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Accurate chromosome segregation relies on kinetochores carrying out multiple functions, including establishing and maintaining microtubule attachments, forming precise bi-oriented attachments between sister chromatids, and activating the spindle assembly checkpoint. Central to these processes is the highly conserved Ndc80 complex. This kinetochore subcomplex interacts directly with microtubules but also serves as a critical platform for recruiting kinetochore-associated factors and as a key substrate for error correction kinases. The precise manner in which these kinetochore factors interact and regulate each other's function remains unknown, considerably hindering our understanding of how Ndc80 complex-dependent processes function together to orchestrate accurate chromosome segregation. Here, we aimed to uncover the role of Nuf2's CH domain, a component of the Ndc80 complex, in ensuring these processes. Through extensive mutational analysis, we identified a conserved interaction domain composed of two segments in Nuf2's CH domain that form the binding site for Mps1 within the yeast Ndc80 complex. Interestingly, this site also associates with the Dam1 complex, suggesting Mps1 recruitment may be subject to regulation by competitive binding with other factors. Mutants disrupting this "interaction hub" exhibit defects in spindle assembly checkpoint function and severe chromosome segregation errors. Significantly, specifically restoring Mps1-Ndc80 complex association rescues these defects. Our findings shed light on the intricate regulation of Ndc80 complex-dependent functions and highlight the essential role of Mps1 in kinetochore bi-orientation and accurate chromosome segregation.
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Proteínas de Ciclo Celular , Segregação de Cromossomos , Cinetocoros , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Cinetocoros/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genéticaRESUMO
Purpose: This study focuses on evaluating the prognostic value of the NDC80 kinetochore complex in ovarian cancer (OC) using the Gene Expression Omnibus (GEO) database and the Cancer Genome Atlas (TCGA) database and reveals the relationship between the NDC80 complex and immune infiltrates in OC. Methods: We collected data on NDC80 complex expression levels in both OC tissues and non-OC ovarian tissues from the University of California Santa Cruz Xena and GEO databases. The clinicopathological characteristics correlated with overall survival were analyzed using Cox regression and the Kaplan-Meier method. Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis, gene set enrichment analysis and CIBERSORT were performed using data from TCGA database. Immunohistochemical staining was used to verify the higher expression level of NUF2 protein in OC in vitro. Meanwhile, we utilized the Tumor Immune Estimation Resource to analyze the correlation between the NDC80 complex and immunocyte infiltration. Results: The NDC80 complex expression level was prominently higher in OC tissues than in non-OC ovarian tissues and correlated with advanced histologic grade characteristics. Gene expression profiling interactive analysis and the Kaplan-Meier survival curve uncovered a close relationship between high expression of the NDC80 complex and poor overall survival in OC patients. The univariate Cox regression hazard model produced age, pathologic stage, tumor status, primary therapy outcome, SPC24 expression level, and Karnofsky performance score as prognostic factors for OC patients. NDC80 complex expression levels were highly associated with immune cell infiltration, showing NK CD56 bright cells and NK cells with a negative correlation and T helper 2 cells with a positive correlation (P<0.05). Conclusion: These findings provide evidence that an increased expression level of the NDC80 complex is closely associated with the progression of OC and could also serve as a novel target of immunotherapy in OC.
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Centromere is the chromosomal site of kinetochore assembly and microtubule attachment for chromosome segregation. Given its importance, markers that allow specific labeling of centromeric chromatin throughout the cell cycle and across all chromosome types are sought for facilitating various centromere studies. Antibodies against the N-terminal region of CENH3 are commonly used for this purpose, since CENH3 is the near-universal marker of functional centromeres. However, because the N-terminal region of CENH3 is highly variable among plant species, antibodies directed against this region usually function only in a small group of closely related species. As a more versatile alternative, we present here antibodies targeted to the conserved domains of two outer kinetochore proteins, KNL1 and NDC80. Sequence comparison of these domains across more than 350 plant species revealed a high degree of conservation, particularly within a six amino acid motif, FFGPVS in KNL1, suggesting that both antibodies would function in a wide range of plant species. This assumption was confirmed by immunolabeling experiments in angiosperm (monocot and dicot) and gymnosperm species, including those with mono-, holo-, and meta-polycentric chromosomes. In addition to centromere labeling on condensed chromosomes during cell division, both antibodies detected the corresponding regions in the interphase nuclei of most species tested. These results demonstrated that KNL1 and NDC80 are better suited for immunolabeling centromeres than CENH3, because antibodies against these proteins offer incomparably greater versatility across different plant species which is particularly convenient for studying the organization and function of the centromere in non-model species.
