancer. To better understand how cells are able to adapt to high levels of chromosomal instability, we previously examined yeast cells that were deleted of the gene BIR1, a member of the chromosomal passenger complex (CPC). We found bir1Δ cells quickly adapted by acquiring specific combinations of beneficial aneuploidies. In this study, we monitored these yeast strains for longer periods of time to determine how cells adapt to high levels of both CIN and aneuploidy in the long term. We identify suppressor mutations that mitigate the chromosome missegregation phenotype. The mutated proteins fall into four main categories: outer kinetochore subunits, the SCFCdc4 ubiquitin ligase complex, the mitotic kinase Mps1, and the CPC itself. The identified suppressor mutations functioned by reducing chromosomal instability rather than alleviating the negative effects of aneuploidy. Following the accumulation of suppressor point mutations, the number of beneficial aneuploidies decreased. These experiments demonstrate a time line of adaptation to high rates of CIN.
Assuntos
Proteínas F-Box , Neoplasias , Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Aneuploidia , Instabilidade Cromossômica/genética , Cinetocoros/metabolismo , Neoplasias/genética , Segregação de Cromossomos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas F-Box/genéticaRESUMO
During asymmetric cell division, cell polarity is coordinated with the cell cycle to allow proper inheritance of cell fate determinants and the generation of cellular diversity. In the Caenorhabditis elegans zygote, polarity is governed by evolutionarily conserved Partitioning-defective (PAR) proteins that segregate to opposing cortical domains to specify asymmetric cell fates. Timely establishment of PAR domains requires a cell cycle kinase, Aurora A (AIR-1 in C. elegans). Aurora A depletion by RNAi causes a spectrum of phenotypes including reversed polarity, excess posterior domains and no posterior domain. How depletion of a single kinase can cause seemingly opposite phenotypes remains obscure. Using an auxin-inducible degradation system and drug treatments, we found that AIR-1 regulates polarity differently at different times of the cell cycle. During meiosis I, AIR-1 acts to prevent later formation of bipolar domains, whereas in meiosis II, AIR-1 is necessary to recruit PAR-2 onto the membrane. Together, these data clarify the origin of multiple polarization phenotypes in RNAi experiments and reveal multiple roles of AIR-1 in coordinating PAR protein localization with cell cycle progression.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Zigoto/metabolismo , Ciclo Celular/genética , Polaridade Celular/genética , Embrião não Mamífero/metabolismoRESUMO
Telomeres regulate DNA damage response (DDR) and DNA repair activity at chromosome ends. How telomere macromolecular structure contributes to ATM regulation and its potential dissociation from control over non-homologous end joining (NHEJ)-dependent telomere fusion is of central importance to telomere-dependent cell aging and tumor suppression. Using super-resolution microscopy, we identify that ATM activation at mammalian telomeres with reduced TRF2 or at human telomeres during mitotic arrest occurs specifically with a structural change from telomere loops (t-loops) to linearized telomeres. Additionally, we find the TRFH domain of TRF2 regulates t-loop formation while suppressing ATM activity. Notably, we demonstrate that ATM activation and telomere linearity occur separately from telomere fusion via NHEJ and that linear DDR-positive telomeres can remain resistant to fusion, even during an extended G1 arrest, when NHEJ is most active. Collectively, these results suggest t-loops act as conformational switches that specifically regulate ATM activation independent of telomere mechanisms to inhibit NHEJ.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Reparo do DNA por Junção de Extremidades , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA , Fibroblastos/citologia , Fibroblastos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Mitose , Domínios Proteicos , Telômero/ultraestrutura , Proteína 2 de Ligação a Repetições Teloméricas/química , Proteína 2 de Ligação a Repetições Teloméricas/metabolismoRESUMO
The mitotic spindle contains many bundles of microtubules (MTs) including midzones and kinetochore fibers, but little is known about how bundled structures are formed. Here, we show that the chromosomal passenger complex (CPC) purified from Escherichia coli undergoes liquid-liquid demixing in vitro. An emergent property of the resultant condensates is to generate parallel MT bundles when incubated with free tubulin and GTP in vitro. We demonstrate that MT bundles emerge from CPC droplets with protruding minus ends that then grow into long and tapered MT structures. During this growth, we found that the CPC in these condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for liquid-liquid demixing or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data demonstrate that an in vitro biochemical activity to produce MT bundles emerges after the concentration of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle. Moreover, these data suggest that cells contain MT-organizing centers that generate MT bundles that emerge with the opposite polarity from centrosomes.