Assuntos
Centrômero , Cinetocoros , Proteínas de Plantas , Sequência de Aminoácidos , Cromatina , Segregação de Cromossomos , Proteínas de Plantas/genéticaRESUMO
During cell division, kinetochores link chromosomes to spindle microtubules. The Ndc80 complex, a crucial microtubule binder, populates each kinetochore with dozens of copies. Whether adjacent Ndc80 complexes cooperate to promote microtubule binding remains unclear. Here we demonstrate that the Ndc80 loop, a short sequence that interrupts the Ndc80 coiled-coil at a conserved position, folds into a more rigid structure than previously assumed and promotes direct interactions between full-length Ndc80 complexes on microtubules. Mutations in the loop impair these Ndc80-Ndc80 interactions, prevent the formation of force-resistant kinetochore-microtubule attachments, and cause cells to arrest in mitosis for hours. This arrest is not due to an inability to recruit the kinetochore-microtubule stabilizing SKA complex and cannot be overridden by mutations in the Ndc80 tail that strengthen microtubule attachment. Thus, loop-mediated organization of adjacent Ndc80 complexes is crucial for stable end-on kinetochore-microtubule attachment and spindle assembly checkpoint satisfaction.
Assuntos
Cinetocoros , Microtúbulos , Segregação de Cromossomos , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Ligação Proteica , AnimaisRESUMO
Mitotic kinetochores are initially captured by dynamic microtubules via a "search-and-capture" mechanism. The microtubule motor, dynein, is critical for kinetochore capture as it has been shown to transport microtubule-attached chromosomes toward the spindle pole during prometaphase. The microtubule-binding nuclear division cycle 80 (Ndc80) complex that is recruited to kinetochores in prophase is known to play a central role in forming kinetochore-microtubule (kMT) attachments in metaphase. It is not yet clear, however, how Ndc80 contributes to initial kMT capture during prometaphase. Here, by combining CRISPR/Cas9-mediated knockout and RNAi technology with assays specific to study kMT capture, we show that mitotic cells lacking Ndc80 exhibit substantial defects in this function during prometaphase. Rescue experiments show that Ndc80 mutants deficient in microtubule-binding are unable to execute proper kMT capture. While cells inhibited of dynein alone are predominantly able to make initial kMT attachments, cells co-depleted of Ndc80 and dynein show severe defects in kMT capture. Further, we use an in vitro total internal reflection fluorescence microscopy assay to reconstitute microtubule capture events, which suggest that Ndc80 and dynein coordinate with each other for microtubule plus-end capture and that the phosphorylation status of Ndc80 is critical for productive kMT capture. A novel interaction between Ndc80 and dynein that we identify in prometaphase extracts might be critical for efficient plus-end capture. Thus, our studies, for the first time, identify a distinct event in the formation of initial kMT attachments, which is directly mediated by Ndc80 and in coordination with dynein is required for efficient kMT capture and chromosome alignment.
Assuntos
Dineínas , Cinetocoros , Dineínas/genética , Dineínas/metabolismo , Cinetocoros/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Microtúbulos/metabolismo , Mitose , Fuso Acromático/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMO
The conserved Ndc80 kinetochore complex, Ndc80c, is the principal link between mitotic spindle microtubules and centromere-associated proteins. We used AlphaFold 2 (AF2) to obtain predictions of the Ndc80 'loop' structure and of the Ndc80 : Nuf2 globular head domains that interact with the Dam1 subunit of the heterodecameric DASH/Dam1 complex (Dam1c). The predictions guided design of crystallizable constructs, with structures close to the predicted ones. The Ndc80 'loop' is a stiff, α-helical 'switchback' structure; AF2 predictions and positions of preferential cleavage sites indicate that flexibility within the long Ndc80c rod occurs instead at a hinge closer to the globular head. Conserved stretches of the Dam1 C terminus bind Ndc80c such that phosphorylation of Dam1 serine residues 257, 265 and 292 by the mitotic kinase Ipl1/Aurora B can release this contact during error correction of mis-attached kinetochores. We integrate the structural results presented here into our current molecular model of the kinetochore-microtubule interface. The model illustrates how multiple interactions between Ndc80c, DASH/Dam1c and the microtubule lattice stabilize kinetochore attachments.
Assuntos
Cinetocoros , Microtúbulos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Aurora Quinase B/metabolismo , Proteínas de Ciclo Celular , Centrômero/genética , Centrômero/metabolismo , Furilfuramida , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Post-translational modifications on histones are well known to regulate chromatin structure and function, but much less information is available on modifications of the centromeric histone H3 variant and their effect at the kinetochore. Here, we report two modifications on the centromeric histone H3 variant CENP-A/Cse4 in the yeast Saccharomyces cerevisiae, methylation at arginine 143 (R143me) and lysine 131 (K131me), that affect centromere stability and kinetochore function. Both R143me and K131me lie in the core region of the centromeric nucleosome, near the entry/exit sites of the DNA from the nucleosome. Unexpectedly, mutation of Cse4-R143 (cse4-R143A) exacerbated the kinetochore defect of mutations in components of the NDC80 complex of the outer kinetochore (spc25-1) and the MIND complex (dsn1-7). The analysis of suppressor mutations of the spc25-1 cse4-R143A growth defect highlighted residues in Spc24, Ndc80, and Spc25 that localize to the tetramerization domain of the NDC80 complex and the Spc24-Spc25 stalk, suggesting that the mutations enhance interactions among NDC80 complex components and thus stabilize the complex. Furthermore, the Set2 histone methyltransferase inhibited kinetochore function in spc25-1 cse4-R143A cells, possibly by methylating Cse4-K131. Taken together, our data suggest that Cse4-R143 methylation and Cse4-K131 methylation affect the stability of the centromeric nucleosome, which is detrimental in the context of defective NDC80 tetramerization and can be compensated for by strengthening interactions among NDC80 complex components.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cinetocoros/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Lisina/genética , Histonas/metabolismo , Metilação , Nucleossomos/genética , Arginina/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Nucleares/genéticaRESUMO
Some interspecific plant hybrids show unequal transmission of chromosomes from parental genomes to the successive generations. It has been suggested that this is due to a differential behavior of parental chromosomes during meiosis. However, underlying mechanism is unknown. We analyzed chromosome composition of the F2 generation of Festuca × Lolium hybrids and reciprocal backcrosses to elucidate effects of male and female meiosis on the shift in parental genome composition. We studied male meiosis, including the attachment of chromosomes to the karyokinetic spindle and gene expression profiling of the kinetochore genes. We found that Lolium and Festuca homoeologues were transmitted differently to the F2 generation. Female meiosis led to the replacement of Festuca chromosomes by their Lolium counterparts. In male meiosis, Festuca univalents were attached less frequently to microtubules than Lolium univalents, lagged in divisions and formed micronuclei, which were subsequently eliminated. Genome sequence analysis revealed a number of non-synonymous mutations between copies of the kinetochore genes from Festuca and Lolium genomes. Furthermore, we found that outer kinetochore proteins NDC80 and NNF1 were exclusively expressed from the Lolium allele. We hypothesize that silencing of Festuca alleles results in improper attachment of Festuca chromosomes to karyokinetic spindle and subsequently their gradual elimination.