Assuntos
Cromossomos , Microtúbulos , Fuso Acromático , Humanos , Células HeLa , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitose , Fuso Acromático/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Animais , Xenopus laevisRESUMO
Among the factors that have been strongly implicated in regulating cancerous transformation, the primary monocilium (cilium) has gained increasing attention. The cilium is a small organelle extending from the plasma membrane, which provides a localized hub for concentration of transmembrane receptors. These receptors transmit signals from soluble factors (including Sonic hedgehog (SHH), platelet-derived growth factor (PDGF-AA), WNT, TGFß, NOTCH, and others) that regulate cell growth, as well as mechanosensory cues provided by flow or extracellular matrix. Ciliation is regulated by cell cycle, with most cells that are in G0 (quiescent) or early G1 ciliation and cilia typically absent in G2/M cells. Notably, while most cells organized in solid tissues are ciliated, cancerous transformation induces significant changes in ciliation. Most cancer cells lose cilia; medulloblastomas and basal cell carcinomas, dependent on an active SHH pathway, rely on ciliary maintenance. Changes in cancer cell ciliation are driven by core oncogenic pathways (EGFR, KRAS, AURKA, PI3K), and importantly ciliation status regulates functionality of those pathways. Ciliation is both influenced by targeted cancer therapies and linked to therapeutic resistance; recent studies suggest ciliation may also influence cancer cell metabolism and stem cell identity. We review recent studies defining the relationship between cilia and cancer.
Assuntos
Proteínas Hedgehog , Neoplasias , Humanos , Proteínas Hedgehog/metabolismo , Transdução de Sinais/fisiologia , Ciclo Celular/fisiologia , Proliferação de Células , Neoplasias/metabolismo , Cílios/metabolismoRESUMO
Cell division involves separating the genetic material and cytoplasm of a mother cell into two daughter cells. The last step of cell division, abscission, consists of cutting the cytoplasmic bridge, a microtubule-rich membranous tube connecting the two cells, which contains the midbody, a dense proteinaceous structure. Canonically, abscission occurs 1-3â h after anaphase. However, in certain cases, abscission can be severely delayed or incomplete. Abscission delays can be caused by mitotic defects that activate the abscission 'NoCut' checkpoint in tumor cells, as well as when cells exert abnormally strong pulling forces on the bridge. Delayed abscission can also occur during normal organism development. Here, we compare the mechanisms triggering delayed and incomplete abscission in healthy and disease scenarios. We propose that NoCut is not a bona fide cell cycle checkpoint, but a general mechanism that can control the dynamics of abscission in multiple contexts.
Assuntos
Microtúbulos , Células-Tronco , Animais , Citoplasma , Citosol , AnáfaseRESUMO
Accurate genome segregation in mitosis requires that all chromosomes are bioriented on the spindle. Cells monitor biorientation by sensing tension across sister centromeres. Chromosomes that are not bioriented have low centromere tension, which allows Aurora B (yeast Ipl1) to perform error correction that locally loosens kinetochore-microtubule attachments to allow detachment of microtubules and fresh attempts at achieving biorientation. However, it is not known whether low tension recruits Aurora B to centromeres or, alternatively, whether low tension directly activates Aurora B already localized at centromeres. In this work, we experimentally induced low tension in metaphase Saccharomyces cerevisiae yeast cells, then monitored Ipl1 localization. We find low tension recruits Ipl1 to centromeres. Furthermore, low tension-induced Ipl1 recruitment depended on Bub1, which is known to provide a binding site for Ipl1. In contrast, Top2, which can also recruit Ipl1 to centromeres, was not required. Our results demonstrate cells are sensitive to low tension at centromeres and respond by actively recruiting Ip1l for error correction.