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Festuca , Lolium , Lolium/genética , Festuca/genética , Hibridização Genética , Genoma de Planta , Cromossomos de Plantas/genética , Meiose/genéticaRESUMO
Background: Glioblastoma is a common malignant neuroepithelial neoplasm with poor clinical outcomes and limited treatment options. It is extremely important to search and confirm diverse hub genes that are effective in the advance and prediction of glioblastoma. Methods: We analyzed GSE50161, GSE4290, and GSE68848, the three microarray datasets retrieved from the GEO database. GO function and KEGG pathway enrichment analyses for differentially expressed genes (DEGs) were performed using DAVID. The PPI network of the DEGs was analyzed using the Search Tool for the Retrieval of Interacting Genes database and visualized by Cytoscape software. Hub genes were identified through the PPI network and a robust rank aggregation method. The Cancer Genome Atlas (TCGA) and the Oncomine database were used to validate the hub genes. In addition, a survival curve analysis was conducted to verify the correlation between the expression of hub genes and patient prognosis. Human glioblastoma cells and normal cells were collected, and then RT-PCR, Western blot, and immunofluorescence were conducted to validate the expression of the NDC80 gene. A cell proliferation assay was used to detect the proliferation of glioma cells. The effects of NDC80 expression on migration and invasion of GBM cell lines were evaluated by conducting scratch and transwell assays. Results: A total of 716 DEGs were common to all three microarray datasets, which included 188 upregulated DEGs and 528 downregulated DEGs. Furthermore, we found that among the common DEGs, 10 hub genes showed a high degree of connectivity. The expression of the 10 hub genes in TCGA and the Oncomine database was significantly overexpressed in glioblastoma compared with normal genes. Additionally, the survival analysis showed that the patients with low expression of six genes (BIR5C, CDC20, NDC80, CDK1, TOP2A, and MELK) had a significantly favorable prognosis (p < 0.01). We discovered that NDC80, which has been shown to be important in other cancers, also has an important role in malignant gliomas. The RT-PCR, Western blot, and immunofluorescence results showed that the expression level of NDC80 was significantly higher in human glioblastoma cells than in normal cells. Moreover, we identified that NDC80 increased the proliferation and invasion abilities of human glioblastoma cells. Conclusion: The six genes identified here may be utilized to form a panel of disease progression and predictive biomarkers of glioblastoma for clinical purposes. NDC80, one of the six genes, was discovered to have a potentially important role in GBM, a finding that needs to be further studied.
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Lung cancer remains the leading cause of malignant mortality worldwide. Hence, the discovery of novel targets that can improve therapeutic effects in lung cancer patients is an urgent need. In this study, we screened differentially expressed genes using isobaric tags for relative and absolute quantitation (iTRAQ) analysis and datasets from the cancer genome atlas database, and found that nuclear division cycle 80 (NDC80) might act as a novel prognostic indicator of lung cancer. The expression of NDC80 was significantly increased in lung cancer tissues, as compared to normal tissues, and high expression levels of NDC80 were correlated with unfavorable survival rates. Furthermore, an in vitro analysis showed that the stable knockdown of NDC80 decreased the cell viability and increased therapeutic sensitivity in two lung cancer cell lines, A549-IRR and H1246-IRR. Moreover, gene set enrichment analysis results showed that NDC80 was enriched in autophagy-related pathways. The downregulation of NDC80 inhibited the formation of autophagosomes, and reduced the expression of autophagy-related proteins such as LC3II, Beclin-1, and p62 in lung cancer cells. To further clarify the role of NDC80 as a downstream regulator of autophagy, we validated autophagic mediators through iTRAQ analysis and real-time polymerase chain reaction arrays. Autophagy-related protein7 (ATG7) was observed to be downregulated after the knockdown of NDC80 in lung cancer cells. Immunohistochemistry assay results revealed that both NDC80 and ATG7 were upregulated in an array of lung adenocarcinoma samples, compared to normal tissues, and the expression of NDC80 was identified to be positively associated with the levels of ATG7. Our findings suggest that NDC80 promotes the development of lung cancer by regulating autophagy, and might serve as a potential target for increasing the therapeutic sensitivity of lung cancer.