Assuntos
Cinetocoros , Saccharomyces cerevisiae , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Centrômero/metabolismo , Segregação de Cromossomos , Proteínas Fúngicas/metabolismo , Cinetocoros/metabolismo , Metáfase , Microtúbulos/metabolismo , Mitose , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
GCN2/eIF2αK4 is exclusively seen as an eIF2α kinase, which regulates reprogramming of protein translation in response to stress. Here, we show that GCN2 has an unexpected role in unstressed cells as a regulator of mitosis. This function is not through its canonical role in translation reprogramming, but through the regulation of two previously unidentified substrates, PP1α and γ. In the absence of GCN2 function, timing and levels of phosphorylation of key mitotic players are altered, leading to aberrant chromosome alignment, missegregating chromosomes, elevated number of tripolar spindles, and a delay in progression through mitosis. Pharmacological inhibition of GCN2 results in similar effects and is synergistic with Aurora A inhibition in causing more severe mitotic errors and cell death. We suggest that GCN2-dependent phosphorylation of PP1α and γ restrains their activity and this is important to ensure the timely regulation of phosphorylation of several PP1 substrates during early mitosis. These findings highlight a druggable PP1 inhibitor and open new avenues of research on the therapeutic potential of GCN2 inhibitors.
Assuntos
Mitose , Proteínas Serina-Treonina Quinases , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fosforilação , Cromossomos/metabolismoRESUMO
Epithelial ovarian cancer (EOC) is a high-risk cancer presenting with heterogeneous tumors. The high incidence of EOC metastasis from primary tumors to nearby tissues and organs is a major driver of EOC lethality. We used cellular models of spheroid formation and readherence to investigate cellular signaling dynamics in each step toward EOC metastasis. In our system, adherent cells model primary tumors, spheroid formation represents the initiation of metastatic spread, and readherent spheroid cells represent secondary tumors. Proteomic and phosphoproteomic analyses show that spheroid cells are hypoxic and show markers for cell cycle arrest. Aurora kinase B abundance and downstream substrate phosphorylation are significantly reduced in spheroids and readherent cells, explaining their cell cycle arrest phenotype. The proteome of readherent cells is most similar to spheroids, yet greater changes in the phosphoproteome show that spheroid cells stimulate Rho-associated kinase 1 (ROCK1)-mediated signaling, which controls cytoskeletal organization. In spheroids, we found significant phosphorylation of ROCK1 substrates that were reduced in both adherent and readherent cells. Application of the ROCK1-specific inhibitor Y-27632 to spheroids increased the rate of readherence and altered spheroid density. The data suggest ROCK1 inhibition increases EOC metastatic potential. We identified novel pathways controlled by Aurora kinase B and ROCK1 as major drivers of metastatic behavior in EOC cells. Our data show that phosphoproteomic reprogramming precedes proteomic changes that characterize spheroid readherence in EOC metastasis.
Assuntos
Neoplasias Ovarianas , Humanos , Feminino , Carcinoma Epitelial do Ovário , Neoplasias Ovarianas/metabolismo , Aurora Quinase B , Proteômica , Esferoides Celulares/metabolismo , Linhagem Celular Tumoral , Metástase Neoplásica , Quinases Associadas a rhoRESUMO
The subcellular events occurring in cells of legume plants as they form transcellular symbiotic-infection structures have been compared with those occurring in premitotic cells. Here, we demonstrate that Aurora kinase 1 (AUR1), a highly conserved mitotic regulator, is required for intracellular infection by rhizobia in Medicago truncatula. AUR1 interacts with microtubule-associated proteins of the TPXL and MAP65 families, which, respectively, activate and are phosphorylated by AUR1, and localizes with them within preinfection structures. MYB3R1, a rhizobia-induced mitotic transcription factor, directly regulates AUR1 through two closely spaced, mitosis-specific activator cis elements. Our data are consistent with a model in which the MYB3R1-AUR1 regulatory module serves to properly orient preinfection structures to direct the transcellular deposition of cell wall material for the growing infection thread, analogous to its role in cell plate formation. Our findings indicate that the eukaryotically conserved MYB3R1-TPXL-AUR1-MAP65 mitotic module was conscripted to support endosymbiotic infection in legumes.
Assuntos
Aurora Quinases , Medicago truncatula , Proteínas de Plantas , Rhizobium , Simbiose , Aurora Quinases/genética , Aurora Quinases/metabolismo , Regulação da Expressão Gênica de Plantas , Medicago truncatula/genética , Medicago truncatula/microbiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Rhizobium/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Cancer continues to be a major health concern globally, although the advent of targeted therapy has revolutionized treatment options. Aurora Kinase B is a serine-threonine kinase that has been explored as an oncology therapeutic target for more than two decades. Aurora Kinase B inhibitors show promising biological results in in-vitro and in-vivo experiments. However, there are no inhibitors approved yet for clinical use, primarily because of the side effects associated with Aurora B inhibitors. Several studies demonstrate that Aurora B inhibitors show excellent synergy with various chemotherapeutic agents, radiation therapy, and targeted therapies. This makes it an excellent choice as an adjuvant therapy to first-line therapies, which greatly improves the therapeutic window and side effect profile. Recent studies indicate the role of Aurora B in some deadly cancers with limited therapeutic options, like triple-negative breast cancer and glioblastoma. Herein, we review the latest developments in Aurora Kinase B targeted research, with emphasis on its potential as an adjuvant therapy and its role in some of the most difficult-to-treat cancers.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Aurora Quinase B/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/uso terapêutico , Neoplasias/tratamento farmacológico , Aurora Quinase A/uso terapêutico , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêuticoRESUMO
Aurora kinases (AURKs) are mitotic kinases important for regulating cell cycle progression. Small-molecule inhibitors of AURK have shown promising antitumor effects in multiple cancers; however, the utility of these inhibitors as inducers of cancer cell death has thus far been limited. Here, we examined the role of the Bcl-2 family proteins in AURK inhibition-induced apoptosis in colon cancer cells. We found that alisertib and danusertib, two small-molecule inhibitors of AURK, are inefficient inducers of apoptosis in HCT116 and DLD-1 colon cancer cells, the survival of which requires at least one of the two antiapoptotic Bcl-2 family proteins, Bcl-xL and Mcl-1. We further identified Bcl-xL as a major suppressor of alisertib- or danusertib-induced apoptosis in HCT116 cells. We demonstrate that combination of a Bcl-2 homology (BH)3-mimetic inhibitor (ABT-737), a selective inhibitor of Bcl-xL, Bcl-2, and Bcl-w, with alisertib or danusertib potently induces apoptosis through the Bcl-2 family effector protein Bax. In addition, we identified Bid, Puma, and Noxa, three BH3-only proteins of the Bcl-2 family, as mediators of alisertib-ABT-737-induced apoptosis. We show while Noxa promotes apoptosis by constitutively sequestering Mcl-1, Puma becomes associated with Mcl-1 upon alisertib treatment. On the other hand, we found that alisertib treatment causes activation of caspase-2, which promotes apoptosis by cleaving Bid into truncated Bid, a suppressor of both Bcl-xL and Mcl-1. Together, these results define the Bcl-2 protein network critically involved in AURK inhibitor-induced apoptosis and suggest that BH3-mimetics targeting Bcl-xL may help overcome resistance to AURK inhibitors in cancer cells.