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Acidic nuclear phosphoprotein 32 family member E (ANP32E) is a histone chaperone that removes H2A.Z from chromatin. ANP32E is implicated in numerous cellular processes, including cell proliferation, apoptosis and cell differentiation. Increasing evidence suggests that dysregulation of ANP32E expression is strongly associated with carcinogenesis. However, the relationship between ANP32E in the development of gastric cancer (GC) is unknown. The present study aimed to explore the potential role of ANP32E in the development of GC using gainoffunction, lossoffunction, CCK8, colony formation, apoptosis, reverse transcriptionquantitative PCR, immunoblotting and luciferase reporter assay. The results of the present study demonstrated that ANP32E expression levels were significantly increased in GC tissues. ANP32E knockdown markedly inhibited GC cell proliferation and colony formation and significantly induced GC cell apoptosis, whereas overexpression of ANP32E significantly induced GC cell malignancy. Furthermore, the results demonstrated that there was a positive association between ANP32E and NUF2 component of NDC80 kinetochore complex (NUF2) expression levels. By assessing NUF2 expression levels, it was demonstrated that ANP32E promoted tumor cell proliferation and inhibited cell apoptosis by increasing NUF2 expression levels in GC cell lines. In conclusion, the present study indicated that ANP32E may function as an efficient oncogene, which promotes tumorigenesis of GC cells by inducing NUF2 expression.
Assuntos
Proteínas de Ciclo Celular , Chaperonas Moleculares , Neoplasias Gástricas , Carcinogênese/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/genética , Histonas/genética , Histonas/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Ativação Transcricional , Regulação para CimaRESUMO
During mitosis and meiosis in the majority of eukaryotes, centromeric chromatin comprised of CENP-A nucleosomes and their reader CENP-C recruits components of the outer kinetochore to build an interface with spindle microtubules.1,2 One exception is C. elegans oocyte meiosis, where outer kinetochore proteins form cup-like structures on chromosomes independently of centromeric chromatin.3 Here, we show that the nucleoporin MEL-28 (ortholog of human ELYS) and CENP-CHCP-4 act in parallel to recruit outer kinetochore components to oocyte meiotic chromosomes. Unexpectedly, co-inhibition of MEL-28 and CENP-CHCP-4 resulted in chromosomes being expelled from the meiotic spindle prior to anaphase onset, a more severe phenotype than what was observed following ablation of the outer kinetochore.4,5 This observation suggested that MEL-28 and the outer kinetochore independently link chromosomes to spindle microtubules. Consistent with this, the chromosome expulsion defect was observed following co-inhibition of MEL-28 and the microtubule-coupling KNL-1/MIS-12/NDC-80 (KMN) network of the outer kinetochore. Use of engineered mutants showed that MEL-28 acts in conjunction with the microtubule-binding NDC-80 complex to keep chromosomes within the oocyte meiotic spindle and that this function likely involves the Y-complex of nucleoporins that associate with MEL-28; by contrast, the ability to dock protein phosphatase 1, shared by MEL-28 and KNL-1, is not involved. These results highlight nuclear pore-independent functions for a conserved nucleoporin and explain two unusual features of oocyte meiotic chromosome segregation in C. elegans: centromeric chromatin-independent outer kinetochore assembly, and dispensability of the outer kinetochore for constraining chromosomes in the acentrosomal meiotic spindle.
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Proteínas de Caenorhabditis elegans , Cinetocoros , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona , Segregação de Cromossomos , Proteínas de Ligação a DNA/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fuso Acromático/metabolismoRESUMO
For correct chromosome segregation in mitosis, eukaryotic cells must establish chromosome biorientation where sister kinetochores attach to microtubules extending from opposite spindle poles. To establish biorientation, any aberrant kinetochore-microtubule interactions must be resolved in the process called error correction. For resolution of the aberrant interactions in error correction, kinetochore-microtubule interactions must be exchanged until biorientation is formed (the SWAP process). At initiation of biorientation, the state of weak kinetochore-microtubule interactions should be converted to the state of stable interactions (the SWITCH process)-the conundrum of this conversion is called the initiation problem of biorientation. Once biorientation is established, tension is applied on kinetochore-microtubule interactions, which stabilizes the interactions (the STABILIZE process). Aurora B kinase plays central roles in promoting error correction, and Mps1 kinase and Stu2 microtubule polymerase also play important roles. In this article, we review mechanisms of error correction by considering the SWAP, SWITCH, and STABILIZE processes. We mainly focus on mechanisms found in budding yeast, where only one microtubule attaches to a single kinetochore at biorientation, making the error correction mechanisms relatively simpler.
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Cinetocoros , Microtúbulos , Aurora Quinase B/genética , Segregação de Cromossomos , MitoseRESUMO
Correct chromosome segregation in mitosis relies on chromosome biorientation, in which sister kinetochores attach to microtubules from opposite spindle poles prior to segregation. To establish biorientation, aberrant kinetochore-microtubule interactions must be resolved through the error correction process. During error correction, kinetochore-microtubule interactions are exchanged (swapped) if aberrant, but the exchange must stop when biorientation is established. In this article, we discuss recent findings in budding yeast, which have revealed fundamental molecular mechanisms promoting this "swap and stop" process for error correction. Where relevant, we also compare the findings in budding yeast with mechanisms in higher eukaryotes. Evidence suggests that Aurora B kinase differentially regulates kinetochore attachments to the microtubule end and its lateral side and switches relative strength of the two kinetochore-microtubule attachment modes, which drives the exchange of kinetochore-microtubule interactions to resolve aberrant interactions. However, Aurora B kinase, recruited to centromeres and inner kinetochores, cannot reach its targets at kinetochore-microtubule interface when tension causes kinetochore stretching, which stops the kinetochore-microtubule exchange once biorientation is established.
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Cinetocoros , Saccharomycetales , Aurora Quinase B/genética , Segregação de Cromossomos , Microtúbulos/fisiologia , MitoseRESUMO
BACKGROUNDS: Chemotherapy is a standard systemic treatment option for triple-negative breast cancer (TNBC). Cisplatin has been used to treat TNBC, but frequently leads to cisplatin resistance in patients. The aim of our study was to investigate cisplatin-resistant mechanism in TNBC. MATERIALS AND METHODS: To identify the potential genes and pathways relative to cisplatin resistance, GSE103115 data were analyzed by the Limma package and Gene set enrichment analysis (GSEA). TNBC data from TCGA, GSE76250 and GSE115275 datasets were used to calculate NDC80 expression. Immunohistochemistry detected NDC80 protein expression in TNBC tissues from patients before and after cisplatin treatment. After expose to cisplatin treatment, the viability and proliferation of TNBC cells were measured by CCK-8 and colony formation assays, respectively. RESULTS: NDC80 was regarded as a cisplatin-resistant gene because after cisplatin treatment NDC80 was downregulated in cisplatin-sensitive cells but was upregulated in cisplatin-resistant cells. NDC80 was over-expressed in TNBC tissues compared to normal tissues. Furthermore, NDC80 expression in TNBC patients was increased after cisplatin treatment. Cisplatin-sensitive TNBC patients showed lower NDC80 expression than cisplatin-resistant patients. Additionally, NDC80 expression was correlated with clinical stages, tumor size and chemotherapy of TNBC patients. Moreover, NDC80 overexpression promoted the viability and proliferation of TNBC cells and enhanced the cells resistance to cisplatin. The potential pathways relative to cisplatin resistance were obtained, such as p53 signaling pathway and Oxidative phosphorylation. CONCLUSION: These findings provide new insights for understanding the mechanism of cisplatin resistance in TNBC, and NDC80 may be a potential therapeutic target for TNBC treatment.