Assuntos
Antineoplásicos , Apoptose , Aurora Quinases , Proteína bcl-X , Humanos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/metabolismo , Aurora Quinases/antagonistas & inibidores , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/fisiopatologia , Ativação Enzimática/efeitos dos fármacos , Células HCT116 , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Mitotic kinesin-like protein 2 (MKLP2; also known as KIF20A) is a motor protein with a well-established function in promoting cytokinesis. However, our results with siRNAs targeting MKLP2 and small-molecule inhibitors of MKLP2 (MKLP2i) suggest that it also has a function earlier in mitosis, prior to anaphase. In this study, we provide direct evidence that MKLP2 facilitates chromosome congression in prometaphase. We employed live imaging to observe HeLa cells with fluorescently tagged histones treated with MKLP2i and discovered a pronounced chromosome congression defect. We show that MKLP2 facilitates error correction, as inhibited cells have a significant increase in unstable, syntelic kinetochore-microtubule attachments. We find that the aberrant attachments are accompanied by elevated Aurora kinase (A and B) activity and phosphorylation of the downstream target HEC1 (also known as NDC80) at Ser55. Finally, we show that MKLP2 inhibition results in aneuploidy, confirming that MKLP2 safeguards cells against chromosomal instability. This article has an associated First Person interview with the first author of the paper.
Assuntos
Cinesinas/metabolismo , Mitose , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Segregação de Cromossomos , Cromossomos/metabolismo , Células HeLa , Humanos , Cinesinas/genética , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitose/genética , Fuso Acromático/metabolismoRESUMO
Chromosome segregation requires that centromeres properly attach to spindle microtubules. This essential step regulates the accuracy of cell division and must therefore be precisely regulated. One of the main centromeric regulatory signaling pathways is the haspin-H3T3ph-chromosomal passenger complex (CPC) cascade, which is responsible for the recruitment of the CPC to the centromeres. During mitosis, the haspin kinase phosphorylates histone H3 at threonine 3 (H3T3ph), an essential epigenetic mark that recruits the CPC, in which the catalytic component is Aurora B kinase (AURKB). However, the centromeric haspin-H3T3ph-CPC pathway remains largely uncharacterized in mammalian male meiosis. We have analyzed haspin functions by either its chemical inhibition with LDN-192960 in cultured spermatocytes, or the ablation of the Haspin gene in Haspin-/- mice. Our studies suggest that haspin kinase activity is required for proper chromosome congression both during meiotic divisions and for the recruitment of Aurora B and kinesin MCAK (also known as KIF2C) to meiotic centromeres. However, the absence of H3T3ph histone mark does not alter borealin (or CDCA8) and SGO2 centromeric localization. These results add new and relevant information regarding the regulation of the haspin-H3T3ph-CPC pathway and centromere function during meiosis.
Assuntos
Aurora Quinase B , Segregação de Cromossomos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Serina-Treonina Quinases , Animais , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinesinas/genética , Masculino , Mamíferos/metabolismo , Meiose/genética , Camundongos , Mitose , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Treonina/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is a highly malignant tumor with a global prevalence. In addition to the existing clinical guidelines, the effectiveness of anlotinib and Aurora-A inhibitors in treating HCC has also been demonstrated. However, Anlotinib, as an anti-angiogenesis therapy, has shown significant benefits in clinical trials but is limited by its single-agent treatment and the development of drug resistance. Aurora-A inhibitors are currently being tested in clinical trials but have limited efficacy. Combination therapy may offer clear advantages over monotherapy in this context. METHODS: In this study, we used HCC cell lines to investigate whether the combination of the two drugs could enhance their individual strengths and mitigate their weaknesses, thereby providing greater clinical benefits both in vitro and in vivo. RESULTS: Our findings confirmed that the Aurora-A inhibitor alisertib and anlotinib exhibited a time-dose-dependent inhibitory effect on HCC cells. In vitro cytological experiments demonstrated that the combination of the two drugs synergistically inhibited cell proliferation, invasion, and metastasis, while promoting cell apoptosis. Furthermore, we identified the underlying molecular mechanism by which the combination of the Aurora-A inhibitor alisertib and anlotinib inhibited HCC through the inhibition of the NF-ĸB signaling pathway. CONCLUSIONS: In summary, we have demonstrated the effectiveness of combining anlotinib with an Aurora-A inhibitor, which expands the potential applications of anlotinib in the clinical treatment of HCC in the future.