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Antineoplásicos , Neoplasias de Mama Triplo Negativas , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Proteínas do Citoesqueleto/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Hec1 (Highly expressed in cancer 1) resides in the outer kinetochore where it works to facilitate proper kinetochore-microtubule interactions during mitosis. Hec1 is overexpressed in various cancers and its expression shows correlation with high tumour grade and poor patient prognosis. Chemical perturbation of Hec1 is anticipated to impair kinetochore-microtubule binding, activate the spindle assembly checkpoint (spindle checkpoint) and thereby suppress cell proliferation. In this study, we performed high-throughput screen to identify novel small molecules that target the Hec1 calponin homology domain (CHD), which is needed for normal microtubule attachments. 4 million compounds were first virtually fitted against the CHD, and the best hit molecules were evaluated in vitro. These approaches led to the identification of VTT-006, a 1,2-disubstituted-tetrahydro-beta-carboline derivative, which showed binding to recombinant Ndc80 complex and modulated Hec1 association with microtubules in vitro. VTT-006 treatment resulted in chromosome congression defects, reduced chromosome oscillations and induced loss of inter-kinetochore tension. Cells remained arrested in mitosis with an active spindle checkpoint for several hours before undergoing cell death. VTT-006 suppressed the growth of several cancer cell lines and enhanced the sensitivity of HeLa cells to Taxol. Our findings propose that VTT-006 is a potential anti-mitotic compound that disrupts M phase, impairs kinetochore-microtubule interactions, and activates the spindle checkpoint.
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Fibroblast growth factor receptor 2 (FGFR2) fusions have emerged as a new therapeutic target for cholangiocarcinoma in clinical practice following the United States Food and Drug Administration (FDA) approval of Pemigatinib in May 2020. FGFR2 fusions can result in a ligand-independent constitutive activation of FGFR2 signaling with a downstream activation of multiple pathways, including the mitogen-activated protein (MAPK) cascade. Until today, only a limited number of fusion partners have been reported, of which the most prevalent is BicC Family RNA Binding Protein (BICC1), representing one-third of all detected FGFR2 fusions. Nonetheless, in the majority of cases rare or yet unreported fusion partners are discovered in next-generation sequencing panels, which confronts clinicians with a challenging decision: Should a therapy be based on these variants or should the course of treatment follow the (limited) standard regime? Here, we present the case of a metastasized intrahepatic cholangiocarcinoma harboring a novel FGFR2-NDC80 fusion, which was discussed in our molecular tumor board. The protein NDC80 kinetochore complex component (NDC80) is an integral part of the outer kinetochore, which is involved in microtubule binding and spindle assembly. For additional therapeutic guidance, an immunohistochemical analysis of the predicted fusion and downstream effector proteins was performed and compared to cholangiocarcinoma samples of a tissue microarray. The FGFR2-NDC80 fusion resulted in strong activation of the FGFR2 signaling pathway. These supporting results led to a treatment recommendation of Pemigatinib. Unfortunately, the patient passed away before the commencement of therapy.
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Neoplasias dos Ductos Biliares , Colangiocarcinoma , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/genética , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/genética , Proteínas do Citoesqueleto , Humanos , Morfolinas , Pirimidinas , Pirróis , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Estados UnidosRESUMO
Chromosome segregation during cell division requires engagement of kinetochores of sister chromatids with microtubules emanating from opposite poles. As the corresponding microtubules shorten, these 'bioriented' sister kinetochores experience tension-dependent stabilization of microtubule attachments. The yeast XMAP215 family member and microtubule polymerase, Stu2, associates with kinetochores and contributes to tension-dependent stabilization in vitro. We show here that a C-terminal segment of Stu2 binds the four-way junction of the Ndc80 complex (Ndc80c) and that residues conserved both in yeast Stu2 orthologs and in their metazoan counterparts make specific contacts with Ndc80 and Spc24. Mutations that perturb this interaction prevent association of Stu2 with kinetochores, impair cell viability, produce biorientation defects, and delay cell cycle progression. Ectopic tethering of the mutant Stu2 species to the Ndc80c junction restores wild-type function in vivo. These findings show that the role of Stu2 in tension-sensing depends on its association with kinetochores by binding with Ndc80c.
Assuntos
Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Segregação de Cromossomos , Proteínas Associadas aos Microtúbulos/genética , Mutação , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: NEK2 has an established involvement in hepatocellular carcinoma (HCC) but the roles of NEK2 and its interacting proteins in HCC have not been systematically explored. METHODS: This study examined NEK2 and its interacting proteins in HCC based on multiple databases. RESULTS: NEK2 mRNA was highly expressed in HCC tissues compared with normal liver tissues. The survival of HCC patients with high NEK2 mRNA expression was shorter than those with low expression. MAD1L1, CEP250, MAPK1, NDC80, PPP1CA, PPP1R2 and NEK11 were the interacting proteins of NEK2. Among them, NDC80 and CEP250 were the key interacting proteins of NEK2. Mitotic prometaphase may be the key pathway that NEK2 and its interacting proteins contributed to HCC pathogenesis. NEK2, NDC80 and CEP250 mRNAs were highly expressed in HCC tissues compared with normal liver tissues. The mRNA levels of NEK2 were positively correlated with those of NDC80 or CEP250. Univariate regression showed that NEK2, NDC80 and CEP250 mRNA expressions were significantly associated with HCC patients' survival. Multivariate regression showed that NDC80 mRNA expression was an independent predictor for HCC patients' survival. Methylations and genetic alterations of NEK2, NDC80 and CEP250 were observed in HCC samples. The alterations of NEK2, NDC80 and CEP250 genes were co-occurrence. Patients with high mRNA expression and genetic alterations of NEK2, NDC80 and CEP250 had poor prognosis. CONCLUSIONS: NEK2 and its interacting proteins NDC80 and CEP250 play important roles in HCC development and progression and thus may be potentially used as biomarkers and therapeutic targets of HCC.
Assuntos
Autoantígenos/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Quinases Relacionadas a NIMA/metabolismo , Domínios e Motivos de Interação entre Proteínas , Autoantígenos/genética , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Estudos de Casos e Controles , Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto/genética , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Quinases Relacionadas a NIMA/genética , Prognóstico , Taxa de SobrevidaRESUMO
To ensure the faithful inheritance of DNA, a macromolecular protein complex called the kinetochore sustains the connection between chromosomes and force-generating dynamic microtubules during cell division. Defects in this process lead to aneuploidy, a common feature of cancer cells and the cause of many developmental diseases [1-4]. One of the major microtubule-binding activities in the kinetochore is mediated by the conserved Ndc80 complex (Ndc80c) [5-7]. In budding yeast, the retention of kinetochores on dynamic microtubule tips also depends on the essential heterodecameric Dam1 complex (Dam1c) [8-15], which binds to the Ndc80c and is proposed to be a functional ortholog of the metazoan Ska complex [16, 17]. The load-bearing activity of the Dam1c depends on its ability to oligomerize, and the purified complex spontaneously self-assembles into microtubule-encircling oligomeric rings, which are proposed to function as collars that allow kinetochores to processively track the plus-end tips of microtubules and harness the forces generated by disassembling microtubules [10-15, 18-22]. However, it is unknown whether there are specific regulatory events that promote Dam1c oligomerization to ensure accurate segregation. Here, we used a reconstitution system to discover that Cdk1, the major mitotic kinase that drives the cell cycle, phosphorylates the Ask1 component of the Dam1c to increase its residence time on microtubules and enhance kinetochore-microtubule attachment strength. We propose that Cdk1 activity promotes Dam1c oligomerization to ensure that kinetochore-microtubule attachments are stabilized as kinetochores come under tension in mitosis.